Team:Freiburg/Project/pRIG15 11

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pRIG15_11

Figure 1: pRIG15_11. BBa_K162003 inserted into the sumission backbone pSB1C3.

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. Although this part is derived from the neurotoxin it is not pathogenic itself.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 and then assembled with the digested pSB1C3 backbone using Gibson assembly. To prove correct insertion of our fragment we did a test digest 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 (figure 2). As verification for our results we use a western blot (figure 3).

Figure 2: 12.5% SDS-PAGE analysis of the purification of C. tetani antigen. Protein purification was performed with gravity flow columns and Ni-NTA Agarose. The protein was eluted by 500mM imidazole. The expected molecular weight is 50 kDa. FT-Flowthrough, W-Wash, E-Elution.

Figure 3: 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.
Link to Registry: BBa_K1621003

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