Team:Dundee/Strategy

Strategy

Type VI Secretion System

We choose to isolate the T6SS from Salmonella enterica typhimurium as only two genes of the 13 involved would require site directed mutagenesis to remove biobrick restriction sites. Each of the 13 genes were cloned individually by PCR and then digested using either BclI or BglII and HindIII. The primers used to clone the genes included BamHI, PstI and HindIII. In addition, several included His tagged to allow characterisation by Western blots. The genes to be cloned are all shown in the diagram above. As indicated by the overlaps, ClpV, VipA, VipB and TssJ, TssK (referred to hereafter as ClpV and TssJK) were cloned as in the original operon.
DH5α cells were transformed with pUNI PROM from stock and grown overnight. We were then able to extract the plasmid using Qiagen miniprep kits and digest them ready for gene insertion. The plasmid was digested HindIII and BamHI to allow the insertion of the genes using suicide ligation between BamHI and BclI or BglII.
Initially all gene fragments were ligated into pUNI PROM and transformed into DH5α E. coli cells. Those that were successful were sent for sequencing. TssA was the first gene to be fully sequenced and as a result this was chosen as the starting point for combinatorial cloning. This plasmid was digested BamHI HindIII to allow the next gene, ClpV, to be added. Following successful transformation the plasmid was extracted and digested EcoRI HindIII. The product was run on a gel to check that an insert of the predicted size was present. Following this, the plasmid containing the TssA-ClpV clone was sent for sequencing using a primer designed to read from near the end of TssA into the next gene and a pUNI PROM reverse primer.
Each gene was sequentially added to this plasmid in the way described above to give the first plasmid displayed in the diagram below. We found that once this plasmid had nine of the 13 genes cloned it was very hard to add any more genes to it. To clone the last five genes we began the second plasmid shown below starting with TssM. The remaining genes were added to this plasmid using the same method as above until all genes were cloned.

Endolysin fusions

A second PCR was performed for Hcp and VgrG. In this reaction, the reverse primer lacked a stop codon and contained an XbaI site, this would allow these genes to be fused with the endolysin. The PCR products were digested BamHI XbaI and ligated into pUNI PROM cut with the same enzymes.
Neil Fairweather from Imperial College London was kind enough to send us a synthetic gene for the endolysin. This was cloned using PCR and the product was digested XbaI and HindIII. The plasmids containing Hcp and VgrG with the XbaI site were digested XbaI HindIII to allow ligation with the endolysin genes. These were transformed into competent E. coli cells and eventually sent for sequencing if successful.
The fused genes could then be cut out of the plasmid Bcl dIII and cloned into the combination plasmids as above.
This procedure was repeated for mCherry.

Biosensor

Biosensor genes were cloned as a single product, digested Bam HI- EcoRI, into pUNI prom. Due to the presence of a PstI site in the genes, site-directed mutagenesis had to be carried out to remove this site before it could be used further.