Team:Freiburg/Project/pRIG15 11
![](https://static.igem.org/mediawiki/2015/8/85/Freiburg_wiki_background_2_parted_left.png)
![](https://static.igem.org/mediawiki/2015/f/f4/Freiburg_wiki_background_2_parted_right.png)
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)
![](https://static.igem.org/mediawiki/2015/c/cb/Freiburg_labjournal-cloning-tetanus_sdspage-1.png)