|
|
| (7 intermediate revisions by 5 users not shown) |
| Line 29: |
Line 29: |
| | <div class="content_box"> | | <div class="content_box"> |
| | | | |
| − | <h1 class="sectionedit1">pRIG15_13</h1> | + | <h1 class="sectionedit1"> </h1> |
| | <div class="level1"> | | <div class="level1"> |
| − |
| |
| − | <div class="image_box right">
| |
| − | <div class="thumb2 trien" style="width:410px">
| |
| − | <div class="thumbinner">
| |
| − | <a href="https://static.igem.org/mediawiki/2015/8/8d/Freiburg_labjournal-cloning-prig15_13.jpg" class="lightbox_trigger">
| |
| − | <img src="https://static.igem.org/mediawiki/2015/8/8d/Freiburg_labjournal-cloning-prig15_13.jpg" width="400px">
| |
| − | <div class="thumbcaption">
| |
| − | </a>
| |
| − | <p><strong>Figure 1: pRIG15_13.</strong><a href="http://parts.igem.org/Part:BBa_K1621007" target="_blank">BBa_K162007</a> inserted into the submission backbone pSB1C3.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − | <p>
| |
| − | To generate a single chain variable fragment against dihydroxyacid dehydratase the group of Prof. Dr. Hust used a human naive antibody gene library. <sup><a class="fn_top" href="#fn__1" id="fnt__1" name="fnt__1">1)</a></sup> They thankfully provided us with the sequence of the anti-dihydroxyacid dehydratase already cloned into an expression vector.<br>
| |
| − | </p>
| |
| − |
| |
| − | <p>
| |
| − | To insert the sequence for the <i>Salmonella</i> antibody (anti-dehydroxyacid dehydratase) into <a href="" target="_blank">pSB1C3</a>, we designed Gibson primers with compatible overhangs that also included the start codon ATG. This fragment was amplified via <a clas="wikilink1" href="https://2015.igem.org/Team:Freiburg/Labjournals/Cloning/August#PCRbb13" title="PCRbb13">PCR</a> from the expression vector we obtained from Prof. Dr. Hust and then assembled with the digested pSB1C3 backbone using Gibson Assembly.
| |
| − | To prove correct insertion of our fragment we performed a test digest (Link Labjournal) and verified the part by sequencing.
| |
| − | </p>
| |
| − |
| |
| − | <p>
| |
| − | We used the plasmids sent to us by Prof. Dr. Hust to express both proteins, <i>Salmonella</i> dihydroxyacid dehydratase and anti-dihydroxyacid dehydratase, in <i>E.coli</i>. We could show overexpression by SDS-PAGE (Fig. 2) verified the interaction of both proteins by Western Blot (Fig. 3).
| |
| − | </p>
| |
| − |
| |
| − | <p>
| |
| − | Get the sequence in GeneBank format: <a class="media" href="https://static.igem.org/mediawiki/2015/9/9b/Freiburg_2015_BBa_K1621007.gb" title="2015_Freiburg_BBa_K1621007" src="https://static.igem.org/mediawiki/2015/9/9b/Freiburg_2015_BBa_K1621007.gb">BBa_K1621007.gb</a>.
| |
| − | </p>
| |
| − | </div>
| |
| − | <div class="image_box left">
| |
| − | <div class="thumb2 trien" style="width:410px">
| |
| − | <div class="thumbinner">
| |
| − | <a href="https://static.igem.org/mediawiki/2015/9/9c/Freiburg_SDS_Salmonella_neu.png" class="lightbox_trigger">
| |
| − | <img src="https://static.igem.org/mediawiki/2015/9/9c/Freiburg_SDS_Salmonella_neu.png" width="400px">
| |
| − | <div class="thumbcaption">
| |
| − | </a>
| |
| − | <p><strong>Figure 2: 12,5% SDS-PAGE analysis of the protein purification of <a href="http://parts.igem.org/Part:BBa_K1621006">S. Typhimurium antigen</a> (dehydroxyacid dehydratase) and <a href="http://parts.igem.org/Part:BBa_K1621007"><i>S.</i> Typhimurium antibody</a> (anti-dehydroxyacid dehydratase).</strong> Protein purification was performed with gravity flow columns and Ni-NTA Agarose. The protein was eluated by 500mM Imidazole. The expected molecular weight for the <i>S.</i> Typhimurium antigen is 63 kDa and 37 kDa for the antibody, respectively. FT-Flowthrough, W-Wash, E-Elution.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | <div class="image_box right">
| |
| − | <div class="thumb2 trien" style="width:410px">
| |
| − | <div class="thumbinner">
| |
| − | <a href="https://static.igem.org/mediawiki/2015/0/0a/Freiburg_labjournal-cloning-salmonella_westernblot-1.png" class="lightbox_trigger">
| |
| − | <img src="https://static.igem.org/mediawiki/2015/0/0a/Freiburg_labjournal-cloning-salmonella_westernblot-1.png" width="400px">
| |
| − | <div class="thumbcaption">
| |
| − | </a>
| |
| − | <p><strong>Figure 3: Western Blot of <a href="http://parts.igem.org/Part:BBa_K1621006"><i>S.</i> Typhimurium antigen</a> (dehydroxyacid dehydratase) and <a href="http://parts.igem.org/Part:BBa_K1621007"><i>S.</i> Typhimurium antibody</a> (anti-dehydroxyacid dehydratase).</strong> (A) Western Blot of the antigen as well as of the antibody with anti-His HRP Conjugate. Both protein are His-tagged. The expected molecular weight for the <i>S.</i> Typhimurium antigen is 63 kDa and 37 kDa for the antibody, respectively. (B) In this Western Blot the <i>S.</i> Typhimurium antigen was analyzed by 12,5% SDS-PAGE. Purified <i>S.</i> Typhimurium antibody was used in a 1:100 dilution. Additionally, this single chain antibody is fused to a c-Myc Tag. We used an anti-c-Myc antibody (1:1000; rabbit). For detection the anti-rabbit HRP antibody (1:5000) was used.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − | </br>
| |
| − | </br>
| |
| − | </br>
| |
| − | </br>
| |
| − | </br>
| |
| − | </br></br></br></br></br></br></br></br></br></br></br></br></br></br>
| |
| − |
| |
| − |
| |
| − | <div class="footnotes">
| |
| − | <div class="fn"><sup><a class="fn_bot" href="#fnt__1" id="fn__1" name="fn__1">1)</a></sup>
| |
| − | <a class="urlextern" href="http://www.ncbi.nlm.nih.gov/pubmed/22703709" rel="nofollow" target="_Blank" title="http://www.ncbi.nlm.nih.gov/pubmed/22703709"> Meyer et al. 2012. Identification of immunogenic proteins and generation of antibodies against Salmonella Typhimurium using phage display. BMC biotechnology</a></div>
| |
| − | </div>
| |
| | | | |
| | </div> | | </div> |
