Difference between revisions of "Team:Freiburg/Project/pRIG15 11"
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− | <p><strong>Figure 2: 12 | + | <p><strong>Figure 2: 12.5% SDS-PAGE analysis of the purification of <a href="http://parts.igem.org/Part:BBa_K1621003">C. tetani antigen</a>.</strong> 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.</p> |
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Revision as of 16:42, 16 September 2015
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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 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).