Team:Freiburg/Project/pRIG15 13
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pRIG15_13
To find the respective antibody against the putative dihydroxyacid dehydratase the group of Prof. Dr. Hust 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 (Link zum Labjournal-Eintrag) from the expression vectors we got from Prof. Dr. Hust 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.
We used the plasmids sent to us by Prof. Dr. Hust to express both proteins, Salmonella dihydroxyacid dehydratase and anti-dihydroxyacid dehydratase, in E.coli. We could show overexpression via a sds-page (Fig. 1) and western blot and we could additionally show interaction of both proteins with a western blot (Fig. 2)
![](https://static.igem.org/mediawiki/2015/c/c3/Freiburg_labjournal-cloning-salmonella_sdspage-1.png)
![](https://static.igem.org/mediawiki/2015/0/0a/Freiburg_labjournal-cloning-salmonella_westernblot-1.png)