Using Type VI Secretion as an Antibacterial Agent

The T6SS we want to use has several difficulties in its current form. First, it is found in P. aeruginosa which can be pathogenic, as well as not being an enteric bacteria. As our model is anti-enteric, the native system would be unfeasible.

Secondly, T6SS is highly regulated at both the transcriptional and post-transcriptional level. We want our toxin delivery system to be expressed in either a constitutive or easily inducible manner.

Heterologous Expression of T6SS

In order to create a probiotic application for this system, we first attempt to express it heterologously in non-pathogenic E. Coli. Starting from a Fosmid containing our T6SS, we are using Recombineering as described by Warming et al (2005) to replace the strict native regulation with robust T7 promoters to create strong expression of the T6SS.

All the essential genes for our T6SS are contained within two putative operons, encoded in opposite directions. The native promoters for both operons are found in the same intergenic region, between fha1 and tssA1. Therefore, we can easily replace the promoter regions for both operons in one step.

The Essential T6SS

Toxin/Antitoxin Inducible Circuit

At the same time, we are creating inducible circuits to allow for production and regulation for the antibacterial toxin and antitoxin system, allowing us to create "assassin cells" upon induction.