Difference between revisions of "Team:Freiburg/Project/pRIG15 11"
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| + | <a class="media" href="https://static.igem.org/mediawiki/2015/1/1e/Freiburg_labjournal-cloning-prig15_11.jpg" title="labjournal:cloning:prig15_11.jpg"><img alt="" class="mediacenter" src="https://static.igem.org/mediawiki/2015/1/1e/Freiburg_labjournal-cloning-prig15_11.jpg" width="300"/></a></div><strong>Figure 1: pRIG15_11.</strong> <a href="http://parts.igem.org/Part:BBa_K1621003" target="_blank">BBa_K162003</a> inserted into the sumission backbone pSB1C3. </div> | ||
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| − | + | The bacterium <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/Diseases#Tetanus" title="Tetanus"><em>Clostridum tetani</em></a> can cause severe illness due to its neurotoxin which causes strong muscular spasms eventual leading to death. For our experiments we decided to use a sequence which codes for the carboxyl fragment of the heavy chain of tetanus neurotoxin.<sup><a class="fn_top" href="#fn__1" id="fnt__1" name="fnt__1">1)</a></sup> | |
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To insert the sequence for TeNT_Hc into pSB1C3 we designed Gibson primers with compatible overhangs that also included the start codon ATG. This fragment was amplified via <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Labjournals/Cloning/August#PCRbb11" title="PCRbb11">PCR</a> and then assembled with the digested pSB1C3 backbone using Gibson assembly. | To insert the sequence for TeNT_Hc into pSB1C3 we designed Gibson primers with compatible overhangs that also included the start codon ATG. This fragment was amplified via <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Labjournals/Cloning/August#PCRbb11" title="PCRbb11">PCR</a> and then assembled with the digested pSB1C3 backbone using Gibson assembly. | ||
To prove correct insertion of our fragment we did a <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Labjournals/Cloning/August#TDbb11" title="TDbb11">test digest</a> and sent the whole plasmid for sequencing. | To prove correct insertion of our fragment we did a <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Labjournals/Cloning/August#TDbb11" title="TDbb11">test digest</a> and sent the whole plasmid for sequencing. | ||
| − | During our project we inserted the sequence coding for TeNT_Hc into pET22b+ and induced overexpression of the protein in <em>E.coli</em>. We could show successfull overexpression of TeNT_Hc (Fig. | + | During our project we inserted the sequence coding for TeNT_Hc into pET22b+ and induced overexpression of the protein in <em>E.coli</em>. We could show successfull overexpression of TeNT_Hc (Fig.2). As verification for our results we use a western blot (Fig.3). |
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| − | <div class="thumb2 trien" style="width: | + | <div class="thumb2 trien" style="width:310px"><div class="thumbinner"><a class="media" href="https://static.igem.org/mediawiki/2015/c/cb/Freiburg_labjournal-cloning-tetanus_sdspage-1.png" title="labjournal:cloning:tetanus_sdspage-1.png"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/c/cb/Freiburg_labjournal-cloning-tetanus_sdspage-1.png" width="300"/></a></div><strong>Figure 2: 12,5% SDS-PAGE analysis of the purification of <a href="http://parts.igem.org/Part:BBa_K1621003">C. tetani antigen</a>.</strong> Protein purification was performed with gravity flow columns and Ni-NTA Agarose. The protein was eluted by 500mM imidazole. The expected molecular weight is 50 kDa. FT-Flowthrough, W-Wash, E-Elution</div> |
| − | The soluble fraction of the overexpressed protein was used for this Western Blot | + | |
| + | <div class="thumb2 trien" style="width:310px"><div class="thumbinner"><a class="media" href="https://static.igem.org/mediawiki/2015/d/d7/Freiburg_labjournal-cloning-tetanus_western-1.png" title="labjournal:cloning:tetanus_western-1.png"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/d/d7/Freiburg_labjournal-cloning-tetanus_western-1.png" width="300"/></a></div><strong>Figure 3: Western Blot of his-tagged <i>C. tetani</i> antigen with anti His Tag HRP Conjugate.</strong> The soluble fraction of the overexpressed protein was used for this Western Blot. Anti- His Tag HRP conjugated was used in a 1:1000 dilution in blocking buffer. The expected molecular weight is 50 kDa.</div> | ||
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| − | + | Get the sequence in genebank format: <a class="media" href="https://static.igem.org/mediawiki/2015/e/ee/Freiburg_2015_BBa_K1621003.gb" title="2015_Freiburg_BBa_K1621003" src="https://static.igem.org/mediawiki/2015/e/ee/Freiburg_2015_BBa_K1621003.gb">BBa_K1621003.gb</a>. | |
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<div class="footnotes"> | <div class="footnotes"> | ||
<div class="fn"><sup><a class="fn_bot" href="#fnt__1" id="fn__1" name="fn__1">1)</a></sup> | <div class="fn"><sup><a class="fn_bot" href="#fnt__1" id="fn__1" name="fn__1">1)</a></sup> | ||
Revision as of 13:23, 14 September 2015
pRIG15_11
The bacterium Clostridum tetani can cause severe illness due to its neurotoxin which causes strong muscular spasms eventual leading to death. For our experiments we decided to use a sequence which codes for the carboxyl fragment of the heavy chain of tetanus neurotoxin.1)
To insert the sequence for TeNT_Hc into pSB1C3 we designed Gibson primers with compatible overhangs that also included the start codon ATG. This fragment was amplified via PCR and then assembled with the digested pSB1C3 backbone using Gibson assembly. To prove correct insertion of our fragment we did a test digest and sent the whole plasmid for sequencing. During our project we inserted the sequence coding for TeNT_Hc into pET22b+ and induced overexpression of the protein in E.coli. We could show successfull overexpression of TeNT_Hc (Fig.2). As verification for our results we use a western blot (Fig.3).
