Difference between revisions of "Team:Freiburg/Project/pRIG15 13"
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− | To generate a single chain variable fragment against dihydroxyacid dehydratase the group of Prof. Dr. | + | To generate a single chain variable fragment against dihydroxyacid dehydratase the group of Prof. Dr. Dübel 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> |
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− | To insert the sequence for the <i>Salmonella</i> antibody (anti-dehydroxyacid dehydratase) into <a href="http://parts.igem.org/Part:pSB1C3" 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. | + | To insert the sequence for the <i>Salmonella</i> antibody (anti-dehydroxyacid dehydratase) into <a href="http://parts.igem.org/Part:pSB1C3" 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. Dübel and then assembled with the digested <a href="http://parts.igem.org/Part:pSB1C3" target="_blank">pSB1C3</a> backbone using <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/Classic_vs_Gibson" title="Gibson">Gibson Assembly</a>. |
To prove correct insertion of our fragment we performed a test digest and verified the part by sequencing. | To prove correct insertion of our fragment we performed a test digest and verified the part by sequencing. | ||
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− | We used the plasmids sent to us by Prof. Dr. | + | We used the plasmids sent to us by Prof. Dr. Dübel 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 (figure 2) verified the interaction of both proteins by Western Blot (figure 3). |
</p> | </p> | ||
Revision as of 14:43, 18 September 2015
pRIG15_13
To generate a single chain variable fragment against dihydroxyacid dehydratase the group of Prof. Dr. Dübel used a human naive antibody gene library. 1) They thankfully provided us with the sequence of the anti-dihydroxyacid dehydratase already cloned into an expression vector.
To insert the sequence for the Salmonella antibody (anti-dehydroxyacid dehydratase) into pSB1C3, we designed Gibson primers with compatible overhangs that also included the start codon ATG. This fragment was amplified via PCR from the expression vector we obtained from Prof. Dr. Dübel and then assembled with the digested pSB1C3 backbone using Gibson Assembly. To prove correct insertion of our fragment we performed a test digest and verified the part by sequencing.
We used the plasmids sent to us by Prof. Dr. Dübel to express both proteins, Salmonella dihydroxyacid dehydratase and anti-dihydroxyacid dehydratase, in E.coli. We could show overexpression by SDS-PAGE (figure 2) verified the interaction of both proteins by Western Blot (figure 3).
Link to Registry: BBa_K1621007