Team:Freiburg/Project/pRIG15 11

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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)

Figure 1. 12,5% SDS-PAGE analysis of the protein purification of C. tetani antigen. 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

Figure 2. Western Blot of his-tagged C. tetani 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.

Link to genebank file: BBa_K1621003.gb.

¹⁾ Yu et al. 2011. Enhanced expression of soluble recombinant tetanus neurotoxin Hc in Escherichia coli as a tetanus vaccine candidate. Immunobiology