Difference between revisions of "Team:Valencia UPV/Notebook/Content"
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− | <h2 | + | <h2>Notebook</h2> |
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<ul class="actions"> | <ul class="actions"> | ||
<li><a href="https://2015.igem.org/Team:Valencia_UPV/Notebook#scroll1" class="button">Protocols</a></li> | <li><a href="https://2015.igem.org/Team:Valencia_UPV/Notebook#scroll1" class="button">Protocols</a></li> | ||
<li><a href="https://2015.igem.org/Team:Valencia_UPV/Notebook/Content#scroll1" class="button">Daily notebook</a></li> | <li><a href="https://2015.igem.org/Team:Valencia_UPV/Notebook/Content#scroll1" class="button">Daily notebook</a></li> | ||
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</ul> | </ul> | ||
</section> | </section> | ||
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<section id="main" class="container"> | <section id="main" class="container"> | ||
− | <div class="row" style="font-size:initial"> | + | <div id="scroll1" class="row" style="font-size:initial"> |
<div class="12u"> | <div class="12u"> | ||
<section class="box"> | <section class="box"> | ||
<header class="major"> | <header class="major"> | ||
− | <h2> | + | <h2>Daily notebook<br /> |
</h2><hr> | </h2><hr> | ||
</header> | </header> | ||
− | <p | + | <p>Here we present you all the procedures we did to develop our project. On this page you can find the notebook contents. If preferred, you can go directly to the protocols by pressing in the buttons above or below. We hope you enjoy reading our incredible journey!</p> |
− | + | ||
− | + | ||
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− | + | ||
− | + | ||
+ | <br/> | ||
<div> | <div> | ||
<details id="scrollsect1"> | <details id="scrollsect1"> | ||
<summary class="button fit">June</summary> | <summary class="button fit">June</summary> | ||
<div class="clsPadding"> | <div class="clsPadding"> | ||
− | <p> | + | |
− | + | <h3 style="color:green">27 May 2015</h3> | |
+ | |||
+ | |||
+ | |||
+ | <p>Starts our work in the lab! </p> | ||
+ | |||
+ | <p>Marta, a lab mate gives us a construction, the red toggle swich (E:PIF6:PhyB:VP16:Etr8:luc), we just have to add the renilla; α2 (GB160) to test it.</p> | ||
+ | |||
+ | <p>Make the ligation (step 2 in the protocol):</p> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>E:PIF6:PhyB:VP16:Etr8:luc+ren; Ω1</td></tr> | ||
+ | |||
+ | <tr><td>1µL E:PIF6:PhyB:VP16:Etr8:luc; α1</td></tr> | ||
+ | |||
+ | <tr><td>1µL renilla; apha2</td></tr> | ||
+ | |||
+ | <tr><td>1µL Ω1</td></tr> | ||
+ | |||
+ | <tr><td>6,8µL H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">28 May 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Electroporation (step 3 in the protocol) of <i>E. coli</i> to insert our first construction.</p> | ||
+ | |||
+ | <p>Make a petri dish culture with a LB-Agar plate with streptomycin.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">29 May 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>There is no white colonies, we electroporate again and make petri dish culture.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">30 May 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>There was just one white colony, make the ligation again.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>E:PIF6:PhyB:VP16:Etr8:luc+ren; Ω1</td></tr> | ||
+ | |||
+ | <tr><td>0.5µL E:PIF6:PhyB:VP16:Etr8:luc; α1</td></tr> | ||
+ | |||
+ | <tr><td>1µL renilla; apha2</td></tr> | ||
+ | |||
+ | <tr><td>1µL Ω1</td></tr> | ||
+ | |||
+ | <tr><td>7.2µL H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">1 June 2015</h3> | ||
+ | |||
+ | <p>Electroporation of the new ligation.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">2 June 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>There are white colonies. Make 2 liquid cultures of them (Step 4 in the protocol). Add 3.5ml of LB and 3.5µL of spectomycin.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make liquid culture of just some glycerinates:</p> | ||
+ | |||
+ | <ul><li>α1</li> | ||
+ | |||
+ | <li>α2</li> | ||
+ | |||
+ | <li>Ω1</li> | ||
+ | |||
+ | <li>Ω2</li> | ||
+ | |||
+ | <li>pUPD2</li> | ||
+ | |||
+ | <li>E:PIF6:NLS; pUPD2 (GB0288)</li> | ||
+ | |||
+ | <li>E:PIF6:NLS; α1 (GB892)</li> | ||
+ | |||
+ | <li>E:PIF6:NLS; Ω2 (GB893)</li> | ||
+ | |||
+ | <li>E:PIF6:NLS:luc:PhyB; α1 (GB896)</li> | ||
+ | |||
+ | <li>Luc:PhyB; Ω1 (GB890)</li> | ||
+ | |||
+ | <li>PhyB:VP16; pUPD2 (GB289)</li> | ||
+ | |||
+ | <li>PhyB:VP16; α2 (GB88E)</li> | ||
+ | |||
+ | <li>Etr8:CMVmini; pUPD2 (GB1097)</li> | ||
+ | |||
+ | <li>OpLexA:mini35S; pUPD2 (GB733)</li> | ||
+ | |||
+ | <li>OpLexA:mini35S:luc:Tnos; α2</li> | ||
+ | |||
+ | <li>LexABD; pUPD2 (GB0732)</li> | ||
+ | |||
+ | <li>LacI for N-Tfusion; pUPD2 (GB858)</li> | ||
+ | |||
+ | <li>Linker:LacIBD; pUPD2 (GB704)</li> | ||
+ | |||
+ | <li>OpLacI:mini35S:luc:Tnos; α2 (GB152)</li> | ||
+ | |||
+ | <li>OpLacI:mini35S; pPUD2 (GB534)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">3 June 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Al cultures have grown except for Ω2. Make minipreps (Step 5 in the protocol).</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Digestion of the minipreps (Step 6 of the protocol).</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>α1</td><td>-</td><td>-</td></tr> | ||
+ | |||
+ | <tr><td>α2</td><td>-</td><td>-</td></tr> | ||
+ | |||
+ | <tr><td>Ω1</td><td>-</td><td>-</td></tr> | ||
+ | |||
+ | <tr><td>pUPD2</td><td>-</td><td>-</td></tr> | ||
+ | |||
+ | <tr><td>E:PIF6:NLS; pUPD2 (GB0288)</td><td>EcoRI</td><td>3000, 1000</td></tr> | ||
+ | |||
+ | <tr><td>E:PIF6:NLS; α1 (GB892)</td><td> EcoRI</td><td>6300, 2500</td></tr> | ||
+ | |||
+ | <tr><td>E:PIF6:NLS; Ω2 (GB893)</td><td>EcoRV</td><td>1800, 6600, 900</td></tr> | ||
+ | |||
+ | <tr><td>E:PIF6:NLS:luc:PhyB; α1 (GB896)</td><td>EcoRI</td><td>3600, 6300, 5600</td></tr> | ||
+ | |||
+ | <tr><td>Luc:PhyB; Ω1 (GB890)</td><td>BamHI</td><td>2300, 6300, 4200</td></tr> | ||
+ | |||
+ | <tr><td>PhyB:VP16; pUPD2 (GB289)</td><td>EcoRI</td><td>3000, 2000, 500</td></tr> | ||
+ | |||
+ | <tr><td>PhyB:VP16; α2 (GB88E)</td><td>HindIII</td><td>2100, 6300, 1800</td></tr> | ||
+ | |||
+ | <tr><td>Etr8:CMVmini; pUPD2 (GB1097)</td><td>EcoRI</td><td>3000, 480</td></tr> | ||
+ | |||
+ | <tr><td>OpLexA:mini35S; pUPD2 (GB733)</td><td>EcoRI</td><td>3000, 460</td></tr> | ||
+ | |||
+ | <tr><td>OpLexA:mini35S:luc:Tnos; α2 (GB151)</td><td>HindIII</td><td>2500</td></tr> | ||
+ | |||
+ | <tr><td>LexABD; pUPD2 (GB0732)</td><td>EcoRI</td><td>3000, 300</td></tr> | ||
+ | |||
+ | <tr><td>LacI for N-Tfusion; pUPD2 (GB858)</td><td>EcoRI</td><td>3000, 1000</td></tr> | ||
+ | |||
+ | <tr><td>Linker:LacIBD; pUPD2 (GB704)</td><td>EcoRI</td><td>3000, 1000</td></tr> | ||
+ | |||
+ | <tr><td>OpLacI:mini35S:luc:Tnos; α2 (GB152)</td><td>HindIII</td><td>2500, 2600</td></tr> | ||
+ | |||
+ | <tr><td>OpLacI:mini35S; pPUD2 (GB534)</td><td>EcoRI</td><td>3000, 560</td></tr> | ||
+ | |||
+ | <tr><td>E:PIF6:PhyB:VP16:Etr8:luc+ren; Ω1</td><td>BamHI</td><td>3700, 6100, 6600, 4200</td></tr> | ||
+ | |||
+ | <tr><td>E:PIF6:PhyB:VP16:Etr8:luc+ren; Ω1</td><td>EcoRV</td><td>11000, 400, 2500, 3000, 4000</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Make the gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>pUPD2</td><td>Alf</td><td>Alpha1</td><td>288</td><td>289</td><td>534</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>?</td></tr> | ||
+ | |||
+ | <tr><td>704</td><td>732</td><td>733</td><td>858</td><td>892</td><td>896</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td></tr> | ||
+ | |||
+ | <tr><td>1097</td><td>Alpha2</td><td>88E</td><td>151</td><td>152</td><td>Omega1</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td></tr> | ||
+ | |||
+ | <tr><td>890</td><td>893</td><td>Red toggle (C1) (EcoRV)</td><td>Red toggle (C1) (BamHI)</td><td>Red toggle (C2) (EcoRV)</td><td>Red toggle (C2) (BamHI)</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td>no</td><td>no</td><td>no</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">4 June 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Ask for the NDronpa sequence. This will be part of our blue toggle. </p> | ||
+ | |||
+ | <p>This piece is known by reading the paper ‘Reversible photoswichable Dronpa-1 monitors nucleocytoplasmic transport of an RNA-binding protein in transgenic plants?(Doi: 10.111/j.1600-0854.2011.01180.lambda.).</p> | ||
+ | |||
+ | <p>The sequence of NDronpa is plant optimised and avoid cryptic sequences. We have domesticated this sequence with a linker in N-terminal to allow us to join it to a binding domain and also we had a NLS in the C-terminal to transport itself to the nucleus. It is domesticated as B5 part for Golden Braid assembling. </p> | ||
+ | |||
+ | <p>After obtaining the sequence we compare the protein in Uniprot and we can observed that our sequence add a V in the position 2. We compare this results with other papers and none of them has this addition. When we compare this sequence with the paper ?Optical control protein activity by fluorescent protein domains?(Doi: 10.1126/science.1226854) we observed that our position 146 is the position 145 and as what we want is the interaction caused by the N145-K145, we eliminate the V. We also eliminate a pair of amino acids at the end of the sequence following the same criteria. </p> | ||
+ | |||
+ | <p>Once obtained both variants of Dronpa, we decided to add the binding domain to KDronpa and the activation to NDronpa as this last one tetramerizes and all operator sequence are repeated in our promoters.</p> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">5 June 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>We had 2 cultures from the last day, corresponding to other 2 colonies of ligation. </p> | ||
+ | |||
+ | <p><i>Agrobacterium</i> culture of promoter less: Luciferase + Renilla </p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Minipreps</p> | ||
+ | |||
+ | <p>Digestion with BamHI and EcoRV</p> | ||
+ | |||
+ | <p>Agarose gel 1%</p> | ||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/1/1c/Valencia_upv_gel_150605.png> | ||
+ | |||
+ | |||
+ | |||
+ | <p>How to ask and make primers?</p> | ||
+ | |||
+ | <ul><li>Select the sequence to amplify and save in FASTA format.</li> | ||
+ | |||
+ | <li>gbCloning, go to Tools-Domesticator-1?Category</li> | ||
+ | |||
+ | <li>Add FASTA and select parts.</li> | ||
+ | |||
+ | <li>On the protocol we have the primers </li> | ||
+ | |||
+ | <li>The oligos they give us:</li> | ||
+ | |||
+ | <ul class="ul_2"><li>4 first nucleotides: so the enzyme can recognize without problems</li> | ||
+ | |||
+ | <li>6 following bingind sites.</li> | ||
+ | |||
+ | <li>1 extra nucleotide.</li> | ||
+ | |||
+ | <li>4 overhangs. </li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Meeting with Daniel Ramón (Biopolis). </p> | ||
+ | |||
+ | <p>Ligations:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PIF6 + PhyB; Ω1</td><td>Etr8(CMV)+BxbI:T35S; α1</td></tr> | ||
+ | |||
+ | <tr><td>1µL (GB892) PIF; α1</td><td>1µL (GB1097) Etr8(CMV); pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1µL (GB88E) PhyB; α2</td><td>1µL BxbI; pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1µL Ω1 </td><td>1µL Tnos pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>6.8µL H<sub>2</sub>O</td><td>1µL α1</td></tr> | ||
+ | |||
+ | <tr><td></td><td>5.8µL H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Digestions:</p> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>(GB160) 35S:Renilla:tNOS-35S:P19:tNOS</td><td>EcoRV</td><td>2475, 381, 4601</td></tr> | ||
+ | |||
+ | <tr><td>(GB896) Luc:PIF6:PhyB</td><td>EcoRV</td><td>11608, 3942</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">6 June 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transform to <i>E. coli</i> from PIF+Phy and BxbI and make petri dish cultures.</p> | ||
+ | |||
+ | <p>Digest of 160, 289 and the two ligations, PIF+phy and Etr8+BxbI. </p> | ||
+ | |||
+ | <p>Agarose gel. </p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>GB160</td><td>289</td><td>PIF+PhyB</td><td>BxbI </td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>no</td><td>?</td><td>?</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/4/46/Valencia_upv_gel_150606.png> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">7 June 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>We’ve got white colonies from PIF+Phy and BxbI!</p> | ||
+ | |||
+ | <p>Pick two colonies from each construction.</p> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">8 June 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Minipreps of the 4 liquid cultures and digestion to see the band patterns.</p> | ||
+ | |||
+ | <p>Digestion:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Etr8(CMV):Bxb1:Tnos; α1</td><td>EcoRI</td><td>6345, 238</td></tr> | ||
+ | |||
+ | <tr><td>EPIF6 + PhyB-PV16; Ω1</td><td>BamHI</td><td>6686, 1439, 2685, 2237</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Agarose gel was made:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>BxbI (C1)</td><td>BxbI (C2)</td><td>E:PIF6+PhyB-VP16 (C1)</td><td>E:PIF6+PhyB-PV16 (C2)</td><td></td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>no</td><td>no</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/5/58/Valencia_upv_gel_150608.png> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Repeat digestion because we are not sure of the last digestions.</p> | ||
+ | |||
+ | <p>We don’t have sure the toggle, so we decide to repeat the digestion with other enzyme tomorrow, noticing that the colony 2 has better bands pattern.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Optimized ligation:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PIF-PhyB-Luc-Renilla-P19</td></tr> | ||
+ | |||
+ | <tr><td>1 µL vector</td></tr> | ||
+ | |||
+ | <tr><td>0.8 µL dilution ?GB160</td></tr> | ||
+ | |||
+ | <tr><td>1.7 µL PIF:PhyB</td></tr> | ||
+ | |||
+ | <tr><td>4.15 µL H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | <tr><td>Ratio 1:2 vector insert</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>As BxbI was good at the digestion we put 1 µL of LB and 1 µL of Kanamicyne on the tube where it had grown and store at 37ºC to glycerinate later.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>We design primers to binding domain (BD) and PIF.</p> | ||
+ | |||
+ | <ul><li>Problem: domesticator is introduced in an old pUPD2. The new one has different bases. </li> | ||
+ | |||
+ | <li>Change manually the pUPD2 bases in the program (Benchling).</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | </br><h3 style="color:green">9 June 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Digestion of the ligation of yesterday containing: EPIF6-PhyB-VP16 (C1 y C2)</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>EPIF6-PhyB-VP16</td><td>PvuII (green buffer)</td><td>3663, 9472pb</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Agarose gel 1%:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>EPIF6-PhyB-VP16 (C1)</td><td>EPIF6-PhyB-VP16 (C2)</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/a/a0/Valencia_upv_gel_150609.png> | ||
+ | |||
+ | <p>We see three bands: 7000, 4000, 1900pb</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transform optimized ligation PIF-Phy-Luc-Renilla-P19 and make petri dish cultures.</p> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">10 June 2015</h3> | ||
+ | |||
+ | <ul><li>Check the primers and order LexA, Gal4, PIF6, LacI, Dronpa.</li> | ||
+ | |||
+ | <li>Check linker VP16 (88E) and make a primer for it.</li> | ||
+ | |||
+ | <li>Take out glycerinate of Ω2.</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>Alfredo’s part is not working.</p> | ||
+ | |||
+ | <ul><li>Make liquid culture of E:PIF6:PhyB:VP16:luc:ren (C1-C3).</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <ul><li>Minipreps of liquid culture (PIF + Phy), colonies C3, C4, C5, C6</li> | ||
+ | |||
+ | <li>Digestion:</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PIF+Phy:VP16</td><td>PvuII (buffer green 10x)</td><td>3663, 9472</td></tr> | ||
+ | |||
+ | <tr><td>PIF+Phy:VP16</td><td>BamHI</td><td>1939, 2685, 2337, 6674</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>Agarose gel 1%:</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/e/ea/Valencia_upv_gel_150610.png> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PIF + Phy (PvuII) C3</td><td>PIF + Phy (PvuII) C4</td><td>PIF + Phy (PvuII) C5</td><td>PIF + Phy (PvuII) C6</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>ok</td><td>no</td><td>No</td></tr> | ||
+ | |||
+ | <tr><td>PIF + Phy (BamHI) C3</td><td>PIF + Phy (BamHI) C4</td><td>PIF + Phy (BamHI) C5</td><td>PIF + Phy (BamHI) C6</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>ok</td><td>no</td><td>No</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>Transformation into <i>Agrobacterium</i>EPIF6-PhyB-VP16 + luciferase (GB896) and make petri dish culture. We are not going to have the positive control (renilla+P19) and we won’t be able to quantify and make ratios.</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | </br><h3 style="color:green">11 June 2015</h3> | ||
+ | |||
+ | <ul><li>Minipreps of the culture:</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <ul><li>Digestion:</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>E:PIF6:PhyB:VP16:luc:ren</td><td>BamHI</td><td>4209, 3756, 6100, 6674</td></tr> | ||
+ | |||
+ | <tr><td></td><td>EcoRV</td><td>3942, 2989, 2475, 381, 10952</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PIF6:PhyB:VP16:luc:ren C1 (BamHI)</td><td>PIF6:PhyB:VP16:luc:ren C3 (BamHI)</td><td>PIF6:PhyB:VP16:luc:ren C1 (EcoRV)</td><td>PIF6:PhyB:VP16:luc:ren C3 (EcoRV)</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>no</td><td>no</td><td></td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/6/6a/Valencia_upv_gel_150611.png> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transformation into <i>Agrobacterium</i>of Renilla (GB160) because we could not join this construction with PIF:PhyB and so we will do a cotransfection of both plasmids.Make petri dish culture.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">12 June 2015</h3> | ||
+ | |||
+ | <p>The petri dish with PIF:PhyB:luc was taken out the 37ºC room and put into the fridge to pick colonies tomorrow.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">13 June 2015</h3> | ||
+ | |||
+ | <p>Pick colonies to make liquid culture:</p> | ||
+ | |||
+ | <ul><li>Renilla in agrobacterium: just one colony, it was made liquid culture but check carefully the gel.</li> | ||
+ | |||
+ | <li>It was noticed that the piece 160, renilla, needs a pSub plasmid to replicate itself so we will transform 160 into a agrobacterium with this plasmid (C58 pSub).</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>BxbI; α1+PhyB; α2</td></tr> | ||
+ | |||
+ | <tr><td>1µl BxbI</td></tr> | ||
+ | |||
+ | <tr><td>1 µl PhyB</td></tr> | ||
+ | |||
+ | <tr><td>1 µl Ω2</td></tr> | ||
+ | |||
+ | <tr><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>Transform renilla (160) with pSub plasmid into agrobacterium and make petri dish culture. </li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | </br><h3 style="color:green">15 June 2015</h3> | ||
+ | |||
+ | <ul><li>Repeat the ligation BxbI+35S:E-PIF6:tnos because PIF was Ω2</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>BxbI + 35S:E-PIF6:tnos; Ω1</td></tr> | ||
+ | |||
+ | <tr><td>1µl BxbI</td></tr> | ||
+ | |||
+ | <tr><td>1 µl PhyB</td></tr> | ||
+ | |||
+ | <tr><td>1 µl Ω1</td></tr> | ||
+ | |||
+ | <tr><td>4.6</td><td>µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>KDronpa has arrived:</li> | ||
+ | |||
+ | <ul class="ul_2"><li>Centrifuge it 2-5sec at maximum velocity.</li> | ||
+ | |||
+ | <li>Add 50 µl to have a concentration of 20ng/µl</li> | ||
+ | |||
+ | <li>Mix it with the vortex and spin.</li> | ||
+ | |||
+ | </ul><li>Ligation:</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>KDronpa; pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1 µl KDronpa</td></tr> | ||
+ | |||
+ | <tr><td>1 µl pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>5.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <ul><li>It was not possible to pick colonies of the <i>Agrobacterium</i> transformed with renilla because they did not grow. Maybe the problem is that with tetraciclyn bacterias grow slowly. Wait 1 day more.</li> | ||
+ | |||
+ | <li>Transformation of the ligation, BxbI+35S:E-PIF6:tnos; Ω1, into <i>E. coli</i>.Make petri dish culture.</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | </br><h3 style="color:green">16 June 2015</h3> | ||
+ | |||
+ | <ul><li>Transformation of the ligation, KDronpa, into <i>E. coli</i>.</li> | ||
+ | |||
+ | <li>Pick colonies of BxbI:E-PIF6 and make liquid culture (C1-C3).</li> | ||
+ | |||
+ | <li>Primers had arrived, it has been done the resuspension (dilution 1:10) of all of them.</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Primers</td><td>Code </td><td>Template</td><td>Working temperature (ºC)</td></tr> | ||
+ | |||
+ | <tr><td>LacI F</td><td>1</td><td>LacI (858)</td><td>69.7</td></tr> | ||
+ | |||
+ | <tr><td>LacI R </td><td>2</td><td></td></tr> | ||
+ | |||
+ | <tr><td>Gal4 F</td><td>3</td><td>We did not take out the glicerynate.</td><td>63.2</td></tr> | ||
+ | |||
+ | <tr><td>Gal4 </td><td>4</td><td></td></tr> | ||
+ | |||
+ | <tr><td>LexA F</td><td>5</td><td>LexA (732)</td><td>62.7</td></tr> | ||
+ | |||
+ | <tr><td>LexA R</td><td>6</td><td></td></tr> | ||
+ | |||
+ | <tr><td>PIF:VP16 F</td><td>7</td><td>PIF6 (288)</td><td>60.1</td></tr> | ||
+ | |||
+ | <tr><td>PIFVP16 R</td><td>8</td><td></td></tr> | ||
+ | |||
+ | <tr><td>NDronpa F1</td><td>9</td><td>Kdronpa</td><td>67.7</td></tr> | ||
+ | |||
+ | <tr><td>NDronpa R1</td><td>10</td><td></td></tr> | ||
+ | |||
+ | <tr><td>Dronpa F2</td><td>11</td><td>58.5</td></tr> | ||
+ | |||
+ | <tr><td>NDronpa R2</td><td>12</td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>A PCR with all the primers and the fragments was done, the samples were put in order following the temperature gradient.</li> | ||
+ | |||
+ | <ul class="ul_2"><li>The templates were in dilution 1:50, exception of KDronpa that was dilution 1:5 and the primers 1:10.</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PCR Fusion Taq (50µl)</td></tr> | ||
+ | |||
+ | <tr><td>DNA template (10 µg/µl)</td></tr> | ||
+ | |||
+ | <tr><td>0.5 µl fusion taq</td></tr> | ||
+ | |||
+ | <tr><td>2.5 µl primer F</td></tr> | ||
+ | |||
+ | <tr><td>2.5 µl primer R</td></tr> | ||
+ | |||
+ | <tr><td>2 µl NTPs</td></tr> | ||
+ | |||
+ | <tr><td>31.5 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">17 June 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>Pick colonies and make liquid culture of:</li> | ||
+ | |||
+ | <ul class="ul_2"><li>KDronpa (C1-C4)</li> | ||
+ | |||
+ | </ul><li>Ligations with the PCR’s products:</li> | ||
+ | |||
+ | <ul class="ul_2"><li>Templates PCR: 1+2, 5+6, 7+8PIF, 7+8VP16, 9+10, 11+12.</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Template PCR; pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>0.5µl template</td></tr> | ||
+ | |||
+ | <tr><td>1µl pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>6.1µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <ul><li>Minipreps of liquid cultures:</li> | ||
+ | |||
+ | <ul class="ul_2"><li>BxbI:E-PIF6 (C1-C3)</li> | ||
+ | |||
+ | </ul><li>Agarose gel with the PCRs:</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/3/3a/Valencia_upv_gel_150617.png> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Template</td><td>1+2</td><td>5+6</td><td>7+8PIF</td><td>7+8VP16</td><td>9+10</td><td>11+12</td></tr> | ||
+ | |||
+ | <tr><td>Band pattern</td><td>1017</td><td>284</td><td>391</td><td>478</td><td>464</td><td>290</td></tr> | ||
+ | |||
+ | <tr><td>Gel result</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>No DNA</td><td>ok</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>Transformation in <i>E. coli</i> of the correct ligations and make petri dishes cultures:</li> | ||
+ | |||
+ | <ul class="ul_2"><li>1+2, 5+6, 7+8PIF, 7+8VP16, 11+12 </li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | </br><h3 style="color:green">18 June 2015</h3> | ||
+ | |||
+ | <ul><li>Minipreps of the liquid cultures:</li> | ||
+ | |||
+ | <ul class="ul_2"><li>KDronpa (C1-C4) </li> | ||
+ | |||
+ | </ul><li>Digestions:</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>KDronpa EcoRI 2800</p> | ||
+ | |||
+ | <ul><li>Gel:</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Kdronpa C1</td><td>Kdronpa C2</td><td>Kdronpa C3</td><td>Kdronpa C4</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>ok</td><td>no</td></tr> | ||
+ | |||
+ | <tr><td>Etr8:BxbI:phyB C1</td><td>Etr8:BxbI:phyB C2</td><td>Etr8:BxbI:phyB C3</td><td></td></tr> | ||
+ | |||
+ | <tr><td>No</td><td>no</td><td>no</td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/1/12/Valencia_upv_gel_150618.png> | ||
+ | |||
+ | |||
+ | |||
+ | <p>We discovered that the construction with BxbI did not go well because our lab college gives us the wrong piece. Thanks Alfredo :)</p> | ||
+ | |||
+ | <ul><li>Take glicerynates out:</li> | ||
+ | |||
+ | <ul class="ul_2"><li>Gal4; pUPD2 (GB731)</li> | ||
+ | |||
+ | <li>Ω2</li> | ||
+ | |||
+ | <li>NoATGPromoter (GB00552)</li> | ||
+ | |||
+ | <li>Renilla (GB160)(GB159)(GB109)</li> | ||
+ | |||
+ | </ul><li>PCR:</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>NDronpa</td></tr> | ||
+ | |||
+ | <tr><td>2.5 µl (9+10) primer F</td></tr> | ||
+ | |||
+ | <tr><td>2.5 µl (11+12) primer R</td></tr> | ||
+ | |||
+ | <tr><td>2 µl NTPs</td></tr> | ||
+ | |||
+ | <tr><td>0.2 µl Taq</td></tr> | ||
+ | |||
+ | <tr><td>10 µl Buffer</td></tr> | ||
+ | |||
+ | <tr><td>31.5</td><td>µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <ul><li>Ligations:</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Etr8:BxbI:T35S; α1</td><td>Template PCR; pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1 µlEtr8</td><td>0.5µl template</td></tr> | ||
+ | |||
+ | <tr><td>1 µl BxbI</td><td>1µl pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1 µl T35S</td><td>6.1µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | <tr><td>1 µl α1</td><td></td></tr> | ||
+ | |||
+ | <tr><td>5.8 µl H<sub>2</sub>O</td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Templates PCR: 1+2, 5+6, 7+8PIF, 7+8VP16</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">19 June 2015</h3> | ||
+ | |||
+ | <ul><li>We do a PCR with the normal Taq polymerase.</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>1µl of DNA’s template (9+10, 9+12 and 11+12)</td></tr> | ||
+ | |||
+ | <tr><td>2µl of specific buffer</td></tr> | ||
+ | |||
+ | <tr><td>2µl of NTPs</td></tr> | ||
+ | |||
+ | <tr><td>1µl primer forward</td></tr> | ||
+ | |||
+ | <tr><td>1µl primer reverse</td></tr> | ||
+ | |||
+ | <tr><td>0.5 µl of Taq</td></tr> | ||
+ | |||
+ | <tr><td>12.5 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>These quantities multiplied by 3.</p> | ||
+ | |||
+ | <ul><li>Minipreps of the yesterday’s glycerinated cultures.</li> | ||
+ | |||
+ | <ul class="ul_2"><li>Gal4; pUPD2 (GB731)</li> | ||
+ | |||
+ | <li>Ω2</li> | ||
+ | |||
+ | <li>NoATGPromoter (GB00552)</li> | ||
+ | |||
+ | <li>Renilla (GB160)(GB159)(GB109)</li> | ||
+ | |||
+ | </ul><li>Do the glycerinates digestions:</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Minipreps:</td><td>Enzime</td><td>Band pattern</td></tr> | ||
+ | |||
+ | <tr><td>(GB159) pDGB1_Ω2 renilla</td><td>EcoRV</td><td>2909, 2475,882, 812, 381</td></tr> | ||
+ | |||
+ | <tr><td>Entry vector, Ω2</td><td>EcoRV</td><td>6652, 621</td></tr> | ||
+ | |||
+ | <tr><td>(GB552) pP35s NoATG; pUPD2</td><td>EcoRI</td><td>2997, 1090</td></tr> | ||
+ | |||
+ | <tr><td>(GB160) renilla pDGB1, α2 </td><td>EcoRV</td><td>4601, 2475, 381</td></tr> | ||
+ | |||
+ | <tr><td>(GB731) Gal4BD (CDS); pUPD2</td><td>EcoRI</td><td>2997, 2493</td></tr> | ||
+ | |||
+ | <tr><td>(GB109)</td><td>355:renilla:Tnos; α1</td><td>EcoRI</td><td>2580, 2493</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>We make an agarose gel with the digestions made before and the PCR of KDronpa. </li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>159</td><td>160</td><td>Ω2</td><td>552</td><td>731</td><td>109</td><td>9+10</td><td>9+12</td><td>11+12</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>no</td><td>ok</td><td>ok</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/0/0d/Valencia_upv_gel_150619.png> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>Transformation into <i>E. coli</i> of ligations:</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>1+2, 5+6, 7+8PIF, 7+8VP16 all in pUPD2</p> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>We made an stack of Cloranfenicol petri dishes</li> | ||
+ | |||
+ | <ul class="ul_2"><li>250ml LB agar</li> | ||
+ | |||
+ | <li>X-Gal (1:500): 500 µl</li> | ||
+ | |||
+ | <li>IPTG (1:1000): 250 µl</li> | ||
+ | |||
+ | <li>Cloranfenicol (1:2000): 125 µl</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | </br><h3 style="color:green">20 june 2015</h3> | ||
+ | |||
+ | <p>We have white colonies of renilla! Also of Etr8+BxbI; α1</p> | ||
+ | |||
+ | <p>We have also pUPD2 colonies but they are so close to the blue ones that we can’t pick anyone.So we make strakes.</p> | ||
+ | |||
+ | <ul><li>We make a liquid culture of <i>Agrobacterium</i>of Renilla (rif/kan/tetr).</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">21 June 2015</h3> | ||
+ | |||
+ | <ul><li>Pick colonies and make liquid culure of (all colonies are in pUPD2):</li> | ||
+ | |||
+ | <ul class="ul_2"><li>Plates : PIF (17/06/15) (C1 and C2)</li> | ||
+ | |||
+ | <li>VP16 (C1 and C3)</li> | ||
+ | |||
+ | <li>LacI (C1-C3)</li> | ||
+ | |||
+ | <li>Plates (19/06/15): BxbI (C1, C2, C3), </li> | ||
+ | |||
+ | <li>VP16 (C4, C5)</li> | ||
+ | |||
+ | <li>LacI (C1, C2)</li> | ||
+ | |||
+ | <li>PIF (C1-C5) </li> | ||
+ | |||
+ | <li>LexA (C1, C2)</li> | ||
+ | |||
+ | </ul><li>We take out two glicerynates of GFP and BFP (of the Alfredo’s box)</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | </br><h3 style="color:green">22 June 2015</h3> | ||
+ | |||
+ | <ul><li>We made minipreps of the liquid culture of the day before:</li> | ||
+ | |||
+ | <ul class="ul_2"><li>LacIBD; pUPD2 (C1-C5)</li> | ||
+ | |||
+ | <li>LexABD; pUPD2 (C1, C2)</li> | ||
+ | |||
+ | <li>Etr8(CMV):Bxb1 (C1-C3)</li> | ||
+ | |||
+ | <li>PIF6; pUPD2 (C1-C5)</li> | ||
+ | |||
+ | <li>VP16; pUPD2 (C1, C4, C5)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <ul><li>Make the digestions of all the minipreps:</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LacIBD, pUPD2</td><td>NotI</td><td>2046, 1053</td></tr> | ||
+ | |||
+ | <tr><td>LexABD, pUPD2 </td><td>NotI</td><td>2046, 321</td></tr> | ||
+ | |||
+ | <tr><td>Etr8(CMV):Bxb1 </td><td>NotI</td><td>1532, 1290, 5896</td></tr> | ||
+ | |||
+ | <tr><td>PIF6,pUPD2 </td><td>NotI</td><td>2046, 407</td></tr> | ||
+ | |||
+ | <tr><td>VP16, pUPD2 </td><td>NotI</td><td>2046, 500</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>Refresh the viral system that a lab mate borrow to us. This is going to be use to agroinfiltrate some plants to make some cool draws to sent to a TV programm so they can watch what are we doing. This cultures consist of three parts divided in three <i>Agrobacterium</i>colonies. They are the citoplasm, the fluerescent protein (GFP, DsRed or YFP) and the integrase, in our case PhiC31.</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>We received the reporter BxbI (RepBxbI)!</li> | ||
+ | |||
+ | <ul class="ul_2"><li>500ng of sample</li> | ||
+ | |||
+ | <li>Centrifuge at 3000rpm for 5 seconds (spin).</li> | ||
+ | |||
+ | <li>Add 50 µl H<sub>2</sub>O</li> | ||
+ | |||
+ | <li>Shake it and let at 50ºC for 20min</li> | ||
+ | |||
+ | </ul><li>Make a PCR of Gal4 and NDronpa (9-10), the primers of NDronpa are aliquoted.</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make an agarose gel with all the digestions:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LacI C1</td><td>LacI C2</td><td>LacI C3</td><td>LacI C4</td><td>LacI C5</td><td>LexA C1</td><td>LexA C2</td><td>BxbI C1</td><td>BxbI C2</td><td>BxbI C3</td></tr> | ||
+ | |||
+ | <tr><td>Ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>no</td><td>no</td><td>ok</td><td>ok</td><td>no</td></tr> | ||
+ | |||
+ | <tr><td>PIF C1</td><td>PIF C2</td><td>PIF C3</td><td>PIF C4</td><td>PIF C5</td><td>VP16 C1</td><td>VP16 C4</td><td>VP16 C5</td><td></td></tr> | ||
+ | |||
+ | <tr><td>No</td><td>no</td><td>-</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td></td></tr> | ||
+ | |||
+ | <tr><td>Gal4</td><td>NDronpa 1</td><td>NDronpa 2</td><td></td><td></td></tr> | ||
+ | |||
+ | <tr><td></td><td></td><td></td><td></td><td></td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>FOTO </p> | ||
+ | |||
+ | <ul><li>We make ligations of:</li> | ||
+ | |||
+ | <ul class="ul_2"><li>Etr8(CMV):BxbI; α1 + PhyB:VP16;α2; Ω1</li> | ||
+ | |||
+ | <li>LacIBD;pUPD2 + PIF6BDPless; pUPD2; α1</li> | ||
+ | |||
+ | <li>KDronpa;pUPD2 + LacIBD; pUPD2; α1</li> | ||
+ | |||
+ | <li>Gal4BD; pUPD2</li> | ||
+ | |||
+ | <li>Reporter of BxbI; pUPD2</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <ul><li>Tomorrow we have to take out pUPD2 of constitutive promoters, terminators and GFP (CDS).</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | </br><h3 style="color:green">23 June 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>Transform into E.Coli the 5 ligations done yesterday and two more transformations of 5+6(1) and 5+6(2) which are the ligations in pUPD2 of the 18/06. </li> | ||
+ | |||
+ | <ul class="ul_2"><li>Etr8(CMV):BxbI; α1 + PhyB:VP16;α2; Ω1</li> | ||
+ | |||
+ | <li>LacIBD;pUPD2 + PIF6BDPless; pUPD2; α1</li> | ||
+ | |||
+ | <li>KDronpa;pUPD2 + LacIBD; pUPD2; α1</li> | ||
+ | |||
+ | <li>Gal4BD; pUPD2</li> | ||
+ | |||
+ | <li>Reporter of BxbI; pUPD2</li> | ||
+ | |||
+ | <li>LexABD (5+6), pUPD2 (1 and 2)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <ul><li>We have taken out of the -80ºC fridge the glycerinate of GFP; pUPD2 (GB0059)/ampicilin.</li> | ||
+ | |||
+ | <li>The liquid culture of Renilla (ryfampicin/kanamycin/tetracyclin) does not grow after the two days required. So we decide to refresh two new colonies, one of them in a tube with the three antibiotics and another with rifampicina and kanamicine. Asun says that the tetracycline slow down the growth of Agro.</li> | ||
+ | |||
+ | <li>The 4 liquid cultures of LexA+IPTG/+gal are all blue: throw them.</li> | ||
+ | |||
+ | <li>We ordered again the primer n?0 (NDronpa R1). Changing one codon in 3?and delete another in 5?</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | </br><h3 style="color:green">24 June 2015</h3> | ||
+ | |||
+ | <p>Pick colonies of the plates done yesterday and pass them into a liquid medium:</p> | ||
+ | |||
+ | <ul><li>LacIBD+PIF; α1 (C1, C2)</li> | ||
+ | |||
+ | <li>Gal4BD; pUPD2 (C1)</li> | ||
+ | |||
+ | <li>RepBxb1; pUPD2 (C1-C3)</li> | ||
+ | |||
+ | <li>LacIBD+KDonpa; α1 (C1, C2)</li> | ||
+ | |||
+ | <li>Etr8(CMV)+BxbI+PhyB+VP16; Ω1 (C1)</li> | ||
+ | |||
+ | <li>LexABD1; pUPD2 (C1-C4)</li> | ||
+ | |||
+ | <li>LexABD2; pUPD2. No colonies.</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>The viral systems of <i>Agrobacterium</i>cultures to make the color mosaics are ready after 2 days at 28ºC. We can make the agroinfiltration.</p> | ||
+ | |||
+ | <p>Protocol to prepare solution to agroinfiltrate in the protocols notebook part.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>Ligation:</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>ETR8(CMV):BxbI; α1+PhyB:VP16; α2; Ω1 </td><td>Gal4BD(pcr) + pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1.5 µl Etr8:BxbI</td><td>1 µl Gal4 PCR</td></tr> | ||
+ | |||
+ | <tr><td>1.5 µl 88E (PhyB:VP16)</td><td>1 µl pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1 µl Ω1</td><td>5,6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | <tr><td>3.6µl H<sub>2</sub>O</td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Quantification of DNA:</p> | ||
+ | |||
+ | <ul><li>GFP (GB0059); pUPD2: 249 ng/µl</li> | ||
+ | |||
+ | <li>Ω2: 238 ng/µl</li> | ||
+ | |||
+ | <li>Alfredo’s pUPD2, domesticator: 102 ng/µl</li> | ||
+ | |||
+ | <li>iGEM704: 405 ng/µl</li> | ||
+ | |||
+ | <li>iGEM735: 403 ng/µl</li> | ||
+ | |||
+ | <li>552 AMP 35S noATG: 45 ng/µl</li> | ||
+ | |||
+ | <li>PIF (C5), pUPD2: 119 ng/µl</li> | ||
+ | |||
+ | <li>pD6B3, Ω2 (22/06): 158 ng/µl</li> | ||
+ | |||
+ | <li>LacIBD (C1); pUPD2 (22/06): 129 ng/µl</li> | ||
+ | |||
+ | <li>109 renillaDC: 49 ng/µl</li> | ||
+ | |||
+ | <li>IGEM 534: 13.6 ng/µl</li> | ||
+ | |||
+ | <li>VP16 (C1); pUPD2:102 ng/µl</li> | ||
+ | |||
+ | <li>IGEM 1097: 409 ng/µl</li> | ||
+ | |||
+ | <li>KDronpa (C3); pUPD2 (18/06): 174 ng/µl</li> | ||
+ | |||
+ | <li>IGEM 858: 487 ng/µl</li> | ||
+ | |||
+ | <li>731AMP Gal4 (19/06): 81 ng/µl</li> | ||
+ | |||
+ | <li>IGEM pUPD2 domesticator: 87 ng/µl</li> | ||
+ | |||
+ | <li>PIF+PhyB (C1) (08/06): 108 ng/µl</li> | ||
+ | |||
+ | <li>160 renilla, α2 (19/06): 46 ng/µl</li> | ||
+ | |||
+ | <li>159 renilla, Ω2 (19/06): 149 ng/µl</li> | ||
+ | |||
+ | <li>Etr8:BxbI (C1)(22/06): 149 ng/µl</li> | ||
+ | |||
+ | <li>IGEM 732: 422 ng/µl</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">25 June 2015</h3> | ||
+ | |||
+ | <p>Minipreps of the liquid culture:</p> | ||
+ | |||
+ | <ul><li>We don’t observed growth in LacIBD+PIF and LacIBD+KDronpa.</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Digestion of the minipreps and do the gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4BD; pUPD2</td><td>NotI</td><td>2046, 282</td></tr> | ||
+ | |||
+ | <tr><td>RepBxbI; pUPD2</td><td>NotI</td><td>2046, 460</td></tr> | ||
+ | |||
+ | <tr><td>Etr8(CMV):BxbI:PhyB; α1</td><td>BamHI</td><td>6674, 2237, 2806, 1174</td></tr> | ||
+ | |||
+ | <tr><td>LexABD; pUPD2</td><td>NotI</td><td>2046, 321</td></tr> | ||
+ | |||
+ | <tr><td>9+10; pUPD2</td><td>NotI</td><td>464</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Etr8:BxbI</td><td>LexA C1</td><td>LexA C2</td><td>LexA C3</td><td>LexA C4</td><td>RepBxbI C1</td><td>RepBxbI C2</td><td>RepBxbI C3</td><td>Gal4 C1</td><td>PCR 9+10</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>no</td><td>no</td><td>no</td><td>ok</td><td>ok</td><td>ok</td><td>no</td><td>ok</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/2/2c/Valencia_upv_gel_150625.png> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>We make a PCR of the Fusion Taq pH (proof-reading) to prove that the primer received number 10. This new one works! Amplify the sequence of NDronpa (R1).</li> | ||
+ | |||
+ | <li>Refresh the cultures of <i>Agrobacterium</i>with the viral system. Add only ryfampicin and kanamycin.</li> | ||
+ | |||
+ | <li>Ligations:</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>N-dronpa; pUPD2</td><td>RepBxbI; α1</td><td>Gal4BD, pUPD2</td><td>LexABD; pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1 µl PCR 9+10</td><td>1 µl Rep Bxb1</td><td>1 µl PCR 3+4</td><td>1 µl PCR 5+6</td></tr> | ||
+ | |||
+ | <tr><td>1 µl PCR11+12</td><td>1 µl Promoter without ATG</td><td>1 µl pUPD2</td><td>1 µl pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1 µl pUPD2</td><td>1 µl Tnos</td><td></td></tr> | ||
+ | |||
+ | <tr><td></td><td>1 µl α1</td></tr> | ||
+ | |||
+ | <tr><td>4,6 µl H<sub>2</sub>O</td><td>3,6 µl H<sub>2</sub>O</td><td>5,6 µl H<sub>2</sub>O</td><td>5,6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Etr8:BxbI+PhyB; Ω1</td><td></td></tr> | ||
+ | |||
+ | <tr><td>1 µl Etr8:BxbI</td><td></td></tr> | ||
+ | |||
+ | <tr><td>1 µl 88E</td><td></td></tr> | ||
+ | |||
+ | <tr><td>1µl Ω1</td><td></td></tr> | ||
+ | |||
+ | <tr><td>3,6 µl H<sub>2</sub>O</td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transform ligations into E.Coli and make petri dish cultures with cloranfenicol for all of them except the ligation of Etr8:Bxb1+PhyB that goes with streptomycin.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">26 June 2015</h3> | ||
+ | |||
+ | <p>Do ligations: </p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>RepBxbI+GFP; α2</td><td>LacIBD+PIF6; α1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl RepBxbI</td><td>1 µl LacIBD, pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1 µl promoter without ATG</td><td>1 µl PIF6, pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1 µl Tnos</td><td>1 µl promoter</td></tr> | ||
+ | |||
+ | <tr><td>1µl GFP (0059)</td><td>1 µl T35</td></tr> | ||
+ | |||
+ | <tr><td>1 µl α2</td><td>1 µl α1</td></tr> | ||
+ | |||
+ | <tr><td>2.6 µl H<sub>2</sub>O</td><td>2.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | <tr><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Digestion:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LacIBD+PIF6; α1</td><td>EcoRI</td><td>6345, 1997, 641</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LacIBD+PIF C1</td><td>LacIBD+PIF C2</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Both of them present the fragment of the vector at 6000 pb but none of them at 2000bp which is the insert one.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/3/35/Valencia_upv_gel_150626.png> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Measurement of the ODs of PhyB:PIF6:luc and renilla+P19.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PhyB:PIF6:luc: 0.35 (1:2)</td><td>0.35</td><td>1.429 µl</td></tr> | ||
+ | |||
+ | <tr><td>Ren+P19: 0.34 (1:2)</td><td>0.34</td><td>1.412 µl</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>Ligation of: </li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LacIBD; pUPD2+KDronpa; pUPD2; α1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl 35S</td></tr> | ||
+ | |||
+ | <tr><td>1 µl LacIBD;pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1 µl KDronpa; pUPD</td></tr> | ||
+ | |||
+ | <tr><td>1 µl T35S</td></tr> | ||
+ | |||
+ | <tr><td>1 µl α1</td></tr> | ||
+ | |||
+ | <tr><td>2.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>1?EXPERIMENT. Red toggle. E:PIF6:PhyB and renilla. For more info, click here.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">27 June 2015</h3> | ||
+ | |||
+ | <p>Transformation into <i>E. coli</i> of LacIBD+KDronpa; α1 and make petri dish culture.</p> | ||
+ | |||
+ | <p>Make petri dish culture of LexABD and Etr8(CMV):Bxb1:GFP.</p> | ||
+ | |||
+ | <p>We make liquid culture of:</p> | ||
+ | |||
+ | <ul><li>RepBxbI:GFP (C1-C4)</li> | ||
+ | |||
+ | <li>LacIBD+PIF6 (C1-C5)</li> | ||
+ | |||
+ | <li>NDronpa (C1-C4)</li> | ||
+ | |||
+ | <li>Gal4BD (C1-C5)</li> | ||
+ | |||
+ | <li>LexABD (C1-C3)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | </br><h3 style="color:green">28 June 2015</h3> | ||
+ | |||
+ | <p>Do the minipreps of the liquid cultures that have grown.</p> | ||
+ | |||
+ | <ul><li>RepBxbI:GFP (C1 and C2)</li> | ||
+ | |||
+ | <li>LacIBD+PIF6 (C1-C4)</li> | ||
+ | |||
+ | <li>NDronpa (C1-C4)</li> | ||
+ | |||
+ | <li>Gal4BD (C1-C5)</li> | ||
+ | |||
+ | <li>LexA: didn’t grow</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Do the digestions of the minipreps:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LacIBD+PIF; α1</td><td>EcoRI</td><td>6345, 1997, 641</td></tr> | ||
+ | |||
+ | <tr><td>RepBxbI:GFP; Ω2</td><td>HindIII</td><td>6345, 2683</td></tr> | ||
+ | |||
+ | <tr><td>Gal4BD; pUPD2</td><td>NotI</td><td>2681, 644</td></tr> | ||
+ | |||
+ | <tr><td>NDronpa; pUPD2</td><td>NotI</td><td>2046, 744</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Make the gel.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>RepBxbI:GFP C1</td><td>RepBxbI:GFP C2</td><td>LacIBD+PIF C1</td><td>LacIBD+PIF C2</td><td>LacIBD+PIF C3</td><td>LacIBD+PIF C4</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>no</td><td>no</td><td>no</td><td>No</td></tr> | ||
+ | |||
+ | <tr><td>Gal4BD C1</td><td>Gal4BD C2</td><td>Gal4BD C3</td><td>Gal4BD C4</td><td>Gal4BD C5</td><td>N-Dronpa C1</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td></tr> | ||
+ | |||
+ | <tr><td>N-Dronpa C2</td><td>N-Dronpa C3</td><td>N-Dronpa C4</td><td></td><td></td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>ok</td><td>ok</td><td></td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/c/c7/Valencia_upv_gel_150628.png> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Take glycerinated:</p> | ||
+ | |||
+ | <ul><li>GB0030: p35S</li> | ||
+ | |||
+ | <li>GB0036: T35S</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <ul><li>Make liquid culture of LexABD (C1-C4).</li> | ||
+ | |||
+ | <li>We transform again LacIBD:KDronpa and RepBxb1:GFP, adding to the agar plates 100 µl of each transformation. </li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | </br><h3 style="color:green">29 June 2015</h3> | ||
+ | |||
+ | <p>Do the minipreps of the 4 colonies of LexABD and both glycerinates, 35S and T35S.</p> | ||
+ | |||
+ | <p>Do the digestion of the minipreps:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LexABD; pPPD2</td><td>NotI</td><td>2358, 312</td></tr> | ||
+ | |||
+ | <tr><td>35S; pUPD2</td><td>NotI</td><td>2981, 1074</td></tr> | ||
+ | |||
+ | <tr><td>T35S; pPUD2</td><td>NotI</td><td>2981, 304</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make the gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LexA C1</td><td>LexA C2</td><td>LexA C3</td><td>LexA C4</td><td>P35S</td><td>T35S</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>Ok?</td><td>Ok?</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/e/e9/Valencia_upv_gel_150629.png> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make ligations:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LacIBD+KDronpa+promoter+termi; α1</td><td>Gal4BD+KDonpa+prom+ter; α1</td><td>LexABD+KDronpa+prom+term; α1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl LacI; pUPD2</td><td>1 µl Gal4; pUPD2</td><td>1 µl Gal4; pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1 µl KDronpa; pUPD2</td><td>1 µl KDronpa; pUPD2</td><td>1 µl KDronpa; pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1 µl 35S (GB0030)</td><td>1 µl 35S (GB0030)</td><td>1 µl 35S (GB0030)</td></tr> | ||
+ | |||
+ | <tr><td>1 µl T35S (GB0036)</td><td>1 µl T35S (GB0036)</td><td>1 µl T35S (GB0036)</td></tr> | ||
+ | |||
+ | <tr><td>2.6 µl H<sub>2</sub>O</td><td>2.6 µl H<sub>2</sub>O</td><td>2.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | <tr><td>1 µl α1</td><td>1 µl α1</td><td>1 µl α1</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>NDronpa+VP16; α2</td><td>Gal4BD+PIF6; α1</td><td>LacIBD+PIF6; α1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl NDronpa; pUPD2</td><td>1 µl Gal4BD; pUPD2</td><td>1 µl LacIBD; pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1 µl VP16; pUPD2</td><td>1 µl PIF6; pUPD2</td><td>1 µl PIF6; pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1 µl 35S (GB0030)</td><td>1 µl 35S (GB0030)</td><td>1 µl 35S (GB0030)</td></tr> | ||
+ | |||
+ | <tr><td>1 µl T35S (GB0036)</td><td>1 µl T35S (GB0036)</td><td>1 µl T35S (GB0036)</td></tr> | ||
+ | |||
+ | <tr><td>2.6 µl H<sub>2</sub>O</td><td>2.6 µl H<sub>2</sub>O</td><td>2.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | <tr><td>1 µl α2</td><td>1 µl α1</td><td>1 µl α1</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LexABD+PIF6; α1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl LexABD; pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1 µl PIF6; pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1 µl 35S (GB0030)</td></tr> | ||
+ | |||
+ | <tr><td>1 µl T35S (GB0036)</td></tr> | ||
+ | |||
+ | <tr><td>2.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | <tr><td>1 µl α2</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>Transform all the ligations into E.Coli. Gal4BD+K-Dronpa and LacIBD+K-Dronpa went wrong and we have to do it again. </li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>Sent N-Dronpa with the primers 9 and 12 to sequence to check if the codon that synthetize for the amino acid K has change to the amino acid N.</p> | ||
+ | |||
+ | <p>Quantification of DNA:</p> | ||
+ | |||
+ | <ul><li>RepBxbI:GFP (C1): 163.8 ng/µl</li> | ||
+ | |||
+ | <li>NDronpa; pUPD2 (C4):113.1 ng/µl</li> | ||
+ | |||
+ | <li>NDronpa (C3): 83.2 ng/µl</li> | ||
+ | |||
+ | <li>NDronpa (C1): 116.6 ng/µl</li> | ||
+ | |||
+ | <li>Gal4BD (C1): 95.2 ng/µl</li> | ||
+ | |||
+ | <li>Gal4BD (C2): 120.7 ng/µl</li> | ||
+ | |||
+ | <li>RepBxbI:GFP (C2): 170.6 ng/µl</li> | ||
+ | |||
+ | <li>RepBxbI (C1): 80.6 ng/µl</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | </br><h3 style="color:green">30 June 2015</h3> | ||
+ | |||
+ | <p>Transform Gal4+KDronpa and LacI+KDronpa and make petri dish culture.</p> | ||
+ | |||
+ | <p>Miniprep of:</p> | ||
+ | |||
+ | <ul><li>RepBxbI+GFP (C1-C3)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>RepBxb1+GFP; Ω2</td><td>HindIII</td><td>6345, 2683</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>RepBxbI+GFP C1</td><td>RepBxbI+GFP C2</td><td>RepBxbI+GFP C3</td></tr> | ||
+ | |||
+ | <tr><td>No</td><td>no</td><td>no</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/8/8a/Valencia_upv_gel_150630.png> | ||
+ | |||
+ | <p>We pick more colonies of RepBxb1+GFP, Ω2 and make liquid cultures.</p> | ||
+ | |||
+ | <p> </p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make liquid culture of:</p> | ||
+ | |||
+ | <p>LexABD+KDronpa+prom+term; α1 (C1 and C2)</p> | ||
+ | |||
+ | <p>NDronpa+VP16; α2 (C1 and C2)</p> | ||
+ | |||
+ | <p>Gal4BD+PIF6; α1 (C1 and C2)</p> | ||
+ | |||
+ | <p>LacIBD+PIF6; α1 (C1 and C2)</p> | ||
+ | |||
+ | <p>LexABD+PIF6; α1 (C1 and C2)</p> | ||
+ | |||
+ | <p>Take out a glycerinate 35S:Luciferase:Tnos (GB0227) and do a miniprep.</p> | ||
+ | |||
+ | <br/> | ||
</div> | </div> | ||
</details> | </details> | ||
Line 49: | Line 1,784: | ||
<summary class="button fit">July</summary> | <summary class="button fit">July</summary> | ||
<div class="clsPadding"> | <div class="clsPadding"> | ||
− | <p> | + | |
− | + | ||
+ | <h3 style="color:green">1 July 2015</h3> | ||
+ | |||
+ | <p>Do the minipreps of the 10 liquid cultures.</p> | ||
+ | |||
+ | <p>Do the digestions:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LexABD+KDronpa;α1</td><td>EcoRI</td><td>6345, 2296</td></tr> | ||
+ | |||
+ | <tr><td>NDonpa+VP16; α2</td><td>HindIII</td><td>6345, 2427</td></tr> | ||
+ | |||
+ | <tr><td>Gal4BD+PIF6; α1</td><td>EcoRI</td><td>6345, 1867</td></tr> | ||
+ | |||
+ | <tr><td>LacI+PIF; α1</td><td>EcoRI</td><td>6345, 2638</td></tr> | ||
+ | |||
+ | <tr><td>LexABD+PIF6; α1</td><td>EcoRI</td><td>6345, 1906</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Do the gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4+PIF C1</td><td>Gal4+PIF C2</td><td>LexA+PIF C1</td><td>LexA+PIF C2</td><td>LexA+KDronpa C1</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>Ok</td></tr> | ||
+ | |||
+ | <tr><td>LexA+Kdronpa C2</td><td>LacI+PIF C1</td><td>LacI+PIF C2</td><td>Ndonpa+VP16 C1</td><td>Ndronpa+VP16 C2</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/1/1f/Valencia_upv_gel_150701.png> | ||
+ | |||
+ | <p>Prepare liquid culture of:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>LexA+PIF; Ω1 (C1)</li> | ||
+ | |||
+ | <li>LacI+PIF; Ω1 (C1)</li> | ||
+ | |||
+ | <li>LexA+K-Dronpa (C1)</li> | ||
+ | |||
+ | <li>Gal4+PIF; Ω1 (C1)</li> | ||
+ | |||
+ | <li>VP16, pUPD2 (C1)</li> | ||
+ | |||
+ | <li>LexABD, pUPD2 (C2)</li> | ||
+ | |||
+ | <li>PIF6, pUPD2 (C5)</li> | ||
+ | |||
+ | <li>LacIBD, pUPD2 (C1)</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>EXPERIMENT 1.</p> | ||
+ | |||
+ | <ul><li>Luciferase essay.</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>Pick colonies and make liquid culture of:</p> | ||
+ | |||
+ | <ul><li>Gal4+K-Dronpa (C1 and C2)</li> | ||
+ | |||
+ | <li>LacI+K-Dronpa (C1 and C2)</li> | ||
+ | |||
+ | <li>RepBxbI+GFP (C4-C6)</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <ul><li>Code:</li> | ||
+ | |||
+ | <li>210.08-249: pUPD2, KDronpa C3</li> | ||
+ | |||
+ | <li>210.08-250: pUPD2, NDronpa C1</li> | ||
+ | |||
+ | <li>210.08-251: pUPD2, NDronpa C3</li> | ||
+ | |||
+ | <li>210.08-252: pUPD2, NDronpa C4</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">2 July 2015</h3> | ||
+ | |||
+ | <p>Minipreps of:</p> | ||
+ | |||
+ | <ul><li>Gal4+KDronpa (C1 and C2)</li> | ||
+ | |||
+ | <li>LacI+KDronpa (C1 and C2)</li> | ||
+ | |||
+ | <li>RepBxbi+GFP (C4-C6)</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4+KDronpa</td><td>EcoRI</td><td>6345, 3028</td></tr> | ||
+ | |||
+ | <tr><td>LacI+KDronpa</td><td>EcoRI</td><td>6345, 2257</td></tr> | ||
+ | |||
+ | <tr><td>RepBxbI+GFP</td><td>HindIII</td><td>6300, 2400</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Do the gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LacI+KDronpa C1</td><td>LacI+KDronpa C2</td><td>Gal4+KDronpa C1</td><td>Gal4+KDronpa C2</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td></td><td></td></tr> | ||
+ | |||
+ | <tr><td>RepBxb1+GFP C4</td><td>RepBxb1+GFP C5</td><td>RepBxb1+GFP C6</td><td></td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make ligations:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LexA:Kdonpa+NDronpa; Ω1</td><td>LacI:Kdronpa+NDronpa; Ω1</td><td>Gal4:Kdronpa+NDronpa; Ω1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl LexA:Kdronpa</td><td>1 µl LacI:Kdronpa</td><td>1 µl Gal4:Kdronpa</td></tr> | ||
+ | |||
+ | <tr><td>1 µl NDronpa</td><td>1 µl NDronpa</td><td>1 µl NDronpa</td></tr> | ||
+ | |||
+ | <tr><td>1 µl Ω1</td><td>1 µl Ω1</td><td>1 µl Ω1</td></tr> | ||
+ | |||
+ | <tr><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LexA:PIF+PhyB:VP16; Ω1</td><td>LacI:PIF+PhyB:VP16; Ω1</td><td>Gal4:PIF+PhyB:VP16; Ω1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl LexA:PIF</td><td>1 µl LacI:PIF</td><td>1 µl Gal4:PIF</td></tr> | ||
+ | |||
+ | <tr><td>1 µl PhyB:VP16 (88E)</td><td>1 µl PhyB:VP16</td><td>1 µl PhyB:VP16</td></tr> | ||
+ | |||
+ | <tr><td>1 µl Ω1</td><td>1 µl Ω1</td><td>1 µl Ω1</td></tr> | ||
+ | |||
+ | <tr><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>35S:BxbI+RepBxbI:GFP; Ω1</td><td>E-PIF+phyB+luc+ren; Ω1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl 35s:Bxb1:T35S (alfredo’s)</td><td>0.5 µl PIF:PhyB:luc (GB0896)</td></tr> | ||
+ | |||
+ | <tr><td>1 µl NoATGProm:RepBxbI:GFP</td><td>1 µl Renilla GB0160</td></tr> | ||
+ | |||
+ | <tr><td>1 µl Ω1</td><td>1 µl Ω1</td></tr> | ||
+ | |||
+ | <tr><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>The samples that we sent to sequence have arrived:</p> | ||
+ | |||
+ | <ul><li>210.08-249: pUPD2, KDronpa C3 - ok</li> | ||
+ | |||
+ | <li>210.08-250: pUPD2, NDronpa C1 - ok</li> | ||
+ | |||
+ | <li>210.08-251: pUPD2, NDronpa C3 - ok</li> | ||
+ | |||
+ | <li>210.08-252: pUPD2, NDronpa C4 - ok</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>The sequences of NDronpa have the desired mutation.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transformation in E.Coli of the 8 ligations and make petri dish cultures.</p> | ||
+ | |||
+ | <p>Refresh the Agro’s cultures Renilla and PIF:phy:luc.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">4 July 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make the 2nd refresh of the culture of <i>Agrobacterium</i>.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make liquid cultures of the 8 colonies of E.Coli. </p> | ||
+ | |||
+ | <ul><li>Red toggle (C1)</li> | ||
+ | |||
+ | <li>Gal4:Kdronpa+NDronpa (C1 and C2)</li> | ||
+ | |||
+ | <li>LexA:Kdonpa+NDronpa (C1 and C2)</li> | ||
+ | |||
+ | <li>LacI:Kdronpa+NDronpa (C1 and C2)</li> | ||
+ | |||
+ | <li>LexA:PIF+PhyB:VP16 (C1 and C2)</li> | ||
+ | |||
+ | <li>35S:BxbI+RepBxbI:GFP (C1 and C2)</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">5 July 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Ligations:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LacI:PIF+PhyB:VP16; Ω1</td><td>Gal4:PIF+PhyB:VP16; Ω1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl LacI:PIF</td><td>1 µl Gal4:PIF</td></tr> | ||
+ | |||
+ | <tr><td>1 µl PhyB:VP16</td><td>1 µl PhyB:VP16</td></tr> | ||
+ | |||
+ | <tr><td>1 µl Ω1</td><td>1 µl Ω1</td></tr> | ||
+ | |||
+ | <tr><td>4.6</td><td>µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Minipreps of the liquid cultures.</p> | ||
+ | |||
+ | <p>Digestion of the minipreps:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LacI:Kdronpa+NDronpa</td><td>BamHI</td><td>6674, 5437</td></tr> | ||
+ | |||
+ | <tr><td>35S:BxbI+RepBxbI:GFP</td><td>BamHI</td><td>6674, 3859, 1782</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:Kdronpa+NDronpa</td><td>BamHI</td><td>6674, 4666</td></tr> | ||
+ | |||
+ | <tr><td>LexA:Kdonpa+NDronpa</td><td>BamHI</td><td>6674, 4705</td></tr> | ||
+ | |||
+ | <tr><td>LexA:PIF+PhyB:VP16</td><td>BamHI</td><td>6674, 3513, 2337</td></tr> | ||
+ | |||
+ | <tr><td>E:PIF6:PhyB:VP16</td><td>BamHI</td><td>4209, 3756, 6100, 6674</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <ul><li>Agarose gel (1%): </li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LacI:KNDronpa C1</td><td>LacI:KNDronpa C2</td><td>BxbI:Rep:GFP C1</td><td>BxbI:Rep:GFP C2</td><td>Gal4:KNDronpa C1</td><td>Gal4:KNDronpa C2</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td></tr> | ||
+ | |||
+ | <tr><td>LexA:KNDronpa C1</td><td>LexA:KNDronpa C2</td><td>LexA:PIF:Phy C1</td><td>LexA:PIF:Phy C2</td><td>Red toggle</td><td></td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>no</td><td>no</td><td>no</td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/c/c5/Valencia_upv_gel_150705_1.png> | ||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/c/c0/Valencia_upv_gel_150705_2.png> | ||
+ | |||
+ | |||
+ | |||
+ | <p>We have to repeat the digestion of: LexA+PIF:phy+VP16.</p> | ||
+ | |||
+ | <ul><li>Take out the glycerinate 88C (GB1098), Etr8:luc:Tnos. We will use it like a negative control in the second luciferase essay.</li> | ||
+ | |||
+ | <li>Calculation of the ODs:</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Renilla</td><td>0.22</td><td>182 µl of sample + 1.818 ml MES</td></tr> | ||
+ | |||
+ | <tr><td>E:PIF6:PhyB:Luc</td><td>0.26</td><td>154 µl of sample + 1.646 ml MES</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>EXPERIMENT 2.</p> | ||
+ | |||
+ | <p>Agroinfiltration of renilla and PIF6:PhyB:luc. For more info, click here.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">6 July 2015</h3> | ||
+ | |||
+ | <p>Transform the negative control into agrobacterium and make petri dish culture of:</p> | ||
+ | |||
+ | <ul><li>Etr8:luc:Tnos</li> | ||
+ | |||
+ | <li>BxbI:ReporterBxbI:GF</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>We were doing dry lab preparing the power point to present our project to the rector and biotecs companies.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">7 July 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Prepare this cultures for agroinfiltration:</p> | ||
+ | |||
+ | <ul><li>Renilla</li> | ||
+ | |||
+ | <li>E:PIF6:PhyB:Luc </li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Renilla</td><td>0.29</td><td>0.138ml of sample + 1.862ml of MES</td></tr> | ||
+ | |||
+ | <tr><td>PhyB+PIF+luc</td><td>0.69</td><td>0.145ml of sample + 1.855ml of MES</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>EXPERIMENT 3. Start. Agroinfiltrate PhyB+PIF+luc and Renilla</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Digestions:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Red toggle</td><td>BamHI</td><td>4209, 3756, 6100, 6674</td><td></td></tr> | ||
+ | |||
+ | <tr><td>LexA+PIF+phy</td><td>BamHI</td><td>3518, 5855, 6674</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Red toggle</td><td>LexA+PIF+Phy C1</td><td>LexA+PIF+Phy C2</td></tr> | ||
+ | |||
+ | <tr><td>No</td><td>Ok</td><td>no</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/b/b6/Valencia_upv_gel_150707.png> | ||
+ | |||
+ | |||
+ | |||
+ | <p>It has arrived a new construction: AsLOVpep.</p> | ||
+ | |||
+ | <ul><li>Resuspended with 50µl of H<sub>2</sub>O.</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>Ligations:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>AsLOVpep; pUPD2</td><td>Red Toggle</td><td>LacI:Kdronpa:Ndronpa:VP16+renilla; α1</td><td>Gal4:Kdronpa:Ndronpa:VP16+renilla; α1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl AsLOVpep</td><td>1 µl GB846</td><td>1 µl LacI:KNdronpa:VP16</td><td>1 µl Gal4:KNdronpa</td></tr> | ||
+ | |||
+ | <tr><td>1 µl pUPD2</td><td>1 µl GB160</td><td>1 µl GB159</td><td>1 µl GB159</td></tr> | ||
+ | |||
+ | <tr><td>5.6 µl H<sub>2</sub>O</td><td>1 µl Ω1</td><td>1 µl α1</td><td>1 µl α1</td></tr> | ||
+ | |||
+ | <tr><td></td><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LexA:Kdronpa:Ndronpa:VP16+renilla; α1</td><td>LexA:PIF:Phy:VP16+renilla; α1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl LexA:Kdronpa:Ndronpa:VP16</td><td>1 µl LexA:PIF:Phy:VP16</td></tr> | ||
+ | |||
+ | <tr><td>1 µl GB159</td><td>1 µl GB159</td></tr> | ||
+ | |||
+ | <tr><td>1 µl α1</td><td>1 µl α1</td></tr> | ||
+ | |||
+ | <tr><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">8 July 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>EXPERIMENT 2. Change the ligth conditions of the plants.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transform into <i>E. coli</i> last ligations.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">9 July 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Pick colonies and make liquid culture of:</p> | ||
+ | |||
+ | <ul><li>AsLOVpep; pUPD2 colonies didn’t grow. Repeat.</li> | ||
+ | |||
+ | <li>Red toggle colonies are all blue. Repeat.</li> | ||
+ | |||
+ | <li>LacI:Kdronpa:Ndronpa:VP16+renilla (C1-C3)</li> | ||
+ | |||
+ | <li>Gal4:Kdronpa:Ndronpa:VP16+renilla (C1-C3)</li> | ||
+ | |||
+ | <li>LexA:Kdronpa:Ndronpa:VP16+renilla (C1 and C2)</li> | ||
+ | |||
+ | <li>LexA:PIF:Phy:VP16+renilla (C1 and C2)</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>Repeat the ligations.</p> | ||
+ | |||
+ | <ul><li>AsLOVpep, as before.</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Red toggle (PIF+PhyB+luc+ren)</td></tr> | ||
+ | |||
+ | <tr><td>0.5 µl PIF+phy+luc (896)</td></tr> | ||
+ | |||
+ | <tr><td>1 µl renilla (160)</td></tr> | ||
+ | |||
+ | <tr><td>1 µl Ω1</td></tr> | ||
+ | |||
+ | <tr><td>5.1 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>EXPERIMENT 2.</p> | ||
+ | |||
+ | <p>Luciferase essay.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transform the ligations of AsLOVpepe and red toggle. This time we make a spin to concentrate the cells to make petri dish culture. </p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">10 July 2015</h3> | ||
+ | |||
+ | <p>Minipreps of:</p> | ||
+ | |||
+ | <ul><li>LexABD:PIF:<T>PhyB:VP16+Renilla; α1 (C1,C2)</li> | ||
+ | |||
+ | <li>LexA:Kdronpa:Ndronpa+renilla; α1 C1, C2)</li> | ||
+ | |||
+ | <li>Gal4:Kdronpa:Ndronpa+renilla; α1 (C1-C3)</li> | ||
+ | |||
+ | <li>LacI:Kdronpa:Ndronpa+renilla; α1 (C1,C2)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Digestions of:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LexABD:PIF:PhyB:VP16+Renilla; α1</td><td>EcoRI</td><td>6345, 5487, 4891</td></tr> | ||
+ | |||
+ | <tr><td>LexA:Kdronpa:Ndronpa+renilla; α1</td><td>EcoRI</td><td>9333, 6345</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:Kdronpa:Ndronpa+renilla; α1</td><td>EcoRI</td><td>6345, 9194</td></tr> | ||
+ | |||
+ | <tr><td>LacI:Kdronpa:Ndronpa+renilla; α1</td><td>EcoRI</td><td>6345, 9965</td></tr> | ||
+ | |||
+ | <tr><td>LacIBD+PIF+PhyB; Ω1</td><td>BamHI</td><td>6674, 4245, 2337</td></tr> | ||
+ | |||
+ | <tr><td>Gal4BD+PIF+PhyB; Ω1</td><td>BamHI</td><td>6674, 3474, 2337</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Make the gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LacIBD+PIF+PhyB</td><td>Gal4BD+PIF+PhyB</td><td>LexA:PIF:PhyB:VP16+Ren C1</td><td>LexA:PIF:PhyB:VP16+Ren C2</td></tr> | ||
+ | |||
+ | <tr><td>Ok</td><td>Ok</td><td>ok</td><td>ok</td></tr> | ||
+ | |||
+ | <tr><td>LexA:KNdronpa+ren C1</td><td>LexA:KNdronpa+ren C2</td><td>Gal4:KNdronpa+ren C1</td><td>Gal4:KNdronpa+ren C2</td></tr> | ||
+ | |||
+ | <tr><td>Ok</td><td>Ok</td><td>ok</td><td>Ok</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:KNdronpa+ren C3</td><td>LacI:Kdronpa:Ndronpa+ren C1</td><td>LacI:Kdronpa:Ndronpa+ren C2</td><td></td></tr> | ||
+ | |||
+ | <tr><td>Ok</td><td>Ok</td><td>ok</td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>We received two constructions:</p> | ||
+ | |||
+ | <ul><li>CDS: phiC31. Resuspended with 100µl.</li> | ||
+ | |||
+ | <li>Reporter Phi31. Resuspended with 50 µl.</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>Pick colonies and make liquid culture of:</p> | ||
+ | |||
+ | <ul><li>AsLOVpep; pUPD2 (C1 and C2)</li> | ||
+ | |||
+ | <li>Red toggle (C1 and C2).</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Ligations:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PhiC31; pUPD2</td><td>Reporter PhiC31; pUPD2</td><td>LacI:PIF:PhyB:VP16+ren; α1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl PhiC31</td><td>1 µl RepPhiC31</td><td>1 µl LacI:PIF:PhyB:VP16</td></tr> | ||
+ | |||
+ | <tr><td>1µl pUPD2</td><td>1µl pUPD2</td><td>1 µl renilla (GB159)</td></tr> | ||
+ | |||
+ | <tr><td>5.6 µl H<sub>2</sub>O</td><td>5.6 µl H<sub>2</sub>O</td><td>1 µl α1</td></tr> | ||
+ | |||
+ | <tr><td></td><td></td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4:PIF:Phy:VP16+ren; α1</td><td>LexA:PIF:PhyB:ren+OpLex:luc; Ω1</td><td>LexA:KNdronpa:ren+OpLex:Luc; Ω1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl Gal4:PIF:phy:VP16</td><td>1 µl LexA:PIF:phy:ren</td><td>1 µl LexA:KNdronpa:ren</td></tr> | ||
+ | |||
+ | <tr><td>1 µl renilla (GB159)</td><td>1 µl opLex:luc (GB151)</td><td>1 µl OpLex:Luc (GB151)</td></tr> | ||
+ | |||
+ | <tr><td>1 µl α1</td><td>1 µl Ω1</td><td>1 µl Ω1</td></tr> | ||
+ | |||
+ | <tr><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4:KNdronpa:ren+UAS:luc; Ω1</td><td>LacI:KNdronpa:ren+OpLacI:luc; Ω1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl Gal4:KNdronpa:ren</td><td>1 µl LacI:KNdronpa:ren</td></tr> | ||
+ | |||
+ | <tr><td>1 µl OpUAS:luc (GB227)</td><td>1 µl OpLacI:luc (GB152)</td></tr> | ||
+ | |||
+ | <tr><td>1 µl Ω1</td><td>1 µl Ω1</td></tr> | ||
+ | |||
+ | <tr><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | <tr><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">11 July 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Prepare glycerinates of:</p> | ||
+ | |||
+ | <ul><li>BxbI+Rep:GFP; Ω1</li> | ||
+ | |||
+ | <li>NDronpa; pUPD2</li> | ||
+ | |||
+ | <li>BxbI:Etr8; pUPD2</li> | ||
+ | |||
+ | <li>Etr8(CMV):BxbI:T35S; α1</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Do miniprep of:</p> | ||
+ | |||
+ | <ul><li>AsLOVpep (C1 and C2)</li> | ||
+ | |||
+ | <li>Red toggle (C2) (C1 was blue)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Digestions:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>E:PIF6:PhyB:luc:ren</td><td>BamHI</td><td>6674, 6100, 4209, 3756</td></tr> | ||
+ | |||
+ | <tr><td>AsLOVpep</td><td>NotI</td><td>2558, 512</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>AsLOVpep C1</td><td>AsLOVpep C2</td><td>Red toggle C2</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>no</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Do transformation of DHSa and yesterday ligations:</p> | ||
+ | |||
+ | <ul><li>phyC31;pUPD2</li> | ||
+ | |||
+ | <li>Reporter PhiC31; pUPD2</li> | ||
+ | |||
+ | <li>LacI:PIF:PhyB:VP16+ren; α1</li> | ||
+ | |||
+ | <li>Gal4:PIF:phy:VP16+ren; α1</li> | ||
+ | |||
+ | <li>LexA:PIF:phy:ren+opLex:luc; Ω1</li> | ||
+ | |||
+ | <li>LexA:KNdronpa:ren+OpLex:luc; Ω1</li> | ||
+ | |||
+ | <li>Gal4:KNdronpa:ren+OpUAS:luc; Ω1</li> | ||
+ | |||
+ | <li>LacI:KNdronpa:ren+OpLacI:luc; Ω1</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | </br><h3 style="color:green">12 July 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Pick colonies and make liquid culture:</p> | ||
+ | |||
+ | <ul><li>PhiC31;pUPD2 (C1-C3)</li> | ||
+ | |||
+ | <li>Reporter PhiC31; pUPD2 (C1-C3)</li> | ||
+ | |||
+ | <li>LacI:PIF:PhyB:VP16+ren; α1 (C1-C3)</li> | ||
+ | |||
+ | <li>Gal4:PIF:phy:VP16+ren; α1 All blue colonies.</li> | ||
+ | |||
+ | <li>LexA:PIF:phy:ren+OpLex:luc; Ω1 (C1)</li> | ||
+ | |||
+ | <li>LexA:KNdronpa:ren+OpLex:Luc; Ω1(C1-C3)</li> | ||
+ | |||
+ | <li>Gal4:KNdronpa:ren+UAS:luc; Ω1 (C1)</li> | ||
+ | |||
+ | <li>LacI:KNdronpa:ren+OpLacI:luc; Ω1 All blue colonies.</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Ligations that were repeated:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4:PIF:phy:VP16+ren; α1</td><td>LacI:KNdronpa:ren+OpLacI:luc; Ω1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl Gal4:PIF:phy:VP16</td><td>1 µl LacI:KNdronpa:ren</td></tr> | ||
+ | |||
+ | <tr><td>1 µl renilla (GB159)</td><td>1 µl OpLacI:luc (GB152)</td></tr> | ||
+ | |||
+ | <tr><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | <tr><td>1 µl α1</td><td>1 µl Ω1</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Refresh the liquid cultures of <i>Agrobacterium</i>:</p> | ||
+ | |||
+ | <ul><li>BxbI:GFP and Etr8:Tnos.</li> | ||
+ | |||
+ | <li>Pnos, it was at the fridge (-4ºC).</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | </br><h3 style="color:green">13 July 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>All the liquid cultures have grown, do minipreps.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Do digestions:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>phyC31;pUPD2</td><td>NotI</td><td>2046, 1899</td></tr> | ||
+ | |||
+ | <tr><td>Reporter phiC31; pUPD2</td><td>NotI</td><td>2046, 475</td></tr> | ||
+ | |||
+ | <tr><td>LacI:PIF:Phy:VP16+ren; α1</td><td>EcoRI</td><td>6345, 5623, 5487</td></tr> | ||
+ | |||
+ | <tr><td>LexA:PIF:phy:ren+opLex:luc; Ω1</td><td>BamHI</td><td>9431, 6674, 3531</td></tr> | ||
+ | |||
+ | <tr><td>LexA:KNdronpa:ren+OpLex:Luc; Ω1</td><td>BamHI</td><td>1199, 6674</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:KNdronpa:ren+UAS:luc; Ω1</td><td>BamHI</td><td>11582, 6674</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Agarose gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PhiC31 C1</td><td>PhiC31 C2</td><td>PhiC31 C3</td><td>RepPhiC31 C1</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>no</td><td>ok</td></tr> | ||
+ | |||
+ | <tr><td>RepPhiC31 C2 </td><td>LacI:PIF:PhyB:VP16+ren C1</td><td>LacI:PIF:PhyB:VP16+ren C2</td><td>LacI:PIF:PhyB:VP16+ren C3</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>ok</td><td>ok</td></tr> | ||
+ | |||
+ | <tr><td>LexA:PIF:phy:ren+OpLex:luc</td><td>LexA:KNdronpa:ren+OpLex:luc C1</td><td>LexA:KNdronpa:ren+OpLex:Luc C2</td><td>LexA:KNdronpa:ren+OpLex:Luc C3</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>ok</td><td>ok</td><td>ok</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:KNdronpa:ren+OpUAS:luc C1</td><td></td><td></td></tr> | ||
+ | |||
+ | <tr><td>no</td><td></td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/8/8e/Valencia_upv_gel_150713.png> | ||
+ | |||
+ | |||
+ | |||
+ | <p>The <i>Agrobacterium</i> cultures refreshed yesterday were store in the fridge.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>It is made another culture of 35S:BxbI+reporterBxbI:GFP to keep it in the fridge.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>EXPERIMENT 4. Recombinase BxbI. </p> | ||
+ | |||
+ | <p>Mesurement of the OD’s:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>35S:BxbI+reporterBxbI:GFP: 0.28</td><td>143 µl of culture+1857 µl of MES</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transformation of the ligation: Gal4:PIF:phy:VP16+ren; α1 and LacI:KNdronpa:ren+OpLacI:luc; Ω1.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>The liquid cultures of this constructions were repeated:</p> | ||
+ | |||
+ | <ul><li>phyC31;pUPD2 (C4 and C5)</li> | ||
+ | |||
+ | <li>Reporter phiC31; pUPD2 (C4)</li> | ||
+ | |||
+ | <li>LacI:PIF:PhyB:VP16+ren; α1 (C4)</li> | ||
+ | |||
+ | <li>LexA:PIF:phy:ren+OpLex:luc; Ω1 (C1)</li> | ||
+ | |||
+ | <li>LexA:KNdronpa:ren+OpLex:Luc; Ω1(C1-C3)</li> | ||
+ | |||
+ | <li>Gal4:KNdronpa:ren+UAS:luc;Ω1 (C1)</li> | ||
+ | |||
+ | <li>AsLOVpep; pUPD2 (C3)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | </br><h3 style="color:green">14 July 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Do minipreps of:</p> | ||
+ | |||
+ | <ul><li>phyC31;pUPD2 (C4 and C5)</li> | ||
+ | |||
+ | <li>Reporter phiC31; pUPD2 (C4)</li> | ||
+ | |||
+ | <li>LacI:PIF:PhyB:VP16+ren; α1 (C4)</li> | ||
+ | |||
+ | <li>LexA:PIF:phy:ren+OpLex:luc; Ω1 (C1)</li> | ||
+ | |||
+ | <li>LexA:KNdronpa:ren+OpLex:Luc; Ω1(C1-C3)</li> | ||
+ | |||
+ | <li>Gal4:KNdronpa:ren+UAS:luc;Ω1 (C1)</li> | ||
+ | |||
+ | <li>AsLOVpep; pUPD2 (C3)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Do digestions:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>phyC31;pUPD2</td><td>NotI</td><td>2046, 1899</td></tr> | ||
+ | |||
+ | <tr><td>Reporter phiC31; pUPD2</td><td>NotI</td><td>2046, 475</td></tr> | ||
+ | |||
+ | <tr><td>LacI:PIF:Phy:VP16+ren; α1</td><td>EcoRI</td><td>6345, 5623, 5487</td></tr> | ||
+ | |||
+ | <tr><td>LexA:PIF:phy:ren+opLex:luc, Ω1</td><td>BamHI</td><td>9431, 6674, 3531</td></tr> | ||
+ | |||
+ | <tr><td>LexA:KNdronpa:ren+OpLex:Luc; Ω1</td><td>BamHI</td><td>1199, 6674</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:KNdronpa:ren+UAS:luc; Ω1</td><td>BamHI</td><td>11582, 6674</td></tr> | ||
+ | |||
+ | <tr><td>AsLOVpep; pUPD2 </td><td>NotI</td><td>2558, 512</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Make an agarose gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>phyC31 (C4)</td><td>phyC31 (C5)</td><td>AsLOVpep (C4)</td><td>LexA:PIF:phy:ren+opLex:luc (C1)</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>No</td><td>No</td></tr> | ||
+ | |||
+ | <tr><td>LexA:KNdronpa:ren+OpLex:Luc (C3)</td><td>Gal4:KNdronpa:ren+OpUAS:luc (C1)</td><td>Gal4:KNdronpa:ren+UAS:luc(C2)</td><td>Gal4:KNdronpa:ren+UAS:luc (C3)</td></tr> | ||
+ | |||
+ | <tr><td>Ok</td><td>no</td><td>no</td><td>No</td></tr> | ||
+ | |||
+ | <tr><td>LacI:PIF:Phy:VP16+ren</td><td>Reporter phiC31 (C1)</td><td></td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>Ok</td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Only LexA:KNdronpa:ren+OpLex:Luc and Reporter phiC31 were correct. Repeat the ligations because is the second digestion of this construction that were made.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>Measurement of DNA concentration:</li> | ||
+ | |||
+ | <ul class="ul_2"><li>Reporter:phyC31: 13.6 ng/µl</li> | ||
+ | |||
+ | <li>PhiC31: 4.6 ng/µl </li> | ||
+ | |||
+ | <li>AsLOVpep: 30.3 ng/µl</li> | ||
+ | |||
+ | <li>OpLexA:luc (GB151): 40 ng/µl</li> | ||
+ | |||
+ | <li>Gal4:PIF:PhyB:ren (C1): 124.4 ng/µl</li> | ||
+ | |||
+ | <li>LacI:PIF:PhyB:VP16 (C1): 126 ng/µl</li> | ||
+ | |||
+ | <li>Renilla (GB159): 42 ng/µl</li> | ||
+ | |||
+ | <li>Gal4:Kdronpa:Ndronpa:renilla (C3): 172.8 ng/µl</li> | ||
+ | |||
+ | <li>OpUAS:luc (GB227): 6.3 ng/µl</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <ul><li>Pick colonies and make liquid culture of LacI:KDronpa:NDronpa:ren:luc (C4 and C5). The colonies of Gal4:PIF:phy:VP16+ren were all blue.</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | </br><h3 style="color:green">15 July 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>Miniprep of LacI:KDronpa:NDronpa:ren:luc (C4 and C5). </li> | ||
+ | |||
+ | <li>Digestion:</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LacI:KDronpa:NDronpa:ren:luc</td><td>EcoRI</td><td>6345, 9965</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <ul><li>Make the gel:</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LacI:K:NDronpa:ren:luc C4</td><td>LacI:K:NDronpa:ren:luc C5</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/a/a6/Valencia_upv_gel_150715.png> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>Measurement of DNA concentrations:</li> | ||
+ | |||
+ | <ul class="ul_2"><li>Gal4BD:PIF:PhyB:VP16: 125.6 ng/µl</li> | ||
+ | |||
+ | <li>LexA:PIF:PhyB:VP16:ren: 322.6 ng/µl</li> | ||
+ | |||
+ | <li>LacI:Kdronpa:NDronpa:ren: 209.0 ng/µl</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <ul><li>Repeat the ligations:</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PhiC31;pUPD2</td><td>AsLOVpep; pUPD2</td><td>LacI:PIF:PhyB:VP16+ren; α1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl PhiC31</td><td>1 µl AsLOVpep</td><td>1.5 µl LacI:PIF:PhyB:VP16</td></tr> | ||
+ | |||
+ | <tr><td>1µl pUPD2</td><td>1µl pUPD2</td><td>2.5 µl renilla (159)</td></tr> | ||
+ | |||
+ | <tr><td>5.6 µl H<sub>2</sub>O</td><td>5.6 µl H<sub>2</sub>O</td><td>0.5 µl α1</td></tr> | ||
+ | |||
+ | <tr><td></td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4:PIF:phy:VP16+ren; α1</td><td>LexA:PIF:phy:ren+opLex:luc; Ω1</td><td>LacI:KNdronpa:ren+OpLex:Luc; Ω1</td></tr> | ||
+ | |||
+ | <tr><td>1.5 µl Gal4:PIF:phy:VP16</td><td>1 µl LexA:PIF:phy:ren</td><td>1 µl LacI:KNdronpa:ren</td></tr> | ||
+ | |||
+ | <tr><td>2 µl renilla (159)</td><td>2.5 µl opLex:luc (151)</td><td>3 µl OpLac:Luc (152)</td></tr> | ||
+ | |||
+ | <tr><td>2.6 µl H<sub>2</sub>O</td><td>2.6 µl H<sub>2</sub>O</td><td>3 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | <tr><td>1 µl α1</td><td>0.5 µl Ω1</td><td>0.5 µl Ω1</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4:KNdronpa:ren+OpLex:Luc; Ω1</td><td>PhyB:VP16+PIF6; Ω1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl Gal4:KNdronpa:ren</td><td>1 µl PhyB:VP16 (88E)</td></tr> | ||
+ | |||
+ | <tr><td>4 µl OpUAS:Luc (227)</td><td>2 µl PIF6 (170)</td></tr> | ||
+ | |||
+ | <tr><td>3 µl H<sub>2</sub>O</td><td>3.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | <tr><td>0.5 µl Ω1</td><td>1 µl Ω1</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <ul><li>These <i>Agrobacterium</i> cultures have been refreshed:</li> | ||
+ | |||
+ | <ul class="ul_2"><li>E:PIF:PhyB:luc</li> | ||
+ | |||
+ | <li>Renilla</li> | ||
+ | |||
+ | <li>Pnos</li> | ||
+ | |||
+ | <li>Etr8</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | </br><h3 style="color:green">16 July 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>Yesterday ligations have been transformed into E. coli:</li> | ||
+ | |||
+ | <ul class="ul_2"><li>phyC31;pUPD2</li> | ||
+ | |||
+ | <li>AsLOVpep; pUPD2</li> | ||
+ | |||
+ | <li>LacI:PIF:Phy:VP16+ren; α1</li> | ||
+ | |||
+ | <li>Gal4:PIF:phy:VP16+ren; α1</li> | ||
+ | |||
+ | <li>LexA:PIF:phy:ren+opLex:luc; Ω1</li> | ||
+ | |||
+ | <li>LacI:KNdronpa:ren+OpLex:Luc; Ω1</li> | ||
+ | |||
+ | <li>Gal4:KNdronpa:ren+OpLex:Luc; Ω1</li> | ||
+ | |||
+ | <li>PhyB:VP16+PIF6; Ω1</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>EXPERIMENT 4.</p> | ||
+ | |||
+ | <ul><li>The agroinfiltrated leaf with BxbI:rep:GFP has been observed in the magnifying glass.</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <ul><li>Second refresh of the <i>Agrobacterium</i> cultures:</li> | ||
+ | |||
+ | <ul class="ul_2"><li>E:PIF:PhyB:luc</li> | ||
+ | |||
+ | <li>Renilla</li> | ||
+ | |||
+ | <li>Pnos</li> | ||
+ | |||
+ | <li>Etr8</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <ul><li>Miniprep of these cultures.</li> | ||
+ | |||
+ | <li>Digestion of the minipreps:</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>E:PIF:PhyB:luc; Ω1</td><td>EcoRI</td><td>??</td></tr> | ||
+ | |||
+ | <tr><td>Renilla; Ω2</td><td>HindIII</td><td>7453</td></tr> | ||
+ | |||
+ | <tr><td>Pnos; α1</td><td>EcoRI</td><td>2997, 353</td></tr> | ||
+ | |||
+ | <tr><td>Etr8; Ω1</td><td>EcoRI</td><td>2742</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>The digestions had positive controls that were included in the gel to compare the results obtained.</p> | ||
+ | |||
+ | <p>The digestions are left overnight in the working table.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">17 July 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Do the gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Pnos</td><td>Etr8:luc </td><td>Etr8:luc C+</td><td>PIF6:PhyB:luc</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>ko</td><td>ok</td><td>ok</td></tr> | ||
+ | |||
+ | <tr><td>PIF6:PhyB:luc C+</td><td>renilla</td><td>renilla C+</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>no</td><td>ok</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/1/16/Valencia_upv_gel_150717.png> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>Make liquid cultures of yesterday ligations, three colonies per cultures.</li> | ||
+ | |||
+ | <li>OD’s mesurement for the agroinfiltration.</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | </br><h3 style="color:green"> </h3> | ||
+ | |||
+ | <tr><td>PIF:PhyB:luc</td><td>0.47</td><td>41 µl/ml</td><td>630 µl</td></tr> | ||
+ | |||
+ | <tr><td>Renilla</td><td>0.28</td><td>71 µl/ml</td><td>1065 µl</td></tr> | ||
+ | |||
+ | <tr><td>Etr8:luc</td><td>0.32</td><td>52 µl/ml</td><td>930 µl</td></tr> | ||
+ | |||
+ | <tr><td>Pnos</td><td>0.34</td><td>59 µl/ml</td><td>885 µl</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>EXPERIMENT 5. Red toggle experiement:</p> | ||
+ | |||
+ | <ul><li>PIF:PhyB:luc</li> | ||
+ | |||
+ | <li>Renilla</li> | ||
+ | |||
+ | <li>Etr8:luc (C-)</li> | ||
+ | |||
+ | <li>Pnos (C+)</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>EXPERIMENT 4.</p> | ||
+ | |||
+ | <p>It was observed another leaf with the recombinase BxbI with GFP in the magnifying glass with fluorescent lights.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">18 July 2015</h3> | ||
+ | |||
+ | <p>23 minipreps of the liquid cultrures. LexA:PIF:PhyB:ren:luc (C3) has not grown.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Digestions of the minipreps:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PhiC31;pUPD2</td><td>NotI</td><td>2046, 1899</td></tr> | ||
+ | |||
+ | <tr><td>AsLOVpep; pUPD2</td><td>NotI</td><td>2046, 521</td></tr> | ||
+ | |||
+ | <tr><td>LacI:PIF:PhyB:VP16+ren; α1</td><td>EcoRI</td><td>6345, 5623, 5487</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:PIF:phy:VP16+ren; α1</td><td>EcoRI</td><td>6345, 5487, 4852</td></tr> | ||
+ | |||
+ | <tr><td>LexA:PIF:phy:ren+OpLex:luc; Ω1</td><td>BamHI</td><td>9431, 6674, 3513</td></tr> | ||
+ | |||
+ | <tr><td>LacI:KNdronpa:ren+OpLex:Luc; Ω1</td><td>BamHI</td><td>12632, 6574</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:KNdronpa:ren+OpLex:Luc; Ω1</td><td>BamHI</td><td>11582, 6674</td></tr> | ||
+ | |||
+ | <tr><td>PhyB:VP16+PIF6; Ω1</td><td>BamHI</td><td>6674, 2685, 2337, 1439</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Gel has been done:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>phiC31 C1</td><td>phiC31 C2</td><td>phiC31 C3</td><td>AsLOVpep C1</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>no</td><td>ok</td></tr> | ||
+ | |||
+ | <tr><td>AsLOVpep C2</td><td>AsLOVpep C3</td><td>Gal4:PIF:phy:VP16:ren C1</td><td>Gal4:PIF:phy:VP16:ren C2</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>no</td><td>no</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:PIF:phy:VP16:ren C3</td><td>LacI:PIF:phy:ren C1</td><td>LacI:PIF:phy:ren C2</td><td>LacI:PIF:phy:ren C3</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>ok</td><td>no</td><td>No</td></tr> | ||
+ | |||
+ | <tr><td>LexA:PIF:phy:ren:luc C1</td><td>LexA:PIF:phy:ren:luc C2</td><td>Gal4:KNdronpa:ren:luc C1</td><td>Gal4:KNdronpa:ren:luc C2</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>ok</td><td>No</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:KNdronpa:ren:luc C3</td><td>LacI:KNdronpa:ren:luc C1</td><td>LacI:KNdronpa:ren:luc C2</td><td>LacI:KNdronpa:ren:luc C3</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>ok</td><td>No</td></tr> | ||
+ | |||
+ | <tr><td>PhyB:VP16:PIF6 C1</td><td>PhyB:VP16:PIF6 C2</td><td>PhyB:VP16:PIF6 C3</td><td></td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>no</td><td>no</td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/4/47/Valencia_upv_gel_150718.png> | ||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/b/b1/Valencia_upv_gel_150718-2.png> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Pick more colonies of:</p> | ||
+ | |||
+ | <ul><li>Gal4:PIF:phy:VP16+ren; α1</li> | ||
+ | |||
+ | <li>LexA:PIF:phy:ren+opLex:luc; Ω1</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>New digestions with new enzymes:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>phiC31;pUPD2</td><td>XhoI (buffer red)</td><td>2119, 934, 894</td></tr> | ||
+ | |||
+ | <tr><td>LacI:PIF:Phy:VP16+ren; a1</td><td>NEB4</td><td>5949, 5653, 3610, 2246</td></tr> | ||
+ | |||
+ | <tr><td>LacI:PIF:Phy:VP16+ren; a1</td><td>HindIII</td><td>11568, 5587</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>After 3 digestions of LacI:PIF:Phy:VP16+ren; α1 is accepted the construction.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>It was observed another leaf with the recombinase BxbI with GFP in the magnifying glass with fluorescent lights. It was not observed a lot of spots, the efficiency is very low. This is the 4th day…</p> | ||
+ | |||
+ | <p>FOTO</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">19 July 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>The incredible mistery of the lost notebook papers...</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">20 July 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Another day...</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">21 July 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>EXPERIMENT 5. Red toggle.</p> | ||
+ | |||
+ | <p>It was picked disc samples: 3 in far red, 3 in darkness and 6 in red (3 of them were in far red and the other 3 in darkness). </p> | ||
+ | |||
+ | <p>Time lapses: </p> | ||
+ | |||
+ | <p>-19:00=t0</p> | ||
+ | |||
+ | <p>-1:00=t1</p> | ||
+ | |||
+ | <p>-7:00= t2</p> | ||
+ | |||
+ | <p>-19:00= t3 (it was taken the controls in natural light)</p> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>Minipreps of the colonies that were in 37ºC.</li> | ||
+ | |||
+ | <ul class="ul_2"><li>LexA:PIF:Phy:ren:luc (C1 and C2)</li> | ||
+ | |||
+ | <li>PhyC31 (C1, C2, C4 and C5)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LexA:PIF:Phy:ren:luc</td><td>BamHI</td><td>9431, 6674, 3513</td></tr> | ||
+ | |||
+ | <tr><td>PhiC31</td><td>NotI</td><td>2046, 1899</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Gel with the digestions:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PhiC31 C1</td><td>PhiC31 C2</td><td>PhiC31 C4</td><td>PhiC31 C5</td></tr> | ||
+ | |||
+ | <tr><td>Ok?</td><td>ok</td><td>ok</td><td>ok</td></tr> | ||
+ | |||
+ | <tr><td>LexA:PIF:PhyB:ren:luc C1</td><td>LexA:PIF:PhyB:ren:luc C2</td><td></td></tr> | ||
+ | |||
+ | <tr><td>No DNA</td><td>No DNA</td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/b/b1/Valencia_upv_gel_1507121.png> | ||
+ | |||
+ | |||
+ | |||
+ | <p>We had problems with some colonies because in the digestion did not appear DNA. The minipreps will be made with a better kit.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transform in <i>Agrobacterium</i> this cultures:</p> | ||
+ | |||
+ | <ul><li>LexABD:KDronpa:NDronpa:ren:luc</li> | ||
+ | |||
+ | <li>Gal4BD:KDronpa:NDronpa:ren:luc</li> | ||
+ | |||
+ | <li>LacIBD:KDronpa:NDronpa:ren:luc</li> | ||
+ | |||
+ | <li>OpLexA:luc (151)</li> | ||
+ | |||
+ | <li>OpUAS:luc (227)</li> | ||
+ | |||
+ | <li>OpLacI:luc (152)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>It was made liquid culture of Gal4:PIF:phyB:ren; Ω1 (C1-C5)</p> | ||
+ | |||
+ | <p>It was made ligations:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>35S:Gal4:AsLOVpep:T35S; α1</td><td>35S:LacI:AsLOVpep:T35S; α1</td><td>35S:LexA:AsLOVpep:T35S; Ω1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl 35S (0030)</td><td>1 µl 35S (0030)</td><td>1 µl 35S (0030)</td></tr> | ||
+ | |||
+ | <tr><td>1 µl Gal4BD</td><td>1 µl LacI</td><td>1 µl LexA</td></tr> | ||
+ | |||
+ | <tr><td>1 µl AsLOVpep </td><td>1 µl AsLOVpep </td><td>1 µl AsLOVpep </td></tr> | ||
+ | |||
+ | <tr><td>1 µl T35S (0036)</td><td>1 µl T35S (0036)</td><td>1 µl T35S (0036)</td></tr> | ||
+ | |||
+ | <tr><td>1 µl α1 </td><td>1 µl α1 </td><td>1 µl α1 </td></tr> | ||
+ | |||
+ | <tr><td>2.6 µl H<sub>2</sub>O</td><td>2.6 µl H<sub>2</sub>O</td><td>2.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PsinATG:RepPhiC31:GFP:T35S; α2</td><td>LacI:PIF:PhyB:ren+luc; Ω1</td><td>PIF:PhyB+renilla; α2</td></tr> | ||
+ | |||
+ | <tr><td>1 µl PsinATG (552)</td><td>1.5 µl LacI:PIF:PhyB:ren; α1</td><td>1.5 µl PIF:PhyB</td></tr> | ||
+ | |||
+ | <tr><td>1 µl ReporterPhiC31</td><td>3 µl OpLacI:luc (152); α2</td><td>2.5 µl renilla (159)</td></tr> | ||
+ | |||
+ | <tr><td>1 µl GFP (0059)</td><td>0.5 µl Ω1</td><td>0.5 µl α2</td></tr> | ||
+ | |||
+ | <tr><td>1 µl T35S (0036)</td><td>2.6 H<sub>2</sub>O</td><td>3 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | <tr><td>1 µl α2 </td><td></td></tr> | ||
+ | |||
+ | <tr><td>2.6 µl H<sub>2</sub>O</td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>EXPERIMENT 5.</p> | ||
+ | |||
+ | <p>Luciferase essay.</p> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">22 July 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Miniprep of Gal4:PIF:PhyB:ren (C1-C3) C4 and C5 did not grow.</p> | ||
+ | |||
+ | <p>Digestion of the miniprep:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4:PIF:PhyB:ren</td><td>BamHI</td><td>16684</td></tr> | ||
+ | |||
+ | <tr><td></td><td>EcoRI</td><td>4852, 5487, 6345</td></tr> | ||
+ | |||
+ | <tr><td></td><td>EcoRV</td><td>1849, 3942, 2475, 381, 8037</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Gel was made:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4:PIF:PhyB:ren (BamHI)</td><td>Gal4:PIF:PhyB:ren (EcoRI)</td><td>Gal4:PIF:PhyB:ren (EcoRV)</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>no</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/7/7f/Valencia_upv_gel_1507122.png> | ||
+ | |||
+ | |||
+ | |||
+ | <p>After doing several digestions with different enzyme all with wrong band patterns, it was decided to revise each part making digestions. The parts are: </p> | ||
+ | |||
+ | <ul><li>PhiC31; pUPD2</li> | ||
+ | |||
+ | <li>Gal4:PIF:PhyB </li> | ||
+ | |||
+ | <li>Renilla (GB159)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Made liquid culture of the ReporterBxbI:GFP in <i>Agrobacterium</i>.</p> | ||
+ | |||
+ | <p>Transform the ligations into E. coli:</p> | ||
+ | |||
+ | <ul><li>35S:Gal4:AsLOVpep:T35S; α1</li> | ||
+ | |||
+ | <li>35S:LacI:AsLOVpep:T35S; α1</li> | ||
+ | |||
+ | <li>35S:LexA:AsLOVpep:T35S; α1</li> | ||
+ | |||
+ | <li>PsinATG:RepPhiC31:GFP:T35S; α2</li> | ||
+ | |||
+ | <li>LacI:PIF:PhyB:ren+luc; Ω1</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>EXPERIMENT 6.</p> | ||
+ | |||
+ | <p>Luciferase essay:</p> | ||
+ | |||
+ | <ul><li>Sampling: samples that have been in red and natural light 48h, 3 samples.</li> | ||
+ | |||
+ | <li>200 µl/sample x 3 sample= 600 µl of passive lissis buffer (5x)</li> | ||
+ | |||
+ | <li>Passive lissis buffer 1x= 120 µl+480 µl water.</li> | ||
+ | |||
+ | <li>2.4 µl of Stop and glow</li> | ||
+ | |||
+ | <li>118 µl of buffer.</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | </br><h3 style="color:green">23 July 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>EXPERIMENT 7.</p> | ||
+ | |||
+ | <p>We decided to infiltrate soybean sprouts. First we decided to infiltrate with dye to observe the characteristics and the capacity of absorption.</p> | ||
+ | |||
+ | <p>FOTO</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make liquid cultures of yesterday transformations.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">24 July 2015</h3> | ||
+ | |||
+ | <p>Minipreps of the 13 liquid culture. LacI:PIF:phyB:ren cultures did not grow.</p> | ||
+ | |||
+ | <p>Digestion of the minipreps:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4:AsLOVpep</td><td>EcoRI</td><td>6345, 1972</td></tr> | ||
+ | |||
+ | <tr><td>LacI:AsLOVpep</td><td>EcoRI</td><td>6345, 2743</td></tr> | ||
+ | |||
+ | <tr><td>LexA:AsLOVpep</td><td>EcoRI</td><td>6345, 2011</td></tr> | ||
+ | |||
+ | <tr><td>PsinATG:RepPhiC31:GFP</td><td>HindIII</td><td>6345, 2691</td></tr> | ||
+ | |||
+ | <tr><td>LexA:PIF:PhyB:ren+luc</td><td>BamHI</td><td>9431, 6674, 3513</td><td></td></tr> | ||
+ | |||
+ | <tr><td>PhyC31; pUPD2</td><td>NotI</td><td>2046, 1899</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:PIF:phyB</td><td>BamHI</td><td>6674, 3474, 2337</td></tr> | ||
+ | |||
+ | <tr><td>Renilla (GB159)</td><td>EcoRV</td><td>2909, 2475, 882, 812, 381</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>It was done 2 gels with ligations:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4:AsLOV C1</td><td>Gal4:AsLOV C2</td><td>Gal4:AsLOV C3</td><td>PsinATG:RepPhiC31:GFP C1</td><td>PsinATG:RepPhiC31:GFP C2</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>no</td></tr> | ||
+ | |||
+ | <tr><td>PsinATG:RepPhiC31:GFP C3</td><td>LacI:AsLOV C1</td><td>LacI:AsLOV C2</td><td>LexA:AsLOV C1</td><td>LexA:AsLOV C2</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>ok</td><td>No</td><td>no</td><td>ok</td></tr> | ||
+ | |||
+ | <tr><td>LexA:AsLOV C3</td><td>LexA:PIF:PhyB:ren+luc C1</td><td>LexA:PIF:PhyB:ren+luc C2</td><td></td><td></td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>Ok</td><td></td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/e/e8/Valencia_upv_gel_150624.png> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PhiC31 (C1)</td><td>PhiC31 (C2)</td><td>PhiC31 (C3)</td><td>PhiC31 (C4)</td><td>PhiC31 (C5)</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>ok</td><td>ok</td><td>ok</td><td>no</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:PIF:phyB</td><td>Renilla (GB159)</td><td></td><td></td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>Ok</td><td></td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">26 July 2015</h3> | ||
+ | |||
+ | <p>Liquid cultures of <i>Agrobacterium</i> were refreshed:</p> | ||
+ | |||
+ | <ul><li>TsinATG:BxbIreporter:GFP</li> | ||
+ | |||
+ | <li>TsinATG:BxbI:reporterBxbI:GFP</li> | ||
+ | |||
+ | <li>Viral vectors (citoplasm, integrase)</li> | ||
+ | |||
+ | <li>GFP</li> | ||
+ | |||
+ | <li>Dsred</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | </br><h3 style="color:green">27 July 2015</h3> | ||
+ | |||
+ | <p>Sent to sequence:</p> | ||
+ | |||
+ | <ul><li>210.08.256: LacIBD; pUPD2 (C1)</li> | ||
+ | |||
+ | <li>210.08.258: Gal4BD; pUPD2 (C2)</li> | ||
+ | |||
+ | <li>210.08.259:LexABD; pUPD2 (C1) </li> | ||
+ | |||
+ | <li>210.08.260: PIF6; pUPD2 (C5)</li> | ||
+ | |||
+ | <li>210.08.261: VP16; pUPD2 (C1)</li> | ||
+ | |||
+ | <li>210.08.262: PhiC31; pUPD2 (C2)</li> | ||
+ | |||
+ | <li>210.08.264: PhiC31; pUPD2 (C3)</li> | ||
+ | |||
+ | <li>210.08.266: PhiC31; pUPD2 (C4)</li> | ||
+ | |||
+ | <li>210.08.268: ReporterBxbI; pUPD2 (C1)</li> | ||
+ | |||
+ | <li>210.08.269: ReporterPhiC31; pUPD2 (C1)</li> | ||
+ | |||
+ | <li>210.08.270: AsLOVpep; pUPD2 (C1)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>The sample had to have 10µl of miniprep (200ng/µl aprox) + 5 µl of primer (dilution 1:3)</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Ligations:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4:PIF:phiB + ren; α1</td><td>LacI:PIF:phiB:ren + luc; Ω2</td><td>PIF:PhyB+renilla; α2</td></tr> | ||
+ | |||
+ | <tr><td>1.5 µl Gal4:PIF:PhyB</td><td>1.5 µl LacI:PIF:PhyB:ren</td><td>1.5 µl PIF:PhyB</td></tr> | ||
+ | |||
+ | <tr><td>2 µl Renilla (GB159)</td><td>2 µl OpLacI:luc</td><td>2.5 µl Renilla (GB159)</td></tr> | ||
+ | |||
+ | <tr><td>0.5 µl α1</td><td>0.5 µl Ω2</td><td>0.5 µl α2</td></tr> | ||
+ | |||
+ | <tr><td>3.6 µl H<sub>2</sub>O</td><td>2.6 µl H<sub>2</sub>O</td><td>3 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>OD’s measurements to agroinfiltrate:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Cytoplasm: 0.39 (viral)</td><td>0.25ml</td></tr> | ||
+ | |||
+ | <tr><td>Integrase: 0.35 (viral)</td><td>0.28ml</td></tr> | ||
+ | |||
+ | <tr><td>GFP: 0.32 (viral)</td><td>0.31ml</td></tr> | ||
+ | |||
+ | <tr><td>Dsred: 0.31 (viral)</td><td>0.32ml</td></tr> | ||
+ | |||
+ | <tr><td>BxbI:reporter: 0.41</td><td>0.49ml</td></tr> | ||
+ | |||
+ | <tr><td>Reporter: 0.26</td><td>0.77ml</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>EXPERIMENT 8.</p> | ||
+ | |||
+ | <ul><li>BxbI:RepBxbI:GFP</li> | ||
+ | |||
+ | <li>RepBxbI:GFP</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>EXPERIMENT 9.</p> | ||
+ | |||
+ | <p>Infiltrate at vaccum the soyabeans sprouts with viral system and GFP.</p> | ||
+ | |||
+ | <p>Ligations to join the negative controls with renilla:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Etr8:luc+staffer(SF)</td><td>OpLexA:luc+SF</td><td>OpLacI:luc+SF</td><td>OpUAS:luc+SF</td></tr> | ||
+ | |||
+ | <tr><td>1.5 µl Etr8:luc (88C o 1098)</td><td>1.5 µl OpLexA:luc (151)</td><td>1.5 µl OpLacI:luc (152)</td><td>1.5 µl UAS:luc (227)</td></tr> | ||
+ | |||
+ | <tr><td>1 µl SF; α2</td><td>1 µl SF; α2</td><td>1 µl SF; α2</td><td>1 µl SF;α2</td></tr> | ||
+ | |||
+ | <tr><td>1 µl Ω1</td><td>1 µl Ω1</td><td>1 µl Ω1</td><td>1 µl Ω1</td></tr> | ||
+ | |||
+ | <tr><td>6.1 µl H<sub>2</sub>O</td><td>6.1 µl H<sub>2</sub>O</td><td>6.1 µl H<sub>2</sub>O</td><td>6.1 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Transformation of E. coli of the ligations and make petri dish cultures:</p> | ||
+ | |||
+ | <ul><li>Gal4:PIF:PhyB + ren; α1</li> | ||
+ | |||
+ | <li>LacI:PIF:PhyB:ren + luc; Ω2</li> | ||
+ | |||
+ | <li>PIF:PhyB+renilla; α2</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Transformation into <i>Agrobacterium</i> with the constructions and make petri dish cultures:</p> | ||
+ | |||
+ | <ul><li>Gal4:KDronpa:NDronpa:ren:luc</li> | ||
+ | |||
+ | <li>LacI:KDronpa:NDronpa:ren:luc</li> | ||
+ | |||
+ | <li>LexA:KDronpa:NDronpa:ren:luc</li> | ||
+ | |||
+ | <li>OpLexA:luc (GB151)</li> | ||
+ | |||
+ | <li>OpLacI:luc (GB152)</li> | ||
+ | |||
+ | <li>OpUAS:luc (GB227)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>It was received a new piece (ePDZ) which is part of the blue toggle (plan A). It arrived in <i>E. coli</i> so it was made a lquid culture and let it grow at 37ºC overnight.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">28 July 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Miniprep of the liquid culture: ePDZ.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transformation into <i>E. coli</i> of the ligations:</p> | ||
+ | |||
+ | <ul><li>Etr8:luc+staffer(SF)</li> | ||
+ | |||
+ | <li>OpLexA:luc+SF</li> | ||
+ | |||
+ | <li>OpLacI:luc+SF</li> | ||
+ | |||
+ | <li>UAS:luc+SF</li> | ||
+ | |||
+ | <li>Gal4:PIF:PhyB:ren</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>EXPERIMENT 9.</p> | ||
+ | |||
+ | <p>It was observed the soybean sprouts that were infiltrated with <i>Agrobacterium</i>with green light and red filter. It was not observed nothing significant, moreover, the damage is evident. </p> | ||
+ | |||
+ | <p>FOTO</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transformation in <i>Agrobacterium</i> the ReporterPhiC31:GFP.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Pick colonies and make liquid cultures adding X-Gal and IPTG because the colonies were little and we can not observe clearly if they were white or blue.</p> | ||
+ | |||
+ | <ul><li>Gal4:PIF:phiB+ren. Did not grow any colony.</li> | ||
+ | |||
+ | <li>LacI:PIF:PhyB:ren+luc (C1-C3)</li> | ||
+ | |||
+ | <li>PIF:PhyB+renilla (C1- C3)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>The medicine LTB (heat labile toxin B subunit) which is part of an enterotoxin of Echerichia coli homologous to the same toxin in Vibrio cholerae that causes diarrhea. </p> | ||
+ | |||
+ | <p>We add 50µl to have a final concentration of 10ng/µl.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Ligation:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LTB; pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1 µl LTB</td></tr> | ||
+ | |||
+ | <tr><td>1 µl pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>5.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">29 July 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Miniprep of yesterday liquid culture:</p> | ||
+ | |||
+ | <ul><li>LacI:PIF:phiB:ren+luc (C1 and C3) C2 turn into blue.</li> | ||
+ | |||
+ | <li>PIF:PhyB+renilla (C2) C1 and C3 did not grow.</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Digestion:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LacI:PIF:phiB:ren:luc</td><td>EcoRV</td><td>882, 968, 1652, 3942, 2475, 381, 3477, 6674</td></tr> | ||
+ | |||
+ | <tr><td>PIF:PhyB+renilla</td><td>HindIII</td><td>4316, 5887, 788, 6345</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LacI:PIF:phiB:ren:luc (C1)</td><td>LacI:PIF:phiB:ren:luc (C3)</td><td>PIF:PhyB+renilla (C2)</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>no</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/e/e9/Valencia_upv_gel_150629.png> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Pick colonies and make liquid culture of:</p> | ||
+ | |||
+ | <ul><li>Etr8:luc:staffer(SF) (C1-C3)</li> | ||
+ | |||
+ | <li>OpLexA:luc:SF (C1-C3)</li> | ||
+ | |||
+ | <li>OpLacI:luc:SF (C1-C3)</li> | ||
+ | |||
+ | <li>UAS:luc:SF (C1-C3)</li> | ||
+ | |||
+ | <li>Gal4:PIF:phyB:ren (C1-C3)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Transformation in <i>E. coli</i> of:</p> | ||
+ | |||
+ | <ul><li>LTB; pUPD2</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">30 July 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Minipreps have been done:</p> | ||
+ | |||
+ | <ul><li>Etr8:luc:staffer(SF) (C1-C3)</li> | ||
+ | |||
+ | <li>OpLexA:luc:SF (C1 and C3) C2 did not grow. </li> | ||
+ | |||
+ | <li>OpLacI:luc:SF (C1-C3)</li> | ||
+ | |||
+ | <li>OpUAS:luc:SF (C1-C3)</li> | ||
+ | |||
+ | <li>Gal4:PIF:phyB:ren (C1-C3)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <ul><li>LacI:PIF:phy:ren:luc (C1-C3)</li> | ||
+ | |||
+ | <li>PIF:PhyB:ren (C1-C3)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Etr8:luc:staffer(SF)</td><td>BamHI</td><td>6674, 2766</td></tr> | ||
+ | |||
+ | <tr><td>OpLexA:luc:SF</td><td>BamHI</td><td>6674, 2746</td></tr> | ||
+ | |||
+ | <tr><td>OpLacI:luc:SF</td><td>BamHI</td><td>6674, 2847</td></tr> | ||
+ | |||
+ | <tr><td>OpUAS:luc:SF</td><td>BamHI</td><td>6674, 2568</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:PIF:phyB:ren</td><td>EcoRI</td><td>6345, 5487, 4852</td></tr> | ||
+ | |||
+ | <tr><td>LacI:PIF:phy:ren:luc</td><td>BamHI</td><td>20451</td></tr> | ||
+ | |||
+ | <tr><td>PIF:PhyB:ren</td><td>HindIII</td><td>6345, 5887, 4316, 788</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Do the gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Primers have arrived:</p> | ||
+ | |||
+ | <ul><li>ePDZ reverse and forward and phiC31.</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Make a PCR with ePDZ and its primers to obtain the desired fragment and put it in pUPD2.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>ePDZ PCR</td></tr> | ||
+ | |||
+ | <tr><td>10µl Buffer HF</td></tr> | ||
+ | |||
+ | <tr><td>31.5 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | <tr><td>2 µl dNTPs</td></tr> | ||
+ | |||
+ | <tr><td>2.5 µl Primer forward</td></tr> | ||
+ | |||
+ | <tr><td>2.5 µl primer reverse</td></tr> | ||
+ | |||
+ | <tr><td>1 µl ePDZ (dilution 1:50)</td></tr> | ||
+ | |||
+ | <tr><td>0.5 µl Taq phunion</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Pick 2 colonies of PhiC31:GFP in <i>Agrobacterium</i> and make liquid culture.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Ligations:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>ePDZ; pUPD2</td><td>PIF:phyB+ren; α2</td><td>OpUAS:luc:SF+ren; α1</td><td>OpLexA:luc:SF+ren; α1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl ePDZ</td><td>1 µl PIF:phyB</td><td>1 µl OpUAS:luc:SF</td><td>1 µl OpLexA:luc:SF</td></tr> | ||
+ | |||
+ | <tr><td>1 µl pUPD2</td><td>1 µl ren (159)</td><td>1 µl ren</td><td>1 µl ren</td></tr> | ||
+ | |||
+ | <tr><td>5.6 µl H<sub>2</sub>O</td><td>1 µl α2</td><td>1 µl α1</td><td>1 µl α1</td></tr> | ||
+ | |||
+ | <tr><td></td><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>OpEtr8:luc:SF+ren; α1</td><td>Op:LacI:luc:SF+ren; α1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl OpEtr8:luc:SF</td><td>1 µl OpLacI:luc:SF</td></tr> | ||
+ | |||
+ | <tr><td>1 µl ren</td><td>1 µl ren</td></tr> | ||
+ | |||
+ | <tr><td>1 µl α1</td><td>1 µl aplha1</td></tr> | ||
+ | |||
+ | <tr><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make liquid culture (<i>E. coli</i>) of:</p> | ||
+ | |||
+ | <ul><li>LTB; pUPD2 (C1-C3)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | </br><h3 style="color:green">31 July 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Ligation:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LacI:PIF:PhyB:ren+OpLacI:luc; Ω2</td></tr> | ||
+ | |||
+ | <tr><td>1.5 µl LacI:PIF:phyB:ren</td></tr> | ||
+ | |||
+ | <tr><td>3 µl OpLacI:luc</td></tr> | ||
+ | |||
+ | <tr><td>0.5 µl Ω2</td></tr> | ||
+ | |||
+ | <tr><td>2.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>EXPERIMENT 8.</p> | ||
+ | |||
+ | <p>After 4 days till the agroinfiltration we have observed again the samples with BxbI and the negative control.</p> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Minipreps of liquid culture LTB (C1 and C2)</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Digestion:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LTB; pUPD2</td><td>NotI</td><td>2046, 474</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LTB (C1)</td><td>LTB (C2)</td><td>PCR ePDZ</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/e/ef/Valencia_upv_gel_150731.png> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Take out glicerynates of Paloma, lab mate:</p> | ||
+ | |||
+ | <ul><li>Sip rotavirus CH<sub>2</sub></li> | ||
+ | |||
+ | <li>Sip rotavirus CH<sub>2</sub>-CH<sub>3</sub></li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>For <i>E. coli</i> and <i>Agrobacterium</i>.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Pick a colony of Asun interferon (IFN) in Agro, make liquid culture.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>We had miniprep of IFN in pUPD. Make ligations.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transform in <i>E. coli</i> the ligations and make petri dishes cultures:</p> | ||
+ | |||
+ | <ul><li>ePDZ; pUPD2</li> | ||
+ | |||
+ | <li>PIF:phyB+ren; α2</li> | ||
+ | |||
+ | <li>OpUAS:luc:SF+ren; α1</li> | ||
+ | |||
+ | <li>OpLexA:luc:SF+ren; α1</li> | ||
+ | |||
+ | <li>OpEtr8:luc:SF+ren; α1</li> | ||
+ | |||
+ | <li>Op:LacI:luc:SF+ren; α1</li> | ||
+ | |||
+ | <li>LacI:PIF:phyB:ren+OpLacI:luc; Ω2</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Refresh agro cultures to agroinfiltrate tomorrow:</p> | ||
+ | |||
+ | <ul><li>PhiC31 (viral system)</li> | ||
+ | |||
+ | <li>ReporterPhiC31</li> | ||
+ | |||
+ | <li>BxbI+reporterBxbI</li> | ||
+ | |||
+ | <li>ReporterBxbI</li> | ||
+ | |||
+ | <li>Gal4:KDronpa:NDronpa:luc:ren (blue toggle)</li> | ||
+ | |||
+ | <li>EPIF:phi:luc</li> | ||
+ | |||
+ | <li>Renilla</li> | ||
+ | |||
+ | <li>P19</li> | ||
+ | |||
+ | <li>Pnos</li> | ||
+ | |||
+ | |||
</p><br/> | </p><br/> | ||
</div> | </div> | ||
Line 57: | Line 4,021: | ||
<summary class="button fit">August and September</summary> | <summary class="button fit">August and September</summary> | ||
<div class="clsPadding"> | <div class="clsPadding"> | ||
− | <p> | + | <h3 style="color:green">1 August 2015</h3> |
− | + | ||
− | + | <p>Prepare the <i>Agrobacterium</i> cultures for agroinfiltration:</p> | |
+ | |||
+ | <p>Gal4B:KDronpa:NDronpa:luc:ren, PhiC31 and its control, BxbI and its control and PIF:PhyB and its control.</p> | ||
+ | |||
+ | <p>Mesurement oof the ODs:</p> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Ren+P19</td><td>0.09</td><td>888.9 µl</td></tr> | ||
+ | |||
+ | <tr><td>RepPhiC31</td><td>0.69</td><td>115.94 µl</td></tr> | ||
+ | |||
+ | <tr><td>BxbI</td><td>0.46</td><td>174 µl</td></tr> | ||
+ | |||
+ | <tr><td>RepBxbI</td><td>0.15</td><td>533.3 µl</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:KNDronpa:ren:luc</td><td>0.51</td><td>156.9 µl</td></tr> | ||
+ | |||
+ | <tr><td>Pnos</td><td>0.29</td><td>275.9 µl</td></tr> | ||
+ | |||
+ | <tr><td>PIF:phy:luc</td><td>0.17</td><td>470.6 µl</td></tr> | ||
+ | |||
+ | <tr><td>PhiC31</td><td>0.32</td><td>250 µl</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>EXPERIMENT 9.</p> | ||
+ | |||
+ | <ul><li>PhiC31</li> | ||
+ | |||
+ | <li>RepPhiC31:GFP</li> | ||
+ | |||
+ | <li>BxbI:RepBxbI:GFP</li> | ||
+ | |||
+ | <li>RepBxbI:GFP</li> | ||
+ | |||
+ | <li>Gal4:NDronpa:KDronpa:luc:ren.</li> | ||
+ | |||
+ | <li>Pnos</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Look the petri dishes culture:</p> | ||
+ | |||
+ | <ul><li>PIF:phyB+ren; α2</li> | ||
+ | |||
+ | <li>OpLexA:luc:SF+ren; α1</li> | ||
+ | |||
+ | <li>Op:LacI:luc:SF+ren; α1</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <ul><li>ePDZ; pUPD2</li> | ||
+ | |||
+ | <li>OpUAS:luc:SF+ren; α1</li> | ||
+ | |||
+ | <li>OpEtr8:luc:SF+ren; α1</li> | ||
+ | |||
+ | <li>LacI:PIF:PhyB:ren+OpLac:luc; α2</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | </br><h3 style="color:green">3 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Miniprep of the liquid culture:</p> | ||
+ | |||
+ | <ul><li>Sip rotavirus CH<sub>2</sub></li> | ||
+ | |||
+ | <li>Sip rotavirus CH<sub>2</sub>-CH<sub>3</sub></li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Measure of DNA concentration of:</p> | ||
+ | |||
+ | <ul><li>OpLexA:luc:SF=169ng/µl</li> | ||
+ | |||
+ | <li>OpacI:luc:SF=291ng/µl</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Pick colonies and make liquid culture of:</p> | ||
+ | |||
+ | <ul><li>ePDZ; pUPD2 (C1-C3)</li> | ||
+ | |||
+ | <li>OpUAS:luc:SF+ren; α1(C1-C3)</li> | ||
+ | |||
+ | <li>OpEtr8:luc:SF+ren; α1(C1-C3)</li> | ||
+ | |||
+ | <li>LacI:PIF:phyB:ren+OpLacI:luc; Ω2 (C1-C3) Added X-gal and IPTG.</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Repeat ligations:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>OpLexA:luc:SF+ren; α1</td><td>Op:LacI:luc:SF+ren; α1</td><td>E-PIF:phyB+ren; α2</td></tr> | ||
+ | |||
+ | <tr><td>1 µl OpLexA:luc:SF</td><td>1 µl OpLacI:luc:SF</td><td>1 µl E-PIF:phy</td></tr> | ||
+ | |||
+ | <tr><td>3 µl ren</td><td>3 µl ren</td><td>3 µl ren</td></tr> | ||
+ | |||
+ | <tr><td>1 µl α1</td><td>1 µl α1</td><td>1 µl α2</td></tr> | ||
+ | |||
+ | <tr><td>2.6 µl H<sub>2</sub>O</td><td>2.6 µl H<sub>2</sub>O</td><td>2.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Refresh agrobacterium cultures of:</p> | ||
+ | |||
+ | <ul><li>Interferon</li> | ||
+ | |||
+ | <li>SIP-CH<sub>2</sub></li> | ||
+ | |||
+ | <li>SIP-CH<sub>2</sub>-CH<sub>3</sub></li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Take the glycerinate of Renilla; Ω2 (GB159) and make liquid culture.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>EXPERIMENT 9.</p> | ||
+ | |||
+ | <p>We have made all the leaf discs and put in order in the plates.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">4 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>We do a Western blot with Asun so we can learn how to do it. Lo copio? De verdad!!?</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Miniprep of the liquid cultures:</p> | ||
+ | |||
+ | <ul><li>ePDZ; pUPD2 (C1-C3)</li> | ||
+ | |||
+ | <li>OpUAS:luc:SF+ren; ?1(C1-C3)</li> | ||
+ | |||
+ | <li>OpEtr8:luc:SF+ren; ?1(C1-C3)</li> | ||
+ | |||
+ | <li>LacI:PIF:phyB+ren; ?2 (C2 and C3) C1 was blue.</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Digestions:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>ePDZ; pUPD2</td><td>NotI</td><td>2046, 642</td></tr> | ||
+ | |||
+ | <tr><td>OpUAS:luc:SF+ren; α1</td><td>EcoRI</td><td>6345, 7096</td></tr> | ||
+ | |||
+ | <tr><td>OpEtr8:luc:SF+ren; α1</td><td>EcoRI</td><td>6345, 7294</td></tr> | ||
+ | |||
+ | <tr><td>LacI:PIF:phyB:ren+OpLacI:luc; Ω2</td><td>EcoRV</td><td>6674, 3477, 381, 2475, 3942, 1652, 968, 882</td></tr> | ||
+ | |||
+ | <tr><td>Renilla 159</td><td>EcoRV</td><td>2909, 2475, 882, 812, 381</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>ePDZ C1</td><td>ePDZ C2</td><td>ePDZ C3</td><td>LacI:PIF:PhyB+ren </td></tr> | ||
+ | |||
+ | <tr><td>Ok</td><td>ok</td><td>ok</td><td></td></tr> | ||
+ | |||
+ | <tr><td>Renilla 159</td><td>OpUAS:luc:SF+ren C1</td><td>OpUAS:luc:SF+ren C2</td><td>OpUAS:luc:SF+ren C3</td></tr> | ||
+ | |||
+ | <tr><td>Ok</td><td>¿?</td><td></td></tr> | ||
+ | |||
+ | <tr><td>OpEtr8:luc:SF+ren C1</td><td>OpEtr8:luc:SF+ren C2</td><td>OpEtr8:luc:SF+ren C3</td><td></td></tr> | ||
+ | |||
+ | <tr><td></td><td></td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Transformation into <i>E. coli</i> of yesterday ligations and make petri dish culture:</p> | ||
+ | |||
+ | <ul><li>OpLexA:luc:SF+ren; α1</li> | ||
+ | |||
+ | <li>Op:LacI:luc:SF+ren; α1</li> | ||
+ | |||
+ | <li>E-PIF:phyB+ren; α2</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Ligations:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>35S+ePDZ+VP16+T35S;α2</td><td>35S+LTB+T35S; α1</td></tr> | ||
+ | |||
+ | <tr><td>1µl ePDZ</td><td>1µl 35S (30)</td></tr> | ||
+ | |||
+ | <tr><td>1 µl VP16</td><td>1µl T35S (36)</td></tr> | ||
+ | |||
+ | <tr><td>1 µl 35S (30)</td><td>1µl LTB</td></tr> | ||
+ | |||
+ | <tr><td>1 µl T35S (36)</td><td>1µl α1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl α2</td><td>3.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | <tr><td>2.6 µl H<sub>2</sub>O</td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Refresh agrobacterium cultures of:</p> | ||
+ | |||
+ | <ul><li>Interferon</li> | ||
+ | |||
+ | <li>SIP-CH<sub>2</sub></li> | ||
+ | |||
+ | <li>SIP-CH<sub>2</sub>-CH<sub>3</sub></li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | </br><h3 style="color:green">5 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transform ligations:</p> | ||
+ | |||
+ | <ul><li>35S+ePDZ+VP16+T35S; α2</li> | ||
+ | |||
+ | <li>35S+LTB+T35S; α1</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>Pick colonies and make liquid cultures cultures of:</p> | ||
+ | |||
+ | <ul><li>OpLexA:luc:SF+ren; α1 (C1-C3)</li> | ||
+ | |||
+ | <li>Op:LacI:luc:SF+ren; α1 (C1-C3)</li> | ||
+ | |||
+ | <li>E-PIF:phyB+ren; α2 (C1)</li> | ||
+ | |||
+ | <li>35S+ePDZ+VP16+T35S; α2 (C1 and C2)</li> | ||
+ | |||
+ | <li>35S+LTB+T35S; α1 (C1 and C2)</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>EXPERIMENT 9.</p> | ||
+ | |||
+ | <p>Make luciferase essay of red and blue toggle:</p> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">6 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Miniprep of the liquid cultures.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Digestion:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>OpLexA:luc:SF+ren</td><td>EcoRI</td><td>6345, 7274</td></tr> | ||
+ | |||
+ | <tr><td>Op:LacI:luc:SF+ren</td><td>EcoRI</td><td>6345, 7375</td></tr> | ||
+ | |||
+ | <tr><td>E-PIF:phyB+ren</td><td>HindIII</td><td>6345, 788, 5887, 4316</td></tr> | ||
+ | |||
+ | <tr><td>35S+ePDZ+VP16+T35S</td><td>HindIII</td><td>6345, 2316</td></tr> | ||
+ | |||
+ | <tr><td>35S+LTB+T35S</td><td>EcoRI</td><td>6345, 1684</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PIF:phyB+ren</td><td>OpLexA:luc:SF+ren C1</td><td>OpLexA:luc:SF+ren C2</td><td>OpLexA:luc:SF+ren C3</td></tr> | ||
+ | |||
+ | <tr><td>No</td><td>Ok, repeat</td><td>No</td><td>Ok, repeat</td></tr> | ||
+ | |||
+ | <tr><td>Op:LacI:luc:SF+ren C1</td><td>Op:LacI:luc:SF+ren C2</td><td>Op:LacI:luc:SF+ren C3</td><td>35S+LTB+T35S C1</td></tr> | ||
+ | |||
+ | <tr><td>Ok, repeat</td><td>Ok</td><td>Ok</td><td>ok</td></tr> | ||
+ | |||
+ | <tr><td>35S+LTB+T35S C2</td><td>35S+ePDZ+VP16+T35S C1</td><td>35S+ePDZ+VP16+T35S C2</td><td></td></tr> | ||
+ | |||
+ | <tr><td>Ok</td><td>ok</td><td>ok</td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Make colony PCR with 6 colonies of PhiC31:</p> | ||
+ | |||
+ | <ul><li>DNA- </li> | ||
+ | |||
+ | <li>JM1: 2 µl (dilution 1:10)</li> | ||
+ | |||
+ | <li>JM2: 2 µl (dilution 1:10)</li> | ||
+ | |||
+ | <li>Buffer Taq (with Mg): 2 µl</li> | ||
+ | |||
+ | <li>dNTPs: 2.5 µl</li> | ||
+ | |||
+ | <li>Taq: 0.5 µl</li> | ||
+ | |||
+ | <li>H<sub>2</sub>O: 10 µl</li> | ||
+ | |||
+ | <li>Program: 96ºC-2min; 96ºC-30min; 55ºC-30min; 72ºC-30min</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Make a gel with the PCR but we did not see the correct bands.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Repeat the colony PCR:</p> | ||
+ | |||
+ | <ul><li>DNA- </li> | ||
+ | |||
+ | <li>JM1: 2.5 µl (dilution 1:10)</li> | ||
+ | |||
+ | <li>JM2: 2.5 µl (dilution 1:10)</li> | ||
+ | |||
+ | <li>Buffer Taq (with Mg): 10 µl</li> | ||
+ | |||
+ | <li>dNTPs: 2 µl</li> | ||
+ | |||
+ | <li>Taq: 0.5 µl</li> | ||
+ | |||
+ | <li>H<sub>2</sub>O: 7.5 µl</li> | ||
+ | |||
+ | <li>Program: : 96ºC-10min; 96ºC-30min; 55ºC-30min; 72ºC-30min</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Ligations:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LexA:AsLOVpep+ePDZ; Ω1</td><td>LacI:AsLOVpep+ePDZ;Ω1</td><td>Gal4:AsLOVpep+ePDZ; Ω1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl LexA:AsLOVpep; α1</td><td>1 µl LacI:AsLOVpep; α1</td><td>1 µl Gal4:AsLOVpep;α1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl ePDZ; α2</td><td>1 µl ePDZ; α2</td><td>1 µl ePDZ; α2</td></tr> | ||
+ | |||
+ | <tr><td>1 µl BsmBI</td><td>1 µl BsmBI</td><td>1 µl BsmBI</td></tr> | ||
+ | |||
+ | <tr><td>1 µl Ω1</td><td>1 µl Ω1</td><td>1 µl Ω1</td></tr> | ||
+ | |||
+ | <tr><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Refresh <i>Agrobacterium</i>cultures to agroinfiltrate:</p> | ||
+ | |||
+ | <ul><li>PhiC31:RepPhiC31:GFP</li> | ||
+ | |||
+ | <li>RepPhiC31:GFP</li> | ||
+ | |||
+ | <li>BxbI:RepBxbI:GFP</li> | ||
+ | |||
+ | <li>RepBxbI:GFP</li> | ||
+ | |||
+ | <li>IFN (interferon)</li> | ||
+ | |||
+ | <li>Sip-CH<sub>2</sub></li> | ||
+ | |||
+ | <li>Sip-CH<sub>2</sub>-CH<sub>3</sub></li> | ||
+ | |||
+ | <li>Pnos</li> | ||
+ | |||
+ | <li>P19 (this culture has 10ml of LB + 10 µl antibiotics + 5 µl culture)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | </br><h3 style="color:green">7 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transformation into <i>Agrobacterium</i>:</p> | ||
+ | |||
+ | <ul><li>35S:LTB:VP16:T35S</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Transformation into <i>E. coli</i> and make petri dishes cultures:</p> | ||
+ | |||
+ | <ul><li>LexA:AsLOVpep+ePDZ</li> | ||
+ | |||
+ | <li>LacI:AsLOVpep+ePDZ</li> | ||
+ | |||
+ | <li>Gal4:AsLOVpep+ePDZ</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Make a gel with the colonies PCRs of PhiC31:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>C7</td><td>C8</td><td>C9</td><td>C10</td><td>C11</td><td>C12</td><td>C13</td><td>C14</td></tr> | ||
+ | |||
+ | <tr><td>No</td><td>no</td><td>no</td><td>no</td><td>no</td><td>no</td><td>no</td><td>no</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/c/c4/Valencia_upv_150807.png> | ||
+ | |||
+ | |||
+ | |||
+ | <p>There was no DNA, there is a problem with the cells or the procedure, it have to be revised.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Ligation:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PhyC31; pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>3 µl PhiC31</td></tr> | ||
+ | |||
+ | <tr><td>1 µl pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1 µl BsmBI</td></tr> | ||
+ | |||
+ | <tr><td>3.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Refresh agrobacterium cultures for tomorrow infiltration:</p> | ||
+ | |||
+ | <ul><li>Sip-CH<sub>2</sub></li> | ||
+ | |||
+ | <li>Sip-CH<sub>2</sub>-CH<sub>3</sub></li> | ||
+ | |||
+ | <li>Pnos</li> | ||
+ | |||
+ | <li>Red toggle (PIF6:PhyB:luc)</li> | ||
+ | |||
+ | <li>Renilla</li> | ||
+ | |||
+ | <li>Blue toggle (LacI:KDronpa:NDronpa:ren:OpLacI:luc)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>EXPERIMENT 10.</p> | ||
+ | |||
+ | <ul><li>PhiC31:RepPhiC31:GFP</li> | ||
+ | |||
+ | <li>RepPhiC31:GFP</li> | ||
+ | |||
+ | <li>BxbI:RepBxbI:GFP</li> | ||
+ | |||
+ | <li>RepBxbI:GFP</li> | ||
+ | |||
+ | <li>IFN (interferon)</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>Measure f the ODs:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Construction</td><td>OD</td><td>Volume (ml)</td></tr> | ||
+ | |||
+ | <tr><td>IFN</td><td>0.13</td><td>1.54</td></tr> | ||
+ | |||
+ | <tr><td>RepBxbI:GFP</td><td>0.13</td><td>1.54</td></tr> | ||
+ | |||
+ | <tr><td>BxbI:RepBxbI:GFP</td><td>0.33</td><td>0.61</td></tr> | ||
+ | |||
+ | <tr><td>PhiC31</td><td>0.19</td><td>1.05</td></tr> | ||
+ | |||
+ | <tr><td>RepPhiC31:GFP</td><td>0.22</td><td>0.91</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">8 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transform the ligation into <i>E. coli</i> and make petri dishes cultures:</p> | ||
+ | |||
+ | <ul><li>PhiC31; pUPD2.</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <ul><li>LexA:AsLOVpep+ePDZ (C1-C5)</li> | ||
+ | |||
+ | <li>LacI:AsLOVpep+ePDZ (C1-C4)</li> | ||
+ | |||
+ | <li>Gal4:AsLOVpep+ePDZ (C1-C4)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>EXPERIMENT 11.</p> | ||
+ | |||
+ | <ul><li>Sip-CH<sub>2</sub></li> | ||
+ | |||
+ | <li>Sip-CH<sub>2</sub>-CH<sub>3</sub></li> | ||
+ | |||
+ | <li>Pnos</li> | ||
+ | |||
+ | <li>Red toggle (PIF6:PhyB:luc)</li> | ||
+ | |||
+ | <li>Renilla</li> | ||
+ | |||
+ | <li>Blue toggle (LacI:KDronpa:NDronpa:ren:OpLacI:luc)</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>Measurement of the ODs:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Construction</td><td>OD</td><td>Volume (ml)</td></tr> | ||
+ | |||
+ | <tr><td>Sip-CH<sub>2</sub></td><td>0.04</td><td>6.667</td></tr> | ||
+ | |||
+ | <tr><td>Sip-CH<sub>2</sub>-CH<sub>3</sub></td><td>0.04</td><td>6.667</td></tr> | ||
+ | |||
+ | <tr><td>Red toggle (PIF6:PhyB:luc)</td><td>0.09</td><td>15</td></tr> | ||
+ | |||
+ | <tr><td>Blue toggle (LacI:KDronpa:NDronpa:ren:luc)</td><td>0.09</td><td>15.00</td></tr> | ||
+ | |||
+ | <tr><td>Renilla</td><td>0.04</td><td>6.667</td></tr> | ||
+ | |||
+ | <tr><td>Pnos</td><td>0.04</td><td>6.667</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">9 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make a colony PCRs with the 10 white colonies of PhiC31 that grow in the petri dishes.</p> | ||
+ | |||
+ | <ul><li>DNA- colony</li> | ||
+ | |||
+ | <li>JM1: 2 µl (dilution 1:10)</li> | ||
+ | |||
+ | <li>JM2: 2 µl (dilution 1:10)</li> | ||
+ | |||
+ | <li>Buffer Taq (with Mg): 2 µl</li> | ||
+ | |||
+ | <li>dNTPs: 2.5 µl</li> | ||
+ | |||
+ | <li>Taq: 0.5 µl</li> | ||
+ | |||
+ | <li>H<sub>2</sub>O: 1 µl</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Minipreps of the liquid cultures:</p> | ||
+ | |||
+ | <ul><li>LexA:AsLOVpep+ePDZ (C1-C4) C5 has turn into blue color.</li> | ||
+ | |||
+ | <li>LacI:AsLOVpep+ePDZ (C1-C4)</li> | ||
+ | |||
+ | <li>Gal4:AsLOVpep+ePDZ (C1-C4)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Digestion of the minipreps:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LexA:AsLOVpep+ePDZ</td><td>BamHI</td><td>6674, 4309</td></tr> | ||
+ | |||
+ | <tr><td>LacI:AsLOVpep+ePDZ</td><td>BamHI</td><td>6674, 5041</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:AsLOVpep+ePDZ</td><td>BamHI</td><td>6674, 4270</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Make the gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LexA:AsLOVpep+ePDZ (C1)</td><td>LexA:AsLOVpep+ePDZ (C2)</td><td>LexA:AsLOVpep+ePDZ (C3)</td><td>LexA:AsLOVpep+ePDZ (C4)</td></tr> | ||
+ | |||
+ | <tr><td>Ok</td><td>ok</td><td>ok</td><td>ok</td></tr> | ||
+ | |||
+ | <tr><td>oLacI:AsLOVpep+ePDZ (C1)</td><td>LacI:AsLOVpep+ePDZ (C2)</td><td>LacI:AsLOVpep+ePDZ (C3)</td><td>LacI:AsLOVpep+ePDZ (C4)</td></tr> | ||
+ | |||
+ | <tr><td>Ok</td><td>ok</td><td>ok</td><td>ok</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:AsLOVpep+ePDZ (C1)</td><td>Gal4:AsLOVpep+ePDZ (C2)</td><td>Gal4:AsLOVpep+ePDZ (C3)</td><td>Gal4:AsLOVpep+ePDZ (C4)</td></tr> | ||
+ | |||
+ | <tr><td>Ok</td><td>ok</td><td>ok</td><td>Ok</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/d/d1/Valencia_upv_150809.png> | ||
+ | |||
+ | |||
+ | |||
+ | <p>We keep in our inventory the colony C1 of each construction</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make a gel of the colonies PCRs of PhiC31: we did not observed any DNA. Still can’t fix the problem. </p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make liquid culture of renilla and PIF6:PhyB:luc in <i>Agrobacterium</i> because the last time that we did an experiment they have grown slowly.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Store in the 4ºC fridge an agrobacterium culture with P19.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Ligations:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LexA:AsLOVpep+ePDZ+ren; α1</td><td>LacI:AsLOVpep+ePDZ+ren; α1</td><td>Gal4:AsLOVpep+ePDZ+ren; α1</td></tr> | ||
+ | |||
+ | <tr><td>1µl LexA:AsLOVpep+ePDZ</td><td>1µl LacI:AsLOVpep+ePDZ</td><td>1µl Gal4:AsLOVpep+ePDZ</td></tr> | ||
+ | |||
+ | <tr><td>1 µl renilla (159)</td><td>1 µl renilla (159)</td><td>1 µl renilla (159)</td></tr> | ||
+ | |||
+ | <tr><td>1 µl α1</td><td>1 µl α1</td><td>1 µl α1</td></tr> | ||
+ | |||
+ | <tr><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">10 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transform into <i>E. coli</i> this ligations and make petri dishes cultures:</p> | ||
+ | |||
+ | <ul><li>LexA:AsLOVpep+ePDZ+ren; α1</li> | ||
+ | |||
+ | <li>LacI:AsLOVpep+ePDZ+ren; α1</li> | ||
+ | |||
+ | <li>Gal4:AsLOVpep+ePDZ+ren; α1</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make liquid culture of 11 colonies of PhiC31 due to that this construction was giving us problems to obtain.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Refresh the agrobacterium cultures of:</p> | ||
+ | |||
+ | <ul><li>BxbI:Rep:BxbI:GFP</li> | ||
+ | |||
+ | <li>Rep:BxbI:GFP</li> | ||
+ | |||
+ | <li>PhiC31</li> | ||
+ | |||
+ | <li>RepPhiC31:GFP</li> | ||
+ | |||
+ | <li>P19</li> | ||
+ | |||
+ | <li>Citoplasm</li> | ||
+ | |||
+ | <li>Integrase</li> | ||
+ | |||
+ | <li>Dsred</li> | ||
+ | |||
+ | <li>GFP</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>EXPERIMENT 10.</p> | ||
+ | |||
+ | <p>Take the samples of the agroinfiltrated plants that were in darkness to change the conditions.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make liquid culture of:</p> | ||
+ | |||
+ | <ul><li>LexA:AsLOVpep+ePDZ+ren (C1 and C2)</li> | ||
+ | |||
+ | <li>LacI:AsLOVpep+ePDZ+ren (C1-C4)</li> | ||
+ | |||
+ | <li>Gal4:AsLOVpep+ePDZ+ren (C1 and C2)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">11 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Primers have arrived, they have been resuspended. They were used to eliminate the recognitions sites of the enzymes in BioBricks. This will make our constructions ready to be send to the iGEM</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Minipreps of the liquid cultures done yesterday:</p> | ||
+ | |||
+ | <ul><li>PhiC31 (C6-C16)</li> | ||
+ | |||
+ | <li>LacI:AsLOVpep+ePDZ+ren (C1-C4)</li> | ||
+ | |||
+ | <li>Gal4:AsLOVpep+ePDZ+ren (C1 and C2)</li> | ||
+ | |||
+ | <li>LexA:AsLOVpep+ePDZ+ren colonies were all blue</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Make PCRs with all the primers and constructions:</p> | ||
+ | |||
+ | <p>All of them follow the same composition which is:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>10 µl </td><td>Buffer HF</td></tr> | ||
+ | |||
+ | <tr><td>26.5 µl</td><td>H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | <tr><td>2 µl</td><td>dNTPs</td></tr> | ||
+ | |||
+ | <tr><td>0.5 µl</td><td>Taq Phusion</td></tr> | ||
+ | |||
+ | <tr><td>1 µl </td><td>DNA (dilution 1:10)</td></tr> | ||
+ | |||
+ | <tr><td>2.5 µl</td><td>Primer forward (F) (dilution 1:10)</td></tr> | ||
+ | |||
+ | <tr><td>2.5 µl</td><td>Primer reverse (R) (dilution 1:10)</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>*The three last lines are the only specify bellow.</p> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>IFN (1)</td><td>IFN (2)</td><td>AsLOVpep (1)</td><td>AsLOVpep (2)</td></tr> | ||
+ | |||
+ | <tr><td>IFN</td><td>IFN</td><td>AsLOVpep</td><td>AsLOVpep</td></tr> | ||
+ | |||
+ | <tr><td>Mys int F</td><td>IFN domBB R</td><td>AsLOVpep F</td><td>AsLOVpep Fint</td></tr> | ||
+ | |||
+ | <tr><td>Mys int R</td><td>IFN domBB F</td><td>AsLOVpep Rint</td><td>AsLOVpep R</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>AsLOVpep (nested)</td><td>PhiC31 (1)</td><td>PhiC31 (2)</td><td>PhiC31 (3)</td></tr> | ||
+ | |||
+ | <tr><td>AsLOVpep</td><td>PhiC31</td><td>PhiC31</td><td>PhiC31</td></tr> | ||
+ | |||
+ | <tr><td>AsLOVpep nested</td><td>PhiC31 Fint 1</td><td>PhiC31 Fint 2</td><td>PhiC31 Fint 3</td></tr> | ||
+ | |||
+ | <tr><td>AsLOVpep</td><td>PhiC31 Rint 2</td><td>PhiC31 Rint 3</td><td>PhiC31 R</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PhiC31</td></tr> | ||
+ | |||
+ | <tr><td>PhiC31</td></tr> | ||
+ | |||
+ | <tr><td>PhiC31 F</td></tr> | ||
+ | |||
+ | <tr><td>PhiC31 Rint</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Digestions of the minipreps:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PhiC31</td><td>NotI</td><td>2046, 1899</td></tr> | ||
+ | |||
+ | <tr><td>LacI:AsLOVpep+ePDZ+ren </td><td>EcoRI</td><td>6345, 8798</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:AsLOVpep+ePDZ+ren </td><td>EcoRI</td><td>6345, 9569</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Make the gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PhiC31 (C6)</td><td>C7…C16</td><td>LacI:AsLOVpep:ePDZ:ren (C1)</td><td>LacI:AsLOVpep:ePDZ:renC2</td><td>LacI:AsLOVpep:ePDZ:ren(C3)</td><td>LacI:AsLOVpep:ePDZ:ren(C4)</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>ok</td><td>no</td><td>ok</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:AsLOVpep+ePDZ+ren (C1)</td><td>Gal4:AsLOVpep+ePDZ+ren (C2)</td><td></td><td></td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td></td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/6/69/Valencia_upv_gel_150811.png> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Mesurement of the ODs:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Citoplasm</td><td>0.21</td><td>7.35 ml</td></tr> | ||
+ | |||
+ | <tr><td>RepPhiC31</td><td>0.26</td><td>9.1ml</td></tr> | ||
+ | |||
+ | <tr><td>35S:LTB:T35S</td><td>0.27</td><td>9.45ml</td></tr> | ||
+ | |||
+ | <tr><td>PhiC31</td><td>0.27</td><td>9.45ml</td></tr> | ||
+ | |||
+ | <tr><td>GFP</td><td>0.20</td><td>10ml</td></tr> | ||
+ | |||
+ | <tr><td>RepBxbI</td><td>0.33</td><td>11.55ml</td><td></td></tr> | ||
+ | |||
+ | <tr><td>PsinATG:RepBxbI:GFP</td><td>0.36</td><td>12.6ml</td></tr> | ||
+ | |||
+ | <tr><td>PhiC31</td><td>0.34</td><td>11.9ml</td></tr> | ||
+ | |||
+ | <tr><td>DsRed</td><td>0.26</td><td>9.1ml</td></tr> | ||
+ | |||
+ | <tr><td>P19</td><td>0.28</td><td>9.8ml</td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>EXPERIMENT 11.</p> | ||
+ | |||
+ | <p>Take the samples of the agroinfiltrated plants that were in darkness to change the conditions.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>EXPERIMENT 12.</p> | ||
+ | |||
+ | <ul><li>Citoplasm</li> | ||
+ | |||
+ | <li>RepPhiC31</li> | ||
+ | |||
+ | <li>35S:LTB:T35S</li> | ||
+ | |||
+ | <li>PhiC31</li> | ||
+ | |||
+ | <li>GFP</li> | ||
+ | |||
+ | <li>RepBxbI</li> | ||
+ | |||
+ | <li>PsinATG:RepBxbI:GFP</li> | ||
+ | |||
+ | <li>PhiC31</li> | ||
+ | |||
+ | <li>DsRed</li> | ||
+ | |||
+ | <li>P19</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | </br><h3 style="color:green">12 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>New digestions to check if the negative control constructions are correct and we can transformed it in agro</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>OpLexA:luc:SF:ren</td><td>EcoRI</td><td>6345, 7274</td></tr> | ||
+ | |||
+ | <tr><td>Op:UAS:luc:SF:ren</td><td>EcoRI</td><td>6345, 7096</td></tr> | ||
+ | |||
+ | <tr><td>OpEtr8.luc:SF:ren</td><td>EcoRI</td><td>6345, 7294</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Op:UAS:luc:SF:ren C1</td><td>Op:UAS:luc:SF:ren C2</td><td>Op:UAS:luc:SF:ren C3</td><td>OpEtr8.luc:SF:ren</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>no</td><td>no</td></tr> | ||
+ | |||
+ | <tr><td>OpLexA:luc:SF:ren C1</td><td>OpLexA:luc:SF:ren C2</td><td>Partner dna</td><td>Partner dna</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/c/c2/Valencia_upv_gel_150812.png> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transform in <i>Agrobacterium</i>and make petri dishes cultures:</p> | ||
+ | |||
+ | <ul><li>LexA:AsLOVpep:ePDZ</li> | ||
+ | |||
+ | <li>Gal4:AsLOVpep:ePDZ</li> | ||
+ | |||
+ | <li>LacI:AsLOVpep:ePDZ</li> | ||
+ | |||
+ | <li>LexA:PIF6:phyB:VP16</li> | ||
+ | |||
+ | <li>Gal4:PIF6:phyB:VP16</li> | ||
+ | |||
+ | <li>LacI:PIF6:phyB:VP16</li> | ||
+ | |||
+ | <li>All of them are in Ω1.</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Ligations:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LexA:AsLOVpep:ePDZ+ren; α1</td><td>Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; Ω1</td><td>LacI:AsLOVpep:ePDZ:ren+OpLacI:luc; Ω1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl LexA:AsLOVpep:ePDZ</td><td>1 µl Gal4:AsLOVpep:ePDZ:ren</td><td>1 µl LacI:AsLOVpep:ePDZ</td></tr> | ||
+ | |||
+ | <tr><td>1 µl renilla (159)</td><td>1 µl OpUAS:luc</td><td>1 µl OpLacI:luc</td></tr> | ||
+ | |||
+ | <tr><td>1 µl α1</td><td>1 µl Ω1</td><td>1 µl Ω1</td></tr> | ||
+ | |||
+ | <tr><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>PROTOPLAST 1: Do for the first time. PROTOCOL, click here.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">13 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Analysis of the AsLOVpep PCR’s products that were set to sequencing:</p> | ||
+ | |||
+ | <ul><li>The primer AsLOVpep F (forward) works well.</li> | ||
+ | |||
+ | <li>The primer AsLOVpep Ri (reverse intern): it is possible that occurs a deletion in the position 147.</li> | ||
+ | |||
+ | <li>The primer AsLOVpep Fi (forward intern): change in one base A to C in the position 446, last base in the codon, the amino acid (alanine) still the same. </li> | ||
+ | |||
+ | <li>The primer AsLOVpep R (reverse): it is ok but the las 50bp are no sequenced.</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <ul><li>The primer PhiC31 Fi (forward intern): did not work well. 4 consecutive deletions and changes in the nucleotides from 470 to 990.</li> | ||
+ | |||
+ | <li>The primer PhiC1 Ri2 (reverse intern 2): corrects the errors in the sequencing of the las primer. There is not deletion in the zone between 470 till 990 but is diffuse.</li> | ||
+ | |||
+ | <li>The primer PhiC31 Fi2 (forward intern 2): possible deletion in 1063.</li> | ||
+ | |||
+ | <li>The primer PhiC31 Fi3 (forward inter 3): ok</li> | ||
+ | |||
+ | <li>The primer PhiC31 R (reverse): ok</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Transform into <i>E. coli</i> yesterday ligations and make petri dishes cultures:</p> | ||
+ | |||
+ | <ul><li>LexA:AsLOVpep:ePDZ+ren; ?1</li> | ||
+ | |||
+ | <li>Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; ?1</li> | ||
+ | |||
+ | <li>LacI:AsLOVpep:ePDZ:ren+OpLacI:luc; ?1</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>PROTOPLAST 1:We try to obtain the protoplast but we make a mistake, repeat. </p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>PROTOPLAST 2. Start the procedure again.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>EXPERIMENT 11. Luciferase essay of blue and red toggle.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Ligations of the PCR products:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>AsLOVpep (BB); pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1µl AsLOVpep1</td></tr> | ||
+ | |||
+ | <tr><td>1µl AsLOVpep2</td></tr> | ||
+ | |||
+ | <tr><td>1µl pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1µl BsmbI</td></tr> | ||
+ | |||
+ | <tr><td>4.6µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">14 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Repeat the digestions of the negative controls:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>OpLexA:luc:SF:ren</td><td>SacII</td><td>13619</td></tr> | ||
+ | |||
+ | <tr><td></td><td>PouI</td><td>2495, 2055, 5937, 3152</td></tr> | ||
+ | |||
+ | <tr><td>OpUAS:luc:SF:ren</td><td>PouI</td><td>2475, 2055, 5937, 2474</td></tr> | ||
+ | |||
+ | <tr><td>OpEtr8.luc:SF:ren</td><td>PouI</td><td>2637, 2055, 5937, 3010</td></tr> | ||
+ | |||
+ | <tr><td>OpLacI:luc:SF:ren</td><td>SacII</td><td>12930, 790</td></tr> | ||
+ | |||
+ | <tr><td>OpLacI:luc:SF:ren</td><td>PouI</td><td>2475, 2055, 5937, 3253</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make the gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>OpUAS:luc:SF:ren C1</td><td>OpUAS:luc:SF:ren C2</td><td>OpUAS:luc:SF:ren C3</td><td>OpEtr8.luc:SF:ren</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>no</td><td>ok</td><td>ok</td></tr> | ||
+ | |||
+ | <tr><td>OpLacI:luc:SF:ren (PouI)</td><td>OpLacI:luc:SF:ren (SacII)</td><td>OpLexA:luc:SF:ren (PouI)</td><td>OpLexA:luc:SF:ren (SacII)</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/8/83/Valencia_upv_gel_150814.png> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transform into <i>E. coli</i> AsLOVpep (BB) and make petri dish culture.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make liquid cultures of:</p> | ||
+ | |||
+ | <ul><li>LexA:AsLOVpep:ePDZ+ren; ?1 (C1-C4)</li> | ||
+ | |||
+ | <li>Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; ?1 (C1-C4)</li> | ||
+ | |||
+ | <li>LacI:AsLOVpep:ePDZ:ren+OpLacI:luc; ?1 (C1-C4)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Make liquid cultures of agrobacterium cultures in petri dishes:</p> | ||
+ | |||
+ | <ul><li>LexA:AsLOVpep:ePDZ</li> | ||
+ | |||
+ | <li>Gal4:AsLOVpep:ePDZ</li> | ||
+ | |||
+ | <li>LacI:AsLOVpep:ePDZ</li> | ||
+ | |||
+ | <li>Gal4:PIF6:phyB:VP16</li> | ||
+ | |||
+ | <li>LacI:PIF6:phyB:VP16</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>We make triple strake method to obtain isolated colonies of LexA:PIF6:PhyB:VP16. </p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>PROTOPLAST 2:</p> | ||
+ | |||
+ | <p>Continue with the procedure to obtain protoplasts. We finally see in the microscope the protoplasts.</p> | ||
+ | |||
+ | <p>Transform protoplasts. How? Click here.</p> | ||
+ | |||
+ | <ul><li>E:PIF6:PhyB:luc+ren</li> | ||
+ | |||
+ | <li>E:PIF6:PhyB:luc+ren</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | |||
+ | |||
+ | <p>EXPERIMENT 12.</p> | ||
+ | |||
+ | <p>Prepare samples of both recombinases and its negative control to see them in the confocal microscope. We see samples of plants agroinfiltrated 7 days ago and other agroinfiltrated 5 days ago with the viral silencing repressor, P19.</p> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>PROTOPLAST 2:</p> | ||
+ | |||
+ | <p>Take samples at time 8h of protoplasts. </p> | ||
+ | |||
+ | <p>Samples of both toggles and in all the conditions. </p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">15 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Minipreps of:</p> | ||
+ | |||
+ | <ul><li>LexA:AsLOVpep:ePDZ+ren; α1</li> | ||
+ | |||
+ | <li>Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; Ω1</li> | ||
+ | |||
+ | <li>LacI:AsLOVpep:ePDZ:ren+OpLacI:luc; Ω1</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Minipreps digestions:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LexA:AsLOVpep:ePDZ+ren; α1</td><td>EcoRI</td><td>8837, 6345</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; Ω1</td><td>BamHI</td><td>6674, 11186</td></tr> | ||
+ | |||
+ | <tr><td>LacI:AsLOVpep:ePDZ:ren+OpLacI:luc; Ω1</td><td>BamHI</td><td>6674, 12236</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make the gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LexA:AsLOVpep:ePDZ+ren C1</td><td>LexA:AsLOVpep:ePDZ+ren C2</td><td>LexA:AsLOVpep:ePDZ+ren C3</td><td>LexA:AsLOVpep:ePDZ+ren C4</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td>Ok</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc C1</td><td>Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc C2</td><td>Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc C3</td><td>Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc C4</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>no</td><td>No</td></tr> | ||
+ | |||
+ | <tr><td>LacI:AsLOVpep:ePDZ:ren+OpLacI:luc C1</td><td>LacI:AsLOVpep:ePDZ:ren+OpLacI:luc C2</td><td>LacI:AsLOVpep:ePDZ:ren+OpLacI:luc C3</td><td>LacI:AsLOVpep:ePDZ:ren+OpLacI:luc C4</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/d/d4/Valencia_upv_gel_150815.png> | ||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/f/fa/Valencia_upv_gel_150815-2.png> | ||
+ | |||
+ | |||
+ | |||
+ | <p>We keep LexA:AsLOVpep:ePDZ+ren C1 and LacI:AsLOVpep:ePDZ:ren+OpLacI:luc C4.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make liquid cultures of (added X-gal + IPTG):</p> | ||
+ | |||
+ | <ul><li>AsLOvpep (BB); pUPD2 (C1-C4)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>PROTOPLASTS 3.</p> | ||
+ | |||
+ | <ul><li>We cut 3 leafs into strips and let this overnight shaking at minimum.</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>PROTOPLAST 2.</p> | ||
+ | |||
+ | <p>Take protoplasts samples at time 16h. Samples of both toggles and in all the conditions. </p> | ||
+ | |||
+ | <p>Take also time 24h.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">16 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>All liquid cultures of AsLOvpep (BB); pUPD2 (C1-C4) were blue.</p> | ||
+ | |||
+ | <p>Pick 4 more colonies also with added X-gal + IPTG</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Ligations:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; Ω1</td><td>AsLOVpep; pUPD2</td><td>LexA:AsLOVpep:ePDZ:ren+OpLexA:luc; Ω1</td></tr> | ||
+ | |||
+ | <tr><td>1µl Gal4:AsLOVpep:ePDZ:ren</td><td>1µl AsLOVpep1</td><td>1µl Lex!:AsLOVpep:ePDZ:ren</td></tr> | ||
+ | |||
+ | <tr><td>1µl OpUAS:luc (227)</td><td>1µl AsLOVpep2</td><td>1µl OpLexA:luc (151)</td></tr> | ||
+ | |||
+ | <tr><td>1µl Ω1</td><td>1µl pUPD2</td><td>1µl Ω1</td></tr> | ||
+ | |||
+ | <tr><td>4.6µl H<sub>2</sub>O</td><td>4.6µl H<sub>2</sub>O</td><td>4.6µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>PROTOPLASTS 3.</p> | ||
+ | |||
+ | <p>Finish the protoplasts started yesterday, we obtain good concentrations.</p> | ||
+ | |||
+ | <p>Transform them:</p> | ||
+ | |||
+ | <ul><li>LacI:AsLOVpep:ePDZ:ren:OpLacI:luc</li> | ||
+ | |||
+ | <li>LacI:KDronpa:NDronpa:ren:OpLacI:luc</li> | ||
+ | |||
+ | <li>OpLacI:luc:ren</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>PROTOPLAST 2. Luciferase essay.</p> | ||
+ | |||
+ | <p>Do a luciferase essay with protoplasts, click here. </p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transform into agro and make petri dish culture of:</p> | ||
+ | |||
+ | <ul><li>LacI:AsLOVpep:ePDZ:ren:OpLacI:luc; Ω1</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Refresh agrobacterium liquid culture of:</p> | ||
+ | |||
+ | <ul><li>Viral system (cytoplasm, integrase, Dsred).</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <ul><li>Pnos, to make a miniprep tomorrow and obtain the DNA construction.</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | </br><h3 style="color:green">17 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>PROTOPLASTS 3.</p> | ||
+ | |||
+ | <p>Take protoplasts samples at time 16h. Big disapoint here, we dscovered hat they were cloroplasts... ha, ha.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Liquid cultures of AsLOVpep (BB); pUPD2 were all blue. The ligation was made again.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Miniprep of Pnos in <i>Agrobacterium</i>.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transform the ligations in <i>E. coli</i>:</p> | ||
+ | |||
+ | <ul><li>Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; Ω1</li> | ||
+ | |||
+ | <li>AsLOVpep; pUPD2</li> | ||
+ | |||
+ | <li>LexA:AsLOVpep:ePDZ:ren+OpLexA:luc; Ω1</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>EXPERIMENT 13.</p> | ||
+ | |||
+ | <p>Prepare the viral system in agrobacterium cultures to infiltrate into sunflowers sprouts by vacuum.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Measurement of the ODs:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>DsRed</td><td>0.14</td><td>71.4ml</td></tr> | ||
+ | |||
+ | <tr><td>Integrase</td><td>0.14</td><td>71.4ml</td></tr> | ||
+ | |||
+ | <tr><td>Citoplasm</td><td>0.12</td><td>83.3ml</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make 60ml of medium for each infiltration.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>New pieces have arrived:</p> | ||
+ | |||
+ | <ul><li>1st part Lactoferrina</li> | ||
+ | |||
+ | <li>Primer PhiC31 F</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | |||
+ | |||
+ | <p>EXPERIMENT 11 and 12.</p> | ||
+ | |||
+ | <p>Take samples of leads agroinfiltrated with LTB, IFN and sip rotavirus CH<sub>2</sub> and CH<sub>2</sub>-CH<sub>3</sub>.</p> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">18 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make PCRs of PhiC31 and AsLOVpep to eliminate the enzyme recognition sites of Biobricks (BB).</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PhiC31 (BB)</td><td>AsLOVpep (BB)</td></tr> | ||
+ | |||
+ | <tr><td>1 µl PhiC31</td><td>1 µl AsLOVpep</td></tr> | ||
+ | |||
+ | <tr><td>2.5 µl PhiC31 F (1:10)</td><td>2.5 µl AsLOVpep nested</td></tr> | ||
+ | |||
+ | <tr><td>2.5 µl PhiC31 Rint (1:10)</td><td>2.5 µl AsLOVpep Fint</td></tr> | ||
+ | |||
+ | <tr><td>Tm=44ºC</td><td>Tm=59ºC</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Program: 96ºC-10’; 96ºC-30’’; Tm-30’’ and 72ºC-30’’</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Ligation of the PCRs products:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PhiC31 (BB); pUPD2</td><td>AsLOVpep (BB); pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1µl Phi1</td><td>1µl AsLOVpep1 </td></tr> | ||
+ | |||
+ | <tr><td>1µl Phi2</td><td>1µl AsLOVpep nested</td></tr> | ||
+ | |||
+ | <tr><td>1µl Phi3</td><td>1µl pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>1µl Phi4</td><td>4.6µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | <tr><td>1µl pUPD2</td><td></td></tr> | ||
+ | |||
+ | <tr><td>2.6µl H<sub>2</sub>O</td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transform in <i>Agrobacterium</i>:</p> | ||
+ | |||
+ | <ul><li>OpLexA:luc:SF:ren; α1</li> | ||
+ | |||
+ | <li>OpUAS:luc:SF:ren; α1</li> | ||
+ | |||
+ | <li>OpEtr8:luc:SF:ren; α1</li> | ||
+ | |||
+ | <li>OpLacI:luc:SF:ren; α1</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Make liquid culture of:</p> | ||
+ | |||
+ | <ul><li>Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; Ω1 (C1 and C2)</li> | ||
+ | |||
+ | <li>LexA:AsLOVpep:ePDZ:ren+OpLexA:luc; Ω1 (C1-C3)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>We start to do the western to see if any drugs had been produced in our plants.</p> | ||
+ | |||
+ | <p>The gel order is:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>IFN 1</td><td>IFN2</td><td>LTB1 (10µL)</td><td>LTB1 (10 µL)</td><td>LTB2 (30 µL)</td></tr> | ||
+ | |||
+ | <tr><td></td><td></td><td></td></tr> | ||
+ | |||
+ | <tr><td>Sip 1 CH<sub>2</sub></td><td>Sip2 CH<sub>2</sub></td><td>Sip 1 CH<sub>2</sub>-CH<sub>3</sub></td><td>Sip 2 CH<sub>2</sub>-CH<sub>3</sub></td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make a gel with the PCRs products: </p> | ||
+ | |||
+ | <p>PhiC31 and AsLOVpep modified for BioBricks. NO SE LAS BANDAS ESPERADAS.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>We change a little bit the protocol to make protoplasts, now we follow a protocol found in a Nature paper. The important changes are that during the digestion of the cell wall there is no agitation.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transform into <i>E. coli</i> the PCRs products:</p> | ||
+ | |||
+ | <ul><li>PhiC31 (BB)</li> | ||
+ | |||
+ | <li>AsLOVpep (BB)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Prepare liquid culture of:</p> | ||
+ | |||
+ | <ul><li>LTB; α1</li> | ||
+ | |||
+ | <li>Gal4:AsLOVpep:ePDZ; Ω1</li> | ||
+ | |||
+ | <li>LacI:AsLOVpep:ePDZ; Ω1</li> | ||
+ | |||
+ | <li>LexA:AsLOVpep:ePDZ; Ω1</li> | ||
+ | |||
+ | <li>ePDZ; pPUD2</li> | ||
+ | |||
+ | <li>AsLOVpep; pUPD2</li> | ||
+ | |||
+ | <li>NDronpa; pUPD2</li> | ||
+ | |||
+ | <li>Gal4:AsLOVpep; α1</li> | ||
+ | |||
+ | <li>RepPhiC31; pPUPD2</li> | ||
+ | |||
+ | <li>LexA:AsLOVpep; α1</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | </br><h3 style="color:green">19 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Miniprep of the liquid cultures:</p> | ||
+ | |||
+ | <ul><li>Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; Ω1 (C1 and C2), did not grow.</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Digestions:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LTB; α1</td><td>EcoRI</td><td>6345, 1684</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:AsLOVpep:ePDZ; Ω1</td><td>BamHI</td><td>6674, 4270</td></tr> | ||
+ | |||
+ | <tr><td>LacI:AsLOVpep:ePDZ; Ω1</td><td>BamHI</td><td>6674, 5041</td></tr> | ||
+ | |||
+ | <tr><td>LexA:AsLOVpep:ePDZ; Ω1</td><td>BamHI</td><td>6674, 4309</td></tr> | ||
+ | |||
+ | <tr><td>ePDZ; pPUD2</td><td>NotI</td><td>2046, 642</td></tr> | ||
+ | |||
+ | <tr><td>AsLOVpep; pPUD2</td><td>NotI</td><td>2046, 521</td></tr> | ||
+ | |||
+ | <tr><td>NDronpa; pPUD2</td><td>NotI</td><td>2046, 735</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:AsLOVpep; α1</td><td>EcoRI</td><td>6345, 1972</td></tr> | ||
+ | |||
+ | <tr><td>RepPhiC31: pPUD2</td><td>NotI</td><td>2046, 475</td></tr> | ||
+ | |||
+ | <tr><td>LexA:AsLOVpep; α1</td><td>EcoRI</td><td>6345, 2016</td></tr> | ||
+ | |||
+ | <tr><td>LexA:AsLOVpep:ePDZ:ren+OpLexA:luc; Ω1</td><td>BamHI</td><td>6674, 11403</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LTB; α1</td><td>NDronpa; pPUD2</td><td>RepPhiC31: pPUD2</td><td>AsLOVpep; pPUD2</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td></tr> | ||
+ | |||
+ | <tr><td>LexA:AsLOVpep</td><td>Gal4:AsLOVpep</td><td>LTB; α1</td><td>LexA:AsLOVpep; α1</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td>Ok</td></tr> | ||
+ | |||
+ | <tr><td>LacI:AsLOVpep:ePDZ</td><td>Gal4:AsLOVpep:ePDZ</td><td>LexA:AsLOVpep:ePDZ:ren+OpLexA:luc C2</td><td>LexA:AsLOVpep:ePDZ:ren+OpLexA:luc C3</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td>Ok</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make liquid culture of:</p> | ||
+ | |||
+ | <ul><li>PhiC31 (BB) (C1-C9)</li> | ||
+ | |||
+ | <li>AsLOVpep (BB) (C1-C3)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Also had grown the liquid cultures:</p> | ||
+ | |||
+ | <ul><li>LTB; pUPD2</li> | ||
+ | |||
+ | <li>LacI:AsLOVpep:ePDZ:ren; α1 (C1 and C2)</li> | ||
+ | |||
+ | <li>Gal4;pUPD2 and RepBxbI; α2 did not grow.</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | </br><h3 style="color:green">20 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>All the liquid cultures of AsLOVpep (BB) and PhiC31 (BB) had grown. Do 12 minipreps.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Digestions of the minipreps:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>AsLOVpep (BB)</td><td>NotI</td><td>2046, 518</td></tr> | ||
+ | |||
+ | <tr><td>PhiC31 (BB)</td><td>NotI</td><td>2046, 1899</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Do the gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>AsLOV C1</td><td>AsLOV C2</td><td>AsLOV C3</td><td>Phi C1</td><td>Phi C2</td><td>Phi C3</td></tr> | ||
+ | |||
+ | <tr><td>Ok</td><td>ok</td><td>ok</td><td>no</td><td>no</td><td>no</td></tr> | ||
+ | |||
+ | <tr><td>Phi C4</td><td>Phi C5</td><td>Phi C6</td><td>Phi C7</td><td>Phi C8</td><td>Phi C9</td></tr> | ||
+ | |||
+ | <tr><td>No</td><td>no</td><td>Ok?</td><td>Ok?</td><td>no</td><td>No</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>We keep the three colonies of AsLOVpep but we make another digestion with different enzymes of PhiC31 colonies C6 and C7.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Optimized ligation:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; Ω1</td></tr> | ||
+ | |||
+ | <tr><td>2.5 µl Gal4:AsLOVpep:ePDZ:ren</td></tr> | ||
+ | |||
+ | <tr><td>6 µl OpUAS:luc</td></tr> | ||
+ | |||
+ | <tr><td>0.5 µl Ω1</td></tr> | ||
+ | |||
+ | <tr><td>0 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make glycerinates of:</p> | ||
+ | |||
+ | <ul><li>ePDZ; pUPD2</li> | ||
+ | |||
+ | <li>NDronpa; pUPD2</li> | ||
+ | |||
+ | <li>RepPhiC31; pUPD2</li> | ||
+ | |||
+ | <li>AsLOVpep; pUPD2</li> | ||
+ | |||
+ | <li>LexA:AsLOVpep; α1</li> | ||
+ | |||
+ | <li>Gal4:AsLOVpep; α1</li> | ||
+ | |||
+ | <li>LexA:AsLOVpep:ePDZ; Ω1</li> | ||
+ | |||
+ | <li>LacI:AsLOVpep:ePDZ; Ω1</li> | ||
+ | |||
+ | <li>Gal4:AsLOVpep:ePDZ; Ω1</li> | ||
+ | |||
+ | <li>LexA:AsLOVpep:ePDZ:ren:OpLexA:luc; Ω1</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>Make liquid culture of positive controls in Agro:</p> | ||
+ | |||
+ | <ul><li>OpUAS:luc:ren</li> | ||
+ | |||
+ | <li>OpLexA:luc:ren</li> | ||
+ | |||
+ | <li>OpLacI:luc:ren</li> | ||
+ | |||
+ | <li>OpEtr8:luc:ren</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Prepare the liquid cultures of <i>Agrobacterium</i>to agroinfiltrate:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>IFN</td><td>0.26</td><td>77 µl/ng</td></tr> | ||
+ | |||
+ | <tr><td>LTB</td><td>0.22</td><td>91 µl/ng</td></tr> | ||
+ | |||
+ | <tr><td>Sip CH<sub>2</sub>-CH<sub>3</sub></td><td>0.12</td><td>167 µl/ng</td></tr> | ||
+ | |||
+ | <tr><td>Sip CH<sub>2</sub></td><td>0.09</td><td>223 µl/ng</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>The total volume of preparation will be 6ml each drug.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>EXPERIMENT 14.</p> | ||
+ | |||
+ | <ul><li>IFN</li> | ||
+ | |||
+ | <li>LTB</li> | ||
+ | |||
+ | <li>Sip CH<sub>2</sub>-CH<sub>3</sub></li> | ||
+ | |||
+ | <li>Sip CH<sub>2</sub></li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>Refresh more colonies to make more glycerinates and pick two colonies of the petri dish culture:</p> | ||
+ | |||
+ | <ul><li>Gal4:KDronpa:NDronpa:ren:OpUAS:luc; Ω1</li> | ||
+ | |||
+ | <li>LexA:KDronpa:NDronpa:ren:OpLexA:luc; Ω1</li> | ||
+ | |||
+ | <li>LTB; pUPD2</li> | ||
+ | |||
+ | <li>LTB; α1</li> | ||
+ | |||
+ | <li>Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1 (C1 and C2)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Miniprep of the liquid culture:</p> | ||
+ | |||
+ | <ul><li>LacI:AsLOVpep; α1 (C1 and C2)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Digestion:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LacI:AsLOVpep</td><td>EcoRI</td><td>8345, 2743</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>New digestion to PhiC31 (BB):</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PhiC31 (BB)</td><td>AatII</td><td>2635, 1310</td></tr> | ||
+ | |||
+ | <tr><td>PhiC31 (BB)</td><td>BanI</td><td>3497, 448</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make the gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PhiC31(BB) C6 (banI)</td><td>PhiC31(BB) C7 (BanI)</td><td>PhiC31(BB) C6 (AatII)</td><td>PhiC31(BB) C7 (AatII)</td></tr> | ||
+ | |||
+ | <tr><td>Ok</td><td>no</td><td>ok</td><td>no</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Transformation in <i>Agrobacterium</i>and make petri dishe culture of :</p> | ||
+ | |||
+ | <ul><li>LexA:AsLOVpep:ePDZ:ren:OpLexA:luc; Ω1</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | </br><h3 style="color:green">21 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Miniprep of:</p> | ||
+ | |||
+ | <ul><li>Gal4:KDronpa:NDronpa:ren:OpUAS:luc</li> | ||
+ | |||
+ | <li>LexA:KDronpa:NDronpa:ren:OpLexA:luc</li> | ||
+ | |||
+ | <li>LTB; pUPD2</li> | ||
+ | |||
+ | <li>LTB; α1</li> | ||
+ | |||
+ | <li>Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1 (C1 and C2)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Digestions of the minipreps:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4:KDronpa:NDronpa:ren:OpUAS:luc; Ω1</td><td>BamHI</td><td>6674, 11582</td></tr> | ||
+ | |||
+ | <tr><td>LexA:KDronpa:NDronpa:ren:OpLexA:luc; Ω1</td><td>BamHI</td><td>6674, 11799</td></tr> | ||
+ | |||
+ | <tr><td>LTB; pUPD2</td><td>NotI</td><td>2046, 474</td></tr> | ||
+ | |||
+ | <tr><td>LTB; α1</td><td>EcoRI</td><td>1684, 6345</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1</td><td>BamHI</td><td>6674, 11186</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Do the gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LTB; pUPD2</td><td>LTB; α1</td><td>LTB; α1</td><td>Gal4:KDronpa:NDronpa:ren:OpUAS:luc; Ω1</td></tr> | ||
+ | |||
+ | <tr><td>No</td><td>no</td><td>no</td><td>ok</td></tr> | ||
+ | |||
+ | <tr><td>LexA:KDronpa:NDronpa:ren:OpLexA:luc; Ω1</td><td>Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1 C1</td><td>Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1 C2</td><td></td></tr> | ||
+ | |||
+ | <tr><td>Ok</td><td>no</td><td>no</td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>We make glicerynates of:</p> | ||
+ | |||
+ | <ul><li>Gal4:KDronpa:NDronpa:ren:OpUAS:luc; Ω1</li> | ||
+ | |||
+ | <li>LexA:KDronpa:NDronpa:ren:OpLexA:luc; Ω1</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Transform into <i>E. coli</i> and make Petri dish culture:</p> | ||
+ | |||
+ | <ul><li>Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1 </li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Refresh this cultures to agroinfiltrate:</p> | ||
+ | |||
+ | <ul><li>Pnos</li> | ||
+ | |||
+ | <li>LacI:AsLOVpep:ePDZ:ren:OpLacI:luc</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>Still trying how to make protoplasts… We will win this battle!</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">22 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make liquid culture of the culture of Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1. There was only one white colony, nothing hopeful…</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>We fount that the culture of Pnos in <i>Agrobacterium</i> did not grow, so we decided not to carry on with the experiment because it was our control. Refresh again two different cultures of Pnos that were found in the fridge and do the experiment tomorrow.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transform into <i>Agrobacterium</i>the following constructions:</p> | ||
+ | |||
+ | <ul><li>E:PIF6:PhyB</li> | ||
+ | |||
+ | <li>LexA:PIF6:PhyB</li> | ||
+ | |||
+ | <li>LacI:PIF6:PhyB</li> | ||
+ | |||
+ | <li>Gal4:PIF6:PhyB</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Miniprep of:</p> | ||
+ | |||
+ | <ul><li>Pnos1 (agro)</li> | ||
+ | |||
+ | <li>Pnos2 (Agro)</li> | ||
+ | |||
+ | <li>PhiC31 (BB); pUPD2 (C6)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Digestion:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PhiC31 (BB); pUPD2</td><td>NotI</td><td>2046, 1899</td></tr> | ||
+ | |||
+ | <tr><td>Pnos</td><td>EcoRI</td><td>7317, 634, 11</td></tr> | ||
+ | |||
+ | <tr><td>LTB; pUPD2</td><td>NotI</td><td>2046, 474</td></tr> | ||
+ | |||
+ | <tr><td>LTB; α1</td><td>EcoRI</td><td>1684, 6345</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LTB; α1</td><td>LTB; pUPD2</td><td>Pnos 1</td><td>Pnos 2</td><td>PhiC31 (BB)</td></tr> | ||
+ | |||
+ | <tr><td>Ok</td><td>no</td><td>?</td><td>?</td><td>Ok</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>As the Pnos construction was given to us form a lab partner, we do not surely that the expected bands were correct because we choose the construction at the golden braid register parts.</p> | ||
+ | |||
+ | <p>We can not make any glicerynate because it was not the necessary amount of liquid culture to make it. Refresh again: PhiC31 (BB); pUPD2; LTB; α1.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">23 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Prepare the <i>Agrobacterium</i>cultures to agroinfiltrate:</p> | ||
+ | |||
+ | <p>Pnos and LacI:AsLOVpep:ePDZ:ren:OpLacI:luc.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Pnos</td><td>0.30</td><td>1ml/15ml</td></tr> | ||
+ | |||
+ | <tr><td>LacI:AsLOVpep:ePDZ:ren:OpLacI:luc</td><td>0.34</td><td></td></tr> | ||
+ | |||
+ | <tr><td>OpLacI:luc:ren</td><td>0.47</td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>EXPERIMENT 15. </p> | ||
+ | |||
+ | <ul><li>Pnos</li> | ||
+ | |||
+ | <li>LacI:AsLOVpep:ePDZ:ren:OpLacI:luc.</li> | ||
+ | |||
+ | <li>OpLacI:luc</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <ul><li>PhiC31 (BB); pUPD2</li> | ||
+ | |||
+ | <li>Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1</li> | ||
+ | |||
+ | <li>LTB; α1 did not grow.</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Make liquid culture of agro colonies:</p> | ||
+ | |||
+ | <ul><li>LexA:AsLOVpep:ePDZ:ren:OpLexA:luc; Ω1</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">24 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Digestion of yesterday minipreps:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc</td><td>BamHI</td><td>11186, 6674</td></tr> | ||
+ | |||
+ | <tr><td>PhiC31 (BB)</td><td>NotI</td><td>2096, 1899</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Do a gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc</td><td>PhiC31 (BB)</td></tr> | ||
+ | |||
+ | <tr><td>Ok</td><td>ok</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/e/ea/Valencia_upv_gel_150824.png> | ||
+ | |||
+ | |||
+ | |||
+ | <p>It had arrived the second part of the lactoferrin.</p> | ||
+ | |||
+ | <ul><li>Resuspend them 100 µl.</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>Ligation into pUPD2 of both parts of the lactoferrin and other ligation:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Lactoferrina; pUPD2</td><td>PhiC31; α1</td></tr> | ||
+ | |||
+ | <tr><td>1 µl Part1 Lactoferrin</td><td>1 µl PhiC31</td></tr> | ||
+ | |||
+ | <tr><td>1 µl Part2 Lactoferrin</td><td>1 µl 35S</td></tr> | ||
+ | |||
+ | <tr><td>1 µl pUPD2</td><td>1 µl T35S</td></tr> | ||
+ | |||
+ | <tr><td>4.6 µl H<sub>2</sub>O</td><td>1 µl α1</td></tr> | ||
+ | |||
+ | <tr><td></td><td>3.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transform into <i>E. coli</i> the ligations.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">25 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Refresh PhiC31 (BB) and Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc to make glycerinates.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transfrorm into <i>Agrobacterium</i>the construction:</p> | ||
+ | |||
+ | <ul><li>Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Keep trying protoplasts, this time we see them in the microscope without coverslip. We can see them alive, maybe this was the error. Also we decided to leave the digestion enzymes overnight because after only 3 hours the amount of protoplasts was very little.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>EXPERIMENT 14.</p> | ||
+ | |||
+ | <p>Take leafs agroinflitrated with the drugs</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>EXPERIMENT 15. </p> | ||
+ | |||
+ | <p>Do discs of the agroinfiltrated lefs with the blue toggle (LacI:AsLOVpep:ePDZ:ren:OpLacI:luc) and the control Pnos.</p> | ||
+ | |||
+ | <p>FOTO DE LA PLACA</p> | ||
+ | |||
+ | <p>Make liquid cultures of yesterday transformations:</p> | ||
+ | |||
+ | <ul><li>Lactoferrina; pUPD2 (C1-C3)</li> | ||
+ | |||
+ | <li>PhiC31; α1 (C1-C3)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | </br><h3 style="color:green">26 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Minipreps of:</p> | ||
+ | |||
+ | <ul><li>Lactoferrina; pUPD2 (C1-C3)</li> | ||
+ | |||
+ | <li>PhiC31; α1 (C1-C3)</li> | ||
+ | |||
+ | <li>PhiC31; pUPD2 (BB)</li> | ||
+ | |||
+ | <li>Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Digestions of the minnipeps:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Lactoferrina; pUPD2 </td><td>NotI</td><td>2046, 2142</td></tr> | ||
+ | |||
+ | <tr><td>PhiC31; α1</td><td>EcoRI</td><td>6345, 3127</td></tr> | ||
+ | |||
+ | <tr><td>PhiC31; pUPD2 (BB)</td><td>NotI</td><td>2046, 1899</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc</td><td>BamHI</td><td>6674, 11191</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PhiC31; α1 C1</td><td>PhiC31; α1 C2</td><td>PhiC31; α1 C3</td><td>PhiC31; α1 C4</td><td>Lactoferrina C1</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>no</td><td>ok</td><td>ok</td><td>ok</td></tr> | ||
+ | |||
+ | <tr><td>Lactoferrina C2</td><td>Lactoferrina C3</td><td>Lactoferrina C4</td><td>PhiC31; pUPD2</td><td>Gal4:AsLOVpep:ePDZ:</td></tr> | ||
+ | |||
+ | <tr><td>ren:OpUAS:luc</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Do glycerinates of:</p> | ||
+ | |||
+ | <ul><li>Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1</li> | ||
+ | |||
+ | <li>PhiC31; pUPD2 (BB)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Ligations:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PhiC31+RepPhiC31:GFP; Ω1</td><td>35S+Lactoferrin+T35S; α1</td></tr> | ||
+ | |||
+ | <tr><td>1µl PhiC31</td><td>1µl 35S</td></tr> | ||
+ | |||
+ | <tr><td>1µl RepPhiC31:GFP</td><td>1µl T35S</td></tr> | ||
+ | |||
+ | <tr><td>1µl Ω1</td><td>1µl Lactoferrin; pUPD2</td></tr> | ||
+ | |||
+ | <tr><td>4.6µl H<sub>2</sub>O</td><td>1µl α1</td></tr> | ||
+ | |||
+ | <tr><td></td><td>3.6µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Take samples of the glycerinates:</p> | ||
+ | |||
+ | <ul><li>35S:GFP:T35S (this is necessary to transform into protoplasts and see if the expressed the constructions that we introduce to them)</li> | ||
+ | |||
+ | <li>Alpha1</li> | ||
+ | |||
+ | <li>Omega1</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Refresh the <i>Agrobacterium</i>culture to make agroinfiltration tomorrow of:</p> | ||
+ | |||
+ | <ul><li>Gal4:KDronpa:NDronpa:ren:OpUAS:luc</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | </br><h3 style="color:green">27 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make liquid culture of the <i>Agrobacterium</i>colony of Gal4:AsLOVpep:ePDZ:ren:OPUAS:luc; Ω1.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Minipreps of:</p> | ||
+ | |||
+ | <ul><li>Gal4:KDronpa:NDronpa:ren:OpUAS:luc</li> | ||
+ | |||
+ | <li>35S:GFP:T35S</li> | ||
+ | |||
+ | <li>Alpha1</li> | ||
+ | |||
+ | <li>Omega1</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Digestions:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4:KDronpa:NDronpa:ren:OpUAS:luc</td><td>BamHI</td><td>6674, 11191</td></tr> | ||
+ | |||
+ | <tr><td>35S:GFP:T35S</td><td>EcoRI</td><td>2580, 2274</td></tr> | ||
+ | |||
+ | <tr><td>Alpha1</td><td>Nothing</td><td>6953</td></tr> | ||
+ | |||
+ | <tr><td>Omega1</td><td>Nothing</td><td>7295</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Alpha1</td><td>Omega1</td><td>35S:GFP:T35S</td><td>Gal4:KDronpa:NDronpa:</td></tr> | ||
+ | |||
+ | <tr><td>ren:OpUAS:luc</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td>Ok</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transform into <i>E. coli</i> yesterday ligations:</p> | ||
+ | |||
+ | <ul><li>PhiC31+RepPhiC31:GFP; Ω1</li> | ||
+ | |||
+ | <li>35S+Lactoferrin+T35S; α1</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Take samples of the glycerinates and make liquid culture:</p> | ||
+ | |||
+ | <ul><li>Signal peptide (GB0393), in <i>E. coli</i></li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>Make a glycerinates of Gal4:KDronpa:NDronpa:ren:OpUAS:luc; Ω1</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>EXPERIMENT 15.</p> | ||
+ | |||
+ | <p>Take samples at 48h and store them at -80ºC in the box “The rebellion of AsLOV”.</p> | ||
+ | |||
+ | <p>PROTOPLASTS 4.</p> | ||
+ | |||
+ | <p>Do protoplasts to transform them tomorrow.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">28 August 2015</h3> | ||
+ | |||
+ | <p>Miniprep of: 35S:GFP:Tnos.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Digestion of the miniprep:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>35S:GFP:Tnos</td><td>NotI</td><td>2981, 140</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Gel:</p> | ||
+ | |||
+ | <p>Correct.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Ligation:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Lactoferrina; α1</td></tr> | ||
+ | |||
+ | <tr><td>1µl Lactoferrina</td></tr> | ||
+ | |||
+ | <tr><td>1µl 35S</td></tr> | ||
+ | |||
+ | <tr><td>1µl T35S</td></tr> | ||
+ | |||
+ | <tr><td>1µl GB393</td></tr> | ||
+ | |||
+ | <tr><td>2.6µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Measure the OD of GFP: 157.5. It the right curve.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make liquid culture of the only white colony of PhiC31+RepPhiC31:GFP; Ω1</p> | ||
+ | |||
+ | <p>PROTOPLASTS 4.</p> | ||
+ | |||
+ | <p>Finish the preparation of protoplasts and transform them with 40 µl of 35S:GFP:Tnos at a concentration of 157.5ng/ml.</p> | ||
+ | |||
+ | <p>After the transformation of the protoplast we see them at the microscope and there were no protoplasts.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Take samples of glycerinates and make liquid cultures of:</p> | ||
+ | |||
+ | <ul><li>OpLexA:mini35S (GB0733)</li> | ||
+ | |||
+ | <li>OpLacI:mini35S (GB534)</li> | ||
+ | |||
+ | <li>OpUAS:mini35S (GB0672)</li> | ||
+ | |||
+ | <li>DsRed (GB0672)</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Transform and make petri dish culture of Lactoferrin; α1 ligation.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">29 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Miniprep of the glycerinates’ liquid cultures:</p> | ||
+ | |||
+ | <ul><li>OpLexA:mini35S (GB0733)</li> | ||
+ | |||
+ | <li>OpLacI:mini35S (GB534)</li> | ||
+ | |||
+ | <li>OpUAS:mini35S (GB0672)</li> | ||
+ | |||
+ | <li>DsRed (GB0672)</li> | ||
+ | |||
+ | <li>35S:GFP:Tnos; α1</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Refresh the culture of Gal4:KDronpa:NDronpa:ren:OpUAS:luc; Ω1 due to that in the last transformation of protoplasts we run out all the DNA.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Refresh and make liquid culture of </p> | ||
+ | |||
+ | <ul><li>Dsred (GB672) in case the DNA concentration of the miniprep was low.</li> | ||
+ | |||
+ | <li>Staffer fragment in Ω2.</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Digestion of the minipreps:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>OpLexA:mini35S (GB0733); pUPD2</td><td>NotI</td><td>2981, 477</td></tr> | ||
+ | |||
+ | <tr><td>OpLacI:mini35S (GB534); pUPD2</td><td>NotI</td><td>2981, 878</td></tr> | ||
+ | |||
+ | <tr><td>OpUAS:mini35S (GB0672); pUPD2</td><td>NotI</td><td>2981, 477</td></tr> | ||
+ | |||
+ | <tr><td>DsRed (GB0672); α1</td><td>EcoRI</td><td>2580, 2274</td></tr> | ||
+ | |||
+ | <tr><td>35S:GFP:Tnos; α1</td><td>EcoRI</td><td>2580, 2274</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Do the gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>OpUAS:mini35S (GB0672); pUPD2</td><td>OpLexA:mini35S (GB0733); pUPD2</td><td>OpLacI:mini35S (GB534); pUPD2</td><td>OpLacI:mini35S (GB534); pUPD2</td><td>35S:GFP:Tnos; α1</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make liquid culture of Lactoferrin; α1 (C1-C4)</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">30 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Miniprep of the liquid cultures, lactoferrin C1 and C4 did not grow. We do 5 minipreps.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Digestion of the minipreps:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4:KDronpa:NDronpa:ren:OpUAS:luc; Ω1</td><td>BamHI</td><td>6674, 11191</td></tr> | ||
+ | |||
+ | <tr><td>Dsred (GB672); α 1</td><td>EcoRI</td><td></td></tr> | ||
+ | |||
+ | <tr><td>Staffer fragment in Ω2</td><td>EcoRV</td><td></td></tr> | ||
+ | |||
+ | <tr><td>Lactoferrin; α1</td><td>EcoRI</td><td>6345, 3436</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | <p>Gel(we repeat it because yesterday gel was very bad):</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>OpLacI:mini35S (GB534); pUPD2</td><td>OpLexA:mini35S (GB0733); pUPD2</td><td>OpUAS:mini35S (GB0672); pUPD2</td><td>DsRed (GB0672); pUPD2</td><td>Mini35S:Dsred; α1</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>Ok</td></tr> | ||
+ | |||
+ | <tr><td>Lactoferrin; α1 (C2)</td><td>Lactoferrin; α1 (C3)</td><td>Gal4:KDronpa:NDronpa:ren:OpUAS:luc; Ω1</td><td>Staffer fragment in Ω2</td><td></td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>ok</td><td>ok</td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Ligations: (we make this because the pieze Operator+mini35S can not be ligated with the other constructions in Benchlig so we will join them all in this new ligations).</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>OpLacI:mini35S+Dsred+T35S; α2</td><td>OpLexA:mini35S+DsRed+T35S; α2</td><td>OpUAS:mini35S+DsRed+T35S; α2</td></tr> | ||
+ | |||
+ | <tr><td>1µl OpLacI</td><td>1µl OpLexA</td><td>1µl OpUAS</td></tr> | ||
+ | |||
+ | <tr><td>1µl Dsred</td><td>1µl Dsred</td><td>1µl Dsred</td></tr> | ||
+ | |||
+ | <tr><td>1µl T35S</td><td>1µl T35S</td><td>1µl T35S</td></tr> | ||
+ | |||
+ | <tr><td>1µl mini35S</td><td>1µl mini35S</td><td>1µl mini35S</td></tr> | ||
+ | |||
+ | <tr><td>1µl α2</td><td>1µl α2</td><td>1µl α2</td></tr> | ||
+ | |||
+ | <tr><td>3.6 µl H<sub>2</sub>O</td><td>3.6 µl H<sub>2</sub>O</td><td>3.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4:KDronpa:NDronpa+SF(Ω2); α1</td><td>LexA:KDronpa:NDronpa+SF(Ω2); α1</td><td>LacI:KDronpa:NDronpa+SF(Ω2); α1</td></tr> | ||
+ | |||
+ | <tr><td>1µl Gal4:KDronpa:NDronpa</td><td>1µl LexA:KDronpa:NDronpa</td><td>1µl LacI:KDronpa:NDronpa</td></tr> | ||
+ | |||
+ | <tr><td>1µl SF(Ω2)</td><td>1µl SF(Ω2)</td><td>1µl SF(Ω2)</td></tr> | ||
+ | |||
+ | <tr><td>1µl α1</td><td>1µl α1</td><td>1µl α1</td></tr> | ||
+ | |||
+ | <tr><td>4.6µl H<sub>2</sub>O</td><td>4.6µl H<sub>2</sub>O</td><td>4.6µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Lactoferrin; α1</td></tr> | ||
+ | |||
+ | <tr><td>1µl Lactoferrin</td></tr> | ||
+ | |||
+ | <tr><td>1µl 35S</td></tr> | ||
+ | |||
+ | <tr><td>1µl T35S</td></tr> | ||
+ | |||
+ | <tr><td>1µl α1</td></tr> | ||
+ | |||
+ | <tr><td>2.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">31 August 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transform the ligations into <i>E. coli</i> adding IPTG and X-Gal.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Ligation:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>PhiC31+RepPhiC31:GFP; Ω1</td></tr> | ||
+ | |||
+ | <tr><td>1µl PhiC31 (BB); α1</td></tr> | ||
+ | |||
+ | <tr><td>1µl RepPhiC31:GFP; α2</td></tr> | ||
+ | |||
+ | <tr><td>1µl Ω1</td></tr> | ||
+ | |||
+ | <tr><td>4.6µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>The collaborations part of the Norwich iGEM team have arrived:</p> | ||
+ | |||
+ | <ul><li>This are the Mofippers (cloranfenicol resistance):</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>0408</td><td>Promoters</td><td>224 ng/µl</td><td>7 µl</td></tr> | ||
+ | |||
+ | <tr><td>0409</td><td>CDs</td><td>77 ng/µl</td><td>3 µl</td></tr> | ||
+ | |||
+ | <tr><td>0410</td><td>terminator</td><td>159 ng/µl</td><td>10 µl</td></tr> | ||
+ | |||
+ | <tr><td>0411</td><td>TUs</td><td>158 ng/µl</td><td>10 µl</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Añadir una foto de las placas en la wiki y enlace de la wiki de los the norwich</p> | ||
+ | |||
+ | |||
+ | |||
+ | <ul><li>This are the constructions (carbenicillin resistance) that we are going to prove in our plant.</li> | ||
+ | |||
+ | <ul class="ul_2"><li>584-MAA with YFP</li> | ||
+ | |||
+ | <li>596-RV3034c with YFP</li> | ||
+ | |||
+ | <li>598-RV3037c with YFP</li> | ||
+ | |||
+ | <li>600-RV3030 with YFP</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Transform the Moflippers into <i>E. coli</i> to have later more DNA to make the ligations and be able to send the parts with the necessary characteristics to the BioBricks requirements.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transform the functional constructions into <i>Agrobacterium</i>and make petri dishes cultures.</p> | ||
+ | |||
+ | <p>Refresh liquid cultures of <i>Agrobacterium</i>to make an experiment with red toggle:</p> | ||
+ | |||
+ | <ul><li>LexA:PIF6:PhyB</li> | ||
+ | |||
+ | <li>Gal4:PIF6:PhyB</li> | ||
+ | |||
+ | <li>LacI:PIF6:PhyB</li> | ||
+ | |||
+ | <li>OpLexA:ren:luc</li> | ||
+ | |||
+ | <li>OpLacI:ren:luc</li> | ||
+ | |||
+ | <li>OpUAS:ren:luc</li> | ||
+ | |||
+ | <li>Pnos</li> | ||
+ | |||
+ | </br><h3 style="color:green"SEPTEMBER</h3> | ||
+ | |||
+ | |||
+ | </br><h3 style="color:green">1 September 2015</h3> | ||
+ | |||
+ | |||
+ | <p>Make liquid culture of the Moflippers, 2 colonies for each culture.</p> | ||
+ | |||
+ | <p>Make liquid culture of yesterday transformations:</p> | ||
+ | |||
+ | <ul><li>OpLacI:mini35S+Dsred+T35S; α2</li> | ||
+ | |||
+ | <li>OpLexA:mini35S+DsRed+T35S; α2</li> | ||
+ | |||
+ | <li>OpUAS:mini35S+DsRed+T35S; α2</li> | ||
+ | |||
+ | <li>Gal4:KDronpa:NDronpa+SF(Ω2); α1</li> | ||
+ | |||
+ | <li>LexA:KDronpa:NDronpa+SF(Ω2); α1</li> | ||
+ | |||
+ | <li>LacI:KDronpa:NDronpa+SF(Ω2); α1</li> | ||
+ | |||
+ | <li>Lactoferrin; α1</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Do the ligations in Moflippers with the miniprep that had arrived. This constructions are the ones that go with our plasmid α.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>35S:BxbI:T35S</td><td>35S:PhiC31:T35S</td><td>PsinATG:RepBxbI:GFP:T35S</td><td>PsinATG:RepPhiC31:GFP:T35S</td></tr> | ||
+ | |||
+ | <tr><td>1µl 35S</td><td>1µl 35S</td><td>1µl PsinATG</td><td>1µl PsinATG</td></tr> | ||
+ | |||
+ | <tr><td>1µl BxbI</td><td>1µl PhiC31</td><td>1µl RepBxbI</td><td>1µl RepPhiC31</td></tr> | ||
+ | |||
+ | <tr><td>1µl T35S</td><td>1µl T35S</td><td>1µl GFP</td><td>1µl GFP</td></tr> | ||
+ | |||
+ | <tr><td>1µl Mo411</td><td>1µl Mo411</td><td>1µl T35S</td><td>1µl T35S</td></tr> | ||
+ | |||
+ | <tr><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td><td>1µl Mo411</td><td>1µl Mo411</td></tr> | ||
+ | |||
+ | <tr><td></td><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>35S:Lactoferrin:T35S</td><td>35S:LTB:T35S</td><td>Gal4:KDronpa</td><td>LacI:KDronpa</td></tr> | ||
+ | |||
+ | <tr><td>1µl 35S</td><td>1µl 35S</td><td>1µl 35S</td><td>1µl 35S</td></tr> | ||
+ | |||
+ | <tr><td>1µl Lactoferrin</td><td>1µl LTB</td><td>1µl Gal4</td><td>1µl LacI</td></tr> | ||
+ | |||
+ | <tr><td>1µl T35S</td><td>1µl T35S</td><td>1µl T35S</td><td>1µl T35S</td></tr> | ||
+ | |||
+ | <tr><td>1µl Mo411</td><td>1µl Mo411</td><td>1µl KDronpa</td><td>1µl KDronpa</td></tr> | ||
+ | |||
+ | <tr><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td><td>1µl Mo411</td><td>1µl Mo411</td></tr> | ||
+ | |||
+ | <tr><td></td><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LexA:KDronpa</td><td>NDronpa:VP16</td><td>Gal4:AsLOVpep</td><td>LacI:AsLOVpep</td></tr> | ||
+ | |||
+ | <tr><td>1µl 35S</td><td>1µl 35S</td><td>1µl 35S</td><td>1µl 35S</td></tr> | ||
+ | |||
+ | <tr><td>1µl LexA</td><td>1µl Gal4</td><td>1µl Gal4</td><td>1µl LacI</td></tr> | ||
+ | |||
+ | <tr><td>1µl T35S</td><td>1µl T35S</td><td>1µl T35S</td><td>1µl T35S</td></tr> | ||
+ | |||
+ | <tr><td>1µl KDronpa</td><td>1µl KDronpa</td><td>1µl AsLOVpep</td><td>1µl AsLOVpep</td></tr> | ||
+ | |||
+ | <tr><td>1µl Mo411</td><td>1µl Mo411</td><td>1µl Mo411</td><td>1µl Mo411</td></tr> | ||
+ | |||
+ | <tr><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LexA:AsLOVpep</td><td>ePDZ:VP16</td></tr> | ||
+ | |||
+ | <tr><td>1µl 35S</td><td>1µl 35S</td></tr> | ||
+ | |||
+ | <tr><td>1µl LexA</td><td>1µl ePDZ</td></tr> | ||
+ | |||
+ | <tr><td>1µl T35S</td><td>1µl T35S</td></tr> | ||
+ | |||
+ | <tr><td>1µl AsLOVpep</td><td>1µl VP16</td></tr> | ||
+ | |||
+ | <tr><td>1µl Mo411</td><td>1µl Mo411</td></tr> | ||
+ | |||
+ | <tr><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">2 September 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Do the minipreps of the liquid cultures:</p> | ||
+ | |||
+ | <ul><li>OpLacI:mini35S+Dsred+T35S; α2</li> | ||
+ | |||
+ | <li>OpLexA:mini35S+DsRed+T35S; α2</li> | ||
+ | |||
+ | <li>OpUAS:mini35S+DsRed+T35S; α2</li> | ||
+ | |||
+ | <li>Gal4:KDronpa:NDronpa+SF(Ω2); α1</li> | ||
+ | |||
+ | <li>LexA:KDronpa:NDronpa+SF(Ω2); α1</li> | ||
+ | |||
+ | <li>LacI:KDronpa:NDronpa+SF(Ω2); α1</li> | ||
+ | |||
+ | <li>Lactoferrin; α1</li> | ||
+ | |||
+ | <li>Mo0408</li> | ||
+ | |||
+ | <li>Mo0409</li> | ||
+ | |||
+ | <li>Mo0410</li> | ||
+ | |||
+ | <li>Mo0411</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4:KNDronpa:VP16:SF; Mo</td><td>LacI:KNDronpa:VP16:SF; Mo</td><td>LexA:KNDronpa:VP16:SF; Mo</td></tr> | ||
+ | |||
+ | <tr><td>1µl Gal4:KNDronpa:VP16</td><td>1µl LacI:KNDronpa:VP16</td><td>1µl LexA:KNDronpa:VP16</td></tr> | ||
+ | |||
+ | <tr><td>1µl SF</td><td>1µl SF</td><td>1µl SF</td></tr> | ||
+ | |||
+ | <tr><td>1µl Mo</td><td>1µl Mo</td><td>1µl Mo</td></tr> | ||
+ | |||
+ | <tr><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4:AsLOVpep:ePDZ:VP16:SF; Mo</td><td>LacI:AsLOVpep:ePDZ:VP16:SF; Mo</td><td>LexA:AsLOVpep:ePDZ:VP16:SF; Mo</td></tr> | ||
+ | |||
+ | <tr><td>1µl Gal4:AsLOVpep:ePDZ:VP16</td><td>1µl LacI:AsLOVpep:ePDZ:VP16</td><td>1µl LexA:AsLOVpep:ePDZ:VP16</td></tr> | ||
+ | |||
+ | <tr><td>1µl SF</td><td>1µl SF</td><td>1µl SF</td></tr> | ||
+ | |||
+ | <tr><td>1µl Mo</td><td>1µl Mo</td><td>1µl Mo</td></tr> | ||
+ | |||
+ | <tr><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4:PIF6:PhyB:VP16:SF; Mo</td><td>LacI:PIF6:PhyB:VP16:SF; Mo</td><td>LexA:PIF6:PhyB:VP16:SF; Mo</td></tr> | ||
+ | |||
+ | <tr><td>1µl Gal4:PIF6:PhyB:VP16</td><td>1µl LacI:PIF6:PhyB:VP16</td><td>1µl LexA:PIF6:PhyB:VP16</td></tr> | ||
+ | |||
+ | <tr><td>1µl SF</td><td>1µl SF</td><td>1µl SF</td></tr> | ||
+ | |||
+ | <tr><td>1µl Mo</td><td>1µl Mo</td><td>1µl Mo</td></tr> | ||
+ | |||
+ | <tr><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>35S:BxbI:RepBxbI:GFP:SF</td><td>35S:PhiC31+RepPhiC31:GFP</td></tr> | ||
+ | |||
+ | <tr><td>1µl 35S:BxbI:RepBxbI:GFP</td><td>1µl 35S:PhiC31</td></tr> | ||
+ | |||
+ | <tr><td>1µl SF</td><td>1µl RepPhiC31:GFP</td></tr> | ||
+ | |||
+ | <tr><td>1µl Mo</td><td>1µl Ω1</td></tr> | ||
+ | |||
+ | <tr><td>4.6 µl H<sub>2</sub>O</td><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>Make liquid culture of the Norwich constructions in Agro:</p> | ||
+ | |||
+ | <ul><li>584-MAA with YFP</li> | ||
+ | |||
+ | <li>596-RV3034c with YFP</li> | ||
+ | |||
+ | <li>598-RV3037c with YFP</li> | ||
+ | |||
+ | <li>600-RV3030 with YFP</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Transform and make petri dish culture of yesterday ligations that have Moflipper411, the α constructions.</p> | ||
+ | |||
+ | <ul><li>35S:BxbI:T35S</li> | ||
+ | |||
+ | <li>35S:PhiC31:T35S</li> | ||
+ | |||
+ | <li>PsinATG:RepBxbI:GFP:T35S</li> | ||
+ | |||
+ | <li>PsinATG:RepPhiC31:GFP:T35S</li> | ||
+ | |||
+ | <li>35S:Lactoferrin:T35S</li> | ||
+ | |||
+ | <li>35S:LTB:T35S</li> | ||
+ | |||
+ | <li>Gal4:KDronpa</li> | ||
+ | |||
+ | <li>LacI:KDronpa</li> | ||
+ | |||
+ | <li>LexA:KDronpa</li> | ||
+ | |||
+ | <li>NDronpa:VP16</li> | ||
+ | |||
+ | <li>Gal4:AsLOVpep</li> | ||
+ | |||
+ | <li>LacI:AsLOVpep</li> | ||
+ | |||
+ | <li>LexA:AsLOVpep</li> | ||
+ | |||
+ | <li>ePDZ:VP16</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | </br><h3 style="color:green">3 September 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transform the 11 ligations into <i>E. coli</i> and make petri dish culture.</p> | ||
+ | |||
+ | <ul><li>Gal4:KNDronpa:VP16:SF; Mo</li> | ||
+ | |||
+ | <li>LacI:KNDronpa:VP16:SF; Mo</li> | ||
+ | |||
+ | <li>LexA:KNDronpa:VP16:SF; Mo</li> | ||
+ | |||
+ | <li>Gal4:AsLOVpep:ePDZ:VP16:SF; Mo</li> | ||
+ | |||
+ | <li>LacI:AsLOVpep:ePDZ:VP16:SF; Mo</li> | ||
+ | |||
+ | <li>LexA:AsLOVpep:ePDZ:VP16:SF; Mo</li> | ||
+ | |||
+ | <li>Gal4:PIF6:PhyB:VP16:SF; Mo</li> | ||
+ | |||
+ | <li>LacI:PIF6:PhyB:VP16:SF; Mo</li> | ||
+ | |||
+ | <li>LexA:PIF6:PhyB:VP16:SF; Mo</li> | ||
+ | |||
+ | <li>35S:BxbI:RepBxbI:GFP:SF</li> | ||
+ | |||
+ | <li>35S:PhiC31+RepPhiC31:GFP; Ω1</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Make liquid culture of the 12 petri dishes cultures with the Moflippers construction, 2 colonies for each construction. </p> | ||
+ | |||
+ | <p>Put the protoplasts in the determine conditions to run a new experiment.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">4 September 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Make liquid culture of yesterday cultures.</p> | ||
+ | |||
+ | <p>Minipreps of yesterday liquid cultures:</p> | ||
+ | |||
+ | <p>Digestion:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>35S:BxbI:T35S</td><td>EcoRI+PstI</td><td>2959</td></tr> | ||
+ | |||
+ | <tr><td>35S:PhiC31:T35S</td><td>EcoRI+PstI</td><td>3105</td></tr> | ||
+ | |||
+ | <tr><td>PsinATG:RepBxbI:GFP:T35S</td><td>EcoRI+PstI</td><td>2388</td></tr> | ||
+ | |||
+ | <tr><td>PsinATG:RepPhiC31:GFP:T35S</td><td>EcoRI+PstI</td><td>2669</td></tr> | ||
+ | |||
+ | <tr><td>35S:Lactoferrin:T35S</td><td>EcoRI+PstI</td><td>3141</td></tr> | ||
+ | |||
+ | <tr><td>35S:LTB:T35S</td><td>EcoRI+PstI</td><td>1662</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:KDronpa</td><td>EcoRI+PstI</td><td>1966</td></tr> | ||
+ | |||
+ | <tr><td>LacI:KDronpa</td><td>EcoRI+PstI</td><td>3006</td></tr> | ||
+ | |||
+ | <tr><td>LexA:KDronpa</td><td>EcoRI+PstI</td><td>2274</td></tr> | ||
+ | |||
+ | <tr><td>NDronpa:VP16</td><td>EcoRI+PstI</td><td>2403</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:AsLOVpep</td><td>EcoRI+PstI</td><td>2235</td></tr> | ||
+ | |||
+ | <tr><td>LacI:AsLOVpep</td><td>EcoRI+PstI</td><td>2721</td></tr> | ||
+ | |||
+ | <tr><td>LexA:AsLOVpep</td><td>EcoRI+PstI</td><td>1989</td></tr> | ||
+ | |||
+ | <tr><td>ePDZ:VP16</td><td>EcoRI+PstI</td><td>2292</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <p>The band write in the digestion table only show the approximately legth of the DNA construction. We can not make a proper digestion in Benchling because we do not have the sequence of the Moflippers.</p> | ||
+ | |||
+ | <p>This digestions are special ones due to that we have to use BioBricks enzimes because the plasmid is a Moflipper. So the mix is:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>1µl DNA</td></tr> | ||
+ | |||
+ | <tr><td>1µl Buffer O</td></tr> | ||
+ | |||
+ | <tr><td>0.5µl EcoRI</td></tr> | ||
+ | |||
+ | <tr><td>0.5µl PstI</td></tr> | ||
+ | |||
+ | <tr><td>7 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LexA:KNDronpa (C1)</td><td>LexA:KNDronpa (C2)</td><td>Gal4:KNDronpa (C1)</td><td>Gal4:KNDronpa (C2)</td><td>LacI:KNDronpa (C1)</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>no</td><td>no</td><td>no</td></tr> | ||
+ | |||
+ | <tr><td>LacI:KNDronpa (C2)</td><td>RepBxbI:GFP (C1)</td><td>RepBxbI:GFP (C2)</td><td>Gal4:AsLOV (C1)</td><td>Gal4:AsLOV (C2)</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>no</td><td>No</td><td>no</td></tr> | ||
+ | |||
+ | <tr><td>LacI:AsLOV (C1)</td><td>LacI:AsLOV (C2)</td><td>LexA:AsLOV (C1)</td><td>LexA:AsLOV (C2)</td><td>NDronpa:VP16 (C1)</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>no</td><td>no</td><td>no</td></tr> | ||
+ | |||
+ | <tr><td>NDronpa:VP16 (C2)</td><td>LTB (C1)</td><td>LTB (C2)</td><td>ePDZ:VP16 (C1)</td><td>ePDZ:VP16 (C2)</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>no</td><td>no</td><td>no</td></tr> | ||
+ | |||
+ | <tr><td>Lactoferrin (C1)</td><td>Lactoferrin (C2)</td><td>BxbI (C1)</td><td>BxbI (C2)</td><td>RepPhiC31 (C1)</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>no</td><td>no</td><td>no</td></tr> | ||
+ | |||
+ | <tr><td>RepPhiC31 (C2)</td><td>PhiC31 (C1)</td><td>PhiC31 (C2)</td><td>LexA:AsLOV (C1)</td><td>LexA:AsLOV (C2)</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>no</td><td>no</td><td>no</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <ul><li>We found a problem because the length of the construction is very similar to the Moflipper length, so we can not distinguish very well the bands. Moreover the total weight do not match with the expected for the plasmid.</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | </br><h3 style="color:green">5 September 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Prepare to agroinfiltrate the Norwich constructions:</p> | ||
+ | |||
+ | <ul><li>584-MAA with YFP 0.27 1.11ml</li> | ||
+ | |||
+ | <li>596-RV3034c with YFP 0.3 1ml</li> | ||
+ | |||
+ | <li>598-RV3037c with YFP 0.28 1.07</li> | ||
+ | |||
+ | <li>600-RV3030 with YFP 0.25 1.2</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>We infiltrate 1 plant per construction, 2 leafs per plant.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Miniprep of the last moflippers constructions, the supposed Ω ones.</p> | ||
+ | |||
+ | <p>Digestion:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4:KNDronpa:VP16:SF; Mo</td><td>EcoRI+PstI</td><td>4804</td></tr> | ||
+ | |||
+ | <tr><td>LacI:KNDronpa:VP16:SF; Mo</td><td>EcoRI+PstI</td><td>5580</td></tr> | ||
+ | |||
+ | <tr><td>LexA:KNDronpa:VP16:SF; Mo</td><td>EcoRI+PstI</td><td>4843</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:AsLOVpep:ePDZ:VP16:SF; Mo</td><td>EcoRI+PstI</td><td>-</td></tr> | ||
+ | |||
+ | <tr><td>LacI:AsLOVpep:ePDZ:VP16:SF; Mo</td><td>EcoRI+PstI</td><td>-</td></tr> | ||
+ | |||
+ | <tr><td>LexA:AsLOVpep:ePDZ:VP16:SF; Mo</td><td>EcoRI+PstI</td><td>-</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:PIF6:PhyB:VP16:SF; Mo</td><td>EcoRI+PstI</td><td>-</td></tr> | ||
+ | |||
+ | <tr><td>LacI:PIF6:PhyB:VP16:SF; Mo</td><td>EcoRI+PstI</td><td>-</td></tr> | ||
+ | |||
+ | <tr><td>LexA:PIF6:PhyB:VP16:SF; Mo</td><td>EcoRI+PstI</td><td>-</td></tr> | ||
+ | |||
+ | <tr><td>35S:BxbI:RepBxbI:GFP:SF</td><td>EcoRI+PstI</td><td>2654</td></tr> | ||
+ | |||
+ | <tr><td>35S:PhiC31+RepPhiC31:GFP; Ω1</td><td>BamHI</td><td>1860, 3550, 6674, 86, 30, 10</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <ul><li>We realized that AsLOVpep and PhyB have recognition sites for BioBricks enzyes so we can not sent them to the iGEM.</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>Gel:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>LacI:KNDronpa (C1)</td><td>LacI:KNDronpa (C2)</td><td>Gal4:KNDronpa (C1)</td><td>Gal4:KNDronpa (C2)</td><td>LexA:KNDronpa (C1)</td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>Ok</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:AsLOVpep:ePDZ (C1)</td><td>Gal4:AsLOVpep:ePDZ (C2)</td><td>LexA:AsLOVpep:ePDZ (C2)</td><td>Gal4:PIF6:PhyB (C1)</td><td>Gal4:PIF6:PhyB (C2)</td></tr> | ||
+ | |||
+ | <tr><td>No</td><td>no</td><td>no</td><td>no</td><td>no</td></tr> | ||
+ | |||
+ | <tr><td>LacI:PIF6:PhyB (C1)</td><td>LacI:PIF6:PhyB (C2)</td><td>LexA:PIF6:PhyB (C1)</td><td>LexA:PIF6:PhyB (C2)</td><td>BxbI:RepBxbI:GFP (C1)</td></tr> | ||
+ | |||
+ | <tr><td>no</td><td>no</td><td>no</td><td>no</td><td>Ok</td></tr> | ||
+ | |||
+ | <tr><td>BxbI:RepBxbI:GFP (C2)</td><td>PhiC31+RepPhiC31:GFP (C1)</td><td>PhiC31+RepPhiC31:GFP (C2)</td><td></td></tr> | ||
+ | |||
+ | <tr><td>ok</td><td>ok</td><td>ok</td><td></td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | <img src=https://static.igem.org/mediawiki/2015/4/4b/Valencia_upv_150905.png> | ||
+ | |||
+ | |||
+ | <p>We keep and send to the iGEM parts: </p> | ||
+ | |||
+ | <ul><li>Gal4:KNDronpa:VP16:SF; Mo</li> | ||
+ | |||
+ | <li>LacI:KNDronpa:VP16:SF; Mo</li> | ||
+ | |||
+ | <li>LexA:KNDronpa:VP16:SF; Mo</li> | ||
+ | |||
+ | <li>35S:BxbI:RepBxbI:GFP:SF; Mo</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>35S:PhiC31:RepPhiC31:GFP+SF</td></tr> | ||
+ | |||
+ | <tr><td>1µl PhiC31:RepPhiC31:GFP</td></tr> | ||
+ | |||
+ | <tr><td>1µl SF (466)</td></tr> | ||
+ | |||
+ | <tr><td>1µl Mo411</td></tr> | ||
+ | |||
+ | <tr><td>4.6 µl H<sub>2</sub>O</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">6 September 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Transform in <i>Agrobacterium</i>PhiC31:RepPhiC31:GFP; Ω1</p> | ||
+ | |||
+ | <p>Transnsform into <i>E. coli</i> 35S:PhiC31:RepPhiC31:GFP+SF; Mo. </p> | ||
+ | |||
+ | <p>Make liquid culture again of the colonies that went wrong last time.</p> | ||
+ | |||
+ | <p>Prepare to agroinfilrate:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>OpLacI:luc:ren</td><td>0.19</td><td>200 µl/1.9ml</td></tr> | ||
+ | |||
+ | <tr><td>OpUAS:luc:ren</td><td>0.18</td><td>211 µl/1.9ml</td></tr> | ||
+ | |||
+ | <tr><td>OpLexA:luc:ren</td><td>0.17</td><td>224 µl/1.9ml</td></tr> | ||
+ | |||
+ | <tr><td>LexA:AsLOVpep:ePDZ:luc:ren</td><td>0.18</td><td>211 µl/1.9ml</td></tr> | ||
+ | |||
+ | <tr><td>LacI:AsLOVpep:ePDZ:luc:ren</td><td>0.15</td><td>253 µl/1.9ml</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:AsLOVpep:ePDZ:luc:ren</td><td>0.21</td><td>181 µl/1.9ml</td></tr> | ||
+ | |||
+ | <tr><td>LexA:KNDronpa:ren:luc</td><td>0.04</td><td>950 µl/1.9ml</td></tr> | ||
+ | |||
+ | <tr><td>Pnos</td><td>0.77</td><td>494 µl/1.9ml</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>PROTOPLASTS 5.</p> | ||
+ | |||
+ | <ul><li>There were infiltrated:</li> | ||
+ | |||
+ | <li>OpLacI:luc:ren</li> | ||
+ | |||
+ | <li>OpUAS:luc:ren</li> | ||
+ | |||
+ | <li>OpLexA:luc:ren</li> | ||
+ | |||
+ | <li>LexA:AsLOVpep:ePDZ:luc:ren</li> | ||
+ | |||
+ | <li>LacI:AsLOVpep:ePDZ:luc:ren</li> | ||
+ | |||
+ | <li>Gal4:AsLOVpep:ePDZ:luc:ren</li> | ||
+ | |||
+ | <li>LexA:KNDronpa:ren:luc</li> | ||
+ | |||
+ | <li>Pnos</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p>Mesurement of concentrations:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>Gal4:KNDronpa:VP16:OpUAS:luc:ren: 327.6 ng/µl</td></tr> | ||
+ | |||
+ | <tr><td>LexA:KNDronpa:VP16:OpLexA:luc:ren: 300.5 ng/µl</td></tr> | ||
+ | |||
+ | <tr><td>Gal4:KNDronpa:VP16:SF; Mo: 183.25 ng/µl</td></tr> | ||
+ | |||
+ | <tr><td>LacI:KNDronpa:VP16:SF; Mo: 221.9 ng/µl</td></tr> | ||
+ | |||
+ | <tr><td>LexA:KNDronpa:VP16:SF; Mo: 266.7 ng/µl</td></tr> | ||
+ | |||
+ | <tr><td>35S:BxbI:RepBxbI:GFP:SF; Mo: 187.5 ng/µl</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Prepare the samples to sent to igem. 3µl of each constructions.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">7 September 2015</h3> | ||
+ | |||
+ | |||
+ | |||
+ | <p>Miniprep of the liquid cultures.</p> | ||
+ | |||
+ | <p>Digestion and gel, that goes wrong again.</p> | ||
+ | |||
+ | <p>Do the ligations again with some new things to optimizied it. So we will boil first the plasmid for 10 minutes adding DTT because BsaI cuts better with an unrolled plasmid. Then we will add ATP into the mix to improve the efficiency of the enzyme. General mix is:</p> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="table-wrapper"><table class="alt"> | ||
+ | |||
+ | <tr><td>DNA construction</td></tr> | ||
+ | |||
+ | <tr><td>1µl DNA1</td></tr> | ||
+ | |||
+ | <tr><td>1µl DNA2</td></tr> | ||
+ | |||
+ | <tr><td>1µl DNA3</td></tr> | ||
+ | |||
+ | <tr><td>0.5µl Mo411</td></tr> | ||
+ | |||
+ | <tr><td>1µl DTT</td></tr> | ||
+ | |||
+ | <tr><td>1µl ATP</td></tr> | ||
+ | |||
+ | <tr><td>1µl T4ligase</td></tr> | ||
+ | |||
+ | <tr><td>1µl BsaI</td></tr> | ||
+ | |||
+ | <tr><td>1.2µl BSA 10x</td></tr> | ||
+ | |||
+ | <tr><td>1.2µl buffer ligase</td></tr> | ||
+ | |||
+ | <tr><td>2.4µl H20</td></tr> | ||
+ | |||
+ | </div></table> | ||
+ | |||
+ | |||
+ | |||
+ | <p>DNA constructions that were ligated with this technique:</p> | ||
+ | |||
+ | <ul><li>35S:BxbI:T35S</li> | ||
+ | |||
+ | <li>35S:PhiC31:T35S</li> | ||
+ | |||
+ | <li>PsinATG:RepBxbI:GFP:T35S</li> | ||
+ | |||
+ | <li>PsinATG:RepPhiC31:GFP:T35S</li> | ||
+ | |||
+ | <li>35S:Lactoferrin:T35S</li> | ||
+ | |||
+ | <li>35S:LTB:T35S</li> | ||
+ | |||
+ | <li>Gal4:KDronpa</li> | ||
+ | |||
+ | <li>LacI:KDronpa</li> | ||
+ | |||
+ | <li>LexA:KDronpa</li> | ||
+ | |||
+ | <li>NDronpa:VP16</li> | ||
+ | |||
+ | <li>Gal4:AsLOVpep</li> | ||
+ | |||
+ | <li>LacI:AsLOVpep</li> | ||
+ | |||
+ | <li>LexA:AsLOVpep</li> | ||
+ | |||
+ | <li>ePDZ:VP16</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p>Put the agroinfiltrated leafs to digest the membrane, let them overnight. We did 9 preparation in total. </p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">8 September 2015</h3> | ||
+ | |||
+ | <p>PROTOPLASTS 5.</p> | ||
+ | |||
+ | <p>Obtained the protoplasts and put into the corresponding plate to make the experiment.</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h3 style="color:green">10 September 2015</h3> | ||
+ | |||
+ | <p>The results of the luciferase essay are not going well, we thougth that this could be caused because the time spent between the agroinfiltration and the take of samples is too long. So we decided to make a isolated experiment only with Pnos, to see its activity. </p> | ||
+ | |||
+ | <p>We agroinfilrate 2 plants..</p> | ||
+ | <br/> | ||
</div> | </div> | ||
</details> | </details> | ||
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<ul style="text-align:right"> | <ul style="text-align:right"> | ||
<li class="button alt"><a href="https://2015.igem.org/Team:Valencia_UPV/Notebook#scroll1">Go to Protocols</a></li> | <li class="button alt"><a href="https://2015.igem.org/Team:Valencia_UPV/Notebook#scroll1">Go to Protocols</a></li> | ||
− | |||
− | |||
</ul> | </ul> | ||
Latest revision as of 20:37, 18 September 2015
Here we present you all the procedures we did to develop our project. On this page you can find the notebook contents. If preferred, you can go directly to the protocols by pressing in the buttons above or below. We hope you enjoy reading our incredible journey! Starts our work in the lab! Marta, a lab mate gives us a construction, the red toggle swich (E:PIF6:PhyB:VP16:Etr8:luc), we just have to add the renilla; α2 (GB160) to test it. Make the ligation (step 2 in the protocol): Electroporation (step 3 in the protocol) of E. coli to insert our first construction. Make a petri dish culture with a LB-Agar plate with streptomycin. There is no white colonies, we electroporate again and make petri dish culture. There was just one white colony, make the ligation again. Electroporation of the new ligation. There are white colonies. Make 2 liquid cultures of them (Step 4 in the protocol). Add 3.5ml of LB and 3.5µL of spectomycin. Make liquid culture of just some glycerinates: Al cultures have grown except for Ω2. Make minipreps (Step 5 in the protocol). Digestion of the minipreps (Step 6 of the protocol). Make the gel: Ask for the NDronpa sequence. This will be part of our blue toggle. This piece is known by reading the paper ‘Reversible photoswichable Dronpa-1 monitors nucleocytoplasmic transport of an RNA-binding protein in transgenic plants?(Doi: 10.111/j.1600-0854.2011.01180.lambda.). The sequence of NDronpa is plant optimised and avoid cryptic sequences. We have domesticated this sequence with a linker in N-terminal to allow us to join it to a binding domain and also we had a NLS in the C-terminal to transport itself to the nucleus. It is domesticated as B5 part for Golden Braid assembling. After obtaining the sequence we compare the protein in Uniprot and we can observed that our sequence add a V in the position 2. We compare this results with other papers and none of them has this addition. When we compare this sequence with the paper ?Optical control protein activity by fluorescent protein domains?(Doi: 10.1126/science.1226854) we observed that our position 146 is the position 145 and as what we want is the interaction caused by the N145-K145, we eliminate the V. We also eliminate a pair of amino acids at the end of the sequence following the same criteria. Once obtained both variants of Dronpa, we decided to add the binding domain to KDronpa and the activation to NDronpa as this last one tetramerizes and all operator sequence are repeated in our promoters. We had 2 cultures from the last day, corresponding to other 2 colonies of ligation. Agrobacterium culture of promoter less: Luciferase + Renilla Minipreps Digestion with BamHI and EcoRV Agarose gel 1% How to ask and make primers? Meeting with Daniel Ramón (Biopolis). Ligations: Digestions: Transform to E. coli from PIF+Phy and BxbI and make petri dish cultures. Digest of 160, 289 and the two ligations, PIF+phy and Etr8+BxbI. Agarose gel. We’ve got white colonies from PIF+Phy and BxbI! Pick two colonies from each construction. Minipreps of the 4 liquid cultures and digestion to see the band patterns. Digestion: Agarose gel was made: Repeat digestion because we are not sure of the last digestions. We don’t have sure the toggle, so we decide to repeat the digestion with other enzyme tomorrow, noticing that the colony 2 has better bands pattern. Optimized ligation: As BxbI was good at the digestion we put 1 µL of LB and 1 µL of Kanamicyne on the tube where it had grown and store at 37ºC to glycerinate later. We design primers to binding domain (BD) and PIF. Digestion of the ligation of yesterday containing: EPIF6-PhyB-VP16 (C1 y C2) Agarose gel 1%: We see three bands: 7000, 4000, 1900pb Transform optimized ligation PIF-Phy-Luc-Renilla-P19 and make petri dish cultures. Alfredo’s part is not working. Gel: Transformation into Agrobacteriumof Renilla (GB160) because we could not join this construction with PIF:PhyB and so we will do a cotransfection of both plasmids.Make petri dish culture. The petri dish with PIF:PhyB:luc was taken out the 37ºC room and put into the fridge to pick colonies tomorrow. Pick colonies to make liquid culture: KDronpa EcoRI 2800 We discovered that the construction with BxbI did not go well because our lab college gives us the wrong piece. Thanks Alfredo :) Templates PCR: 1+2, 5+6, 7+8PIF, 7+8VP16 These quantities multiplied by 3. 1+2, 5+6, 7+8PIF, 7+8VP16 all in pUPD2 We have white colonies of renilla! Also of Etr8+BxbI; α1 We have also pUPD2 colonies but they are so close to the blue ones that we can’t pick anyone.So we make strakes. Make an agarose gel with all the digestions: FOTO Pick colonies of the plates done yesterday and pass them into a liquid medium: The viral systems of Agrobacteriumcultures to make the color mosaics are ready after 2 days at 28ºC. We can make the agroinfiltration. Protocol to prepare solution to agroinfiltrate in the protocols notebook part. Quantification of DNA: Minipreps of the liquid culture: Digestion of the minipreps and do the gel: Gel: Transform ligations into E.Coli and make petri dish cultures with cloranfenicol for all of them except the ligation of Etr8:Bxb1+PhyB that goes with streptomycin. Do ligations: Digestion: Gel: Both of them present the fragment of the vector at 6000 pb but none of them at 2000bp which is the insert one. Measurement of the ODs of PhyB:PIF6:luc and renilla+P19. 1?EXPERIMENT. Red toggle. E:PIF6:PhyB and renilla. For more info, click here. Transformation into E. coli of LacIBD+KDronpa; α1 and make petri dish culture. Make petri dish culture of LexABD and Etr8(CMV):Bxb1:GFP. We make liquid culture of: Do the minipreps of the liquid cultures that have grown. Do the digestions of the minipreps: Make the gel. Take glycerinated: Do the minipreps of the 4 colonies of LexABD and both glycerinates, 35S and T35S. Do the digestion of the minipreps: Make the gel: Make ligations: Sent N-Dronpa with the primers 9 and 12 to sequence to check if the codon that synthetize for the amino acid K has change to the amino acid N. Quantification of DNA: Transform Gal4+KDronpa and LacI+KDronpa and make petri dish culture. Miniprep of: Gel: We pick more colonies of RepBxb1+GFP, Ω2 and make liquid cultures. Make liquid culture of: LexABD+KDronpa+prom+term; α1 (C1 and C2) NDronpa+VP16; α2 (C1 and C2) Gal4BD+PIF6; α1 (C1 and C2) LacIBD+PIF6; α1 (C1 and C2) LexABD+PIF6; α1 (C1 and C2) Take out a glycerinate 35S:Luciferase:Tnos (GB0227) and do a miniprep. Do the minipreps of the 10 liquid cultures. Do the digestions: Do the gel: Prepare liquid culture of: EXPERIMENT 1. Pick colonies and make liquid culture of: Minipreps of: Do the gel: Make ligations: The samples that we sent to sequence have arrived: The sequences of NDronpa have the desired mutation. Transformation in E.Coli of the 8 ligations and make petri dish cultures. Refresh the Agro’s cultures Renilla and PIF:phy:luc. Make the 2nd refresh of the culture of Agrobacterium. Make liquid cultures of the 8 colonies of E.Coli. Ligations: Minipreps of the liquid cultures. Digestion of the minipreps: We have to repeat the digestion of: LexA+PIF:phy+VP16. EXPERIMENT 2. Agroinfiltration of renilla and PIF6:PhyB:luc. For more info, click here. Transform the negative control into agrobacterium and make petri dish culture of: We were doing dry lab preparing the power point to present our project to the rector and biotecs companies. Prepare this cultures for agroinfiltration: EXPERIMENT 3. Start. Agroinfiltrate PhyB+PIF+luc and Renilla Digestions: Gel: It has arrived a new construction: AsLOVpep. Ligations: EXPERIMENT 2. Change the ligth conditions of the plants. Transform into E. coli last ligations. Pick colonies and make liquid culture of: Repeat the ligations. EXPERIMENT 2. Luciferase essay. Transform the ligations of AsLOVpepe and red toggle. This time we make a spin to concentrate the cells to make petri dish culture. Minipreps of: Digestions of: Make the gel: We received two constructions: Pick colonies and make liquid culture of: Ligations: Prepare glycerinates of: Do miniprep of: Digestions: Do transformation of DHSa and yesterday ligations: Pick colonies and make liquid culture: Ligations that were repeated: Refresh the liquid cultures of Agrobacterium: All the liquid cultures have grown, do minipreps. Do digestions: Agarose gel: The Agrobacterium cultures refreshed yesterday were store in the fridge. It is made another culture of 35S:BxbI+reporterBxbI:GFP to keep it in the fridge. EXPERIMENT 4. Recombinase BxbI. Mesurement of the OD’s: Transformation of the ligation: Gal4:PIF:phy:VP16+ren; α1 and LacI:KNdronpa:ren+OpLacI:luc; Ω1. The liquid cultures of this constructions were repeated: Do minipreps of: Do digestions: Make an agarose gel: Only LexA:KNdronpa:ren+OpLex:Luc and Reporter phiC31 were correct. Repeat the ligations because is the second digestion of this construction that were made. EXPERIMENT 4. The digestions had positive controls that were included in the gel to compare the results obtained. The digestions are left overnight in the working table. Do the gel: EXPERIMENT 5. Red toggle experiement: EXPERIMENT 4. It was observed another leaf with the recombinase BxbI with GFP in the magnifying glass with fluorescent lights. 23 minipreps of the liquid cultrures. LexA:PIF:PhyB:ren:luc (C3) has not grown. Digestions of the minipreps: Gel has been done: Pick more colonies of: New digestions with new enzymes: After 3 digestions of LacI:PIF:Phy:VP16+ren; α1 is accepted the construction. It was observed another leaf with the recombinase BxbI with GFP in the magnifying glass with fluorescent lights. It was not observed a lot of spots, the efficiency is very low. This is the 4th day… FOTO The incredible mistery of the lost notebook papers... Another day... EXPERIMENT 5. Red toggle. It was picked disc samples: 3 in far red, 3 in darkness and 6 in red (3 of them were in far red and the other 3 in darkness). Time lapses: -19:00=t0 -1:00=t1 -7:00= t2 -19:00= t3 (it was taken the controls in natural light) Gel with the digestions: We had problems with some colonies because in the digestion did not appear DNA. The minipreps will be made with a better kit. Transform in Agrobacterium this cultures: It was made liquid culture of Gal4:PIF:phyB:ren; Ω1 (C1-C5) It was made ligations: EXPERIMENT 5. Luciferase essay. Miniprep of Gal4:PIF:PhyB:ren (C1-C3) C4 and C5 did not grow. Digestion of the miniprep: Gel was made: After doing several digestions with different enzyme all with wrong band patterns, it was decided to revise each part making digestions. The parts are: Made liquid culture of the ReporterBxbI:GFP in Agrobacterium. Transform the ligations into E. coli: EXPERIMENT 6. Luciferase essay: EXPERIMENT 7. We decided to infiltrate soybean sprouts. First we decided to infiltrate with dye to observe the characteristics and the capacity of absorption. FOTO Make liquid cultures of yesterday transformations. Minipreps of the 13 liquid culture. LacI:PIF:phyB:ren cultures did not grow. Digestion of the minipreps: It was done 2 gels with ligations: Liquid cultures of Agrobacterium were refreshed: Sent to sequence: The sample had to have 10µl of miniprep (200ng/µl aprox) + 5 µl of primer (dilution 1:3) Ligations: OD’s measurements to agroinfiltrate: EXPERIMENT 8. EXPERIMENT 9. Infiltrate at vaccum the soyabeans sprouts with viral system and GFP. Ligations to join the negative controls with renilla: Transformation of E. coli of the ligations and make petri dish cultures: Transformation into Agrobacterium with the constructions and make petri dish cultures: It was received a new piece (ePDZ) which is part of the blue toggle (plan A). It arrived in E. coli so it was made a lquid culture and let it grow at 37ºC overnight. Miniprep of the liquid culture: ePDZ. Transformation into E. coli of the ligations: EXPERIMENT 9. It was observed the soybean sprouts that were infiltrated with Agrobacteriumwith green light and red filter. It was not observed nothing significant, moreover, the damage is evident. FOTO Transformation in Agrobacterium the ReporterPhiC31:GFP. Pick colonies and make liquid cultures adding X-Gal and IPTG because the colonies were little and we can not observe clearly if they were white or blue. The medicine LTB (heat labile toxin B subunit) which is part of an enterotoxin of Echerichia coli homologous to the same toxin in Vibrio cholerae that causes diarrhea. We add 50µl to have a final concentration of 10ng/µl. Ligation: Miniprep of yesterday liquid culture: Digestion: Gel: Pick colonies and make liquid culture of: Transformation in E. coli of: Minipreps have been done: Do the gel: Primers have arrived: Make a PCR with ePDZ and its primers to obtain the desired fragment and put it in pUPD2. Pick 2 colonies of PhiC31:GFP in Agrobacterium and make liquid culture. Ligations: Make liquid culture (E. coli) of: Ligation: EXPERIMENT 8. After 4 days till the agroinfiltration we have observed again the samples with BxbI and the negative control. Minipreps of liquid culture LTB (C1 and C2) Digestion: Gel: Take out glicerynates of Paloma, lab mate: For E. coli and Agrobacterium. Pick a colony of Asun interferon (IFN) in Agro, make liquid culture. We had miniprep of IFN in pUPD. Make ligations. Transform in E. coli the ligations and make petri dishes cultures: Refresh agro cultures to agroinfiltrate tomorrow: Prepare the Agrobacterium cultures for agroinfiltration: Gal4B:KDronpa:NDronpa:luc:ren, PhiC31 and its control, BxbI and its control and PIF:PhyB and its control. Mesurement oof the ODs: EXPERIMENT 9. Look the petri dishes culture: Miniprep of the liquid culture: Measure of DNA concentration of: Pick colonies and make liquid culture of: Repeat ligations: Refresh agrobacterium cultures of: Take the glycerinate of Renilla; Ω2 (GB159) and make liquid culture. EXPERIMENT 9. We have made all the leaf discs and put in order in the plates. We do a Western blot with Asun so we can learn how to do it. Lo copio? De verdad!!? Miniprep of the liquid cultures: Digestions: Gel: Transformation into E. coli of yesterday ligations and make petri dish culture: Ligations: Refresh agrobacterium cultures of: Transform ligations: Pick colonies and make liquid cultures cultures of: EXPERIMENT 9. Make luciferase essay of red and blue toggle: Miniprep of the liquid cultures. Digestion: Gel: Make colony PCR with 6 colonies of PhiC31: Make a gel with the PCR but we did not see the correct bands. Repeat the colony PCR: Ligations: Refresh Agrobacteriumcultures to agroinfiltrate: Transformation into Agrobacterium: Transformation into E. coli and make petri dishes cultures: Make a gel with the colonies PCRs of PhiC31: There was no DNA, there is a problem with the cells or the procedure, it have to be revised. Ligation: Refresh agrobacterium cultures for tomorrow infiltration: EXPERIMENT 10. Measure f the ODs: Transform the ligation into E. coli and make petri dishes cultures: EXPERIMENT 11. Measurement of the ODs: Make a colony PCRs with the 10 white colonies of PhiC31 that grow in the petri dishes. Minipreps of the liquid cultures: Digestion of the minipreps: Make the gel: We keep in our inventory the colony C1 of each construction Make a gel of the colonies PCRs of PhiC31: we did not observed any DNA. Still can’t fix the problem. Make liquid culture of renilla and PIF6:PhyB:luc in Agrobacterium because the last time that we did an experiment they have grown slowly. Store in the 4ºC fridge an agrobacterium culture with P19. Ligations: Transform into E. coli this ligations and make petri dishes cultures: Make liquid culture of 11 colonies of PhiC31 due to that this construction was giving us problems to obtain. Refresh the agrobacterium cultures of: EXPERIMENT 10. Take the samples of the agroinfiltrated plants that were in darkness to change the conditions. Make liquid culture of: Primers have arrived, they have been resuspended. They were used to eliminate the recognitions sites of the enzymes in BioBricks. This will make our constructions ready to be send to the iGEM Minipreps of the liquid cultures done yesterday: Make PCRs with all the primers and constructions: All of them follow the same composition which is: *The three last lines are the only specify bellow. Digestions of the minipreps: Make the gel: Mesurement of the ODs: EXPERIMENT 11. Take the samples of the agroinfiltrated plants that were in darkness to change the conditions. EXPERIMENT 12. New digestions to check if the negative control constructions are correct and we can transformed it in agro Gel: Transform in Agrobacteriumand make petri dishes cultures: Ligations: PROTOPLAST 1: Do for the first time. PROTOCOL, click here. Analysis of the AsLOVpep PCR’s products that were set to sequencing: Transform into E. coli yesterday ligations and make petri dishes cultures: PROTOPLAST 1:We try to obtain the protoplast but we make a mistake, repeat. PROTOPLAST 2. Start the procedure again. EXPERIMENT 11. Luciferase essay of blue and red toggle. Ligations of the PCR products: Repeat the digestions of the negative controls: Make the gel: Transform into E. coli AsLOVpep (BB) and make petri dish culture. Make liquid cultures of: Make liquid cultures of agrobacterium cultures in petri dishes: We make triple strake method to obtain isolated colonies of LexA:PIF6:PhyB:VP16. PROTOPLAST 2: Continue with the procedure to obtain protoplasts. We finally see in the microscope the protoplasts. Transform protoplasts. How? Click here. EXPERIMENT 12. Prepare samples of both recombinases and its negative control to see them in the confocal microscope. We see samples of plants agroinfiltrated 7 days ago and other agroinfiltrated 5 days ago with the viral silencing repressor, P19. PROTOPLAST 2: Take samples at time 8h of protoplasts. Samples of both toggles and in all the conditions. Minipreps of: Minipreps digestions: Make the gel: We keep LexA:AsLOVpep:ePDZ+ren C1 and LacI:AsLOVpep:ePDZ:ren+OpLacI:luc C4. Make liquid cultures of (added X-gal + IPTG): PROTOPLASTS 3. PROTOPLAST 2. Take protoplasts samples at time 16h. Samples of both toggles and in all the conditions. Take also time 24h. All liquid cultures of AsLOvpep (BB); pUPD2 (C1-C4) were blue. Pick 4 more colonies also with added X-gal + IPTG Ligations: PROTOPLASTS 3. Finish the protoplasts started yesterday, we obtain good concentrations. Transform them: PROTOPLAST 2. Luciferase essay. Do a luciferase essay with protoplasts, click here. Transform into agro and make petri dish culture of: Refresh agrobacterium liquid culture of: PROTOPLASTS 3. Take protoplasts samples at time 16h. Big disapoint here, we dscovered hat they were cloroplasts... ha, ha. Liquid cultures of AsLOVpep (BB); pUPD2 were all blue. The ligation was made again. Miniprep of Pnos in Agrobacterium. Transform the ligations in E. coli: EXPERIMENT 13. Prepare the viral system in agrobacterium cultures to infiltrate into sunflowers sprouts by vacuum. Measurement of the ODs: Make 60ml of medium for each infiltration. New pieces have arrived: EXPERIMENT 11 and 12. Take samples of leads agroinfiltrated with LTB, IFN and sip rotavirus CH2 and CH2-CH3. Make PCRs of PhiC31 and AsLOVpep to eliminate the enzyme recognition sites of Biobricks (BB). Program: 96ºC-10’; 96ºC-30’’; Tm-30’’ and 72ºC-30’’ Ligation of the PCRs products: Transform in Agrobacterium: Make liquid culture of: We start to do the western to see if any drugs had been produced in our plants. The gel order is: Make a gel with the PCRs products: PhiC31 and AsLOVpep modified for BioBricks. NO SE LAS BANDAS ESPERADAS. We change a little bit the protocol to make protoplasts, now we follow a protocol found in a Nature paper. The important changes are that during the digestion of the cell wall there is no agitation. Transform into E. coli the PCRs products: Prepare liquid culture of: Miniprep of the liquid cultures: Digestions: Gel: Make liquid culture of: Also had grown the liquid cultures: All the liquid cultures of AsLOVpep (BB) and PhiC31 (BB) had grown. Do 12 minipreps. Digestions of the minipreps: Do the gel: We keep the three colonies of AsLOVpep but we make another digestion with different enzymes of PhiC31 colonies C6 and C7. Optimized ligation: Make glycerinates of: Make liquid culture of positive controls in Agro: Prepare the liquid cultures of Agrobacteriumto agroinfiltrate: The total volume of preparation will be 6ml each drug. EXPERIMENT 14. Refresh more colonies to make more glycerinates and pick two colonies of the petri dish culture: Miniprep of the liquid culture: Digestion: New digestion to PhiC31 (BB): Make the gel: Transformation in Agrobacteriumand make petri dishe culture of : Miniprep of: Digestions of the minipreps: Do the gel: We make glicerynates of: Transform into E. coli and make Petri dish culture: Refresh this cultures to agroinfiltrate: Still trying how to make protoplasts… We will win this battle! Make liquid culture of the culture of Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1. There was only one white colony, nothing hopeful… We fount that the culture of Pnos in Agrobacterium did not grow, so we decided not to carry on with the experiment because it was our control. Refresh again two different cultures of Pnos that were found in the fridge and do the experiment tomorrow. Transform into Agrobacteriumthe following constructions: Miniprep of: Digestion: Gel: As the Pnos construction was given to us form a lab partner, we do not surely that the expected bands were correct because we choose the construction at the golden braid register parts. We can not make any glicerynate because it was not the necessary amount of liquid culture to make it. Refresh again: PhiC31 (BB); pUPD2; LTB; α1. Prepare the Agrobacteriumcultures to agroinfiltrate: Pnos and LacI:AsLOVpep:ePDZ:ren:OpLacI:luc. EXPERIMENT 15. Make liquid culture of agro colonies: Digestion of yesterday minipreps: Do a gel: It had arrived the second part of the lactoferrin. Ligation into pUPD2 of both parts of the lactoferrin and other ligation: Transform into E. coli the ligations. Refresh PhiC31 (BB) and Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc to make glycerinates. Transfrorm into Agrobacteriumthe construction: Keep trying protoplasts, this time we see them in the microscope without coverslip. We can see them alive, maybe this was the error. Also we decided to leave the digestion enzymes overnight because after only 3 hours the amount of protoplasts was very little. EXPERIMENT 14. Take leafs agroinflitrated with the drugs EXPERIMENT 15. Do discs of the agroinfiltrated lefs with the blue toggle (LacI:AsLOVpep:ePDZ:ren:OpLacI:luc) and the control Pnos. FOTO DE LA PLACA Make liquid cultures of yesterday transformations: Minipreps of: Digestions of the minnipeps: Gel: Do glycerinates of: Ligations: Take samples of the glycerinates: Refresh the Agrobacteriumculture to make agroinfiltration tomorrow of: Make liquid culture of the Agrobacteriumcolony of Gal4:AsLOVpep:ePDZ:ren:OPUAS:luc; Ω1. Minipreps of: Digestions: Gel: Transform into E. coli yesterday ligations: Take samples of the glycerinates and make liquid culture: Make a glycerinates of Gal4:KDronpa:NDronpa:ren:OpUAS:luc; Ω1 EXPERIMENT 15. Take samples at 48h and store them at -80ºC in the box “The rebellion of AsLOV”. PROTOPLASTS 4. Do protoplasts to transform them tomorrow. Miniprep of: 35S:GFP:Tnos. Digestion of the miniprep: Gel: Correct. Ligation: Measure the OD of GFP: 157.5. It the right curve. Make liquid culture of the only white colony of PhiC31+RepPhiC31:GFP; Ω1 PROTOPLASTS 4. Finish the preparation of protoplasts and transform them with 40 µl of 35S:GFP:Tnos at a concentration of 157.5ng/ml. After the transformation of the protoplast we see them at the microscope and there were no protoplasts. Take samples of glycerinates and make liquid cultures of: Transform and make petri dish culture of Lactoferrin; α1 ligation. Miniprep of the glycerinates’ liquid cultures: Refresh the culture of Gal4:KDronpa:NDronpa:ren:OpUAS:luc; Ω1 due to that in the last transformation of protoplasts we run out all the DNA. Refresh and make liquid culture of Digestion of the minipreps: Do the gel: Make liquid culture of Lactoferrin; α1 (C1-C4) Miniprep of the liquid cultures, lactoferrin C1 and C4 did not grow. We do 5 minipreps. Digestion of the minipreps: Gel(we repeat it because yesterday gel was very bad): Ligations: (we make this because the pieze Operator+mini35S can not be ligated with the other constructions in Benchlig so we will join them all in this new ligations). Transform the ligations into E. coli adding IPTG and X-Gal. Ligation: The collaborations part of the Norwich iGEM team have arrived: Añadir una foto de las placas en la wiki y enlace de la wiki de los the norwich Transform the Moflippers into E. coli to have later more DNA to make the ligations and be able to send the parts with the necessary characteristics to the BioBricks requirements. Transform the functional constructions into Agrobacteriumand make petri dishes cultures. Refresh liquid cultures of Agrobacteriumto make an experiment with red toggle: Make liquid culture of the Moflippers, 2 colonies for each culture. Make liquid culture of yesterday transformations: Do the ligations in Moflippers with the miniprep that had arrived. This constructions are the ones that go with our plasmid α. Do the minipreps of the liquid cultures: Make liquid culture of the Norwich constructions in Agro: Transform and make petri dish culture of yesterday ligations that have Moflipper411, the α constructions. Transform the 11 ligations into E. coli and make petri dish culture. Make liquid culture of the 12 petri dishes cultures with the Moflippers construction, 2 colonies for each construction. Put the protoplasts in the determine conditions to run a new experiment. Make liquid culture of yesterday cultures. Minipreps of yesterday liquid cultures: Digestion: The band write in the digestion table only show the approximately legth of the DNA construction. We can not make a proper digestion in Benchling because we do not have the sequence of the Moflippers. This digestions are special ones due to that we have to use BioBricks enzimes because the plasmid is a Moflipper. So the mix is: Gel: Prepare to agroinfiltrate the Norwich constructions: We infiltrate 1 plant per construction, 2 leafs per plant. Miniprep of the last moflippers constructions, the supposed Ω ones. Digestion: Gel: We keep and send to the iGEM parts: Transform in AgrobacteriumPhiC31:RepPhiC31:GFP; Ω1 Transnsform into E. coli 35S:PhiC31:RepPhiC31:GFP+SF; Mo. Make liquid culture again of the colonies that went wrong last time. Prepare to agroinfilrate: PROTOPLASTS 5. Mesurement of concentrations: Prepare the samples to sent to igem. 3µl of each constructions. Miniprep of the liquid cultures. Digestion and gel, that goes wrong again. Do the ligations again with some new things to optimizied it. So we will boil first the plasmid for 10 minutes adding DTT because BsaI cuts better with an unrolled plasmid. Then we will add ATP into the mix to improve the efficiency of the enzyme. General mix is: DNA constructions that were ligated with this technique: Put the agroinfiltrated leafs to digest the membrane, let them overnight. We did 9 preparation in total. PROTOPLASTS 5. Obtained the protoplasts and put into the corresponding plate to make the experiment. The results of the luciferase essay are not going well, we thougth that this could be caused because the time spent between the agroinfiltration and the take of samples is too long. So we decided to make a isolated experiment only with Pnos, to see its activity. We agroinfilrate 2 plants..Daily notebook
June
27 May 2015
E:PIF6:PhyB:VP16:Etr8:luc+ren; Ω1 1µL E:PIF6:PhyB:VP16:Etr8:luc; α1 1µL renilla; apha2 1µL Ω1 6,8µL H2O 28 May 2015
29 May 2015
30 May 2015
E:PIF6:PhyB:VP16:Etr8:luc+ren; Ω1 0.5µL E:PIF6:PhyB:VP16:Etr8:luc; α1 1µL renilla; apha2 1µL Ω1 7.2µL H2O 1 June 2015
2 June 2015
3 June 2015
α1 - - α2 - - Ω1 - - pUPD2 - - E:PIF6:NLS; pUPD2 (GB0288) EcoRI 3000, 1000 E:PIF6:NLS; α1 (GB892) EcoRI 6300, 2500 E:PIF6:NLS; Ω2 (GB893) EcoRV 1800, 6600, 900 E:PIF6:NLS:luc:PhyB; α1 (GB896) EcoRI 3600, 6300, 5600 Luc:PhyB; Ω1 (GB890) BamHI 2300, 6300, 4200 PhyB:VP16; pUPD2 (GB289) EcoRI 3000, 2000, 500 PhyB:VP16; α2 (GB88E) HindIII 2100, 6300, 1800 Etr8:CMVmini; pUPD2 (GB1097) EcoRI 3000, 480 OpLexA:mini35S; pUPD2 (GB733) EcoRI 3000, 460 OpLexA:mini35S:luc:Tnos; α2 (GB151) HindIII 2500 LexABD; pUPD2 (GB0732) EcoRI 3000, 300 LacI for N-Tfusion; pUPD2 (GB858) EcoRI 3000, 1000 Linker:LacIBD; pUPD2 (GB704) EcoRI 3000, 1000 OpLacI:mini35S:luc:Tnos; α2 (GB152) HindIII 2500, 2600 OpLacI:mini35S; pPUD2 (GB534) EcoRI 3000, 560 E:PIF6:PhyB:VP16:Etr8:luc+ren; Ω1 BamHI 3700, 6100, 6600, 4200 E:PIF6:PhyB:VP16:Etr8:luc+ren; Ω1 EcoRV 11000, 400, 2500, 3000, 4000
pUPD2 Alf Alpha1 288 289 534 ok ok ok ok ok ? 704 732 733 858 892 896 ok ok ok ok ok ok 1097 Alpha2 88E 151 152 Omega1 ok ok ok ok ok ok 890 893 Red toggle (C1) (EcoRV) Red toggle (C1) (BamHI) Red toggle (C2) (EcoRV) Red toggle (C2) (BamHI) ok ok ok no no no 4 June 2015
5 June 2015
PIF6 + PhyB; Ω1 Etr8(CMV)+BxbI:T35S; α1 1µL (GB892) PIF; α1 1µL (GB1097) Etr8(CMV); pUPD2 1µL (GB88E) PhyB; α2 1µL BxbI; pUPD2 1µL Ω1 1µL Tnos pUPD2 6.8µL H2O 1µL α1 5.8µL H2O
(GB160) 35S:Renilla:tNOS-35S:P19:tNOS EcoRV 2475, 381, 4601 (GB896) Luc:PIF6:PhyB EcoRV 11608, 3942 6 June 2015
GB160 289 PIF+PhyB BxbI ok no ? ?
7 June 2015
8 June 2015
Etr8(CMV):Bxb1:Tnos; α1 EcoRI 6345, 238 EPIF6 + PhyB-PV16; Ω1 BamHI 6686, 1439, 2685, 2237
BxbI (C1) BxbI (C2) E:PIF6+PhyB-VP16 (C1) E:PIF6+PhyB-PV16 (C2) ok ok no no
PIF-PhyB-Luc-Renilla-P19 1 µL vector 0.8 µL dilution ?GB160 1.7 µL PIF:PhyB 4.15 µL H2O Ratio 1:2 vector insert 9 June 2015
EPIF6-PhyB-VP16 PvuII (green buffer) 3663, 9472pb
EPIF6-PhyB-VP16 (C1) EPIF6-PhyB-VP16 (C2) no no
10 June 2015
PIF+Phy:VP16 PvuII (buffer green 10x) 3663, 9472 PIF+Phy:VP16 BamHI 1939, 2685, 2337, 6674
PIF + Phy (PvuII) C3 PIF + Phy (PvuII) C4 PIF + Phy (PvuII) C5 PIF + Phy (PvuII) C6 no ok no No PIF + Phy (BamHI) C3 PIF + Phy (BamHI) C4 PIF + Phy (BamHI) C5 PIF + Phy (BamHI) C6 no ok no No 11 June 2015
E:PIF6:PhyB:VP16:luc:ren BamHI 4209, 3756, 6100, 6674 EcoRV 3942, 2989, 2475, 381, 10952
PIF6:PhyB:VP16:luc:ren C1 (BamHI) PIF6:PhyB:VP16:luc:ren C3 (BamHI) PIF6:PhyB:VP16:luc:ren C1 (EcoRV) PIF6:PhyB:VP16:luc:ren C3 (EcoRV) no no no no
12 June 2015
13 June 2015
BxbI; α1+PhyB; α2 1µl BxbI 1 µl PhyB 1 µl Ω2 4.6 µl H2O 15 June 2015
BxbI + 35S:E-PIF6:tnos; Ω1 1µl BxbI 1 µl PhyB 1 µl Ω1 4.6 µl H2O
KDronpa; pUPD2 1 µl KDronpa 1 µl pUPD2 5.6 µl H2O 16 June 2015
Primers Code Template Working temperature (ºC) LacI F 1 LacI (858) 69.7 LacI R 2 Gal4 F 3 We did not take out the glicerynate. 63.2 Gal4 4 LexA F 5 LexA (732) 62.7 LexA R 6 PIF:VP16 F 7 PIF6 (288) 60.1 PIFVP16 R 8 NDronpa F1 9 Kdronpa 67.7 NDronpa R1 10 Dronpa F2 11 58.5 NDronpa R2 12
PCR Fusion Taq (50µl) DNA template (10 µg/µl) 0.5 µl fusion taq 2.5 µl primer F 2.5 µl primer R 2 µl NTPs 31.5 µl H2O 17 June 2015
Template PCR; pUPD2 0.5µl template 1µl pUPD2 6.1µl H2O
Template 1+2 5+6 7+8PIF 7+8VP16 9+10 11+12 Band pattern 1017 284 391 478 464 290 Gel result ok ok ok ok No DNA ok 18 June 2015
Kdronpa C1 Kdronpa C2 Kdronpa C3 Kdronpa C4 no no ok no Etr8:BxbI:phyB C1 Etr8:BxbI:phyB C2 Etr8:BxbI:phyB C3 No no no
NDronpa 2.5 µl (9+10) primer F 2.5 µl (11+12) primer R 2 µl NTPs 0.2 µl Taq 10 µl Buffer 31.5 µl H2O
Etr8:BxbI:T35S; α1 Template PCR; pUPD2 1 µlEtr8 0.5µl template 1 µl BxbI 1µl pUPD2 1 µl T35S 6.1µl H2O 1 µl α1 5.8 µl H2O 19 June 2015
1µl of DNA’s template (9+10, 9+12 and 11+12) 2µl of specific buffer 2µl of NTPs 1µl primer forward 1µl primer reverse 0.5 µl of Taq 12.5 µl H2O
Minipreps: Enzime Band pattern (GB159) pDGB1_Ω2 renilla EcoRV 2909, 2475,882, 812, 381 Entry vector, Ω2 EcoRV 6652, 621 (GB552) pP35s NoATG; pUPD2 EcoRI 2997, 1090 (GB160) renilla pDGB1, α2 EcoRV 4601, 2475, 381 (GB731) Gal4BD (CDS); pUPD2 EcoRI 2997, 2493 (GB109) 355:renilla:Tnos; α1 EcoRI 2580, 2493
159 160 Ω2 552 731 109 9+10 9+12 11+12 ok ok ok ok ok ok no ok ok
20 june 2015
21 June 2015
22 June 2015
LacIBD, pUPD2 NotI 2046, 1053 LexABD, pUPD2 NotI 2046, 321 Etr8(CMV):Bxb1 NotI 1532, 1290, 5896 PIF6,pUPD2 NotI 2046, 407 VP16, pUPD2 NotI 2046, 500
LacI C1 LacI C2 LacI C3 LacI C4 LacI C5 LexA C1 LexA C2 BxbI C1 BxbI C2 BxbI C3 Ok ok ok ok ok no no ok ok no PIF C1 PIF C2 PIF C3 PIF C4 PIF C5 VP16 C1 VP16 C4 VP16 C5 No no - ok ok ok ok ok Gal4 NDronpa 1 NDronpa 2 23 June 2015
24 June 2015
ETR8(CMV):BxbI; α1+PhyB:VP16; α2; Ω1 Gal4BD(pcr) + pUPD2 1.5 µl Etr8:BxbI 1 µl Gal4 PCR 1.5 µl 88E (PhyB:VP16) 1 µl pUPD2 1 µl Ω1 5,6 µl H2O 3.6µl H2O 25 June 2015
Gal4BD; pUPD2 NotI 2046, 282 RepBxbI; pUPD2 NotI 2046, 460 Etr8(CMV):BxbI:PhyB; α1 BamHI 6674, 2237, 2806, 1174 LexABD; pUPD2 NotI 2046, 321 9+10; pUPD2 NotI 464
Etr8:BxbI LexA C1 LexA C2 LexA C3 LexA C4 RepBxbI C1 RepBxbI C2 RepBxbI C3 Gal4 C1 PCR 9+10 no no no no no ok ok ok no ok
N-dronpa; pUPD2 RepBxbI; α1 Gal4BD, pUPD2 LexABD; pUPD2 1 µl PCR 9+10 1 µl Rep Bxb1 1 µl PCR 3+4 1 µl PCR 5+6 1 µl PCR11+12 1 µl Promoter without ATG 1 µl pUPD2 1 µl pUPD2 1 µl pUPD2 1 µl Tnos 1 µl α1 4,6 µl H2O 3,6 µl H2O 5,6 µl H2O 5,6 µl H2O
Etr8:BxbI+PhyB; Ω1 1 µl Etr8:BxbI 1 µl 88E 1µl Ω1 3,6 µl H2O 26 June 2015
RepBxbI+GFP; α2 LacIBD+PIF6; α1 1 µl RepBxbI 1 µl LacIBD, pUPD2 1 µl promoter without ATG 1 µl PIF6, pUPD2 1 µl Tnos 1 µl promoter 1µl GFP (0059) 1 µl T35 1 µl α2 1 µl α1 2.6 µl H2O 2.6 µl H2O
LacIBD+PIF6; α1 EcoRI 6345, 1997, 641
LacIBD+PIF C1 LacIBD+PIF C2 no no
PhyB:PIF6:luc: 0.35 (1:2) 0.35 1.429 µl Ren+P19: 0.34 (1:2) 0.34 1.412 µl
LacIBD; pUPD2+KDronpa; pUPD2; α1 1 µl 35S 1 µl LacIBD;pUPD2 1 µl KDronpa; pUPD 1 µl T35S 1 µl α1 2.6 µl H2O 27 June 2015
28 June 2015
LacIBD+PIF; α1 EcoRI 6345, 1997, 641 RepBxbI:GFP; Ω2 HindIII 6345, 2683 Gal4BD; pUPD2 NotI 2681, 644 NDronpa; pUPD2 NotI 2046, 744
RepBxbI:GFP C1 RepBxbI:GFP C2 LacIBD+PIF C1 LacIBD+PIF C2 LacIBD+PIF C3 LacIBD+PIF C4 no no no no no No Gal4BD C1 Gal4BD C2 Gal4BD C3 Gal4BD C4 Gal4BD C5 N-Dronpa C1 ok ok ok ok ok ok N-Dronpa C2 N-Dronpa C3 N-Dronpa C4 no ok ok
29 June 2015
LexABD; pPPD2 NotI 2358, 312 35S; pUPD2 NotI 2981, 1074 T35S; pPUD2 NotI 2981, 304
LexA C1 LexA C2 LexA C3 LexA C4 P35S T35S ok ok ok ok Ok? Ok?
LacIBD+KDronpa+promoter+termi; α1 Gal4BD+KDonpa+prom+ter; α1 LexABD+KDronpa+prom+term; α1 1 µl LacI; pUPD2 1 µl Gal4; pUPD2 1 µl Gal4; pUPD2 1 µl KDronpa; pUPD2 1 µl KDronpa; pUPD2 1 µl KDronpa; pUPD2 1 µl 35S (GB0030) 1 µl 35S (GB0030) 1 µl 35S (GB0030) 1 µl T35S (GB0036) 1 µl T35S (GB0036) 1 µl T35S (GB0036) 2.6 µl H2O 2.6 µl H2O 2.6 µl H2O 1 µl α1 1 µl α1 1 µl α1
NDronpa+VP16; α2 Gal4BD+PIF6; α1 LacIBD+PIF6; α1 1 µl NDronpa; pUPD2 1 µl Gal4BD; pUPD2 1 µl LacIBD; pUPD2 1 µl VP16; pUPD2 1 µl PIF6; pUPD2 1 µl PIF6; pUPD2 1 µl 35S (GB0030) 1 µl 35S (GB0030) 1 µl 35S (GB0030) 1 µl T35S (GB0036) 1 µl T35S (GB0036) 1 µl T35S (GB0036) 2.6 µl H2O 2.6 µl H2O 2.6 µl H2O 1 µl α2 1 µl α1 1 µl α1
LexABD+PIF6; α1 1 µl LexABD; pUPD2 1 µl PIF6; pUPD2 1 µl 35S (GB0030) 1 µl T35S (GB0036) 2.6 µl H2O 1 µl α2 30 June 2015
RepBxb1+GFP; Ω2 HindIII 6345, 2683
RepBxbI+GFP C1 RepBxbI+GFP C2 RepBxbI+GFP C3 No no no
July
1 July 2015
LexABD+KDronpa;α1 EcoRI 6345, 2296 NDonpa+VP16; α2 HindIII 6345, 2427 Gal4BD+PIF6; α1 EcoRI 6345, 1867 LacI+PIF; α1 EcoRI 6345, 2638 LexABD+PIF6; α1 EcoRI 6345, 1906
Gal4+PIF C1 Gal4+PIF C2 LexA+PIF C1 LexA+PIF C2 LexA+KDronpa C1 ok ok ok ok Ok LexA+Kdronpa C2 LacI+PIF C1 LacI+PIF C2 Ndonpa+VP16 C1 Ndronpa+VP16 C2 ok ok ok ok ok
2 July 2015
Gal4+KDronpa EcoRI 6345, 3028 LacI+KDronpa EcoRI 6345, 2257 RepBxbI+GFP HindIII 6300, 2400
LacI+KDronpa C1 LacI+KDronpa C2 Gal4+KDronpa C1 Gal4+KDronpa C2 ok ok ok ok RepBxb1+GFP C4 RepBxb1+GFP C5 RepBxb1+GFP C6 ok ok ok
LexA:Kdonpa+NDronpa; Ω1 LacI:Kdronpa+NDronpa; Ω1 Gal4:Kdronpa+NDronpa; Ω1 1 µl LexA:Kdronpa 1 µl LacI:Kdronpa 1 µl Gal4:Kdronpa 1 µl NDronpa 1 µl NDronpa 1 µl NDronpa 1 µl Ω1 1 µl Ω1 1 µl Ω1 4.6 µl H2O 4.6 µl H2O 4.6 µl H2O
LexA:PIF+PhyB:VP16; Ω1 LacI:PIF+PhyB:VP16; Ω1 Gal4:PIF+PhyB:VP16; Ω1 1 µl LexA:PIF 1 µl LacI:PIF 1 µl Gal4:PIF 1 µl PhyB:VP16 (88E) 1 µl PhyB:VP16 1 µl PhyB:VP16 1 µl Ω1 1 µl Ω1 1 µl Ω1 4.6 µl H2O 4.6 µl H2O 4.6 µl H2O
35S:BxbI+RepBxbI:GFP; Ω1 E-PIF+phyB+luc+ren; Ω1 1 µl 35s:Bxb1:T35S (alfredo’s) 0.5 µl PIF:PhyB:luc (GB0896) 1 µl NoATGProm:RepBxbI:GFP 1 µl Renilla GB0160 1 µl Ω1 1 µl Ω1 4.6 µl H2O 4.6 µl H2O 4 July 2015
5 July 2015
LacI:PIF+PhyB:VP16; Ω1 Gal4:PIF+PhyB:VP16; Ω1 1 µl LacI:PIF 1 µl Gal4:PIF 1 µl PhyB:VP16 1 µl PhyB:VP16 1 µl Ω1 1 µl Ω1 4.6 µl H2O 4.6 µl H2O
LacI:Kdronpa+NDronpa BamHI 6674, 5437 35S:BxbI+RepBxbI:GFP BamHI 6674, 3859, 1782 Gal4:Kdronpa+NDronpa BamHI 6674, 4666 LexA:Kdonpa+NDronpa BamHI 6674, 4705 LexA:PIF+PhyB:VP16 BamHI 6674, 3513, 2337 E:PIF6:PhyB:VP16 BamHI 4209, 3756, 6100, 6674
LacI:KNDronpa C1 LacI:KNDronpa C2 BxbI:Rep:GFP C1 BxbI:Rep:GFP C2 Gal4:KNDronpa C1 Gal4:KNDronpa C2 ok ok ok ok ok ok LexA:KNDronpa C1 LexA:KNDronpa C2 LexA:PIF:Phy C1 LexA:PIF:Phy C2 Red toggle ok ok no no no
Renilla 0.22 182 µl of sample + 1.818 ml MES E:PIF6:PhyB:Luc 0.26 154 µl of sample + 1.646 ml MES 6 July 2015
7 July 2015
Renilla 0.29 0.138ml of sample + 1.862ml of MES PhyB+PIF+luc 0.69 0.145ml of sample + 1.855ml of MES
Red toggle BamHI 4209, 3756, 6100, 6674 LexA+PIF+phy BamHI 3518, 5855, 6674
Red toggle LexA+PIF+Phy C1 LexA+PIF+Phy C2 No Ok no
AsLOVpep; pUPD2 Red Toggle LacI:Kdronpa:Ndronpa:VP16+renilla; α1 Gal4:Kdronpa:Ndronpa:VP16+renilla; α1 1 µl AsLOVpep 1 µl GB846 1 µl LacI:KNdronpa:VP16 1 µl Gal4:KNdronpa 1 µl pUPD2 1 µl GB160 1 µl GB159 1 µl GB159 5.6 µl H2O 1 µl Ω1 1 µl α1 1 µl α1 4.6 µl H2O 4.6 µl H2O 4.6 µl H2O
LexA:Kdronpa:Ndronpa:VP16+renilla; α1 LexA:PIF:Phy:VP16+renilla; α1 1 µl LexA:Kdronpa:Ndronpa:VP16 1 µl LexA:PIF:Phy:VP16 1 µl GB159 1 µl GB159 1 µl α1 1 µl α1 4.6 µl H2O 4.6 µl H2O 8 July 2015
9 July 2015
Red toggle (PIF+PhyB+luc+ren) 0.5 µl PIF+phy+luc (896) 1 µl renilla (160) 1 µl Ω1 5.1 µl H2O 10 July 2015
LexABD:PIF:PhyB:VP16+Renilla; α1 EcoRI 6345, 5487, 4891 LexA:Kdronpa:Ndronpa+renilla; α1 EcoRI 9333, 6345 Gal4:Kdronpa:Ndronpa+renilla; α1 EcoRI 6345, 9194 LacI:Kdronpa:Ndronpa+renilla; α1 EcoRI 6345, 9965 LacIBD+PIF+PhyB; Ω1 BamHI 6674, 4245, 2337 Gal4BD+PIF+PhyB; Ω1 BamHI 6674, 3474, 2337
LacIBD+PIF+PhyB Gal4BD+PIF+PhyB LexA:PIF:PhyB:VP16+Ren C1 LexA:PIF:PhyB:VP16+Ren C2 Ok Ok ok ok LexA:KNdronpa+ren C1 LexA:KNdronpa+ren C2 Gal4:KNdronpa+ren C1 Gal4:KNdronpa+ren C2 Ok Ok ok Ok Gal4:KNdronpa+ren C3 LacI:Kdronpa:Ndronpa+ren C1 LacI:Kdronpa:Ndronpa+ren C2 Ok Ok ok
PhiC31; pUPD2 Reporter PhiC31; pUPD2 LacI:PIF:PhyB:VP16+ren; α1 1 µl PhiC31 1 µl RepPhiC31 1 µl LacI:PIF:PhyB:VP16 1µl pUPD2 1µl pUPD2 1 µl renilla (GB159) 5.6 µl H2O 5.6 µl H2O 1 µl α1 4.6 µl H2O
Gal4:PIF:Phy:VP16+ren; α1 LexA:PIF:PhyB:ren+OpLex:luc; Ω1 LexA:KNdronpa:ren+OpLex:Luc; Ω1 1 µl Gal4:PIF:phy:VP16 1 µl LexA:PIF:phy:ren 1 µl LexA:KNdronpa:ren 1 µl renilla (GB159) 1 µl opLex:luc (GB151) 1 µl OpLex:Luc (GB151) 1 µl α1 1 µl Ω1 1 µl Ω1 4.6 µl H2O 4.6 µl H2O 4.6 µl H2O
Gal4:KNdronpa:ren+UAS:luc; Ω1 LacI:KNdronpa:ren+OpLacI:luc; Ω1 1 µl Gal4:KNdronpa:ren 1 µl LacI:KNdronpa:ren 1 µl OpUAS:luc (GB227) 1 µl OpLacI:luc (GB152) 1 µl Ω1 1 µl Ω1 4.6 µl H2O 4.6 µl H2O 11 July 2015
E:PIF6:PhyB:luc:ren BamHI 6674, 6100, 4209, 3756 AsLOVpep NotI 2558, 512
AsLOVpep C1 AsLOVpep C2 Red toggle C2 no no no 12 July 2015
Gal4:PIF:phy:VP16+ren; α1 LacI:KNdronpa:ren+OpLacI:luc; Ω1 1 µl Gal4:PIF:phy:VP16 1 µl LacI:KNdronpa:ren 1 µl renilla (GB159) 1 µl OpLacI:luc (GB152) 4.6 µl H2O 4.6 µl H2O 1 µl α1 1 µl Ω1 13 July 2015
phyC31;pUPD2 NotI 2046, 1899 Reporter phiC31; pUPD2 NotI 2046, 475 LacI:PIF:Phy:VP16+ren; α1 EcoRI 6345, 5623, 5487 LexA:PIF:phy:ren+opLex:luc; Ω1 BamHI 9431, 6674, 3531 LexA:KNdronpa:ren+OpLex:Luc; Ω1 BamHI 1199, 6674 Gal4:KNdronpa:ren+UAS:luc; Ω1 BamHI 11582, 6674
PhiC31 C1 PhiC31 C2 PhiC31 C3 RepPhiC31 C1 no no no ok RepPhiC31 C2 LacI:PIF:PhyB:VP16+ren C1 LacI:PIF:PhyB:VP16+ren C2 LacI:PIF:PhyB:VP16+ren C3 no no ok ok LexA:PIF:phy:ren+OpLex:luc LexA:KNdronpa:ren+OpLex:luc C1 LexA:KNdronpa:ren+OpLex:Luc C2 LexA:KNdronpa:ren+OpLex:Luc C3 no ok ok ok Gal4:KNdronpa:ren+OpUAS:luc C1 no
35S:BxbI+reporterBxbI:GFP: 0.28 143 µl of culture+1857 µl of MES 14 July 2015
phyC31;pUPD2 NotI 2046, 1899 Reporter phiC31; pUPD2 NotI 2046, 475 LacI:PIF:Phy:VP16+ren; α1 EcoRI 6345, 5623, 5487 LexA:PIF:phy:ren+opLex:luc, Ω1 BamHI 9431, 6674, 3531 LexA:KNdronpa:ren+OpLex:Luc; Ω1 BamHI 1199, 6674 Gal4:KNdronpa:ren+UAS:luc; Ω1 BamHI 11582, 6674 AsLOVpep; pUPD2 NotI 2558, 512
phyC31 (C4) phyC31 (C5) AsLOVpep (C4) LexA:PIF:phy:ren+opLex:luc (C1) no no No No LexA:KNdronpa:ren+OpLex:Luc (C3) Gal4:KNdronpa:ren+OpUAS:luc (C1) Gal4:KNdronpa:ren+UAS:luc(C2) Gal4:KNdronpa:ren+UAS:luc (C3) Ok no no No LacI:PIF:Phy:VP16+ren Reporter phiC31 (C1) no Ok 15 July 2015
LacI:KDronpa:NDronpa:ren:luc EcoRI 6345, 9965
LacI:K:NDronpa:ren:luc C4 LacI:K:NDronpa:ren:luc C5 no no
PhiC31;pUPD2 AsLOVpep; pUPD2 LacI:PIF:PhyB:VP16+ren; α1 1 µl PhiC31 1 µl AsLOVpep 1.5 µl LacI:PIF:PhyB:VP16 1µl pUPD2 1µl pUPD2 2.5 µl renilla (159) 5.6 µl H2O 5.6 µl H2O 0.5 µl α1 4.6 µl H2O
Gal4:PIF:phy:VP16+ren; α1 LexA:PIF:phy:ren+opLex:luc; Ω1 LacI:KNdronpa:ren+OpLex:Luc; Ω1 1.5 µl Gal4:PIF:phy:VP16 1 µl LexA:PIF:phy:ren 1 µl LacI:KNdronpa:ren 2 µl renilla (159) 2.5 µl opLex:luc (151) 3 µl OpLac:Luc (152) 2.6 µl H2O 2.6 µl H2O 3 µl H2O 1 µl α1 0.5 µl Ω1 0.5 µl Ω1
Gal4:KNdronpa:ren+OpLex:Luc; Ω1 PhyB:VP16+PIF6; Ω1 1 µl Gal4:KNdronpa:ren 1 µl PhyB:VP16 (88E) 4 µl OpUAS:Luc (227) 2 µl PIF6 (170) 3 µl H2O 3.6 µl H2O 0.5 µl Ω1 1 µl Ω1 16 July 2015
E:PIF:PhyB:luc; Ω1 EcoRI ?? Renilla; Ω2 HindIII 7453 Pnos; α1 EcoRI 2997, 353 Etr8; Ω1 EcoRI 2742 17 July 2015
Pnos Etr8:luc Etr8:luc C+ PIF6:PhyB:luc no ko ok ok PIF6:PhyB:luc C+ renilla renilla C+ ok no ok
PIF:PhyB:luc 0.47 41 µl/ml 630 µl Renilla 0.28 71 µl/ml 1065 µl Etr8:luc 0.32 52 µl/ml 930 µl Pnos 0.34 59 µl/ml 885 µl 18 July 2015
PhiC31;pUPD2 NotI 2046, 1899 AsLOVpep; pUPD2 NotI 2046, 521 LacI:PIF:PhyB:VP16+ren; α1 EcoRI 6345, 5623, 5487 Gal4:PIF:phy:VP16+ren; α1 EcoRI 6345, 5487, 4852 LexA:PIF:phy:ren+OpLex:luc; Ω1 BamHI 9431, 6674, 3513 LacI:KNdronpa:ren+OpLex:Luc; Ω1 BamHI 12632, 6574 Gal4:KNdronpa:ren+OpLex:Luc; Ω1 BamHI 11582, 6674 PhyB:VP16+PIF6; Ω1 BamHI 6674, 2685, 2337, 1439
phiC31 C1 phiC31 C2 phiC31 C3 AsLOVpep C1 no no no ok AsLOVpep C2 AsLOVpep C3 Gal4:PIF:phy:VP16:ren C1 Gal4:PIF:phy:VP16:ren C2 no no no no Gal4:PIF:phy:VP16:ren C3 LacI:PIF:phy:ren C1 LacI:PIF:phy:ren C2 LacI:PIF:phy:ren C3 no ok no No LexA:PIF:phy:ren:luc C1 LexA:PIF:phy:ren:luc C2 Gal4:KNdronpa:ren:luc C1 Gal4:KNdronpa:ren:luc C2 no no ok No Gal4:KNdronpa:ren:luc C3 LacI:KNdronpa:ren:luc C1 LacI:KNdronpa:ren:luc C2 LacI:KNdronpa:ren:luc C3 no no ok No PhyB:VP16:PIF6 C1 PhyB:VP16:PIF6 C2 PhyB:VP16:PIF6 C3 ok no no
phiC31;pUPD2 XhoI (buffer red) 2119, 934, 894 LacI:PIF:Phy:VP16+ren; a1 NEB4 5949, 5653, 3610, 2246 LacI:PIF:Phy:VP16+ren; a1 HindIII 11568, 5587 19 July 2015
20 July 2015
21 July 2015
LexA:PIF:Phy:ren:luc BamHI 9431, 6674, 3513 PhiC31 NotI 2046, 1899
PhiC31 C1 PhiC31 C2 PhiC31 C4 PhiC31 C5 Ok? ok ok ok LexA:PIF:PhyB:ren:luc C1 LexA:PIF:PhyB:ren:luc C2 No DNA No DNA
35S:Gal4:AsLOVpep:T35S; α1 35S:LacI:AsLOVpep:T35S; α1 35S:LexA:AsLOVpep:T35S; Ω1 1 µl 35S (0030) 1 µl 35S (0030) 1 µl 35S (0030) 1 µl Gal4BD 1 µl LacI 1 µl LexA 1 µl AsLOVpep 1 µl AsLOVpep 1 µl AsLOVpep 1 µl T35S (0036) 1 µl T35S (0036) 1 µl T35S (0036) 1 µl α1 1 µl α1 1 µl α1 2.6 µl H2O 2.6 µl H2O 2.6 µl H2O
PsinATG:RepPhiC31:GFP:T35S; α2 LacI:PIF:PhyB:ren+luc; Ω1 PIF:PhyB+renilla; α2 1 µl PsinATG (552) 1.5 µl LacI:PIF:PhyB:ren; α1 1.5 µl PIF:PhyB 1 µl ReporterPhiC31 3 µl OpLacI:luc (152); α2 2.5 µl renilla (159) 1 µl GFP (0059) 0.5 µl Ω1 0.5 µl α2 1 µl T35S (0036) 2.6 H2O 3 µl H2O 1 µl α2 2.6 µl H2O 22 July 2015
Gal4:PIF:PhyB:ren BamHI 16684 EcoRI 4852, 5487, 6345 EcoRV 1849, 3942, 2475, 381, 8037
Gal4:PIF:PhyB:ren (BamHI) Gal4:PIF:PhyB:ren (EcoRI) Gal4:PIF:PhyB:ren (EcoRV) no no no
23 July 2015
24 July 2015
Gal4:AsLOVpep EcoRI 6345, 1972 LacI:AsLOVpep EcoRI 6345, 2743 LexA:AsLOVpep EcoRI 6345, 2011 PsinATG:RepPhiC31:GFP HindIII 6345, 2691 LexA:PIF:PhyB:ren+luc BamHI 9431, 6674, 3513 PhyC31; pUPD2 NotI 2046, 1899 Gal4:PIF:phyB BamHI 6674, 3474, 2337 Renilla (GB159) EcoRV 2909, 2475, 882, 812, 381
Gal4:AsLOV C1 Gal4:AsLOV C2 Gal4:AsLOV C3 PsinATG:RepPhiC31:GFP C1 PsinATG:RepPhiC31:GFP C2 ok ok ok ok no PsinATG:RepPhiC31:GFP C3 LacI:AsLOV C1 LacI:AsLOV C2 LexA:AsLOV C1 LexA:AsLOV C2 no ok No no ok LexA:AsLOV C3 LexA:PIF:PhyB:ren+luc C1 LexA:PIF:PhyB:ren+luc C2 no no Ok
PhiC31 (C1) PhiC31 (C2) PhiC31 (C3) PhiC31 (C4) PhiC31 (C5) no ok ok ok no Gal4:PIF:phyB Renilla (GB159) ok Ok 26 July 2015
27 July 2015
Gal4:PIF:phiB + ren; α1 LacI:PIF:phiB:ren + luc; Ω2 PIF:PhyB+renilla; α2 1.5 µl Gal4:PIF:PhyB 1.5 µl LacI:PIF:PhyB:ren 1.5 µl PIF:PhyB 2 µl Renilla (GB159) 2 µl OpLacI:luc 2.5 µl Renilla (GB159) 0.5 µl α1 0.5 µl Ω2 0.5 µl α2 3.6 µl H2O 2.6 µl H2O 3 µl H2O
Cytoplasm: 0.39 (viral) 0.25ml Integrase: 0.35 (viral) 0.28ml GFP: 0.32 (viral) 0.31ml Dsred: 0.31 (viral) 0.32ml BxbI:reporter: 0.41 0.49ml Reporter: 0.26 0.77ml
Etr8:luc+staffer(SF) OpLexA:luc+SF OpLacI:luc+SF OpUAS:luc+SF 1.5 µl Etr8:luc (88C o 1098) 1.5 µl OpLexA:luc (151) 1.5 µl OpLacI:luc (152) 1.5 µl UAS:luc (227) 1 µl SF; α2 1 µl SF; α2 1 µl SF; α2 1 µl SF;α2 1 µl Ω1 1 µl Ω1 1 µl Ω1 1 µl Ω1 6.1 µl H2O 6.1 µl H2O 6.1 µl H2O 6.1 µl H2O 28 July 2015
LTB; pUPD2 1 µl LTB 1 µl pUPD2 5.6 µl H2O 29 July 2015
LacI:PIF:phiB:ren:luc EcoRV 882, 968, 1652, 3942, 2475, 381, 3477, 6674 PIF:PhyB+renilla HindIII 4316, 5887, 788, 6345
LacI:PIF:phiB:ren:luc (C1) LacI:PIF:phiB:ren:luc (C3) PIF:PhyB+renilla (C2) no no no
30 July 2015
Etr8:luc:staffer(SF) BamHI 6674, 2766 OpLexA:luc:SF BamHI 6674, 2746 OpLacI:luc:SF BamHI 6674, 2847 OpUAS:luc:SF BamHI 6674, 2568 Gal4:PIF:phyB:ren EcoRI 6345, 5487, 4852 LacI:PIF:phy:ren:luc BamHI 20451 PIF:PhyB:ren HindIII 6345, 5887, 4316, 788
ePDZ PCR 10µl Buffer HF 31.5 µl H2O 2 µl dNTPs 2.5 µl Primer forward 2.5 µl primer reverse 1 µl ePDZ (dilution 1:50) 0.5 µl Taq phunion
ePDZ; pUPD2 PIF:phyB+ren; α2 OpUAS:luc:SF+ren; α1 OpLexA:luc:SF+ren; α1 1 µl ePDZ 1 µl PIF:phyB 1 µl OpUAS:luc:SF 1 µl OpLexA:luc:SF 1 µl pUPD2 1 µl ren (159) 1 µl ren 1 µl ren 5.6 µl H2O 1 µl α2 1 µl α1 1 µl α1 4.6 µl H2O 4.6 µl H2O 4.6 µl H2O
OpEtr8:luc:SF+ren; α1 Op:LacI:luc:SF+ren; α1 1 µl OpEtr8:luc:SF 1 µl OpLacI:luc:SF 1 µl ren 1 µl ren 1 µl α1 1 µl aplha1 4.6 µl H2O 4.6 µl H2O 31 July 2015
LacI:PIF:PhyB:ren+OpLacI:luc; Ω2 1.5 µl LacI:PIF:phyB:ren 3 µl OpLacI:luc 0.5 µl Ω2 2.6 µl H2O
LTB; pUPD2 NotI 2046, 474
LTB (C1) LTB (C2) PCR ePDZ ok ok ok
August and September
1 August 2015
Ren+P19 0.09 888.9 µl RepPhiC31 0.69 115.94 µl BxbI 0.46 174 µl RepBxbI 0.15 533.3 µl Gal4:KNDronpa:ren:luc 0.51 156.9 µl Pnos 0.29 275.9 µl PIF:phy:luc 0.17 470.6 µl PhiC31 0.32 250 µl 3 August 2015
OpLexA:luc:SF+ren; α1 Op:LacI:luc:SF+ren; α1 E-PIF:phyB+ren; α2 1 µl OpLexA:luc:SF 1 µl OpLacI:luc:SF 1 µl E-PIF:phy 3 µl ren 3 µl ren 3 µl ren 1 µl α1 1 µl α1 1 µl α2 2.6 µl H2O 2.6 µl H2O 2.6 µl H2O 4 August 2015
ePDZ; pUPD2 NotI 2046, 642 OpUAS:luc:SF+ren; α1 EcoRI 6345, 7096 OpEtr8:luc:SF+ren; α1 EcoRI 6345, 7294 LacI:PIF:phyB:ren+OpLacI:luc; Ω2 EcoRV 6674, 3477, 381, 2475, 3942, 1652, 968, 882 Renilla 159 EcoRV 2909, 2475, 882, 812, 381
ePDZ C1 ePDZ C2 ePDZ C3 LacI:PIF:PhyB+ren Ok ok ok Renilla 159 OpUAS:luc:SF+ren C1 OpUAS:luc:SF+ren C2 OpUAS:luc:SF+ren C3 Ok ¿? OpEtr8:luc:SF+ren C1 OpEtr8:luc:SF+ren C2 OpEtr8:luc:SF+ren C3
35S+ePDZ+VP16+T35S;α2 35S+LTB+T35S; α1 1µl ePDZ 1µl 35S (30) 1 µl VP16 1µl T35S (36) 1 µl 35S (30) 1µl LTB 1 µl T35S (36) 1µl α1 1 µl α2 3.6 µl H2O 2.6 µl H2O 5 August 2015
6 August 2015
OpLexA:luc:SF+ren EcoRI 6345, 7274 Op:LacI:luc:SF+ren EcoRI 6345, 7375 E-PIF:phyB+ren HindIII 6345, 788, 5887, 4316 35S+ePDZ+VP16+T35S HindIII 6345, 2316 35S+LTB+T35S EcoRI 6345, 1684
PIF:phyB+ren OpLexA:luc:SF+ren C1 OpLexA:luc:SF+ren C2 OpLexA:luc:SF+ren C3 No Ok, repeat No Ok, repeat Op:LacI:luc:SF+ren C1 Op:LacI:luc:SF+ren C2 Op:LacI:luc:SF+ren C3 35S+LTB+T35S C1 Ok, repeat Ok Ok ok 35S+LTB+T35S C2 35S+ePDZ+VP16+T35S C1 35S+ePDZ+VP16+T35S C2 Ok ok ok
LexA:AsLOVpep+ePDZ; Ω1 LacI:AsLOVpep+ePDZ;Ω1 Gal4:AsLOVpep+ePDZ; Ω1 1 µl LexA:AsLOVpep; α1 1 µl LacI:AsLOVpep; α1 1 µl Gal4:AsLOVpep;α1 1 µl ePDZ; α2 1 µl ePDZ; α2 1 µl ePDZ; α2 1 µl BsmBI 1 µl BsmBI 1 µl BsmBI 1 µl Ω1 1 µl Ω1 1 µl Ω1 4.6 µl H2O 4.6 µl H2O 4.6 µl H2O 7 August 2015
C7 C8 C9 C10 C11 C12 C13 C14 No no no no no no no no
PhyC31; pUPD2 3 µl PhiC31 1 µl pUPD2 1 µl BsmBI 3.6 µl H2O
Construction OD Volume (ml) IFN 0.13 1.54 RepBxbI:GFP 0.13 1.54 BxbI:RepBxbI:GFP 0.33 0.61 PhiC31 0.19 1.05 RepPhiC31:GFP 0.22 0.91 8 August 2015
Construction OD Volume (ml) Sip-CH2 0.04 6.667 Sip-CH2-CH3 0.04 6.667 Red toggle (PIF6:PhyB:luc) 0.09 15 Blue toggle (LacI:KDronpa:NDronpa:ren:luc) 0.09 15.00 Renilla 0.04 6.667 Pnos 0.04 6.667 9 August 2015
LexA:AsLOVpep+ePDZ BamHI 6674, 4309 LacI:AsLOVpep+ePDZ BamHI 6674, 5041 Gal4:AsLOVpep+ePDZ BamHI 6674, 4270
LexA:AsLOVpep+ePDZ (C1) LexA:AsLOVpep+ePDZ (C2) LexA:AsLOVpep+ePDZ (C3) LexA:AsLOVpep+ePDZ (C4) Ok ok ok ok oLacI:AsLOVpep+ePDZ (C1) LacI:AsLOVpep+ePDZ (C2) LacI:AsLOVpep+ePDZ (C3) LacI:AsLOVpep+ePDZ (C4) Ok ok ok ok Gal4:AsLOVpep+ePDZ (C1) Gal4:AsLOVpep+ePDZ (C2) Gal4:AsLOVpep+ePDZ (C3) Gal4:AsLOVpep+ePDZ (C4) Ok ok ok Ok
LexA:AsLOVpep+ePDZ+ren; α1 LacI:AsLOVpep+ePDZ+ren; α1 Gal4:AsLOVpep+ePDZ+ren; α1 1µl LexA:AsLOVpep+ePDZ 1µl LacI:AsLOVpep+ePDZ 1µl Gal4:AsLOVpep+ePDZ 1 µl renilla (159) 1 µl renilla (159) 1 µl renilla (159) 1 µl α1 1 µl α1 1 µl α1 4.6 µl H2O 4.6 µl H2O 4.6 µl H2O 10 August 2015
11 August 2015
10 µl Buffer HF 26.5 µl H2O 2 µl dNTPs 0.5 µl Taq Phusion 1 µl DNA (dilution 1:10) 2.5 µl Primer forward (F) (dilution 1:10) 2.5 µl Primer reverse (R) (dilution 1:10)
IFN (1) IFN (2) AsLOVpep (1) AsLOVpep (2) IFN IFN AsLOVpep AsLOVpep Mys int F IFN domBB R AsLOVpep F AsLOVpep Fint Mys int R IFN domBB F AsLOVpep Rint AsLOVpep R
AsLOVpep (nested) PhiC31 (1) PhiC31 (2) PhiC31 (3) AsLOVpep PhiC31 PhiC31 PhiC31 AsLOVpep nested PhiC31 Fint 1 PhiC31 Fint 2 PhiC31 Fint 3 AsLOVpep PhiC31 Rint 2 PhiC31 Rint 3 PhiC31 R
PhiC31 PhiC31 PhiC31 F PhiC31 Rint
PhiC31 NotI 2046, 1899 LacI:AsLOVpep+ePDZ+ren EcoRI 6345, 8798 Gal4:AsLOVpep+ePDZ+ren EcoRI 6345, 9569
PhiC31 (C6) C7…C16 LacI:AsLOVpep:ePDZ:ren (C1) LacI:AsLOVpep:ePDZ:renC2 LacI:AsLOVpep:ePDZ:ren(C3) LacI:AsLOVpep:ePDZ:ren(C4) no no ok no ok Gal4:AsLOVpep+ePDZ+ren (C1) Gal4:AsLOVpep+ePDZ+ren (C2) ok ok
Citoplasm 0.21 7.35 ml RepPhiC31 0.26 9.1ml 35S:LTB:T35S 0.27 9.45ml PhiC31 0.27 9.45ml GFP 0.20 10ml RepBxbI 0.33 11.55ml PsinATG:RepBxbI:GFP 0.36 12.6ml PhiC31 0.34 11.9ml DsRed 0.26 9.1ml P19 0.28 9.8ml 12 August 2015
OpLexA:luc:SF:ren EcoRI 6345, 7274 Op:UAS:luc:SF:ren EcoRI 6345, 7096 OpEtr8.luc:SF:ren EcoRI 6345, 7294
Op:UAS:luc:SF:ren C1 Op:UAS:luc:SF:ren C2 Op:UAS:luc:SF:ren C3 OpEtr8.luc:SF:ren no no no no OpLexA:luc:SF:ren C1 OpLexA:luc:SF:ren C2 Partner dna Partner dna no no
LexA:AsLOVpep:ePDZ+ren; α1 Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; Ω1 LacI:AsLOVpep:ePDZ:ren+OpLacI:luc; Ω1 1 µl LexA:AsLOVpep:ePDZ 1 µl Gal4:AsLOVpep:ePDZ:ren 1 µl LacI:AsLOVpep:ePDZ 1 µl renilla (159) 1 µl OpUAS:luc 1 µl OpLacI:luc 1 µl α1 1 µl Ω1 1 µl Ω1 4.6 µl H2O 4.6 µl H2O 4.6 µl H2O 13 August 2015
AsLOVpep (BB); pUPD2 1µl AsLOVpep1 1µl AsLOVpep2 1µl pUPD2 1µl BsmbI 4.6µl H2O 14 August 2015
OpLexA:luc:SF:ren SacII 13619 PouI 2495, 2055, 5937, 3152 OpUAS:luc:SF:ren PouI 2475, 2055, 5937, 2474 OpEtr8.luc:SF:ren PouI 2637, 2055, 5937, 3010 OpLacI:luc:SF:ren SacII 12930, 790 OpLacI:luc:SF:ren PouI 2475, 2055, 5937, 3253
OpUAS:luc:SF:ren C1 OpUAS:luc:SF:ren C2 OpUAS:luc:SF:ren C3 OpEtr8.luc:SF:ren ok no ok ok OpLacI:luc:SF:ren (PouI) OpLacI:luc:SF:ren (SacII) OpLexA:luc:SF:ren (PouI) OpLexA:luc:SF:ren (SacII) ok ok ok ok
15 August 2015
LexA:AsLOVpep:ePDZ+ren; α1 EcoRI 8837, 6345 Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; Ω1 BamHI 6674, 11186 LacI:AsLOVpep:ePDZ:ren+OpLacI:luc; Ω1 BamHI 6674, 12236
LexA:AsLOVpep:ePDZ+ren C1 LexA:AsLOVpep:ePDZ+ren C2 LexA:AsLOVpep:ePDZ+ren C3 LexA:AsLOVpep:ePDZ+ren C4 ok ok ok Ok Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc C1 Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc C2 Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc C3 Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc C4 no no no No LacI:AsLOVpep:ePDZ:ren+OpLacI:luc C1 LacI:AsLOVpep:ePDZ:ren+OpLacI:luc C2 LacI:AsLOVpep:ePDZ:ren+OpLacI:luc C3 LacI:AsLOVpep:ePDZ:ren+OpLacI:luc C4 ok ok ok ok
16 August 2015
Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; Ω1 AsLOVpep; pUPD2 LexA:AsLOVpep:ePDZ:ren+OpLexA:luc; Ω1 1µl Gal4:AsLOVpep:ePDZ:ren 1µl AsLOVpep1 1µl Lex!:AsLOVpep:ePDZ:ren 1µl OpUAS:luc (227) 1µl AsLOVpep2 1µl OpLexA:luc (151) 1µl Ω1 1µl pUPD2 1µl Ω1 4.6µl H2O 4.6µl H2O 4.6µl H2O 17 August 2015
DsRed 0.14 71.4ml Integrase 0.14 71.4ml Citoplasm 0.12 83.3ml 18 August 2015
PhiC31 (BB) AsLOVpep (BB) 1 µl PhiC31 1 µl AsLOVpep 2.5 µl PhiC31 F (1:10) 2.5 µl AsLOVpep nested 2.5 µl PhiC31 Rint (1:10) 2.5 µl AsLOVpep Fint Tm=44ºC Tm=59ºC
PhiC31 (BB); pUPD2 AsLOVpep (BB); pUPD2 1µl Phi1 1µl AsLOVpep1 1µl Phi2 1µl AsLOVpep nested 1µl Phi3 1µl pUPD2 1µl Phi4 4.6µl H2O 1µl pUPD2 2.6µl H2O
IFN 1 IFN2 LTB1 (10µL) LTB1 (10 µL) LTB2 (30 µL) Sip 1 CH2 Sip2 CH2 Sip 1 CH2-CH3 Sip 2 CH2-CH3 19 August 2015
LTB; α1 EcoRI 6345, 1684 Gal4:AsLOVpep:ePDZ; Ω1 BamHI 6674, 4270 LacI:AsLOVpep:ePDZ; Ω1 BamHI 6674, 5041 LexA:AsLOVpep:ePDZ; Ω1 BamHI 6674, 4309 ePDZ; pPUD2 NotI 2046, 642 AsLOVpep; pPUD2 NotI 2046, 521 NDronpa; pPUD2 NotI 2046, 735 Gal4:AsLOVpep; α1 EcoRI 6345, 1972 RepPhiC31: pPUD2 NotI 2046, 475 LexA:AsLOVpep; α1 EcoRI 6345, 2016 LexA:AsLOVpep:ePDZ:ren+OpLexA:luc; Ω1 BamHI 6674, 11403
LTB; α1 NDronpa; pPUD2 RepPhiC31: pPUD2 AsLOVpep; pPUD2 ok ok ok ok LexA:AsLOVpep Gal4:AsLOVpep LTB; α1 LexA:AsLOVpep; α1 ok ok ok Ok LacI:AsLOVpep:ePDZ Gal4:AsLOVpep:ePDZ LexA:AsLOVpep:ePDZ:ren+OpLexA:luc C2 LexA:AsLOVpep:ePDZ:ren+OpLexA:luc C3 ok ok ok Ok 20 August 2015
AsLOVpep (BB) NotI 2046, 518 PhiC31 (BB) NotI 2046, 1899
AsLOV C1 AsLOV C2 AsLOV C3 Phi C1 Phi C2 Phi C3 Ok ok ok no no no Phi C4 Phi C5 Phi C6 Phi C7 Phi C8 Phi C9 No no Ok? Ok? no No
Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; Ω1 2.5 µl Gal4:AsLOVpep:ePDZ:ren 6 µl OpUAS:luc 0.5 µl Ω1 0 µl H2O
IFN 0.26 77 µl/ng LTB 0.22 91 µl/ng Sip CH2-CH3 0.12 167 µl/ng Sip CH2 0.09 223 µl/ng
LacI:AsLOVpep EcoRI 8345, 2743
PhiC31 (BB) AatII 2635, 1310 PhiC31 (BB) BanI 3497, 448
PhiC31(BB) C6 (banI) PhiC31(BB) C7 (BanI) PhiC31(BB) C6 (AatII) PhiC31(BB) C7 (AatII) Ok no ok no 21 August 2015
Gal4:KDronpa:NDronpa:ren:OpUAS:luc; Ω1 BamHI 6674, 11582 LexA:KDronpa:NDronpa:ren:OpLexA:luc; Ω1 BamHI 6674, 11799 LTB; pUPD2 NotI 2046, 474 LTB; α1 EcoRI 1684, 6345 Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1 BamHI 6674, 11186
LTB; pUPD2 LTB; α1 LTB; α1 Gal4:KDronpa:NDronpa:ren:OpUAS:luc; Ω1 No no no ok LexA:KDronpa:NDronpa:ren:OpLexA:luc; Ω1 Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1 C1 Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1 C2 Ok no no 22 August 2015
PhiC31 (BB); pUPD2 NotI 2046, 1899 Pnos EcoRI 7317, 634, 11 LTB; pUPD2 NotI 2046, 474 LTB; α1 EcoRI 1684, 6345
LTB; α1 LTB; pUPD2 Pnos 1 Pnos 2 PhiC31 (BB) Ok no ? ? Ok 23 August 2015
Pnos 0.30 1ml/15ml LacI:AsLOVpep:ePDZ:ren:OpLacI:luc 0.34 OpLacI:luc:ren 0.47 24 August 2015
Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc BamHI 11186, 6674 PhiC31 (BB) NotI 2096, 1899
Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc PhiC31 (BB) Ok ok
Lactoferrina; pUPD2 PhiC31; α1 1 µl Part1 Lactoferrin 1 µl PhiC31 1 µl Part2 Lactoferrin 1 µl 35S 1 µl pUPD2 1 µl T35S 4.6 µl H2O 1 µl α1 3.6 µl H2O 25 August 2015
26 August 2015
Lactoferrina; pUPD2 NotI 2046, 2142 PhiC31; α1 EcoRI 6345, 3127 PhiC31; pUPD2 (BB) NotI 2046, 1899 Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc BamHI 6674, 11191
PhiC31; α1 C1 PhiC31; α1 C2 PhiC31; α1 C3 PhiC31; α1 C4 Lactoferrina C1 ok no ok ok ok Lactoferrina C2 Lactoferrina C3 Lactoferrina C4 PhiC31; pUPD2 Gal4:AsLOVpep:ePDZ: ren:OpUAS:luc ok ok ok ok ok
PhiC31+RepPhiC31:GFP; Ω1 35S+Lactoferrin+T35S; α1 1µl PhiC31 1µl 35S 1µl RepPhiC31:GFP 1µl T35S 1µl Ω1 1µl Lactoferrin; pUPD2 4.6µl H2O 1µl α1 3.6µl H2O 27 August 2015
Gal4:KDronpa:NDronpa:ren:OpUAS:luc BamHI 6674, 11191 35S:GFP:T35S EcoRI 2580, 2274 Alpha1 Nothing 6953 Omega1 Nothing 7295
Alpha1 Omega1 35S:GFP:T35S Gal4:KDronpa:NDronpa: ren:OpUAS:luc ok ok ok Ok 28 August 2015
35S:GFP:Tnos NotI 2981, 140
Lactoferrina; α1 1µl Lactoferrina 1µl 35S 1µl T35S 1µl GB393 2.6µl H2O 29 August 2015
OpLexA:mini35S (GB0733); pUPD2 NotI 2981, 477 OpLacI:mini35S (GB534); pUPD2 NotI 2981, 878 OpUAS:mini35S (GB0672); pUPD2 NotI 2981, 477 DsRed (GB0672); α1 EcoRI 2580, 2274 35S:GFP:Tnos; α1 EcoRI 2580, 2274
OpUAS:mini35S (GB0672); pUPD2 OpLexA:mini35S (GB0733); pUPD2 OpLacI:mini35S (GB534); pUPD2 OpLacI:mini35S (GB534); pUPD2 35S:GFP:Tnos; α1 ok ok ok ok ok 30 August 2015
Gal4:KDronpa:NDronpa:ren:OpUAS:luc; Ω1 BamHI 6674, 11191 Dsred (GB672); α 1 EcoRI Staffer fragment in Ω2 EcoRV Lactoferrin; α1 EcoRI 6345, 3436
OpLacI:mini35S (GB534); pUPD2 OpLexA:mini35S (GB0733); pUPD2 OpUAS:mini35S (GB0672); pUPD2 DsRed (GB0672); pUPD2 Mini35S:Dsred; α1 ok ok ok ok Ok Lactoferrin; α1 (C2) Lactoferrin; α1 (C3) Gal4:KDronpa:NDronpa:ren:OpUAS:luc; Ω1 Staffer fragment in Ω2 no no ok ok
OpLacI:mini35S+Dsred+T35S; α2 OpLexA:mini35S+DsRed+T35S; α2 OpUAS:mini35S+DsRed+T35S; α2 1µl OpLacI 1µl OpLexA 1µl OpUAS 1µl Dsred 1µl Dsred 1µl Dsred 1µl T35S 1µl T35S 1µl T35S 1µl mini35S 1µl mini35S 1µl mini35S 1µl α2 1µl α2 1µl α2 3.6 µl H2O 3.6 µl H2O 3.6 µl H2O
Gal4:KDronpa:NDronpa+SF(Ω2); α1 LexA:KDronpa:NDronpa+SF(Ω2); α1 LacI:KDronpa:NDronpa+SF(Ω2); α1 1µl Gal4:KDronpa:NDronpa 1µl LexA:KDronpa:NDronpa 1µl LacI:KDronpa:NDronpa 1µl SF(Ω2) 1µl SF(Ω2) 1µl SF(Ω2) 1µl α1 1µl α1 1µl α1 4.6µl H2O 4.6µl H2O 4.6µl H2O
Lactoferrin; α1 1µl Lactoferrin 1µl 35S 1µl T35S 1µl α1 2.6 µl H2O 31 August 2015
PhiC31+RepPhiC31:GFP; Ω1 1µl PhiC31 (BB); α1 1µl RepPhiC31:GFP; α2 1µl Ω1 4.6µl H2O
0408 Promoters 224 ng/µl 7 µl 0409 CDs 77 ng/µl 3 µl 0410 terminator 159 ng/µl 10 µl 0411 TUs 158 ng/µl 10 µl
1 September 2015
35S:BxbI:T35S 35S:PhiC31:T35S PsinATG:RepBxbI:GFP:T35S PsinATG:RepPhiC31:GFP:T35S 1µl 35S 1µl 35S 1µl PsinATG 1µl PsinATG 1µl BxbI 1µl PhiC31 1µl RepBxbI 1µl RepPhiC31 1µl T35S 1µl T35S 1µl GFP 1µl GFP 1µl Mo411 1µl Mo411 1µl T35S 1µl T35S 4.6 µl H2O 4.6 µl H2O 1µl Mo411 1µl Mo411 4.6 µl H2O 4.6 µl H2O
35S:Lactoferrin:T35S 35S:LTB:T35S Gal4:KDronpa LacI:KDronpa 1µl 35S 1µl 35S 1µl 35S 1µl 35S 1µl Lactoferrin 1µl LTB 1µl Gal4 1µl LacI 1µl T35S 1µl T35S 1µl T35S 1µl T35S 1µl Mo411 1µl Mo411 1µl KDronpa 1µl KDronpa 4.6 µl H2O 4.6 µl H2O 1µl Mo411 1µl Mo411 4.6 µl H2O 4.6 µl H2O
LexA:KDronpa NDronpa:VP16 Gal4:AsLOVpep LacI:AsLOVpep 1µl 35S 1µl 35S 1µl 35S 1µl 35S 1µl LexA 1µl Gal4 1µl Gal4 1µl LacI 1µl T35S 1µl T35S 1µl T35S 1µl T35S 1µl KDronpa 1µl KDronpa 1µl AsLOVpep 1µl AsLOVpep 1µl Mo411 1µl Mo411 1µl Mo411 1µl Mo411 4.6 µl H2O 4.6 µl H2O 4.6 µl H2O 4.6 µl H2O
LexA:AsLOVpep ePDZ:VP16 1µl 35S 1µl 35S 1µl LexA 1µl ePDZ 1µl T35S 1µl T35S 1µl AsLOVpep 1µl VP16 1µl Mo411 1µl Mo411 4.6 µl H2O 4.6 µl H2O 2 September 2015
Gal4:KNDronpa:VP16:SF; Mo LacI:KNDronpa:VP16:SF; Mo LexA:KNDronpa:VP16:SF; Mo 1µl Gal4:KNDronpa:VP16 1µl LacI:KNDronpa:VP16 1µl LexA:KNDronpa:VP16 1µl SF 1µl SF 1µl SF 1µl Mo 1µl Mo 1µl Mo 4.6 µl H2O 4.6 µl H2O 4.6 µl H2O
Gal4:AsLOVpep:ePDZ:VP16:SF; Mo LacI:AsLOVpep:ePDZ:VP16:SF; Mo LexA:AsLOVpep:ePDZ:VP16:SF; Mo 1µl Gal4:AsLOVpep:ePDZ:VP16 1µl LacI:AsLOVpep:ePDZ:VP16 1µl LexA:AsLOVpep:ePDZ:VP16 1µl SF 1µl SF 1µl SF 1µl Mo 1µl Mo 1µl Mo 4.6 µl H2O 4.6 µl H2O 4.6 µl H2O
Gal4:PIF6:PhyB:VP16:SF; Mo LacI:PIF6:PhyB:VP16:SF; Mo LexA:PIF6:PhyB:VP16:SF; Mo 1µl Gal4:PIF6:PhyB:VP16 1µl LacI:PIF6:PhyB:VP16 1µl LexA:PIF6:PhyB:VP16 1µl SF 1µl SF 1µl SF 1µl Mo 1µl Mo 1µl Mo 4.6 µl H2O 4.6 µl H2O 4.6 µl H2O
35S:BxbI:RepBxbI:GFP:SF 35S:PhiC31+RepPhiC31:GFP 1µl 35S:BxbI:RepBxbI:GFP 1µl 35S:PhiC31 1µl SF 1µl RepPhiC31:GFP 1µl Mo 1µl Ω1 4.6 µl H2O 4.6 µl H2O 3 September 2015
4 September 2015
35S:BxbI:T35S EcoRI+PstI 2959 35S:PhiC31:T35S EcoRI+PstI 3105 PsinATG:RepBxbI:GFP:T35S EcoRI+PstI 2388 PsinATG:RepPhiC31:GFP:T35S EcoRI+PstI 2669 35S:Lactoferrin:T35S EcoRI+PstI 3141 35S:LTB:T35S EcoRI+PstI 1662 Gal4:KDronpa EcoRI+PstI 1966 LacI:KDronpa EcoRI+PstI 3006 LexA:KDronpa EcoRI+PstI 2274 NDronpa:VP16 EcoRI+PstI 2403 Gal4:AsLOVpep EcoRI+PstI 2235 LacI:AsLOVpep EcoRI+PstI 2721 LexA:AsLOVpep EcoRI+PstI 1989 ePDZ:VP16 EcoRI+PstI 2292
1µl DNA 1µl Buffer O 0.5µl EcoRI 0.5µl PstI 7 µl H2O
LexA:KNDronpa (C1) LexA:KNDronpa (C2) Gal4:KNDronpa (C1) Gal4:KNDronpa (C2) LacI:KNDronpa (C1) no no no no no LacI:KNDronpa (C2) RepBxbI:GFP (C1) RepBxbI:GFP (C2) Gal4:AsLOV (C1) Gal4:AsLOV (C2) no no no No no LacI:AsLOV (C1) LacI:AsLOV (C2) LexA:AsLOV (C1) LexA:AsLOV (C2) NDronpa:VP16 (C1) no no no no no NDronpa:VP16 (C2) LTB (C1) LTB (C2) ePDZ:VP16 (C1) ePDZ:VP16 (C2) no no no no no Lactoferrin (C1) Lactoferrin (C2) BxbI (C1) BxbI (C2) RepPhiC31 (C1) no no no no no RepPhiC31 (C2) PhiC31 (C1) PhiC31 (C2) LexA:AsLOV (C1) LexA:AsLOV (C2) no no no no no 5 September 2015
Gal4:KNDronpa:VP16:SF; Mo EcoRI+PstI 4804 LacI:KNDronpa:VP16:SF; Mo EcoRI+PstI 5580 LexA:KNDronpa:VP16:SF; Mo EcoRI+PstI 4843 Gal4:AsLOVpep:ePDZ:VP16:SF; Mo EcoRI+PstI - LacI:AsLOVpep:ePDZ:VP16:SF; Mo EcoRI+PstI - LexA:AsLOVpep:ePDZ:VP16:SF; Mo EcoRI+PstI - Gal4:PIF6:PhyB:VP16:SF; Mo EcoRI+PstI - LacI:PIF6:PhyB:VP16:SF; Mo EcoRI+PstI - LexA:PIF6:PhyB:VP16:SF; Mo EcoRI+PstI - 35S:BxbI:RepBxbI:GFP:SF EcoRI+PstI 2654 35S:PhiC31+RepPhiC31:GFP; Ω1 BamHI 1860, 3550, 6674, 86, 30, 10
LacI:KNDronpa (C1) LacI:KNDronpa (C2) Gal4:KNDronpa (C1) Gal4:KNDronpa (C2) LexA:KNDronpa (C1) ok ok ok ok Ok Gal4:AsLOVpep:ePDZ (C1) Gal4:AsLOVpep:ePDZ (C2) LexA:AsLOVpep:ePDZ (C2) Gal4:PIF6:PhyB (C1) Gal4:PIF6:PhyB (C2) No no no no no LacI:PIF6:PhyB (C1) LacI:PIF6:PhyB (C2) LexA:PIF6:PhyB (C1) LexA:PIF6:PhyB (C2) BxbI:RepBxbI:GFP (C1) no no no no Ok BxbI:RepBxbI:GFP (C2) PhiC31+RepPhiC31:GFP (C1) PhiC31+RepPhiC31:GFP (C2) ok ok ok
35S:PhiC31:RepPhiC31:GFP+SF 1µl PhiC31:RepPhiC31:GFP 1µl SF (466) 1µl Mo411 4.6 µl H2O 6 September 2015
OpLacI:luc:ren 0.19 200 µl/1.9ml OpUAS:luc:ren 0.18 211 µl/1.9ml OpLexA:luc:ren 0.17 224 µl/1.9ml LexA:AsLOVpep:ePDZ:luc:ren 0.18 211 µl/1.9ml LacI:AsLOVpep:ePDZ:luc:ren 0.15 253 µl/1.9ml Gal4:AsLOVpep:ePDZ:luc:ren 0.21 181 µl/1.9ml LexA:KNDronpa:ren:luc 0.04 950 µl/1.9ml Pnos 0.77 494 µl/1.9ml
Gal4:KNDronpa:VP16:OpUAS:luc:ren: 327.6 ng/µl LexA:KNDronpa:VP16:OpLexA:luc:ren: 300.5 ng/µl Gal4:KNDronpa:VP16:SF; Mo: 183.25 ng/µl LacI:KNDronpa:VP16:SF; Mo: 221.9 ng/µl LexA:KNDronpa:VP16:SF; Mo: 266.7 ng/µl 35S:BxbI:RepBxbI:GFP:SF; Mo: 187.5 ng/µl 7 September 2015
DNA construction 1µl DNA1 1µl DNA2 1µl DNA3 0.5µl Mo411 1µl DTT 1µl ATP 1µl T4ligase 1µl BsaI 1.2µl BSA 10x 1.2µl buffer ligase 2.4µl H20 8 September 2015
10 September 2015