Difference between revisions of "Team:Freiburg/Project/pRIG15 11"
(Created page with " {{Freiburg/CSS}} {{Freiburg/Menubar}} {{Freiburg/wiki_content_start}} <html> <!-- Labjournal content goes in here --> <div class="content_box"> <h1 class="sectionedit1">pRIG...") |
|||
| Line 1: | Line 1: | ||
| − | |||
{{Freiburg/CSS}} | {{Freiburg/CSS}} | ||
{{Freiburg/Menubar}} | {{Freiburg/Menubar}} | ||
| Line 14: | Line 13: | ||
</p> | </p> | ||
<p> | <p> | ||
| + | <div class="flexbox"> | ||
<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="500"/></a> | <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="500"/></a> | ||
| + | </div> | ||
</p> | </p> | ||
<p> | <p> | ||
| Line 21: | Line 22: | ||
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.1). As verification for our results we use a western blot (Fig.2) | 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.1). As verification for our results we use a western blot (Fig.2) | ||
</p> | </p> | ||
| + | <div class="flexbox"> | ||
<div class="thumb2 trien" style="width:410px"><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="400"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/c/cb/Freiburg_labjournal-cloning-tetanus_sdspage-1.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Figure 1. 12,5% SDS-PAGE analysis of the protein purification of <a href="http://parts.igem.org/Part:BBa_K1621003">C. tetani antigen</a>. The protein purification was performed with gravity flow columns and Ni-NTA Agarose. The protein was eluated by 500mM imidazole. The expected molecular weight is 50 kDa.FT-Flowthrough, W-Wash, E-Elution</div></div></div><div class="thumb2 trien" style="width:410px"><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="400"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/d/d7/Freiburg_labjournal-cloning-tetanus_western-1.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Figure 2. Western Blot of his-tagged <em>C. tetani</em> antigen with anti His Tag HRP Conjugate. | <div class="thumb2 trien" style="width:410px"><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="400"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/c/cb/Freiburg_labjournal-cloning-tetanus_sdspage-1.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Figure 1. 12,5% SDS-PAGE analysis of the protein purification of <a href="http://parts.igem.org/Part:BBa_K1621003">C. tetani antigen</a>. The protein purification was performed with gravity flow columns and Ni-NTA Agarose. The protein was eluated by 500mM imidazole. The expected molecular weight is 50 kDa.FT-Flowthrough, W-Wash, E-Elution</div></div></div><div class="thumb2 trien" style="width:410px"><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="400"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/d/d7/Freiburg_labjournal-cloning-tetanus_western-1.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Figure 2. Western Blot of his-tagged <em>C. tetani</em> antigen with anti His Tag HRP Conjugate. | ||
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></div></div> | 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></div></div> | ||
| + | </div> | ||
</div> | </div> | ||
<div class="footnotes"> | <div class="footnotes"> | ||
Revision as of 13:28, 6 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 (Link zum Labjournal-Eintrag) and then assembled with the digested pSB1C3 backbone using Gibson assembly. To prove correct insertion of our fragment we did a test digest (Link Labjournal) 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.1). As verification for our results we use a western blot (Fig.2)
