The thirteen gene components that make up the type VI secretion system (T6SS) were successfully cloned into two separate pUNI-PROM vectors and are as follows:
Formation of the fuse genes was also successful, with endolysin fused to both Hcp and VgrG being produced. However, upon characterisation of these fusions, bands were not visible on a western blot. It was therefore assumed that expression of these genes was not substantial enough to be visible and thus IPTG was added to the cells to induce overexpression of the proteins via the T7 promoter located on the pUNI prom plasmid. This was successful, as bands for these fusions are clearly visible on the SDS-PAGE gels and western blots shown below.
Upon successful fusion of the endolysin to Hcp and VgrG, it was decided to also try fusing a reporter onto the proteins. The fluorescent protein m-Cherry was used for this experiment and strong bands were gained on the western blot after IPTG- induced expression of the genes.
Cascales et.al (2012) documented that the proteins VgrG and Hcp can be identified in the supernatant, possibly as a result of their fragility. From this knowledge, the supernatant from overnight samples were tested for fluorescence but the evidence obtained for the presence of these proteins was not convincing enough to say that they were released into the supernatant from this system.
As the combinatorial clones (pUNI-TssA etc.) were being constructed, radiolabelling experiments were carried out to confirm the heterlogous expression of each gene in
E.coli. As can be seen in the autoradiograph below (12% gel, 15µl of sample loaded) provides evidence that TssA, VipA, VipB, TssF, ClpV and TssK are transcribed and translated successfully in
E. coli. TssM was labelled in a separate experiment. 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. It was not possible to radiolabel TssJ with this method as the only methionines present are found in the the signal sequence, which is removed upon transport to the membrane. Additional radiolabelling experiments are being carried out on final clones to check expression of all remaining genes (TssL, VgrG, HCP etc.). These results will be added to the Biobrick experience pages
Upon completion of the two pUNI-PROM plasmid constructs, both induced and uninduced samples of all the proteins were run on SDS-PAGE gels. Additional bands were visible in the induced samples, which were identified as VipA (first plasmid), VipB (first plasmid) and TssM (second plasmid). The protein band believed to be LacI, the lac operon inhibitor, has been labelled with a yellow arrow. This was not derived from the plasmid constructs but is a feature of pUNI prom and so this explains its presence in both the non-induced and induced lanes. These gels were produced through the transformation of BL21 (DE3) cells which are renowned for their high-efficiency expression of genes under the control of the T7 promoter and 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, indicating that these bands are these proteins. 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 and protein identification.
The genes in the first pUNI-PROM plasmid: TssA, VipA, VipB, ClpA, TssK, TssE and TssF, are all his-tagged. A future experiment will involve western blot analysis of an induced culture as another means of characterisation of the proteins.
In order to create a complete secretion system the cell must contain all of the thirteen genes cloned in this project. For a single cell this would require transformation with more than one plasmid as this number of genes could not be cloned into a single plasmid. A third plasmid, confering chloramphenicol resistance, pACY-Duet2, was thus created:
The co-expression of one of the above plasmids with the first pUNI-PROM plasmid containing TssA to TssF will be carried out in order to test if Hcp or Hcp-endo and VgrG and or VgrG-endo can be secreted by
E. coli.
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