Difference between revisions of "Team:EPF Lausanne/Notebook/Ecoli"

Line 38: Line 38:
 
           <ul id="nav" class="nav hidden-xs hidden-sm" data-spy="affix">
 
           <ul id="nav" class="nav hidden-xs hidden-sm" data-spy="affix">
 
             <li class="active"><a href="#pdcas9w">Construction of pdCas9-w</a>
 
             <li class="active"><a href="#pdcas9w">Construction of pdCas9-w</a>
−
            <ul class="nav">
+
              <ul class="nav">
−
              <li><a href="#PCRpdcas9">Open pdCas9 by PCR</a></li>
+
                <li><a href="#PCRpdcas9">Open pdCas9 by PCR</a></li>
−
              <li><a href="#PCRpwj66">Extract w subunit from pWJ66 by PCR</a></li>
+
                <li><a href="#PCRpwj66">Extract w subunit from pWJ66 by PCR</a></li>
−
              <li><a href="#gibsonpdcas9w">Gibson assembly of pdCas9-w</a></li>
+
                <li><a href="#gibsonpdcas9w">Gibson assembly of pdCas9-w</a></li>
−
              <li><a href="#mutagenesis">Site-directed mutagenesis of pdCas9-w</a></li>
+
                <li><a href="#mutagenesis">Site-directed mutagenesis of pdCas9-w</a></li>
−
              <li><a href="#biobrickpdcas9w">BioBrick mutated pdCas9-w</a></li>
+
                <li><a href="#biobrickpdcas9w">BioBrick mutated pdCas9-w</a></li>
−
            </ul>
+
              </ul>
 
             </li>
 
             </li>
 
             <li><a href="#pdcas9wsgrna">Construction of pdCas9-w-sgRNAs</a>
 
             <li><a href="#pdcas9wsgrna">Construction of pdCas9-w-sgRNAs</a>
−
            <ul class="nav">
+
              <ul class="nav">
−
              <li><a href="#openpdcas9w">Open pdCas9-w by restriction digest</a></li>
+
                <li><a href="#openpdcas9w">Open pdCas9-w by restriction digest</a></li>
−
              <li><a href="#pcrsgrna">Amplify sgRNA cassettes by PCR</a></li>
+
                <li><a href="#pcrsgrna">Amplify sgRNA cassettes by PCR</a></li>
−
              <li><a href="#gibsonpdcas9wsgrna">Assemble pdCas9-w-sgRNA constructs by Gibson assembly</a></li>
+
                <li><a href="#gibsonpdcas9wsgrna">Assemble pdCas9-w-sgRNA constructs by Gibson assembly</a></li>
−
            </ul>
+
              </ul>
 
             </li>
 
             </li>
 
             <li><a href="#pwj89alt">Construction of pWJ89alt</a>
 
             <li><a href="#pwj89alt">Construction of pWJ89alt</a>
−
            <ul class="nav">
+
              <ul class="nav">
−
              <li><a href="#pcrpwj89">PCR pWJ89</a></li>
+
                <li><a href="#pcrpwj89">PCR pWJ89</a></li>
−
              <li><a href="#pcrj23117alt">PCR J23117alt promoter</a></li>
+
                <li><a href="#pcrj23117alt">PCR J23117alt promoter</a></li>
−
              <li><a href="#gibsonpwj89alt">Gibson assembly of pWJ89alt</a></li>
+
                <li><a href="#gibsonpwj89alt">Gibson assembly of pWJ89alt</a></li>
−
              <li><a href="#bbj23117alt">BioBrick J23117alt promoter</a></li>
+
                <li><a href="#bbj23117alt">BioBrick J23117alt promoter</a></li>
−
             </ul>
+
              </ul>
 +
             </li>
 +
            <li><a href="#double_reporter">Construction of pWJ89_mCherry</a>
 +
              <ul>
 +
                <li><a href="#openpwj89">Open pWJ89 by restriction digest</a></li>
 +
                <li><a href="#pcrpwj89alt">PCR pWJ89alt</a></li>
 +
                <li><a href="#cutmcherry">Extract mCherry by restriction digest</a></li>
 +
                <li><a href="#pcrmcherry">PCR mCherry</a></li>
 +
                <li><a href="#gibsonpwj89_mcherry">Gibson assembly of pWJ89_mCherry</a></li>
 +
              </ul>
 
             </li>
 
             </li>
 
             <li><a href="#activityassay">Transistor activity assays</a>
 
             <li><a href="#activityassay">Transistor activity assays</a>
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<!-- CONSTRUCTION PDCAS9-W-->
 
<!-- CONSTRUCTION PDCAS9-W-->
−
          <section id="pdcas9w" class="panel">
+
    <section id="pdcas9w" class="panel">
−
            <h1>Construction of pdCas9-w</h1>
+
      <h1>Construction of pdCas9-w</h1>
−
            <p>pdCas9-w was constructed using PCR and Gibson assembly from the following plasmids:</p>
+
      <p>pdCas9-w was constructed using PCR and Gibson assembly from the following plasmids:</p>
−
            <ul>
+
      <ul>
−
                <li>pdCas9 (pdCas9-bacteria was a gift from Stanley Qi, Addgene plasmid # 44249): dCas9 controlled by a Tetracyclin inducible promoter and a Chlorampheicol resistance gene</li>
+
        <li>pdCas9 (pdCas9-bacteria was a gift from Stanley Qi, Addgene plasmid # 44249): dCas9 controlled by a Tetracyclin inducible promoter and a Chlorampheicol resistance gene</li>
−
                <li>pWJ66 (pWJ66 was a gift from Luciano Marraffini, Addgene plasmid # 46570): tracrRNA, w-dCas9 (dCas9 fused at its C-terminal to rpoZ, which encodes for the w subunit of RNA polymerase) and CRISPR array. (The w subunit recruits RNAP)</li>
+
        <li>pWJ66 (pWJ66 was a gift from Luciano Marraffini, Addgene plasmid # 46570): tracrRNA, w-dCas9 (dCas9 fused at its C-terminal to rpoZ, which encodes for the w subunit of RNA polymerase) and CRISPR array. (The w subunit recruits RNAP)</li>
−
            </ul>
+
      </ul>
−
            <p>pdCas9 was opened by PCR and the w subunit was extracted from pWJ66 by PCR. The w subunit was then fused to the C-terminal of dCas9 by Gibson assembly, the final product is pdCas9-w.</br>A site-directed mutagenesis was done on this plasmid to eliminate an EcoRI restriction site. This will allow us to submit pdCas9-w as a BioBrick.</p>
+
      <p>pdCas9 was opened by PCR and the w subunit was extracted from pWJ66 by PCR. The w subunit was then fused to the C-terminal of dCas9 by Gibson assembly, the final product is pdCas9-w.</br>A site-directed mutagenesis was done on this plasmid to eliminate an EcoRI restriction site. This will allow us to submit pdCas9-w as a BioBrick.</p>
−
          </section>
+
  
 
<!-- PCR PDCAS9-->
 
<!-- PCR PDCAS9-->
−
          <section id="PCRpdcas9" class="panel">
+
      <div id="PCRpdcas9">
−
            <h1><small>Construction of pdCas9-w</small></br>Open pdCas9 by PCR</h1>
+
        <h2><small>Construction of pdCas9-w</small>Open pdCas9 by PCR</h2>
−
            <p>We received plasmid pdCas9 in bacteria and did a Miniprep (cf. Protocols) on overnight cultures to isolate it. This step consists of linearizing pdCas9 by PCR.</p>
+
        <p>We received plasmid pdCas9 in bacteria and did a Miniprep (cf. Protocols) on overnight cultures to isolate it. This step consists of linearizing pdCas9 by PCR.</p>
 +
        <h3>Materials and method</h3>
 +
        <ul>
 +
          <li>20 µL Phusion PCR (cf. Protocols) of pdCas9 with primers f_Gbs_pdCas9 and r_Gbs_pdCas9</li>
 +
          <li>PCR product purification (cf. Protocols)</li>
 +
          <li>Agarose gel electrophoresis of 2 µL purified PCR products (with 1kb Generuler ladder)</li>
 +
        </ul>
 +
        <h3>Results</h3>
 +
        <div class="row">
 +
          <div class="col-md-8">
 +
            <p>Linearized pdCas9-w is expected to be 6705 bp.</br>The first try of this PCR was unsuccessful (gel is not shown here). For our second try, we tested many parameters: HF vs. GC buffer, different annealing temperatures and different extension times. This time, many, but not all, of our samples were successfully amplified (cf. Fig.1). The difficulty of this PCR is probably due to the fact that the size of the ampicon is very long, almost 7 kb.</br>For next steps, sample from lane 1 (cf. Fig.1) was used.</p>
 +
          </div>
 +
          <div class="col-md-4">
 +
            <figure>
 +
              <a href="https://static.igem.org/mediawiki/2015/d/d4/Lab_nb_ecoli_fig1.jpg"><img src="https://static.igem.org/mediawiki/2015/d/d4/Lab_nb_ecoli_fig1.jpg" alt="Figure 1"></a>
 +
              <figcaption><b>Fig.1</b> - Gel of linearized pdCas9</br><small>Click on the figure to view larger</small></gcaption>
 +
            </figure>
 +
          </div>
 +
        </div>
 +
      </div>
  
−
            <h2>Materials and method</h2>
+
<!-- PCR PWJ66-->
−
            <ul>
+
      <div id="PCRpwj66">
−
                <li>20 µL Phusion PCR (cf. Protocols) of pdCas9 with primers f_Gbs_pdCas9 and r_Gbs_pdCas9</li>
+
        <h2><small>Construction of pdCas9-w</small></br>Extract w subunit from pWJ66 by PCR</h2>
−
                <li>PCR product purification (cf. Protocols)</li>
+
          <p>We received plasmid pWJ66 in bacteria and did a Miniprep (cf. Protocols) on overnight cultures to isolate it. This step consists of extracting rpoZ, the w subunit, from pW66 by PCR.</p>
−
                <li>Agarose gel electrophoresis of 2 µL purified PCR products (with 1kb Generuler ladder)</li>
+
          <h3>Materials and method</h3>
−
            </ul>
+
          <ul>
−
 
+
            <li>20 µL Phusion PCR (cf. Protocols) of pWJ66 using primers f_Gbs_w and r_Gbs_w.</li>
−
            <h2>Results</h2>
+
            <li>PCR product purification (cf. Protocols)</li>
−
            <div class="row">
+
            <li>Agarose gel electrophoresis of 2 µL purified PCR products (with 1kb Generuler)</li>
−
                <div class="col-md-8">
+
          </ul>
−
                    <p>Linearized pdCas9-w is expected to be 6705 bp.</br>The first try of this PCR was unsuccessful (gel is not shown here). For our second try, we tested many parameters: HF vs. GC buffer, different annealing temperatures and different extension times. This time, many, but not all, of our samples were successfully amplified (cf. Fig.1). The difficulty of this PCR is probably due to the fact that the size of the ampicon is very long, almost 7 kb.</br>For next steps, sample from lane 1 (cf. Fig.1) was used.</p>
+
          <h3>Results</h3>
−
                </div>
+
          <div class="row">
−
                <div class="col-md-4">
+
            <div class="col-md-8">
−
                  <figure>
+
              <p>Successful PCR reactions are expected to yield 340 bp fragments.</br>PCR was succesful for sample visible gel. (cf. Fig.2)</p>
−
                    <a href="https://static.igem.org/mediawiki/2015/d/d4/Lab_nb_ecoli_fig1.jpg"><img src="https://static.igem.org/mediawiki/2015/d/d4/Lab_nb_ecoli_fig1.jpg" alt="Figure 1"></a>
+
−
                    <figcaption><b>Fig.1</b> - Gel of linearized pdCas9</br><small>Click on the figure to view larger</small></figcaption>
+
−
                  </figure>
+
−
                </div>
+
 
             </div>
 
             </div>
−
          </section>
+
             <div class="col-md-4">
−
 
+
               <figure>
−
<!-- PCR PWJ66-->
+
                 <a href="https://static.igem.org/mediawiki/2015/e/ef/Lab_nb_ecoli_fig2.jpg"><img src="https://static.igem.org/mediawiki/2015/e/ef/Lab_nb_ecoli_fig2.jpg" alt="Figure 2"></a>
−
          <section id="PCRpwj66" class="panel">
+
                <figcaption><b>Fig.2</b> - Gel of w subunit extracted from pWJ66</br><small>Click on the figure to view larger</small></figcaption>
−
            <h1><small>Construction of pdCas9-w</small></br>Extract w subunit from pWJ66 by PCR</h1>
+
              </figure>
−
            <p>We received plasmid pWJ66 in bacteria and did a Miniprep (cf. Protocols) on overnight cultures to isolate it. This step consists of extracting rpoZ, the w subunit, from pW66 by PCR.</p>
+
−
 
+
−
            <h2>Materials and method</h2>
+
−
              <ul>
+
−
                <li>20 µL Phusion PCR (cf. Protocols) of pWJ66 using primers f_Gbs_w and r_Gbs_w.</li>
+
−
                <li>PCR product purification (cf. Protocols)</li>
+
−
                <li>Agarose gel electrophoresis of 2 µL purified PCR products (with 1kb Generuler)</li>
+
−
              </ul>
+
−
 
+
−
            <h2>Results</h2>
+
−
             <div class="row">
+
−
              <div class="col-md-8">
+
−
                <p>Successful PCR reactions are expected to yield 340 bp fragments.</br>PCR was succesful for sample visible gel. (cf. Fig.2)</p>
+
−
               </div>
+
−
              <div class="col-md-4">
+
−
                 <figure>
+
−
                  <a href="https://static.igem.org/mediawiki/2015/e/ef/Lab_nb_ecoli_fig2.jpg"><img src="https://static.igem.org/mediawiki/2015/e/ef/Lab_nb_ecoli_fig2.jpg" alt="Figure 2"></a>
+
−
                  <figcaption><b>Fig.2</b> - Gel of w subunit extracted from pWJ66</br><small>Click on the figure to view larger</small></figcaption>
+
−
                </figure>
+
−
              </div>
+
 
             </div>
 
             </div>
−
           </section>
+
           </div>
 +
        </div>
  
 
<!-- GIBSON PDCAS9-W-->
 
<!-- GIBSON PDCAS9-W-->
−
         <section id="gibsonpdcas9w" class="panel">
+
         <div id="gibsonpdcas9w" class="panel">
−
            <h1><small>Construction of pdCas9-w</small></br>Gibson assembly of pdCas9-w</h1>
+
          <h2><small>Construction of pdCas9-w</small></br>Gibson assembly of pdCas9-w</h2>
−
            <p>This step consists of fusing the w subunit to dCas9 by Gibson assembly, using the PCR products from previous steps. We transformed cells with the Gibson assembly product and tested for colonies that contain the construct by colony PCR, restriction digest and sequencing.</p>
+
          <p>This step consists of fusing the w subunit to dCas9 by Gibson assembly, using the PCR products from previous steps. We transformed cells with the Gibson assembly product and tested for colonies that contain the construct by colony PCR, restriction digest and sequencing.</p>
−
 
+
          <h3>Materials and method</h3>
−
            <h2>Materials and method</h2>
+
−
            <ul>
+
−
              <li>Gibson assembly (cf. Protocols) with purified PCR products:</li>
+
−
              <ul>
+
−
                <li>Open pdCas9: 0.02 pmol = 95 ng</li>
+
−
                <li>w subunit extracted from pWJ66: 0.06 pmol = 12.5 ng</li>
+
−
              </ul>
+
−
              <li>Transformation (cf. Protocols) of ultra-competent DH5a cells (NEB) with Gibson assembly product, spreading on Chloramphenicol plates</li>
+
−
              <li>Colony PCR (cf. Protocols) of colonies from plate used for culture of transformed cells with primers f_Gbs_w and r_Scq_pdCas9_w_sgRNA primers + agarose gel electrophoresis of 2 µL PCR prodcuts (with 1kb Generuler)</li>
+
−
              <li>Miniprep (cf. Protocols) of overnight liquid cultures in 5 mL LB with Chloramphenicol of colonies from plate used for culture of transformed cells to isolate pdCas9-w plasmids</li>
+
−
              <li>Restriction digest (Cf. Protocols) of pdCas9-w plasmids with BamHI and KpnI seperately + agarose gel electrophoresis of 2 µL digested products (with 1kb Generuler)</li>
+
−
              <li>Sequencing (Microsynth) of one colony for which Gibson assembly worked according to colony PCR and restriction analysis</li>
+
−
            </ul>
+
−
 
+
−
            <h2>Results</h2>
+
−
 
+
−
            <h3>Colony PCR</h3>
+
−
            <div class="row">
+
−
                <div class="col-md-6">
+
−
                    <p>Primers were placed such as amplicons are 666 bp if Gibson assembly worked and 396 bp if the plasmid self-ligated.</br>Lane "C" is a negative control: PCR was run with all components except template DNA, ie. cells. It is empty which means there is no contamination.</br>Gibson assembly seems to have worked for some of our samples. (cf. Fig.3)</br>To avoid working with too many samples, we kept the ones from lanes 16, 22, 25, 31, 37 and 43 for the Minipreps and restriction digest. We did overnight liquid cultures of these colonies.</p>
+
−
                </div>
+
−
                <div class="col-md-6">
+
−
                  <div class="row">
+
−
                    <div class="col-md-6">
+
−
                      <figure>
+
−
                        <a href="https://static.igem.org/mediawiki/2015/f/f9/Lab_nb_ecoli_fig3a.jpg"><img src="https://static.igem.org/mediawiki/2015/f/f9/Lab_nb_ecoli_fig3a.jpg" alt="Figure 3a" style="width:90%"></a>
+
−
                        <a href="https://static.igem.org/mediawiki/2015/7/77/Lab_nb_ecoli_fib3c.jpg"><img src="https://static.igem.org/mediawiki/2015/7/77/Lab_nb_ecoli_fib3c.jpg" alt="Figure 3c" style="width:90%"></a>
+
−
                      </figure>
+
−
                    </div>
+
−
                    <div class="col-md-6">
+
−
                      <figure>
+
−
                        <a href="https://static.igem.org/mediawiki/2015/1/12/Lab_nb_ecoli_fig3b.jpg"><img src="https://static.igem.org/mediawiki/2015/1/12/Lab_nb_ecoli_fig3b.jpg" alt="Figure 3b" style="width:90%"></a>
+
−
                        <a href="https://static.igem.org/mediawiki/2015/f/ff/Lab_nb_ecoli_fig4d.jpg"><img src="https://static.igem.org/mediawiki/2015/f/ff/Lab_nb_ecoli_fig4d.jpg" alt="Figure 3d" style="width:90%"></a>
+
−
                      </figure>
+
−
                    </div>
+
−
                  </div>
+
−
                  <figure>
+
−
                    <figcaption><b>Fig.3</b> - Gels of colony PCR of dCas9-w Gibson assembly products</br><small>Click on the figure to view larger</small></figcaption>
+
−
                  </figure>
+
−
                </div>
+
−
            </div>
+
−
 
+
−
            <h3>Restriction digest</h3>
+
−
            <div class="row">
+
−
                <div class="col-md-8">
+
−
                    <p>pdCas9-w samples from different colonies are present on gels in triplicates in the following order:</p>
+
−
                    <ul>
+
−
                        <li>Undigested - expected to yield 7 kb circular plasmid (migrates faster than linear fragments of the same size)</li>
+
−
                        <li>Digested by BamHI - expected to yield two fragments of 6147 bp and 834 bp if insert is present or one 6705 bp fragment if it is not</li>
+
−
                        <li>Digested by KpnI - expected to yield two fragments of 45334 bp and 2447 bp if insert is present or one 6705 bp fragment if it is not</li>
+
−
                    </ul>
+
−
                    <p>BamHI and KpnI are both unique cutters in pdCas9 (without the insert) and double cutters in pdCas9-w (with the insert).</br>Too much ladder was loaded so it is difficult to estimate the size of the fragments. The smaller fragments are also very difficult to see.</br>By looking at the relative heights, we can say that all colonies seem to have the w subunit insert. There is only the undigested sample for colony 16 that is not visible, probably due to an error while loading the gel. </br>Sequencing confirmed that sample 22 is in fact pdCas9-w. We used this sample for the next steps and stored it as a glycerol stock (c.f. Protocols).</p>
+
−
                </div>
+
−
                <div class="col-md-4">
+
−
                    <figure>
+
−
                        <a href="https://static.igem.org/mediawiki/2015/0/0a/Lab_nb_ecoli_fig4a.jpg"><img src="https://static.igem.org/mediawiki/2015/0/0a/Lab_nb_ecoli_fig4a.jpg" alt="Figure 4a" style="width:70%"></a>
+
−
                        <a href="https://static.igem.org/mediawiki/2015/0/0a/Lab_nb_ecoli_fig4b.jpg"><img src="https://static.igem.org/mediawiki/2015/0/0a/Lab_nb_ecoli_fig4b.jpg" alt="Figure 4b" style="width:70%"></a>
+
−
                        <a href="https://static.igem.org/mediawiki/2015/9/9c/Lab_nb_ecoli_fig4c.jpg"><img src="https://static.igem.org/mediawiki/2015/9/9c/Lab_nb_ecoli_fig4c.jpg" alt="Figure 4c" style="width:70%"></a>
+
−
                        <figcaption><b>Fig.4</b> - Gels of restriction digest of pdCas9-w Gibson assembly products</br><small>Click on the figures to view larger</small></figcaption>
+
−
                    </figure>
+
−
                </div>
+
−
            </div>
+
−
 
+
−
            <h3>Sequencing</h3>
+
−
            <p>As dCas9-w is very long, only part of it was sequenced (the w subunit and its surrounding base pairs). No mutations were detected.</p>
+
−
        </section>
+
−
 
+
−
<!-- MUTAGENESIS A COMPLETER-->
+
−
          <section id="mutagenesis" class="panel">
+
−
          <h1><small>Construction of pdCas9-w</small></br>Site-directed mutagenesis of dCas9-w</h1>
+
−
          <p>It was noticed after assembly of pdCas9-w that dCas9 contains an EcoRI restriction site, which makes it "un-BioBrick-able". Since it is an important part for our project, we decided to remove the restriction site by site-directed mutagenesis to be able to submit it as a functional BioBrick.</p>
+
−
 
+
−
          <h2>Materials and method</h2>
+
 
           <ul>
 
           <ul>
−
             <li>Site-directed mutagenesis (cf. Protocols) of pdCas9-w with primers f_Mt_A2080C_pdCas9-w and r_Mt_A2080C_pdCas9-w</li>
+
             <li>Gibson assembly (cf. Protocols) with purified PCR products:</li>
−
            <li>Miniprep (cf. Protocols) of overnight liquid cultures in 7 mL LB with Chloramphenicol of colonies from site-directed mutagenesis plate to isolate mutated pdCas9-w plasmids</li>
+
−
            <li>Restriction digest (cf. Protocols) of mutated pdCas9-w plasmids with EcoRI to identify colonies for which the mutagenesis was successful</li>
+
−
            <li>Sequencing (Microsynth) of one pdCas9-w for which the mutageneis worked according to the restriction digest</li>
+
−
 
+
−
          <h2>Results</h2>
+
−
              <p>Coming soon</p>
+
−
          </section>
+
−
 
+
−
<!--BIOBRICK A COMPLETER-->
+
−
        <section id="biobrickpdcas9w" class="panel">
+
−
            <h1><small>Construction of pdCas9-w</small></br>BioBrick mutated dCas9-w</h1>
+
−
            <p>To submit dCas9-w as a BioBrick, we extracted dCas9-w from the mutated pdCas9-w and inserted it into pSB1C3, a BioBrick assembly plasmid.</p>
+
−
 
+
−
            <h2>Materials and method</h2>
+
−
            <p>Coming soon</p>
+
−
 
+
−
            <h2>Results</h2>
+
−
            <p>Coming soon</p>
+
−
 
+
−
        </section>
+
−
 
+
−
 
+
−
<!--CONSTRUCTION PDCAS9-W-SGRNAS-->
+
−
        <section id="pdcas9wsgrna" class="panel">
+
−
            <h1>Construction of pdCas9-w-sgRNAs</h1>
+
−
            <div class="row">
+
−
                <div class="col-md-6">
+
−
                    <p>These experiments consist of inserting one or two sgRNA producing cassettes into pdCas9-w.</br>pdCas9-w was linearized by restriction digest. The sgRNA cassettes were synthesized (IDT) and necessary overlaps were added by PCR. The sgRNAs were inserted into pdCas9-w by Gibson assembly.</p>
+
−
                    <p>The sgRNA cassettes were synthesized with sequences "A" and "B" at its ends (cf. Fig.8a). These are the same sequences as those found at the ends of the linearized pdCas9-w.</br>When one cassette is inserted, no overlaps need to be added.</br>When two or three cassettes are added, sequences "C" and/or "D" need to be added at the ends of the cassettes (cf. Fig.8b), thus obtaining unique overlaps for the Gibson assembly.<p>
+
−
                </div>
+
−
                <div class="col-md-6">
+
−
                    <figure>
+
−
                        <a href="https://static.igem.org/mediawiki/2015/3/35/Lab_nb_ecoli_fig8a.jpg"><img src="https://static.igem.org/mediawiki/2015/3/35/Lab_nb_ecoli_fig8a.jpg" alt="Figure ¨8a" style="width:100%"></a>
+
−
                        <a href="https://static.igem.org/mediawiki/2015/archive/a/a0/20150907074344%21Lab_nb_ecoli_fig8b.jpg"><img src="https://static.igem.org/mediawiki/2015/archive/a/a0/20150907074344%21Lab_nb_ecoli_fig8b.jpg" alt="Figure ¨8b" style="width:100%"></a>
+
−
                        <figcaption><b>Fig.8:Schematics of structure and assembly of sgRNA cassettes</b></br><small><b>Fig.8a</b> - Structure of a sgRNA cassette</br><b>Fig.8b</b> - Assembly of three sgRNA cassettes</small></br><small>Click on the figures to view larger</small></figcaption>
+
−
                    </figure>
+
−
                </div>
+
−
            </div>
+
−
        </section>
+
−
 
+
−
<!--OPEN PDCAS9-W-->
+
−
        <section id="openpdcas9w" class="panel">
+
−
            <h1><small>Construction of pdCas9-w-sgRNAs</small></br>Open pdCas9-w by restriction digest</h1>
+
−
 
+
−
            <h2>Materials and method</h2>
+
 
             <ul>
 
             <ul>
−
                <li>Miniprep (cf. Protocols) of overnight liquid cultures in 5 mL LB with Chloramphenicol of pdCas9-w containing bacteria</li>
+
              <li>Open pdCas9: 0.02 pmol = 95 ng</li>
−
                <li>Restriction digest (Cf. Protocols) of pdCas9-w with BsrBI (blunt ends)</li>
+
              <li>w subunit extracted from pWJ66: 0.06 pmol = 12.5 ng</li>
−
                <li>Agarose gel electrophoresis of 2 µL of digested and undigested product (with 1kb Generuler)</li>
+
 
             </ul>
 
             </ul>
−
 
+
             <li>Transformation (cf. Protocols) of ultra-competent DH5a cells (NEB) with Gibson assembly product, spreading on Chloramphenicol plates</li>
−
             <h2>Results</h2>
+
             <li>Colony PCR (cf. Protocols) of colonies from plate used for culture of transformed cells with primers f_Gbs_w and r_Scq_pdCas9_w_sgRNA primers + agarose gel electrophoresis of 2 µL PCR prodcuts (with 1kb Generuler)</li>
−
             <div class="row">
+
            <li>Miniprep (cf. Protocols) of overnight liquid cultures in 5 mL LB with Chloramphenicol of colonies from plate used for culture of transformed cells to isolate pdCas9-w plasmids</li>
−
                <div class="col-md-8">
+
            <li>Restriction digest (Cf. Protocols) of pdCas9-w plasmids with BamHI and KpnI seperately + agarose gel electrophoresis of 2 µL digested products (with 1kb Generuler)</li>
−
                    <p>The digested and undigested plasmids should have the same size. Circular fragments migrates faster than  linear fragments of the same size. This means that the undigested plasmid should migrate faster than the digested plasmid.</br>Even though the ladder is not clear, by looking at the relative heights we can see that the digested sample migrated more slowly than the undigested sample (cf. Fig.9). The restriction digest was successful.</p>
+
            <li>Sequencing (Microsynth) of one colony for which Gibson assembly worked according to colony PCR and restriction analysis</li>
−
                    <p>Note that this was repeated several times since we needed a lot of linearized pdCas9-w.</p>
+
          </ul>
−
                </div>
+
          <h3>Results</h3>
−
                <div class="col-md-4">
+
          <h4>Colony PCR</h4>
−
                    <figure>
+
          <div class="row">
−
                        <a href="https://static.igem.org/mediawiki/2015/2/2e/Lab_nb_ecoli_fig9.jpg"><img src="https://static.igem.org/mediawiki/2015/2/2e/Lab_nb_ecoli_fig9.jpg" alt="Figure 9"></a>
+
            <div class="col-md-6">
−
                        <figcaption><b>Fig.9</b> - Gel of digested pdCas9-w (lane 1) and undigested pdCas9-w (lane 2)</br><small>Click on the figure to view larger</small></figcaption>
+
              <p>Primers were placed such as amplicons are 666 bp if Gibson assembly worked and 396 bp if the plasmid self-ligated.</br>Lane "C" is a negative control: PCR was run with all components except template DNA, ie. cells. It is empty which means there is no contamination.</br>Gibson assembly seems to have worked for some of our samples. (cf. Fig.3)</br>To avoid working with too many samples, we kept the ones from lanes 16, 22, 25, 31, 37 and 43 for the Minipreps and restriction digest. We did overnight liquid cultures of these colonies.</p>
−
                    </figure>
+
−
                </div>
+
 
             </div>
 
             </div>
−
        </section>
+
            <div class="col-md-6">
−
 
+
              <div class="row">
−
<!--PCR SGRNAS A COMPLETER-->
+
−
        <section id="pcrsgrna" class="panel">
+
−
            <h1><small>Construction of pdCas9-w-sgRNAs</small></br>Amplify sgRNA cassettes by PCR</h1>
+
−
 
+
−
            <h2>Materials and method</h2>
+
−
            <ul>
+
−
                <li>20 µL Phusion or Q5 PCR (cf. Protocols) of sgRNA cassette with primers as indicated in table below</li>
+
−
                <li>Agarose gel electrophoresis of 2 µL purified PCR products (with 1kb Generuler)</li>
+
−
                <li>PCR product purification (cf. Protocols)</li>
+
−
            </ul>
+
−
            <table>
+
−
                <tr>
+
−
                    <th>Construct</th>
+
−
                    <th>sgRNA cassette(s)</th>
+
−
                    <th>Forward primer(s) / Reverse primer(s)</th>
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>pdCas9-w-Z0</td>
+
−
                    <td>Z0</td>
+
−
                    <td>f_Gbs_sgRNA-A / r_Gbs_sgRNA-B</td>
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>pdCas9-w-Z4</td>
+
−
                    <td>Z4</td>
+
−
                    <td>f_Gbs_sgRNA-A / r_Gbs_sgRNA-B</td>
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>pdCas9-w-Z35</td>
+
−
                    <td>Z35</td>
+
−
                    <td>f_Gbs_sgRNA-A / r_Gbs_sgRNA-B</td>
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>pdCas9-w-Z0-Z4</td>
+
−
                    <td>Z0</br>Z4</td>
+
−
                    <td>f_Gbs_sgRNA-A / r_Gbs_sgRNA-BtoC</br>f_Gbs_sgRNA-CtoA / r_Gbs_sgRNA-B</td>
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>pdCas9-w-Z0-Z35</td>
+
−
                    <td>Z0</br>Z35</td>
+
−
                    <td>f_Gbs_sgRNA-A / r_Gbs_sgRNA-BtoC</br>f_Gbs_sgRNA-CtoA / r_Gbs_sgRNA-B</td>
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>pdCas9-w-Z4-Z35</td>
+
−
                    <td>Z4</br>Z35</td>
+
−
                    <td>f_Gbs_sgRNA-A / r_Gbs_sgRNA-BtoC</br>f_Gbs_sgRNA-CtoA / r_Gbs_sgRNA-B</td>
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>pdCas9-w-Z0-Z4-Z35</td>
+
−
                    <td>Z0</br>Z4</br>Z35</td>
+
−
                    <td>f_Gbs_sgRNA-A / r_Gbs_sgRNA-BtoC</br>f_Gbs_sgRNA-CtoA / r_Gbs_sgRNA-BtoD</br>f_Gbs_sgRNA-DtoA / r_Gbs_sgRNA-B</td>
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>pdCas9-w-X0</td>
+
−
                    <td>X0</td>
+
−
                    <td>f_Gbs_sgRNA-A / r_Gbs_sgRNA-B</td>
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>pdCas9-w-X4</td>
+
−
                    <td>X4</td>
+
−
                    <td>f_Gbs_sgRNA-A / r_Gbs_sgRNA-B</td>
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>pdCas9-w-X35</td>
+
−
                    <td>X35</td>
+
−
                    <td>f_Gbs_sgRNA-A / r_Gbs_sgRNA-B</td>
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>pdCas9-w-X0-X4</td>
+
−
                    <td>X0</br>X4</td>
+
−
                    <td>f_Gbs_sgRNA-A / r_Gbs_sgRNA-BtoC</br>f_Gbs_sgRNA-CtoA / r_Gbs_sgRNA-B</td>
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>pdCas9-w-X0-X35</td>
+
−
                    <td>X0</br>X35</td>
+
−
                    <td>f_Gbs_sgRNA-A / r_Gbs_sgRNA-BtoC</br>f_Gbs_sgRNA-CtoA / r_Gbs_sgRNA-B</td>
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>pdCas9-w-X4-X35</td>
+
−
                    <td>X4</br>X35</td>
+
−
                    <td>f_Gbs_sgRNA-A / r_Gbs_sgRNA-BtoC</br>f_Gbs_sgRNA-CtoA / r_Gbs_sgRNA-B</td>
+
−
                </tr>
+
−
                <tr>
+
−
                    <td>pdCas9-w-X0-X4-X35</td>
+
−
                    <td>X0</br>X4</br>X35</td>
+
−
                    <td>f_Gbs_sgRNA-A / r_Gbs_sgRNA-BtoC</br>f_Gbs_sgRNA-CtoA / r_Gbs_sgRNA-BtoD</br>f_Gbs_sgRNA-DtoA / r_Gbs_sgRNA-B</td>
+
−
                </tr>
+
−
            </table>
+
−
 
+
−
            <h2>Results</h2>
+
−
            <div class="row">
+
 
                 <div class="col-md-6">
 
                 <div class="col-md-6">
−
                    <p>All amplified sgRNA cassettes are expected to be about 370 bp.</p>
 
−
                    <p>sgRNA cassette Z0 was amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-BtoD and primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Q5 PCR. Both samples were successfully amplified (cf. Fig.10a).</p>
 
−
                    <p>sgRNA cassette Z4 was amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Phusion PCR. Samples 3 and 4 were successfuly amplified (cf. Fig.10b).</p>
 
−
                    <p>sgRNA cassette Z35 was successfuly amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Phusion PCR. However, it is not easily visible on the gel. (cf. Fig.10c)</p>
 
−
                    <p>sgRNA cassette X0 was successfuly amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Phusion PCR. (cf. Fig.10d)</p>
 
−
                    <p>sgRNA cassette X4 was successfuly amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Phusion PCR. (cf. Fig.10e)</p>
 
−
                    <p>sgRNA cassette X35 was successfuly amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B with Phusion PCR. (cf. Fig.10f)</p>
 
−
                </div>
 
−
                <div class="col-md-6">
 
−
                  <div class="row">
 
−
                    <div class="col-md-6">
 
−
                      <figure>
 
−
                        <a href="https://static.igem.org/mediawiki/2015/3/3b/Lab_nb_ecoli_fig10a.jpg"><img src="https://static.igem.org/mediawiki/2015/3/3b/Lab_nb_ecoli_fig10a.jpg" alt="Figure 10a" style="width:60%"></a>
 
−
                        <a href="https://static.igem.org/mediawiki/2015/9/94/Lab_nb_ecoli_fig10c.jpg"><img src="https://static.igem.org/mediawiki/2015/9/94/Lab_nb_ecoli_fig10c.jpg" alt="Figure 10c" style="width:60%"></a>
 
−
                        <a href="https://static.igem.org/mediawiki/2015/9/9a/Lab_nb_ecoli_fig10e.jpg"><img src="https://static.igem.org/mediawiki/2015/9/9a/Lab_nb_ecoli_fig10e.jpg" alt="Figure 10e" style="width:60%"></a>
 
−
                      </figure>
 
−
                    </div>
 
−
                    <div class="col-md-6">
 
−
                      <figure>
 
−
                        <a href="https://static.igem.org/mediawiki/2015/8/80/Lab_nb_ecoli_fig10b.jpg"><img src="https://static.igem.org/mediawiki/2015/8/80/Lab_nb_ecoli_fig10b.jpg" alt="Figure 10b" style="width:50%"></a>
 
−
                        <a href="https://static.igem.org/mediawiki/2015/5/57/Lab_nb_ecoli_fig10d.jpg"><img src="https://static.igem.org/mediawiki/2015/5/57/Lab_nb_ecoli_fig10d.jpg" alt="Figure 10d" style="width:80%"></a>
 
−
                        <a href="https://static.igem.org/mediawiki/2015/e/ef/Lab_nb_ecoli_fig10f.jpg"><img src="https://static.igem.org/mediawiki/2015/e/ef/Lab_nb_ecoli_fig10f.jpg" alt="Figure 10f" style="width 30%"></a>
 
−
                      </figure>
 
−
                    </div>
 
−
                  </div>
 
 
                   <figure>
 
                   <figure>
−
                     <figcaption><b>Fig.10: Gels of sgRNA cassettes amplified by PCR</b></br><small><b>Fig.10a</b> - Gel of sgRNA cassette Z0 amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-BtoD (lane 1) and primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B (lane 2)</br><b>Fig.10b</b> - Gel of sgRNA cassette Z4 amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B</br><b>Fig.10c</b> - Gel of sgRNA cassette Z35 amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B</br><b>Fig.10d</b> - Gel of sgRNA cassette X0 amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B</br><b>Fig.10e</b> - Gel of sgRNA cassette X4 amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B</br><b>Fig.10f</b> - Gel of sgRNA cassette X35 amplified with primers f_Gbs_sgRNA-A + r_Gbs_sgRNA-B</small></br><small>Click on the figures to view larger</small></figcaption>
+
                     <a href="https://static.igem.org/mediawiki/2015/f/f9/Lab_nb_ecoli_fig3a.jpg"><img src="https://static.igem.org/mediawiki/2015/f/f9/Lab_nb_ecoli_fig3a.jpg" alt="Figure 3a" style="width:90%"></a>
 +
                    <a href="https://static.igem.org/mediawiki/2015/7/77/Lab_nb_ecoli_fib3c.jpg"><img src="https://static.igem.org/mediawiki/2015/7/77/Lab_nb_ecoli_fib3c.jpg" alt="Figure 3c" style="width:90%"></a>
 
                   </figure>
 
                   </figure>
 
                 </div>
 
                 </div>
−
            </div>
+
                 <div class="col-md-6">
−
 
+
−
        </section>
+
−
 
+
−
<!--GIBSON PDCAS9-W-SGRNAS A COMPLETER-->
+
−
        <section id="gibsonpdcas9wsgrna" class="panel">
+
−
            <h1><small>Construction of pdCas9-w-sgRNAs</small></br>Assemble pdCas9-w-sgRNA constructs by Gibson assembly</h1>
+
−
           
+
−
            <h2>Materials and method</h2>
+
−
            <h3>Part 1: pdCas9-w + sgRNA assembly</h3>
+
−
            <ul>
+
−
              <li>Gibson assembly (cf. Protocols) with linearized pdCas9-w and purified sgRNA PCR products:</li>
+
−
              <ol>
+
−
                <li>Z0 / Z4 / Z35 / X0 / X4 / X35 (overlaps A and B)</li>
+
−
                <li>Z0+Z4 / Z0+Z35 / Z4+Z35 / X0+X4 / X0+X35 / X4+X35 (overlaps A and C/D for 1st cassette and C/D and B for 2nd cassette)</li>
+
−
              </ul>
+
−
              <li>Transformation (cf. Protocols) of ultra-competent DH5a cells (NEB) with Gibson assembly product, spreading on Chloramphenicol plates</li>
+
−
              <li>Colony PCR (cf. Protocols) of colonies from plate used for culture of transformed cells with primers f_ColPCR_sgRNAs and r_ColPCR_sgRNAs primers + agarose gel electrophoresis of 2 µL PCR prodcuts (with 1kb Generuler)</br>One colony PCR (cf. Fig11a) was done with primers f_Gbs_pdCas9 and r_Scq_pdCas9_w_sgRNA</li>
+
−
              <li>Miniprep (cf. Protocols) of overnight liquid cultures in 5 mL LB with Chloramphenicol of colonies from plate used for culture of transformed cells to isolate pdCas9-w plasmids</li>
+
−
              <li>Sequencing (Microsynth) of one colony for which Gibson assembly worked according to colony PCR and restriction analysis</li>
+
−
            </ul>
+
−
            <h3>Part 2: pdCas9-w-sgRNA + sgRNA assembly</h3>
+
−
            <ul>
+
−
              <li>Coming soon</li>
+
−
            </ul>
+
−
 
+
−
            <h2>Results</h2>
+
−
 
+
−
            <h3>Part 1: pdCas9-w + sgRNA assembly</h3>
+
−
            <div class="row">
+
−
                 <div class="col-md-7">
+
−
                    <p>In colony PCR, primers were placed such as amplicons are 445 bp if the plasmid self-ligated, 723 bp if Gibson assembly of 1 sgRNA cassette worked and 1120 bp if Gibson assembly of 2 sgRNA cassettes worked.</p>
+
−
                    <p>pdCas9-w-Z0 was obtained by a faulty assembly of pdCas9-w-Z0-Z4 that only took up 1 of the 2 sgRNA cassettes. Sample 20 of the colony PCR (cf. Fig.11a) was confirmed by sequencing to be pdCas9-w-Z0 and to not contain any mutations that may have an effect on its activity. Sample 13 seems to be pdCas9-w-Z0-Z4 according to colony PCR (cf. Fig.11a), but sequencing showed that it is pdCas9-w-X0-Z0, probably due to contamination of a tube. (This sample will be used again in Part 2.)</br>Colony PCR showed that all colonies tested for pdCas9-w-Z4 have the inserted sgRNA cassette (cf. Fig.11b). Sequencing comfirmed that sample 5 is in fact pdCas9-w-Z4. Colony PCR showed that the Gibson assembly for sample 5 of pdCas9-w-Z35 seems to have worked (cf. Fig.11c) and sequencing confirmed that is the case. Sequencing also showed some mutations or deletions in the promoter and/or terminator for these 2 samples. We decided to keep working with these and test whether these mutations are significative with an activity assay.</br>Colony PCR and sequencing showed that Gibson assembly was successful for sample 5 of pdCas9-w-X0 (cf. Fig.11d). However, it contains a mutation in the promoter. A 2nd pdCas9-w-X0 without mutations will be constructed in part 2, after which we will be able to compare the activity of this promoter with/without a mutation.</br>Colony PCR of pdCas9-w-X4 showed many potentially good samples (cf. Fig.11e). Sequencing of sample 10 confirmed that it is in fact pdCas9-w-X4 and that it does not contain any mutations.</br>Colony PCR and sequencing showed that Gibson assembly was successful for the colony of pdCas9-w-X35 (cf. Fig.11d) and no mutations were found in the sequence.</p>
+
−
                </div>
+
−
                <div class="col-md-5">
+
−
                  <div class="row">
+
−
                    <div class="col-md-6">
+
−
                      <figure>
+
−
                        <a href="https://static.igem.org/mediawiki/2015/f/f8/Lab_nb_ecoli_fig11a.jpg"><img src="https://static.igem.org/mediawiki/2015/f/f8/Lab_nb_ecoli_fig11a.jpg" alt="Figure 11a" style="width:100%"></a>
+
−
                        <a href="https://static.igem.org/mediawiki/2015/e/e0/Lab_nb_ecoli_fig11c.jpg"><img src="https://static.igem.org/mediawiki/2015/e/e0/Lab_nb_ecoli_fig11c.jpg" alt="Figure 11c" style="width:100%"></a>
+
−
                        <a href="https://static.igem.org/mediawiki/2015/a/ae/Lab_nb_ecoli_fig11e.jpg"><img src="https://static.igem.org/mediawiki/2015/a/ae/Lab_nb_ecoli_fig11e.jpg" alt="Figure 11e" style="width:80%"></a>
+
−
                      </figure> 
+
−
                    </div>
+
−
                    <div class="col-md-6">
+
−
                      <figure>
+
−
                        <a href="https://static.igem.org/mediawiki/2015/c/c4/Lab_nb_ecoli_fig11b.jpg"><img src="https://static.igem.org/mediawiki/2015/c/c4/Lab_nb_ecoli_fig11b.jpg" alt="Figure 11b" style="width:80%"></a>
+
−
                        <a href="https://static.igem.org/mediawiki/2015/archive/6/64/20150902094918%21Lab_nb_ecoli_fig11d.jpg.jpg"><img src="https://static.igem.org/mediawiki/2015/archive/6/64/20150902094918%21Lab_nb_ecoli_fig11d.jpg" alt="Figure 11d" style="width:100%"></a>
+
−
                      </figure>
+
−
                    </div>
+
−
                  </div> 
+
 
                   <figure>
 
                   <figure>
−
                     <figcaption><b>Fig.11: Gels of colony PCR of pdCas9-w-sgRNA Gibson assembly products</b></br><small><b>Fig.11a</b> - Gel of colony PCR of pdCas9-w-Z0-Z4 (lanes 1-10) and self-ligation control pdCas9-w (lane "C"). Lane 2 seems to be pdCas9-w-Z0-Z4 and lanes 5, 8 and 9 seem to only have 1 inserted sgRNA cassette, being either pdCas9-w-Z0 or pdCas9-w-Z4.</br><b>Fig.11b</b> - Gel of colony PCR of pdCas9-w-Z4 (lanes 1-11). Gibson assembly seems to have worked for all colonies</br><b>Fig.11c</b> - Gel of colony PCR of pdCas9-w-Z35 (lanes 1-8), seems to have worked for lane 5, self-ligation for all others</br><b>Fig.11d</b> - Gel of colony PCR of pdCas9-w-X0 (lanes 1-5) and pdCas9-w-X35 (lane 6). Gibson assembly of pdCas9-w-X0 seems to have worked for lanes 2-5, but sample in lane 5 looks slightly better than the others. The faint band in lane 1 is self-ligation. Gibson assembly of pdCas9-w-X35 seems to have worked.</br><b>Fig.11e</b> - Gel of colony PCR of pdCas9-w-X4 (lanes 1-11). All samples seems to have the sgRNA insert, even though the bands are not very precise.</small></br><small>Click on the figures to view larger</small></figcaption>
+
                     <a href="https://static.igem.org/mediawiki/2015/1/12/Lab_nb_ecoli_fig3b.jpg"><img src="https://static.igem.org/mediawiki/2015/1/12/Lab_nb_ecoli_fig3b.jpg" alt="Figure 3b" style="width:90%"></a>
 +
                    <a href="https://static.igem.org/mediawiki/2015/f/ff/Lab_nb_ecoli_fig4d.jpg"><img src="https://static.igem.org/mediawiki/2015/f/ff/Lab_nb_ecoli_fig4d.jpg" alt="Figure 3d" style="width:90%"></a>
 
                   </figure>
 
                   </figure>
 
                 </div>
 
                 </div>
 
               </div>
 
               </div>
−
        </section>
+
              <figure>
−
 
+
                <figcaption><b>Fig.3</b> - Gels of colony PCR of dCas9-w Gibson assembly products</br><small>Click on the figure to view larger</small></figcaption>
−
 
+
              </figure>
−
<!--CONSTRUCTION PWJ89ALT-->
+
            </div>
−
    <section id="pwj89alt" class="panel">
+
−
      <h1>Construction of pWJ89alt</h1>
+
−
      <p>pWJ89alt was constructed using PCR and Gibson assembly from the following DNA fragments:</p>
+
−
            <ul>
+
−
                <li>pWJ89: low copy plasmid with GFP under a J23117 promoter with and GG-rich upstream regulating sequence (URS) and a Kanamycin resistance gene, received from David Bikard's lab</li>
+
−
                <li>J23117alt: alternative promoter with randomly generated sequences except for the -35 and -10 sequences (synthesized by IDT)</li>
+
−
            </ul>
+
−
            <p>pWJ89 was amplified without its J23117 promoter by PCR and the J23117alt promoter was amplified by PCR. These fragments were assembled by Gibson assembly, the final product is pWj89alt.</br>The J23117alt promoter was BioBricked.</p>
+
−
    </section>
+
−
 
+
−
<!--PCR PWJ89-->
+
−
    <section id="pcrpwj89" class="panel">
+
−
      <h1><small>Construction of pWJ89alt</small></br>PCR pWJ89</h1>
+
−
      <p>We received plasmid pWJ89 in bacteria and did a Miniprep (cf. Protocols) on overnight cultures to isolate it. This step consists of amplifying pWJ89 without the J23117 promoter by PCR.</p>
+
−
 
+
−
      <h2>Materials and method</h2>
+
−
        <ul>
+
−
          <li>20 µL Phusion PCR and 25 µL Q5 PCR (cf. Protocols) of pWJ89 using primers f_Rmv_J23117_of_pWJ89 and r_Rmv_J23117_of_pWJ89.</li>
+
−
          <li>Agarose gel electrophoresis of 2 µL PCR products (with 1kb Generuler)</li>
+
−
          <li>PCR product purification (cf. Protocols)</li>
+
−
        </ul>
+
−
 
+
−
      <h2>Results</h2>
+
−
        <div class="row">
+
−
          <div class="col-md-6">
+
−
            <p>Successful PCR reactions are expected to yield 4400 bp amplicons.</br>Both negative control lanes (cf. Fig.13) are empty, which means there is no contamination. Phusion PCR did not work, but Q5 PCR did (cf. Fig.13). We will work with this sample for next steps.</p>
+
 
           </div>
 
           </div>
−
           <div class="col-md-6">
+
          <h4>Restriction digest</h4>
−
             <figure>
+
           <div class="row">
−
               <a href="https://static.igem.org/mediawiki/2015/7/71/Lab_nb_ecoli_fig13.jpg"><img src="https://static.igem.org/mediawiki/2015/7/71/Lab_nb_ecoli_fig13.jpg" alt="Figure 13" style="width:40%"></a>
+
            <div class="col-md-8">
−
              <figcaption><b>Fig.13</b> - Gel of pWJ89 amplified without J23117 promoter by PCR. Lane 1: Phusion PCR, lane 2: Phusion PCR negative control, lane 3: Q5 PCR, lane 4: Q5 PCR negative control</br><small>Click on the figure to view larger</small></figcaption>
+
              <p>pdCas9-w samples from different colonies are present on gels in triplicates in the following order:</p>
−
            </figure>
+
              <ul>
 +
                <li>Undigested - expected to yield 7 kb circular plasmid (migrates faster than linear fragments of the same size)</li>
 +
                <li>Digested by BamHI - expected to yield two fragments of 6147 bp and 834 bp if insert is present or one 6705 bp fragment if it is not</li>
 +
                <li>Digested by KpnI - expected to yield two fragments of 45334 bp and 2447 bp if insert is present or one 6705 bp fragment if it is not</li>
 +
              </ul>
 +
              <p>BamHI and KpnI are both unique cutters in pdCas9 (without the insert) and double cutters in pdCas9-w (with the insert).</br>Too much ladder was loaded so it is difficult to estimate the size of the fragments. The smaller fragments are also very difficult to see.</br>By looking at the relative heights, we can say that all colonies seem to have the w subunit insert. There is only the undigested sample for colony 16 that is not visible, probably due to an error while loading the gel. </br>Sequencing confirmed that sample 22 is in fact pdCas9-w. We used this sample for the next steps and stored it as a glycerol stock (c.f. Protocols).</p>
 +
             </div>
 +
            <div class="col-md-4">
 +
               <figure>
 +
                <a href="https://static.igem.org/mediawiki/2015/0/0a/Lab_nb_ecoli_fig4a.jpg"><img src="https://static.igem.org/mediawiki/2015/0/0a/Lab_nb_ecoli_fig4a.jpg" alt="Figure 4a" style="width:70%"></a>
 +
                <a href="https://static.igem.org/mediawiki/2015/0/0a/Lab_nb_ecoli_fig4b.jpg"><img src="https://static.igem.org/mediawiki/2015/0/0a/Lab_nb_ecoli_fig4b.jpg" alt="Figure 4b" style="width:70%"></a>
 +
                <a href="https://static.igem.org/mediawiki/2015/9/9c/Lab_nb_ecoli_fig4c.jpg"><img src="https://static.igem.org/mediawiki/2015/9/9c/Lab_nb_ecoli_fig4c.jpg" alt="Figure 4c" style="width:70%"></a>
 +
                <figcaption><b>Fig.4</b> - Gels of restriction digest of pdCas9-w Gibson assembly products</br><small>Click on the figures to view larger</small></figcaption>
 +
              </figure>
 +
            </div>
 
           </div>
 
           </div>
 +
          <h4>Sequencing</h4>
 +
          <p>As dCas9-w is very long, only part of it was sequenced (the w subunit and its surrounding base pairs). No mutations were detected.</p>
 
         </div>
 
         </div>
−
    </section>
+
       </section>
−
 
+
−
<!--PCR J23117ALT-->
+
−
    <section id="pcrj23117alt" class="panel">
+
−
       <h1><small>Construction of pWJ89alt</small></br>PCR J23117alt promoter</h1>
+
−
      <p>This step consists of amplifying J23117alt promoter by primer extension PCR to use it for a Gibson assembly in a further step.</p>
+
−
 
+
−
      <h2>Materials and method</h2>
+
−
        <ul>
+
−
          <li>20 µL Phusion PCR and 25 µL Q5 PCR (cf. Protocols) of J23117alt using primers f_G_J23117Alt1IDT and r_G_J23117Alt1IDT.</li>
+
−
          <li>Agarose gel electrophoresis of 2 µL PCR products (with 1kb Generuler)</li>
+
−
          <li>PCR product purification (cf. Protocols)</li>
+
−
        </ul>
+
−
 
+
−
      <h2>Results</h2>
+
−
        <div class="row">
+
−
          <div class="col-md-6">
+
−
            <p>Successful PCR reactions are expected to yield 340 bp amplicons.</br>Both negative control lanes (cf. Fig.14) are empty, which means there is no contamination. Both Phusion and Q5 PCR worked (cf. Fig.13). We will work with the sample amplified by Q5 PCR.</p>
+
−
          </div>
+
−
          <div class="col-md-6">
+
−
            <figure>
+
−
              <a href="https://static.igem.org/mediawiki/2015/1/19/Lab_nb_ecoli_fig14.jpg"><img src="https://static.igem.org/mediawiki/2015/1/19/Lab_nb_ecoli_fig14.jpg" alt="Figure 13" style="width:30%"></a>
+
−
              <figcaption><b>Fig.14</b> - Gel of pWJ89 amplified without J23117 promoter by PCR. Lane 1: Phusion PCR, lane 2: Phusion PCR negative control, lane 3: Q5 PCR, lane 4: Q5 PCR negative control</br><small>Click on the figure to view larger</small></figcaption>
+
−
            </figure>
+
−
          </div>
+
−
        </div>
+
−
    </section>
+
−
 
+
−
<!--GIBSON PWJ89ALT-->
+
−
    <section id="gibsonpwj89alt" class="panel">
+
−
      <h1><small>Construction of pWJ89alt</small></br>Gibson assembly of pWJ89alt</h1>
+
−
        <p>This step consists of using Gibson assembly to insert the J23117alt promoter into pWJ89alt from which its original promoter J23117 was extracted by PCR. We transformed cells with the Gibson assembly product and tested for colonies that contain the construct by colony PCR, restriction digest and sequencing.</p>
+
−
 
+
−
        <h2>Materials and method</h2>
+
−
        <ul>
+
−
          <li>Gibson assembly (cf. Protocols) with purified PCR products:
+
−
          <ul>
+
−
            <li>Amplified pWJ89 (wihtout J23117): 0.05 pmol = 143 ng</li>
+
−
            <li>J23117alt promoter: 0.1 pmol = 22.1 ng</li>
+
−
          </ul>
+
−
          </li>
+
−
          <li>Transformation (cf. Protocols) of ultra-competent DH5a cells (NEB) with Gibson assembly product, spreading on Kanamycin plates</li>
+
−
          <li>Colony PCR (cf. Protocols) of colonies from plate used for culture of transformed cells with primers f_Cl_pWJ89 and r_Sq_J23117alt primers + agarose gel electrophoresis of 2 µL PCR prodcuts (with 1kb Generuler)</li>
+
−
          <li>Miniprep (cf. Protocols) of overnight liquid cultures in 5 mL LB with Kanamycin and sequencing (Microsynth) of one colony for which Gibson assembly worked according to colony PCR</li>
+
−
        </ul>
+
−
 
+
−
        <h2>Results</h2>
+
−
        <p>Colony PCR showed two colonies for which Gibson assembly worked. However, images of gels of colony PCR were lost for technical reasons.</br>Sequencing confirmed that both colonies were in fact pWJ89alt and that one had a deletion but the other one did not have any mutations.</br>We kept the mutation-free colony and stored it in a Glycerol stock (cf. Protocols).</p>
+
−
 
+
−
    </section>
+
−
 
+
−
<!--BIOBRICK J23117ALT-->
+
−
    <section id="bbj23117alt" class="panel">
+
−
      <h1><small>Construction of pWJ89alt</small></br>BioBrick J23117alt promoter</h1>
+
−
      <p>Coming soon</p>
+
−
    </section>
+
−
 
+
−
 
+
−
 
+
−
<!--ACTIVITY ASSAY-->
+
−
    <section id="activityassay" class="panel">
+
−
      <h1>Transistor activity assay</h1>
+
−
      <p>Coming soon</p>
+
−
    </section>
+
−
 
+
  
−
<!--PRIMER TABLE-->
 
−
    <section id="primers" class="panel">
 
−
      <h1>Primer table</h1>
 
−
      <table>
 
−
        <tr>
 
−
          <th>Name</th>
 
−
          <th>Sequence</th>
 
−
          <th>Associated part</th>
 
−
        </tr>
 
−
        <tr>
 
−
          <td>f_Gbs_pdCas9</td>
 
−
          <td>CTCGAGTAAGGATCTCCAG</td>
 
−
          <td>pdCas9</td>
 
−
        </tr>
 
−
        <tr>
 
−
          <td>f_Gbs_sgRNA-CtoA</td>
 
−
          <td>GTCGGCGATGGTGGTAGCTAATTATGTTCCctcgctcactgactcgctac</td>
 
−
          <td>sgRNA cassettes</td>
 
−
        </tr>
 
−
        <tr>
 
−
          <td>f_Gbs_sgRNA-DtoA</td>
 
−
          <td>CTAGACCTAACTGAGATACTGTCATAGACGctcgctcactgactcgctac</td>
 
−
          <td>sgRNA cassettes</td>
 
−
        </tr>
 
−
        <tr>
 
−
          <td>f_Gbs_sgRNA-A</td>
 
−
          <td>ctcgctcactgactcgctac</td>
 
−
          <td>sgRNA cassettes</td>
 
−
        </tr>
 
−
          <td>f_Gbs_w</td>
 
−
          <td>ACACGCATTGATTTGAGTCA</td>
 
−
          <td>pWJ66</td>
 
−
        </tr>
 
−
        <tr>
 
−
          <td>f_Mt_A2080C_pdCas9-w</td>
 
−
          <td>TGACTTTTCGcATTCCTTATTATGTTG</td>
 
−
          <td>pdCas9-w</td>
 
−
        </tr>
 
−
        <tr>
 
−
          <td>r_Gbs_pdCas9</td>
 
−
          <td>GTCACCTCCTAGCTGACTC</td>
 
−
          <td>pdCas9</td>
 
−
        </tr>
 
−
        <tr>
 
−
          <td>r_Gbs_sgRNA-B</td>
 
−
          <td>tggcatcttccaggaaatc</td>
 
−
          <td>sgRNA cassettes/td>
 
−
        </tr>
 
−
        <tr>
 
−
          <td>r_Gbs_sgRNA-BtoC</td>
 
−
          <td>GGAACATAATTAGCTACCACCATCGCCGACtggcatcttccaggaaatc</td>
 
−
          <td>sgRNA cassettes</td>
 
−
        </tr>
 
−
        <tr>
 
−
          <td>r_Gbs_sgRNA-BtoD</td>
 
−
          <td>CGTCTATGACAGTATCTCAGTTAGGTCTAGtggcatcttccaggaaatc</td>
 
−
          <td>sgRNA cassettes/td>
 
−
        </tr>
 
−
        <tr>
 
−
          <td>r_Gbs_w</td>
 
−
          <td>atttgatgcctggagatccttactcgagTTAACGACGACCTTCAGCA</td>
 
−
          <td>pWJ66</td>
 
−
        </tr>
 
−
        <tr>
 
−
          <td>r_Mt_A2080C_pdCas9-w</td>
 
−
          <td>AGATTTTTTCAATCTTCTCACG</td>
 
−
          <td>pdCas9-w</td>
 
−
        </tr>
 
−
        <tr>
 
−
          <td>r_Scq_pdCas9_w_sgRNA</td>
 
−
          <td>ctgatttgagcgtcagat</td>
 
−
          <td>pdCas9-w-sgRNA</td>
 
−
        </tr>
 
−
      </table>
 
−
    </section>
 
  
 
</div>
 
</div>

Revision as of 10:54, 8 September 2015

EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits

E. Coli Laboratory Notebook

Construction of pdCas9-w

pdCas9-w was constructed using PCR and Gibson assembly from the following plasmids:

  • pdCas9 (pdCas9-bacteria was a gift from Stanley Qi, Addgene plasmid # 44249): dCas9 controlled by a Tetracyclin inducible promoter and a Chlorampheicol resistance gene
  • pWJ66 (pWJ66 was a gift from Luciano Marraffini, Addgene plasmid # 46570): tracrRNA, w-dCas9 (dCas9 fused at its C-terminal to rpoZ, which encodes for the w subunit of RNA polymerase) and CRISPR array. (The w subunit recruits RNAP)

pdCas9 was opened by PCR and the w subunit was extracted from pWJ66 by PCR. The w subunit was then fused to the C-terminal of dCas9 by Gibson assembly, the final product is pdCas9-w.
A site-directed mutagenesis was done on this plasmid to eliminate an EcoRI restriction site. This will allow us to submit pdCas9-w as a BioBrick.

Construction of pdCas9-wOpen pdCas9 by PCR

We received plasmid pdCas9 in bacteria and did a Miniprep (cf. Protocols) on overnight cultures to isolate it. This step consists of linearizing pdCas9 by PCR.

Materials and method

  • 20 µL Phusion PCR (cf. Protocols) of pdCas9 with primers f_Gbs_pdCas9 and r_Gbs_pdCas9
  • PCR product purification (cf. Protocols)
  • Agarose gel electrophoresis of 2 µL purified PCR products (with 1kb Generuler ladder)

Results

Linearized pdCas9-w is expected to be 6705 bp.
The first try of this PCR was unsuccessful (gel is not shown here). For our second try, we tested many parameters: HF vs. GC buffer, different annealing temperatures and different extension times. This time, many, but not all, of our samples were successfully amplified (cf. Fig.1). The difficulty of this PCR is probably due to the fact that the size of the ampicon is very long, almost 7 kb.
For next steps, sample from lane 1 (cf. Fig.1) was used.

Figure 1
Fig.1 - Gel of linearized pdCas9
Click on the figure to view larger

Construction of pdCas9-w
Extract w subunit from pWJ66 by PCR

We received plasmid pWJ66 in bacteria and did a Miniprep (cf. Protocols) on overnight cultures to isolate it. This step consists of extracting rpoZ, the w subunit, from pW66 by PCR.

Materials and method

  • 20 µL Phusion PCR (cf. Protocols) of pWJ66 using primers f_Gbs_w and r_Gbs_w.
  • PCR product purification (cf. Protocols)
  • Agarose gel electrophoresis of 2 µL purified PCR products (with 1kb Generuler)

Results

Successful PCR reactions are expected to yield 340 bp fragments.
PCR was succesful for sample visible gel. (cf. Fig.2)

Figure 2
Fig.2 - Gel of w subunit extracted from pWJ66
Click on the figure to view larger

Construction of pdCas9-w
Gibson assembly of pdCas9-w

This step consists of fusing the w subunit to dCas9 by Gibson assembly, using the PCR products from previous steps. We transformed cells with the Gibson assembly product and tested for colonies that contain the construct by colony PCR, restriction digest and sequencing.

Materials and method

  • Gibson assembly (cf. Protocols) with purified PCR products:
    • Open pdCas9: 0.02 pmol = 95 ng
    • w subunit extracted from pWJ66: 0.06 pmol = 12.5 ng
  • Transformation (cf. Protocols) of ultra-competent DH5a cells (NEB) with Gibson assembly product, spreading on Chloramphenicol plates
  • Colony PCR (cf. Protocols) of colonies from plate used for culture of transformed cells with primers f_Gbs_w and r_Scq_pdCas9_w_sgRNA primers + agarose gel electrophoresis of 2 µL PCR prodcuts (with 1kb Generuler)
  • Miniprep (cf. Protocols) of overnight liquid cultures in 5 mL LB with Chloramphenicol of colonies from plate used for culture of transformed cells to isolate pdCas9-w plasmids
  • Restriction digest (Cf. Protocols) of pdCas9-w plasmids with BamHI and KpnI seperately + agarose gel electrophoresis of 2 µL digested products (with 1kb Generuler)
  • Sequencing (Microsynth) of one colony for which Gibson assembly worked according to colony PCR and restriction analysis

Results

Colony PCR

Primers were placed such as amplicons are 666 bp if Gibson assembly worked and 396 bp if the plasmid self-ligated.
Lane "C" is a negative control: PCR was run with all components except template DNA, ie. cells. It is empty which means there is no contamination.
Gibson assembly seems to have worked for some of our samples. (cf. Fig.3)
To avoid working with too many samples, we kept the ones from lanes 16, 22, 25, 31, 37 and 43 for the Minipreps and restriction digest. We did overnight liquid cultures of these colonies.

Figure 3a Figure 3c
Figure 3b Figure 3d
Fig.3 - Gels of colony PCR of dCas9-w Gibson assembly products
Click on the figure to view larger

Restriction digest

pdCas9-w samples from different colonies are present on gels in triplicates in the following order:

  • Undigested - expected to yield 7 kb circular plasmid (migrates faster than linear fragments of the same size)
  • Digested by BamHI - expected to yield two fragments of 6147 bp and 834 bp if insert is present or one 6705 bp fragment if it is not
  • Digested by KpnI - expected to yield two fragments of 45334 bp and 2447 bp if insert is present or one 6705 bp fragment if it is not

BamHI and KpnI are both unique cutters in pdCas9 (without the insert) and double cutters in pdCas9-w (with the insert).
Too much ladder was loaded so it is difficult to estimate the size of the fragments. The smaller fragments are also very difficult to see.
By looking at the relative heights, we can say that all colonies seem to have the w subunit insert. There is only the undigested sample for colony 16 that is not visible, probably due to an error while loading the gel.
Sequencing confirmed that sample 22 is in fact pdCas9-w. We used this sample for the next steps and stored it as a glycerol stock (c.f. Protocols).

Figure 4a Figure 4b Figure 4c
Fig.4 - Gels of restriction digest of pdCas9-w Gibson assembly products
Click on the figures to view larger

Sequencing

As dCas9-w is very long, only part of it was sequenced (the w subunit and its surrounding base pairs). No mutations were detected.

EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits

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