Difference between revisions of "Team:Valencia UPV/Notebook"

Revision as of 13:30, 14 September 2015

Valencia UPV iGEM 2015

Notebook

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 	<p>Be patient, we are under construction</p>
 	<ul class="actions">
-		<li><a href="#scroll1" class="button">Go to notebook</a></li>
+<li><a href="#scroll1" class="button">Protocols/a></li>
+<li><a href="#scroll1" class="button">Notebook</a></li>
+<li><a href="#scroll1" class="button"><i>Nicotiana experiments</i></a></li>
+<li><a href="#scroll1" class="button">Protoplasts</a></li>
 	</ul>
 </section>
 <!-- Main -->
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 					</h2><hr>
 				</header>
-<!--Beginning of notebook-->
-<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; &alpha;2 (GB160) to test it.</p>
-<p>Make the ligation (<a href="https://2015.igem.org/Team:Valencia_UPV/Notebook/Protocols#scrollsect1"target="blank">step 2 in the protocol</a>):</p>
-<div class="table-wrapper"><table class="alt">
-<tr><td>E:PIF6:PhyB:VP16:Etr8:luc+ren; &Omega;1</td></tr>
-<tr><td>1µL E:PIF6:PhyB:VP16:Etr8:luc; &alpha;1</td></tr>
-<tr><td>1µL renilla; apha2</td></tr>
-<tr><td>1µL &Omega;1</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; &Omega;1</td></tr>
-<tr><td>0.5µL E:PIF6:PhyB:VP16:Etr8:luc; &alpha;1</td></tr>
-<tr><td>1µL renilla; apha2</td></tr>
-<tr><td>1µL &Omega;1</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>&alpha;1</li>
-<li>&alpha;2</li>
-<li>&Omega;1</li>
-<li>&Omega;2</li>
-<li>pUPD2</li>
-<li>E:PIF6:NLS; pUPD2 (GB0288)</li>
-<li>E:PIF6:NLS; &alpha;1 (GB892)</li>
-<li>E:PIF6:NLS; &Omega;2 (GB893)</li>
-<li>E:PIF6:NLS:luc:PhyB; &alpha;1 (GB896)</li>
-<li>Luc:PhyB; &Omega;1 (GB890)</li>
-<li>PhyB:VP16; pUPD2 (GB289)</li>
-<li>PhyB:VP16; &alpha;2 (GB88E)</li>
-<li>Etr8:CMVmini; pUPD2 (GB1097)</li>
-<li>OpLexA:mini35S; pUPD2 (GB733)</li>
-<li>OpLexA:mini35S:luc:Tnos; &alpha;2</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; &alpha;2 (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 &Omega;2. 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>&alpha;1</td><td>-</td><td>-</td></tr>
-<tr><td>&alpha;2</td><td>-</td><td>-</td></tr>
-<tr><td>&Omega;1</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; &alpha;1 (GB892)</td><td> EcoRI</td><td>6300, 2500</td></tr>
-<tr><td>E:PIF6:NLS; &Omega;2 (GB893)</td><td>EcoRV</td><td>1800, 6600, 900</td></tr>
-<tr><td>E:PIF6:NLS:luc:PhyB; &alpha;1 (GB896)</td><td>EcoRI</td><td>3600, 6300, 5600</td></tr>
-<tr><td>Luc:PhyB; &Omega;1 (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; &alpha;2 (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; &alpha;2 (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; &alpha;2 (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; &Omega;1</td><td>BamHI</td><td>3700, 6100, 6600, 4200</td></tr>
-<tr><td>E:PIF6:PhyB:VP16:Etr8:luc+ren; &Omega;1</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; &Omega;1</td><td>Etr8(CMV)+BxbI:T35S; &alpha;1</td></tr>
-<tr><td>1µL (GB892) PIF; &alpha;1</td><td>1µL (GB1097) Etr8(CMV); pUPD2</td></tr>
-<tr><td>1µL (GB88E) PhyB; &alpha;2</td><td>1µL BxbI; pUPD2</td></tr>
-<tr><td>1µL &Omega;1 </td><td>1µL Tnos pUPD2</td></tr>
-<tr><td>6.8µL H<sub>2</sub>O</td><td>1µL &alpha;1</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; &alpha;1</td><td>EcoRI</td><td>6345, 238</td></tr>
-<tr><td>EPIF6 + PhyB-PV16; &Omega;1</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
-ºC 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 &Omega;2.</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; &alpha;1+PhyB; &alpha;2</td></tr>
-<tr><td>1µl BxbI</td></tr>
-<tr><td>1 µl PhyB</td></tr>
-<tr><td>1 µl &Omega;2</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 &Omega;2</li>
-</ul>
-<div class="table-wrapper"><table class="alt">
-<tr><td>BxbI + 35S:E-PIF6:tnos; &Omega;1</td></tr>
-<tr><td>1µl BxbI</td></tr>
-<tr><td>1 µl PhyB</td></tr>
-<tr><td>1 µl &Omega;1</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; &Omega;1, 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
-: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>&Omega;2</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; &alpha;1</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 &alpha;1</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>&Omega;2</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_&Omega;2 renilla</td><td>EcoRV</td><td>2909, 2475,882, 812, 381</td></tr>
-<tr><td>Entry vector, &Omega;2</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, &alpha;2 </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; &alpha;1</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>&Omega;2</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; &alpha;1</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; &alpha;1 + PhyB:VP16;&alpha;2; &Omega;1</li>
-<li>LacIBD;pUPD2 + PIF6BDPless; pUPD2; &alpha;1</li>
-<li>KDronpa;pUPD2 + LacIBD; pUPD2; &alpha;1</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; &alpha;1 + PhyB:VP16;&alpha;2; &Omega;1</li>
-<li>LacIBD;pUPD2 + PIF6BDPless; pUPD2; &alpha;1</li>
-<li>KDronpa;pUPD2 + LacIBD; pUPD2; &alpha;1</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; &alpha;1 (C1, C2)</li>
-<li>Gal4BD; pUPD2 (C1)</li>
-<li>RepBxb1; pUPD2 (C1-C3)</li>
-<li>LacIBD+KDonpa; &alpha;1 (C1, C2)</li>
-<li>Etr8(CMV)+BxbI+PhyB+VP16; &Omega;1 (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; &alpha;1+PhyB:VP16; &alpha;2; &Omega;1 </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 &Omega;1</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>&Omega;2: 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, &Omega;2 (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, &alpha;2 (19/06): 46 ng/µl</li>
-<li>159 renilla, &Omega;2 (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; &alpha;1</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; &alpha;1</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 &alpha;1</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; &Omega;1</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 &Omega;1</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; &alpha;2</td><td>LacIBD+PIF6; &alpha;1</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 &alpha;2</td><td>1 µl &alpha;1</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; &alpha;1</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; &alpha;1</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 &alpha;1</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; &alpha;1 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; &alpha;1</td><td>EcoRI</td><td>6345, 1997, 641</td></tr>
-<tr><td>RepBxbI:GFP; &Omega;2</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; &alpha;1</td><td>Gal4BD+KDonpa+prom+ter; &alpha;1</td><td>LexABD+KDronpa+prom+term;
-&alpha;1</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 &alpha;1</td><td>1 µl &alpha;1</td><td>1 µl &alpha;1</td></tr>
-</div></table>
-<div class="table-wrapper"><table class="alt">
-<tr><td>NDronpa+VP16; &alpha;2</td><td>Gal4BD+PIF6; &alpha;1</td><td>LacIBD+PIF6; &alpha;1</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 &alpha;2</td><td>1 µl &alpha;1</td><td>1 µl &alpha;1</td></tr>
-</div></table>
-<div class="table-wrapper"><table class="alt">
-<tr><td>LexABD+PIF6; &alpha;1</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 &alpha;2</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; &Omega;2</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, &Omega;2 and make liquid cultures.</p>
-<p> </p>
-<p>Make liquid culture of:</p>
-<p>LexABD+KDronpa+prom+term; &alpha;1 (C1 and C2)</p>
-<p>NDronpa+VP16; &alpha;2 (C1 and C2)</p>
-<p>Gal4BD+PIF6; &alpha;1 (C1 and C2)</p>
-<p>LacIBD+PIF6; &alpha;1 (C1 and C2)</p>
-<p>LexABD+PIF6; &alpha;1 (C1 and C2)</p>
-<p>Take out a glycerinate 35S:Luciferase:Tnos (GB0227) and do a miniprep.</p>
-<!--End of notebook-->
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