Building a Type VI Dependent Probiotic

One of the major hurdles of creating our probiotic is the high level of regulation that this system is under in its native host. Several transcriptional and post-transcriptional regulators are present in both promoter regions, which prevented the system from expressing in E. coli. This problem was complicated by the large size of the secretion system, which made standard cloning impossible. We also had to create a set of vectors which would contain the toxin/antitoxin system that would activate in the presence of our signal molecule.

Creating the Toxin(Tse2)/Antitoxin(Tsi2) system

The Tse2/Tsi2 locus was amplified using PCR with primers that amplified the region from the start codon of Tse2 to the stop codon of Tsi2. The primers were designed to flank the Tse2/Tsi2 product with approximately 40bp regions of homology to the psb3k3-F2620 plasmid. The forward primer added 44 base pairs of homology to the 3' end of F2620 to the 5' end of the construct. The reverse primer added 38 base pairs of homology to the suffix end of psB3k3 to the 3' end of the construct. The Tse2/Tsi2 locus was then placed downstream from F2620 via Gibson Cloning. Gibson cloning is a method that joins regions of homology found on the 5' and 3' ends of linearized molecules. The end result of this Gibson cloning reaction was a circularized plasmid containing the final F2620-Tse2/Tsi2 construct in psb3k3. The Gibson reaction mixture was transformed into electrocompetent Dh5a, and the resulting colonies were screened for insert of expected length via double restriction digest followed by agarose gel electrophoresis. The plasmids that showed inserts of expected length were sequenced, and it was determined that multiple plasmids had F2620-Tse2/Tsi2 inserts with expected sequences.

Recombineering the type 6 secretion system

In order to replace the native promoters with more robust T7 promoters, we used a technique called Recombineering to flip out the region containing both promoters. We designed primers to create a cassette using PCR, containing the galK galactose metabolism gene flanked by 50 base pairs of homology with the promoter region. The cassette was inserted, giving us a selection factor for our final insertion of our new promoters. A new promoter cassette was designed in total using oligos that encoded the two T7 promoters flanked by 50 base pairs of homology, and inserted using recombineering to create a robust Type VI Secretion System.