Difference between revisions of "Team:Freiburg/Project/pRIG15 15"
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| − | To insert the sequence for the <i>Salmonella</i> dehydroxyacid dehydratase 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#PCRSal_mut" title="PCRSal_mut">PCR</a> from the expression vector we obtained from Prof. Dr. Hust and then assembled with the digested pSB1C3 backbone using Gibson Assembly. | + | To insert the sequence for the <i>Salmonella</i> dehydroxyacid dehydratase into <a class="urlextern" href="http://parts.igem.org/Part:pSB1C3" target="_blank" title="pSB1C3">pSB1C3</a> 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#PCRSal_mut" title="PCRSal_mut">PCR</a> from the expression vector we obtained from Prof. Dr. Hust and then assembled with the digested <a class="urlextern" href="http://parts.igem.org/Part:pSB1C3" target="_blank" title="pSB1C3">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 <a class=wikilink1" href="https://2015.igem.org/Team:Freiburg/Labjournals/Cloning/August#TDbb15" title="TDbb15">test digest</a> and verified the part by sequencing. | To prove correct insertion of our fragment we performed a <a class=wikilink1" href="https://2015.igem.org/Team:Freiburg/Labjournals/Cloning/August#TDbb15" title="TDbb15">test digest</a> and verified the part by sequencing. | ||
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Revision as of 21:52, 16 September 2015
pRIG15_15
Salmonella Typhimurium is a pathogen causing gastrointestinal infections and which is particularly dangerous because it already gained resistence against multiple antibiotics. 1) In collaboration with the group from Prof. Dr. Hust from the University of Braunschweig we were able to get an expression vector containing the sequence for a putative dihydroxyacid dehydratase and an additional vector bearing the sequence for a respective single chain variable fragment, anti-putative dihydroxyacid dehydratase (Bba_K1621007). 2)
To insert the sequence for the Salmonella 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. Hust 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 performed analyses by SDS-PAGE and Western Blot to show successful overexpression in E. coli and the specific binding properties of the antigen and respective antibody.
Link to GeneBank file: BBa_K1621006.gb.
Link to Registry: BBa_K1621006
