Difference between revisions of "Team:Glasgow/Notebook"

 
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             <p>We went to Dundee University to attend the Scotland IGEM team's meeting. We had so much fun in the meeting, sharing different ideas with each team.</p>
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             <h1><strong>James’ muffins</strong></h1><p>Talked to Professors Marshall Stark and John Christie about our project. </p>
 
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           <span class="date">9th</span>
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           <span class="month">Jun</span>
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               <p>The 4 strains of E. coli used are: MGI655ZI (which has tetR, and lacIq, DS941 (recF-), TOP10 (recA-), DH5α (recA-). We designed Phlf and SrpR promoters as BioBricks to be ordered from IDT as if their prefix and suffix were cut with EcoRI and SpeI.</p>
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          <h1><strong>Repressors</strong></h1><p>Ordered P<sub><i>PhlF</i></sub> and P<sub><i>SrpR</i></sub> with BioBrick prefix and suffix as oligos. Ordered; PhlF and SrpR as G-blocks, with ribosome binding site B0032; Terminator B0010+ (a longer version of B0010, that we will characterise and submit as a new BioBrick) with BioBrick prefix and suffix.  Got E5501 and I13500 – GFP (E0040) with ribosome binding sites B0032 and B0034 respectively – in pSB1C3 in DH5α from glycerol stocks from last year’s iGEM team. </p>
 
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           <span class="date">15th</span>
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           <span class="month">Jun</span>
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             <h1><strong>UVA sensor</strong></h1><p>Designed primers to PCR <i>uirS</i>, <i>uirR</i>, and P<sub><i>lsiR</i></sub> from <i>Synechocystis</i> PCC6803 genomic DNA with ribosome binding site B0032 and BioBrick prefix/suffix. Tested UVB sensitivity of strains TOP10 and DH5α (<i>recA<sup>-</sup></i> mutants - very poor DNA repair), DS941 (<i>recF<sup>-</sup></i> - limited DNA repair), and MG1655.Z1 (wild type for DNA repair).</p>
 
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             <p>          We grew bacteria from a single colony from an agar plate in liquid culture to prepare for the dilution series. We designed the Phlf and SrpR respressor sequences with a prefix, B0032 ribosome binding site, TACTAG scar sequence, the repressor itself, TACTAGAG scar sequence, B0010 extended terminator with the help of Steve, and suffix.</p>
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           <span class="date">16th</span>
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           <span class="month">Jun</span>
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          <h1><strong>Bioluminescence</strong></h1><p>Retrieved plasmid DNA of pBAD.LuxCDABEG (Cambridge Luxbrick: K325909) and pBAD.LuxCDABEG.LumP (K769020) from 2015 iGEM distribution plates and transformed into TOP10 and DH5α strains. Designed primers to PCR LuxA, B, C, D, E, and G individually with ribosome binding site B0032 and BioBrick prefix/suffix from K325909. </p>
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            <p>We made TOP10 (which lacks methylation and restriction system, which makes it difficult to ‘cross’ with strains that have the systems) and DH5α (which lacks a restriction system, but has a modification system, ergo, DNA from it can be transferred to other cells) competent for transformation. Since we needed to have the cells in exponential growth phase, we pipetted 4 mL of overnight culture into 20 mL of fresh broth. We made chloramphenicol agar plates. We are also tested our dilution series on non-selective agar plates.</p>
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          <h1><strong>Vilija’s carrot cake</strong></h1><p>So good, honestly.</p>
 
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           <span class="date">17th</span>
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           <span class="month">Jun</span>
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           <p>For TOP10 we had a transformation efficiency of 1.26 x 106 colonies/µg of DNA. For DH5α the control was not transformed. DH5α can be 40 times less competent than TOP10, which would explain our result.
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           <h1><strong>Bioluminescence</strong></h1><p>Tested K325909 and K769020 in TOP10 to see how long until there is expression of bioluminescence after addition of arabinose inducer, how bright the bioluminescence is, and how LumP affects it in K769020.</p>
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          TOP10 is mutant in the repressor for lacI, while DH5α has one copy, so they cannot fully repress the (multiply copied) plasmid. Therefore with IPTG there is no difference in GFP expression in TOP10, and there may be some difference in DH5α.
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           <span class="date">18th</span>
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           <span class="month">Jun</span>
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            <p>We did MiniPrep of the BioBrick transformants and GFP without a promoter. We kept a gel over the weekend in the refrigerator.</p>
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        <h1><strong>Repressors</strong></h1><p>Ligated both P<i><sub>PhlF</sub></i> and P<i><sub>SrpR</sub></i> with E5501 and I13500 in pSB1C3, respectively, and transformed into TOP10 and DH5α. Retrieved C0040 (TetR), R0040 (P<i><sub>TetR</sub></i>) and backbone pSB3K3 (with insert J04450) off distribution plates – to use C0040 as a control for our repressors, and to use pSB3K3 as a second plasmid to have the promoters and repressors on separate plasmids. Ordered G-Block of R0011.B0032 (P<i><sub>LacI</sub></i> + RBS) for C0040 – as the plasmid on the plate has to RBS.</p>
 
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           <span class="date">19th</span>
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           <span class="month">Jun</span>
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           <p>LuxD encodes a transferase, luxE is a synthetase, and luxC is the reductase.
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           <h1><strong>UVA sensor</strong></h1><p>Found genomic DNA from <i>Synechocystis</i> PCC6803 to use for PCR. Ordered P<sub><i>LsiR</i></sub> as a G-Block with BioBrick prefix and suffix.</p>
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If the association map on photobiology of the DEG complex is correct, the whole tetrameric complex may be temperature-sensitive.
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           <span class="date">22th</span>
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           <span class="month">Jun</span>
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          <p>We transformed bacteria that glowed (F1 and F3 are glowing, while F2 and F4 are controls). We are going to measure their glow tomorrow with a more appropriate exposure configuration.We made competent cells.We were going over primer design.
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          <h1><strong>Mhairi’s flapjacks</strong></h1><p>Talked to Amber Jones from the Glasgow School of Art about our project – changed to nightlight toys.</p>
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           <span class="date">23th</span>
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           <span class="month">Jun</span>
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            <p>We transformed cultures with BioBricks (for us and for the master’s lab) and ligations. To make bacteria glow, we have repeated the same experiment as yesterday. After 1h in the 25°C incubator we did not observe bacteria to glow. After 5h in the incubator bacteria were glowing bright.
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          <h1><strong>Bioluminescence</strong></h1><p>Started PCR of Lux genes from K325909 (twice), only LuxB worked.</p>
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We ordered primers for uirR, uirS and all of the lux genes: LuxA, LuxB, LuxC, LuxD, LuxE, LuxG. For the forward primers we included B0032 ribosome binding site and scar. In addition, we ordered a g-block for plsiR. Sean gave us recipes for PCR and Restriction.
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           <span class="date">24th</span>
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           <span class="month">Jun</span>
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          <p>We made overnight cultures.</p>
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        <h1><strong>Repressors</strong></h1><p>Ligated R0040 with I13500 and E5501 separately in pSB1C3, R0011.B0032 oligo with C0040 in pSB1C3, and B0032.PhlF.B0010+ and B0032.SrpR.B0010+ G-Blocks separately in pSB1C3. Attempted transformations into TOP10 and DH5α with both electrocompetent cells, and chemically competent cells, but there was no growth, except on the control plates. Repeated this week’s ligations.</p>
 
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           <span class="date">25th</span>
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           <span class="month">Jun</span>
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           <p>We made glycerol stocks from the BioBricks as well as MiniPreps </p>
+
           <h1><strong>UVA sensor</strong></h1><p>Found <i>Synechocystis</i> PCC6803 genomic DNA in Sean's freezer. Started PCR of <i>uirR</I> and <i>uirS</I> (twice), didn’t work. Ligated P<sub><I>LsiR</i></sub> G-Block into pSB1C3.</p>
 
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           <span class="month">Jun</span>
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           <p>We made restriction digests of the minipreps and annealed R0011. B0032 oligos. After we ran the gel electrophoresis and extracted fragments from the gel. We left ligations overnight and made overnight cultures for making electrocompetent cells.</p>
+
           <h1><strong>Cara’s lemon drizzle cake</strong></h1><p>Pretty cool,^0^</p>
 
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           <span class="date">29th</span>
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           <span class="month">Jun</span>
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           <p>PCR was done to amplify UirR and lux genes (three separate reactions were done) and attempted to make electrocompetent cells. We ligated G-blocks of PhlF and SrpR into pSB1C3.</p>
+
           <h1><strong>Bioluminescence</strong></h1><p>PCR from K325909 worked for LuxA, B, C, D, E and G worked. Ligated into pSB1C3 and transformed into TOP10. No growth.</p>
 
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           <span class="date">30th</span>
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           <span class="month">Jun</span>
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          <p>We made 2 other PCR reactions and tested all PCR products on gel. We found out that none of the amplifications worked except for the one of luxB. We attempted to transform electrocompetent with ligations and we made overnights for making of chemically competent cells in case our transformations fail.</p>
+
        <h1><strong>Repressors</strong></h1><p>Transformed redone ligations of R0040.I13500, R0040.E5501, R0011.B0032.C0040, B0032.PhlF.B0010+, and B0032.SrpR.B0010+ all in pSB1C3 into TOP10.</p>
 
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          <p></p>
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           <span class="date">1st</span>
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           <span class="month">Jul</span>
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           <p>We redid UirS PCR and checked it on gel. This time our reaction successfully yielded a product of the expected size. (What did we change, was it primer concentrations?). We attempted to transform cells again, only this time with chemically competent cells. The large centrifuge was broken, so we divided the work and used a smaller centrifuge in the cold room. We redid digests of PhlF and SrpR G-block, checked them on gel, and redid ligations into pSB1C3.</p>
+
           <h1><strong>UVA sensor</strong></h1><p>PCR of <i>uirS</i> and <i>uirR</i> from Synechocystis PCC6803 genomic DNA worked, ligated <i>uirR</i> into pSB1C3 and transformed into TOP10. PCR of <i>uirS</i> was ligated into TOPO TA cloning plasmid and transformed into TOP10. Designed primers for site-directed mutagenesis of <i>uirS</i> to remove incompatible restriction sites.</p>
 
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           <span class="date">2nd</span>
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           <span class="month">Jul</span>
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           <p>The transformation of chemically competent cells also failed (no growth of positive controls, no growth of anything we hoped to grow) and we do not know why. The plan for today is to insert UirS in pCR2.1TOPO. We are also planning to check gel purified PhlF and SrpR on gel. PLSIR G-block is to be resuspended and digested.</p>
+
           <h1><strong>Adele’s millionaire shortcake</strong></h1><p>Feel like I'm millionaire, LOL.</p>
 
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           <p></p>
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           <span class="date">3th</span>
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           <span class="date"></span>
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           <span class="month">Jul</span>
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           <p>We ran the PCR products of UirR and luxA-G on a gel to check if our PCR worked. We also ligated PLSIR into pSB1C3. We made competent cells and set up overnight cultures and made buffer for the ligation of UirS into TOPO TA.</p>
+
           <h1><strong>Bioluminescence</strong></h1><p>Redid PCR of LuxA, B, C, D, E and G from K325909 and gel purified the PCR product before ligating into pSB1C3 and transforming into TOP10. Designed primers for ribosome binding site library.</p>
 
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           <span class="date">6th</span>
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           <span class="month">Jul</span>
+
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       </div>
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          <p>We made competent cells and transformed ligations (which ligations?). We ligated the purified UirS into TOPO TA and redid the luxE PCR. We ran digested UirR, luxA,B,C,D,G on a gel and extracted them. The digests were then ligated into pSB1C3. We set up overnights of registry order</p>
+
        <h1><strong>Repressors</strong></h1><p>Ligated R0011N with B0032.PhlF.B0010+ and B0032.SrpR.B0010+, separately, and transformed into TOP10.</p>
 
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          <p></p>
+
            <img src="https://static.igem.org/mediawiki/2015/6/67/2015-Glasgow-timeline18.jpg" style="max-width:350px;">
 
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         <div class="meta-date">
 
         <div class="meta-date">
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           <span class="date">7th</span>
+
           <span class="date"></span>
−
           <span class="month">Jul</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
−
<div class="timeline-article">
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           <p>We ran a gel of the PCR product extract and set up ligations (which ligations?). We made minipreps of the overnight cultures and made competent cells and transformed ligations. The transformants were set up as overnights.</p>
+
           <h1><strong>UVA sensor</strong></h1><p>Purified oligos for site-directed mutagenesis. Ligated P<sub><i>LsiR</i></sub> in pSB1C3 with E5501 and I13500, and transformed into TOP10.</p>
 
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         <div class="meta-date">
 
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           <span class="date">8th</span>
+
           <span class="date"></span>
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           <span class="month">Jul</span>
+
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         </div>
 
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       </div>
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<div id="Week6"></div>
 +
</br>
 +
</br>
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</br>
 +
<div class="timeline-start">Week6</div>
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        <p>We made minipreps from the overnight cultures: TOPO TA, control, lig. 17, lig. 18, lig. 20) and we ordered primers for site-directed mutagenesis. We digested (our minipreps?), ran them on a gel, extracted them, and set up ligations. We transformed cells with the luxE ligation and set up overnights of transformants.</p>
+
          <h1><strong>.</strong></h1><p>.</p>
 +
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          <p></p>
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           <p></p>
+
           <h1><strong>.</p>
 
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         <div class="meta-date">
 
         <div class="meta-date">
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           <span class="date">9th</span>
+
           <span class="date"></span>
−
           <span class="month">Jul</span>
+
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−
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        <h1><strong>Repressors</strong></h1><p>.</p>
 
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          <p>We did minipreps and digested ligations 21, 22, 24, and ran them on a gel. We checked yesterday’s extractions on a gel.</p>
+
            <p></p>
 
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         <div class="meta-date">
 
         <div class="meta-date">
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           <span class="date">10th</span>
+
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−
           <span class="month">Jul</span>
+
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       </div>
−
<div class="timeline-start" id="Week5">Week5</div>
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           <p>We digested and ran a gel to check for inversion of the UirS PCR product in the TOPO TA plasmid. We made TOP10 overnights to make competent cells for UirS/TOPO TA, PLSIR.I13500/pSB1C3, PLSIR.E5501/pSB1C3, R0011N.Phlf/pSB1C3, R0011N. SrpR/pSB1C3, and controls (lig. 36, 38, 41, and 43. We also made multiple overnights of UirR/pSB1C3, LuxA/pSB1C3, LuxB/pSB1C3, LuxC/pSB1C3, LuxD/pSB1C3, LuxE/pSB1C3, LuxG/pSB1C3. We ran luxA-G PCR products on a gel and purified them.</p>
+
           <p></p>
 
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           <p></p>
+
           <h1><strong>UVA sensor</strong></h1><p>.</p>
 
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         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">13th</span>
+
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−
           <span class="month">Jul</span>
+
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<div class="timeline-article">
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<div id="Week7"></div>
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</br>
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<div class="timeline-start">Week7</div>
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          <h1><strong>.</strong></h1><p>.</p>
 
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           <p>We checked ligations with digests and transformed ligations of R0011N with Phlf and SrpR in pSB1C3 into TOP10 (these are ligations 35, 36, 37, and 38)</p>
+
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           <span class="date">14th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
−
<div class="timeline-article">
+
      <div class="timeline-article">
 
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           <div class="content-left">
 
           <div class="content-left">
 +
          <p></p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
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           <div class="content-right">
 
           <div class="content-right">
−
           <p></p>
+
           <h1><strong>Bioluminescence</strong></h1><p>.</p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">30th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
−
<div class="timeline-article">
+
      <div class="timeline-article">
 
         <div class="content-left-container">
 
         <div class="content-left-container">
 
           <div class="content-left">
 
           <div class="content-left">
 +
        <h1><strong>Repressors</strong></h1><p>.</p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
           <div class="content-right">
−
          <p></p>
+
            <p></p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">30th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
−
<div class="timeline-article">
+
      <div class="timeline-article">
 
         <div class="content-left-container">
 
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           <div class="content-left">
 
           <div class="content-left">
 +
          <p></p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
           <div class="content-right">
−
           <p></p>
+
           <h1><strong>UVA sensor</strong></h1><p>.</p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">30th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
−
<div class="timeline-article">
+
<div id="Week8"></div>
 +
</br>
 +
</br>
 +
</br>
 +
<div class="timeline-start" >Week8</div>
 +
      <div class="timeline-article">
 
         <div class="content-left-container">
 
         <div class="content-left-container">
 
           <div class="content-left">
 
           <div class="content-left">
 +
          <h1><strong>Ye’s cakes</strong></h1><p></p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
           <div class="content-right">
−
           <p></p>
+
           <img src="https://static.igem.org/mediawiki/2015/c/ca/2015-Glasgow-timeline20.jpg" style="max-width:350px;">
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">30th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
−
<div class="timeline-article">
+
      <div class="timeline-article">
 
         <div class="content-left-container">
 
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           <div class="content-left">
 
           <div class="content-left">
 +
          <img src="https://static.igem.org/mediawiki/2015/9/92/2015-Glasgow-timeline21.jpg" style="max-width:350px;">
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
           <div class="content-right">
−
           <p></p>
+
           <h1><strong>Bioluminescence</strong></h1><ul>
 +
<li> Ligating promoter (pBAD) upstream of LuxAB – test by spraying with decanal</li>
 +
<li> Transformed LuxCD into DH5a and DS941.Z1 – ligate in R0011N (will be repressed in those strains); ligate Lux E downstream</li>
 +
<li> checking if intermediates of tetradecanal synthesis is toxic to cells (hence a repressed promoter)</li>
 +
<li> Checking sequenced of Lux genes with mutations (different from genomic Vibrio sequence) – if both colonies are the same, may be mutations in luxoperon (K325909) – need to design primers</li>
 +
<li> Ribosome binding site library</li>
 +
<ul>
 +
<li> PCR products of LuxA-G individually, transforming into</li>
 +
<li> LuxA ligated into pBAD-pSB1C3</li>
 +
<li> LuxC ligated into R0011N-pSB1C3</li>
 +
<li> LuxB,D,E, and G ligated into pSB1C3 with no promoter</li>
 +
<li> Ligate pBAD-(library)-LuxA with (library)-LuxB and spray with decanal</li>
 +
</ul>
 +
</ul>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">30th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
−
<div class="timeline-article">
+
      <div class="timeline-article">
 
         <div class="content-left-container">
 
         <div class="content-left-container">
 
           <div class="content-left">
 
           <div class="content-left">
 +
        <h1><strong>Repressors</strong></h1>
 +
<ul>
 +
<li> Have promoters+GFP in pSB3K3 in DS941.Z1</li>
 +
<li> Have R0011N+repressors in pSB1C3 in DS941.Z1 – make competent cells, and transform in promoters+GFP in pSB3K3</li>
 +
<li> Growth curve and fluorescence to test promoters</li>
 +
<li>Bi-stable switch: have PPhlF+SrpR, PSrpR+PhlF in pSB1C3 – need to make and check miniprep, then transform promoters+GFP in pSB3K3</li>
 +
<li>Make one promoter with GFP, one with RFP on same plasmid (one needs terminator)</li>
 +
<li>Terminators: ligate “steve” (B0010+), B0010, and B0015 into pSB1A10</li>
 +
 +
</ul>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
           <div class="content-right">
−
          <p></p>
+
            <img src="https://static.igem.org/mediawiki/2015/b/b8/2015-Glasgow-timeline22.jpg" style="max-width:350px;">
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">30th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
−
<div class="timeline-article">
+
      <div class="timeline-article">
 
         <div class="content-left-container">
 
         <div class="content-left-container">
 
           <div class="content-left">
 
           <div class="content-left">
 +
          <img src="https://static.igem.org/mediawiki/2015/9/91/2015-Glasgow-timeline23.jpg" style="max-width:350px">
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
           <div class="content-right">
−
           <p></p>
+
           <h1><strong>UVA sensor</strong></h1>
 +
<ul>
 +
<li>3rd PCR mutagenesis of UirS – so hopefully fixed, needs to be sequenced</li>
 +
<li>Chromophore (PCB) BioBricks as miniprep and checked by digest (has a constitutive promoter)</li>
 +
<li>Confirmed P<sub><i>LsiR</i></sub> is off when not activated (tested by GFP with growth curve)</li>
 +
</ul>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">30th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
−
<div class="timeline-article">
+
<div id="Week9"></div>
 +
</br>
 +
</br>
 +
</br>
 +
<div class="timeline-start">Week9</div>
 +
      <div class="timeline-article">
 
         <div class="content-left-container">
 
         <div class="content-left-container">
 
           <div class="content-left">
 
           <div class="content-left">
 +
          <h1><strong>.</strong></h1><p>.</p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
           <div class="content-right">
−
           <p></p>
+
           <img src="">
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">30th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
−
<div class="timeline-article">
+
      <div class="timeline-article">
 
         <div class="content-left-container">
 
         <div class="content-left-container">
 
           <div class="content-left">
 
           <div class="content-left">
 +
          <p></p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
           <div class="content-right">
−
           <p></p>
+
           <h1><strong>.</strong></h1><p>.</p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">30th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
−
<div class="timeline-article">
+
      <div class="timeline-article">
 
         <div class="content-left-container">
 
         <div class="content-left-container">
 
           <div class="content-left">
 
           <div class="content-left">
 +
        <h1><strong>.</p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
           <div class="content-right">
−
          <p></p>
+
            <p></p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">30th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
−
<div class="timeline-article">
+
      <div class="timeline-article">
 
         <div class="content-left-container">
 
         <div class="content-left-container">
 
           <div class="content-left">
 
           <div class="content-left">
 +
          <p></p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
           <div class="content-right">
−
           <p></p>
+
           <h1><strong>.</strong></h1><p>.</p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">30th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
−
<div class="timeline-article">
+
<div id="Week10"></div>
 +
</br>
 +
</br>
 +
</br>
 +
<div class="timeline-start">Week10</div>
 +
      <div class="timeline-article">
 
         <div class="content-left-container">
 
         <div class="content-left-container">
 
           <div class="content-left">
 
           <div class="content-left">
 +
          <h1><strong>.</strong></h1><p>.</p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
           <div class="content-right">
−
           <p></p>
+
           <img src="">
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">30th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
−
<div class="timeline-article">
+
      <div class="timeline-article">
 
         <div class="content-left-container">
 
         <div class="content-left-container">
 
           <div class="content-left">
 
           <div class="content-left">
 +
          <p></p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
           <div class="content-right">
−
           <p></p>
+
           <h1><strong>.</strong></h1><p>.</p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">30th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
−
<div class="timeline-article">
+
      <div class="timeline-article">
 
         <div class="content-left-container">
 
         <div class="content-left-container">
 
           <div class="content-left">
 
           <div class="content-left">
 +
        <h1><strong>.</p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
           <div class="content-right">
−
          <p></p>
+
            <p></p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">30th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
−
<div class="timeline-article">
+
      <div class="timeline-article">
 
         <div class="content-left-container">
 
         <div class="content-left-container">
 
           <div class="content-left">
 
           <div class="content-left">
 +
          <p></p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
           <div class="content-right">
−
           <p></p>
+
           <h1><strong>.</strong></h1><p>.</p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">30th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
−
<div class="timeline-article">
+
<div id="Week11"></div>
 +
</br>
 +
</br>
 +
</br>
 +
<div class="timeline-start">Week11</div>
 +
      <div class="timeline-article">
 
         <div class="content-left-container">
 
         <div class="content-left-container">
 
           <div class="content-left">
 
           <div class="content-left">
 +
          <h1><strong>.</strong></h1><p>.</p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
           <div class="content-right">
−
           <p></p>
+
           <img src="">
 
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           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">30th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 +
        </div>
 +
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 +
      <div class="timeline-article">
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          <p></p>
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 +
        </div>
 +
        <div class="content-right-container">
 +
          <div class="content-right">
 +
          <h1><strong>.</strong></h1><p>.</p>
 +
          </div>
 +
        </div>
 +
        <div class="meta-date">
 +
          <span class="date"></span>
 +
          <span class="month"></span>
 +
        </div>
 +
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 +
      <div class="timeline-article">
 +
        <div class="content-left-container">
 +
          <div class="content-left">
 +
        <h1><strong>.</p>
 +
          </div>
 +
        </div>
 +
        <div class="content-right-container">
 +
          <div class="content-right">
 +
            <p></p>
 +
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 +
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 +
        <div class="meta-date">
 +
          <span class="date"></span>
 +
          <span class="month"></span>
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 +
      <div class="timeline-article">
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        <div class="content-left-container">
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 +
          <p></p>
 +
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 +
        </div>
 +
        <div class="content-right-container">
 +
          <div class="content-right">
 +
          <h1><strong>.</strong></h1><p>.</p>
 +
          </div>
 +
        </div>
 +
        <div class="meta-date">
 +
          <span class="date"></span>
 +
          <span class="month"></span>
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        </div>
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 +
<div id="Week12"></div>
 +
</br>
 +
</br>
 +
</br>
 +
<div class="timeline-start">Week12</div>
 +
      <div class="timeline-article">
 +
        <div class="content-left-container">
 +
          <div class="content-left">
 +
          <h1><strong>.</strong></h1><p>.</p>
 +
          </div>
 +
        </div>
 +
        <div class="content-right-container">
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          <div class="content-right">
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 +
        <div class="meta-date">
 +
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          <span class="month"></span>
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      <div class="timeline-article">
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        <div class="content-right-container">
 +
          <div class="content-right">
 +
          <h1><strong>.</strong></h1><p>.</p>
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 +
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 +
        <div class="meta-date">
 +
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          <span class="month"></span>
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 +
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 +
        <h1><strong>.</p>
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        <div class="content-right-container">
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        <div class="content-right-container">
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 +
          <h1><strong>.</strong></h1><p>.</p>
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        <div class="meta-date">
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          <span class="date"></span>
 +
          <span class="month readMore"></span>
 
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       </div>
 
       </div>
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     </div>
 
     </div>
 
   </section>
 
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+
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+
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   <script src="https://2015.igem.org/Team:Glasgow/bootstrap.min.js?action=raw&ctype=text/css"></script>
 
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   <style>
 
   <style>
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<body data-spy="scroll" data-target="#myScrollspy" data-offset="20">
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<body data-spy="scroll" data-target="#myScrollspy" data-offset="20">
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−
     <nav class="col-sm-3" id="myScrollspy">
+
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         <li><a href="#Week10">Week10</a></li>
 
         <li><a href="#Week10">Week10</a></li>
 
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<div class="container">
+
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<footer>
+
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−
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−
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−
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.social-button:before {
+
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<script type="text/javascript" >
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var obj = document.getElementById(id);
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var _getHeight = obj.offsetTop;
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+
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 +
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.social-button.facebook .fa {
+
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−
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+
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+
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+
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+
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+
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+
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+
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+
 
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+
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+
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+
−
</style>
+
−
<div class="container">
+
−
<div class="col-md-4">
+
−
                    <h4><strong>Location</storng></h4>
+
−
                    <p>
+
−
                    Bower Building, Wilkins Teaching Laboratory<br>
+
−
                    University of Glasgow  <br>
+
−
                    University Avenue <br>
+
−
                    G12 8QQ
+
−
                    </p>
+
−
</div>
+
−
<div class="col-md-4">
+
−
  <h4>
+
−
  <strong>Follow Us On</strong>
+
−
  </h4>
+
−
  <p>
+
−
        <div class="social-buttons">
+
−
        <a href="https://www.facebook.com/igemglasgow2015" target="_blank" class="social-button facebook"><i class="fa fa-facebook"></i></a>
+
−
        <a href="https://twitter.com/2015iGEMglasgow" target="_blank" class="social-button twitter"><i class="fa fa-twitter"></i></a>
+
−
        <a href="#" class="social-button google"><i class="fa fa-google"></i></a>
+
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</html>
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{{Glasgowfooter}}

Latest revision as of 18:34, 15 November 2015

Responsive Vertical Timeline



Week1

James’ muffins

Talked to Professors Marshall Stark and John Christie about our project.

Repressors

Ordered PPhlF and PSrpR with BioBrick prefix and suffix as oligos. Ordered; PhlF and SrpR as G-blocks, with ribosome binding site B0032; Terminator B0010+ (a longer version of B0010, that we will characterise and submit as a new BioBrick) with BioBrick prefix and suffix. Got E5501 and I13500 – GFP (E0040) with ribosome binding sites B0032 and B0034 respectively – in pSB1C3 in DH5α from glycerol stocks from last year’s iGEM team.

UVA sensor

Designed primers to PCR uirS, uirR, and PlsiR from Synechocystis PCC6803 genomic DNA with ribosome binding site B0032 and BioBrick prefix/suffix. Tested UVB sensitivity of strains TOP10 and DH5α (recA- mutants - very poor DNA repair), DS941 (recF- - limited DNA repair), and MG1655.Z1 (wild type for DNA repair).

Bioluminescence

Retrieved plasmid DNA of pBAD.LuxCDABEG (Cambridge Luxbrick: K325909) and pBAD.LuxCDABEG.LumP (K769020) from 2015 iGEM distribution plates and transformed into TOP10 and DH5α strains. Designed primers to PCR LuxA, B, C, D, E, and G individually with ribosome binding site B0032 and BioBrick prefix/suffix from K325909.




Week2

Vilija’s carrot cake

So good, honestly.

Bioluminescence

Tested K325909 and K769020 in TOP10 to see how long until there is expression of bioluminescence after addition of arabinose inducer, how bright the bioluminescence is, and how LumP affects it in K769020.

Repressors

Ligated both PPhlF and PSrpR with E5501 and I13500 in pSB1C3, respectively, and transformed into TOP10 and DH5α. Retrieved C0040 (TetR), R0040 (PTetR) and backbone pSB3K3 (with insert J04450) off distribution plates – to use C0040 as a control for our repressors, and to use pSB3K3 as a second plasmid to have the promoters and repressors on separate plasmids. Ordered G-Block of R0011.B0032 (PLacI + RBS) for C0040 – as the plasmid on the plate has to RBS.

UVA sensor

Found genomic DNA from Synechocystis PCC6803 to use for PCR. Ordered PLsiR as a G-Block with BioBrick prefix and suffix.




Week3

Mhairi’s flapjacks

Talked to Amber Jones from the Glasgow School of Art about our project – changed to nightlight toys.

Bioluminescence

Started PCR of Lux genes from K325909 (twice), only LuxB worked.

Repressors

Ligated R0040 with I13500 and E5501 separately in pSB1C3, R0011.B0032 oligo with C0040 in pSB1C3, and B0032.PhlF.B0010+ and B0032.SrpR.B0010+ G-Blocks separately in pSB1C3. Attempted transformations into TOP10 and DH5α with both electrocompetent cells, and chemically competent cells, but there was no growth, except on the control plates. Repeated this week’s ligations.

UVA sensor

Found Synechocystis PCC6803 genomic DNA in Sean's freezer. Started PCR of uirR and uirS (twice), didn’t work. Ligated PLsiR G-Block into pSB1C3.




Week4

Cara’s lemon drizzle cake

Pretty cool,^0^

Bioluminescence

PCR from K325909 worked for LuxA, B, C, D, E and G worked. Ligated into pSB1C3 and transformed into TOP10. No growth.

Repressors

Transformed redone ligations of R0040.I13500, R0040.E5501, R0011.B0032.C0040, B0032.PhlF.B0010+, and B0032.SrpR.B0010+ all in pSB1C3 into TOP10.

UVA sensor

PCR of uirS and uirR from Synechocystis PCC6803 genomic DNA worked, ligated uirR into pSB1C3 and transformed into TOP10. PCR of uirS was ligated into TOPO TA cloning plasmid and transformed into TOP10. Designed primers for site-directed mutagenesis of uirS to remove incompatible restriction sites.




Week5

Adele’s millionaire shortcake

Feel like I'm millionaire, LOL.

Bioluminescence

Redid PCR of LuxA, B, C, D, E and G from K325909 and gel purified the PCR product before ligating into pSB1C3 and transforming into TOP10. Designed primers for ribosome binding site library.

Repressors

Ligated R0011N with B0032.PhlF.B0010+ and B0032.SrpR.B0010+, separately, and transformed into TOP10.

UVA sensor

Purified oligos for site-directed mutagenesis. Ligated PLsiR in pSB1C3 with E5501 and I13500, and transformed into TOP10.




Week6

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Repressors

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UVA sensor

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Week7

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Bioluminescence

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Repressors

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UVA sensor

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Week8

Ye’s cakes

Bioluminescence

  • Ligating promoter (pBAD) upstream of LuxAB – test by spraying with decanal
  • Transformed LuxCD into DH5a and DS941.Z1 – ligate in R0011N (will be repressed in those strains); ligate Lux E downstream
  • checking if intermediates of tetradecanal synthesis is toxic to cells (hence a repressed promoter)
  • Checking sequenced of Lux genes with mutations (different from genomic Vibrio sequence) – if both colonies are the same, may be mutations in luxoperon (K325909) – need to design primers
  • Ribosome binding site library
    • PCR products of LuxA-G individually, transforming into
    • LuxA ligated into pBAD-pSB1C3
    • LuxC ligated into R0011N-pSB1C3
    • LuxB,D,E, and G ligated into pSB1C3 with no promoter
    • Ligate pBAD-(library)-LuxA with (library)-LuxB and spray with decanal

Repressors

  • Have promoters+GFP in pSB3K3 in DS941.Z1
  • Have R0011N+repressors in pSB1C3 in DS941.Z1 – make competent cells, and transform in promoters+GFP in pSB3K3
  • Growth curve and fluorescence to test promoters
  • Bi-stable switch: have PPhlF+SrpR, PSrpR+PhlF in pSB1C3 – need to make and check miniprep, then transform promoters+GFP in pSB3K3
  • Make one promoter with GFP, one with RFP on same plasmid (one needs terminator)
  • Terminators: ligate “steve” (B0010+), B0010, and B0015 into pSB1A10

UVA sensor

  • 3rd PCR mutagenesis of UirS – so hopefully fixed, needs to be sequenced
  • Chromophore (PCB) BioBricks as miniprep and checked by digest (has a constitutive promoter)
  • Confirmed PLsiR is off when not activated (tested by GFP with growth curve)



Week9

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Week10

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Week11

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Week12

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Location

Bower Building, Wilkins Teaching Laboratory
University of Glasgow
University Avenue
G12 8QQ

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