"
Team:Dundee/Results
From 2012.igem.org
Revision as of 10:28, 25 September 2012 by Nici Morrice (Talk | contribs)
Characterisation
The thirteen gene components that make up the type VI secretion system (T6SS) were successfully cloned into two separate PT7.5 pUNI-PROM vectors and are as follows:
The overall aim of this project is that of targeting C. difficile for removal through the attachment of an endolysin specific towards C. difficile onto the T6SS the team had cloned. Not only was this endolysin specifically attached onto the tip of the T6SS (VgrG) but it was also possible to fuse the endolysin gene onto the shaft (Hcp) of the T6SS too. Upon characterisation of these fusions, bands were not visible. It was then assumed that expression of these genes was not substantial enough to be visible and thus a T7 promoter was used and induced through the addition of IPTG and then bands for these fusions were clearly seen on SDS-PAGE gels and western blots.
Upon successful fusion it was questioned as to whether it was possible to fuse a completely different substrate onto VgrG and Hcp like that of the fluorescence protein m-Cherry. This was in fact possible and again through induced expression with IPTG, bands were clearly visible. Cascales et.al 2012 documented that the proteins, VgrG and Hcp can be identified in the supernatant possibly due to their fragility. From this knowledge, the supernatant from overnight samples were tested for fluorescence but unconvincing evidence for its presence was obtained.
As the combinatorial clones were being constructed, characterisation experiments were carried out in the form of 35S-radiolabelling. As seen in the autoradiograph below (12% gel, 15µl of sample loaded), evidence to the expression of TssA, vipA, vipB, TssF, ClpV (faint band) and TssK were witnessed. Two faint bands are visible in the lane with TssM alone (new plasmid). Due to its size (143kDa) the top band is believed to be TssM. TssJ was unable to be characterised through this method as the only methionine’s present are those found in the signal sequence which are removed upon transport to the membrane.
