Team:Dundee/Results

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.


Upon completion of the two pUNI-PROM plasmid constructs, protein samples were run on SDS-PAGE gels as well as induced samples. Through identification of additional bands visible in the induced samples vipA along with vipB were identified from the first p-UNI PROM plasmid and TssM from the second. The protein band believed to be LacI, the lac operon inhibitor has been labelled with a yellow arrow and is present in both the non-induced and induced lanes (not derived from the plasmid constructs). These gels were produced through the transformation of BL21 (DE3) cells which are renowned for their high-efficiency protein expression of genes under the control of the T7 promoter. These samples were validated via Tryptic peptide mass fingerprinting, the data from which is shown below. The known protein sequences are given with the matching peptides shown in red. The bands vipA and vipB show almost perfect sequence matching, this indicates that these bands are that of vipA and vipB. In the case of TssF only a small fragment is gained from the whole peptide sequence matching that of the N terminus. The band initially thought to be TssF was consequently labelled as TssF’ on the gel below. It is thought that this protein is unstable and is a broken down fragment of TssF. The mass spec data gained for TssM illustrates a fairly high match of peptides which is convincing enough to label this band as that of TssM.


In order to identify TssE, a gradient SDS-PAGE gel will be performed which should separate it from vipA which has a very similar size. Due to many of the proteins having a molecular weight of between ~37 to 100kDa, distinguishing between these bands was difficult. It is hoped that another autoradiograph will be carried out with a lower percentage (10% rather than 12%) and will be run for a much longer period of time until the 37kDa marker is at the base of the gel in order to separate this chunk of large proteins for clearer labelling e.g. to identify the protein TssL. In the case of the first pUNI-PROM plasmid (TssA to Hcp) TssA, vipB, TssK, TssE and TssF are all his-tagged thus a future experiment will involve western blot analysis of an induced culture as another means of characterisation.

In order to create a complete secretion system the cell must contain Hcp and all thirteen genes. For a single cell this would require transformation with more than one plasmid thus a third plasmid was created on the chloramphenicol plasmid, pACY-Duet2 as seen below.