Western blotting for proper Tse2 expression

In order to determine that Tse2 and Tsi2 were only being produced in the presence of 3OC6HSL, E. coli MG1655 containing the F2620-Tse2-Tsi2 construct was cultured in liquid LB containing either 10,000nM 3OC6HSL, or no HSL. the cultures were pelleted, and western blotted for Tse2. The cultures grown in 3OC6HSL+ media showed bands on the western blot (figure x) indicative of Tse2 being produced when 3OC6HSl is present. The cultures grown in media without 3OC6HSL showed no bands ( figure X), meaning that Tse2 is not being produced unless HSL is present. This is exactly the behavior that was expected if the Tse2/tsi2 system was working properly. The survival of cells in the HSL+ media despite the production of Tse2, combined with the sequence confirmation of Tse2 in the construct implies that tsi2 is working as an antitoxin.

T7 recombinant cassette insertion

SDS-PAGE Protein Array

SDS-PAGE for Type VI Secretion Protein. Expression was induced using 0.5 mM IPTG

Fha1 (Forkhead-associated protein) is an essential component of the Type VI Secretion System. We confirmed the utility of our promoter insertion by using an SDS-PAGE assay with anti-Fha antibody to probe for expressed protein. The recombinant fosmid was transformed into a T7 expression strain, BL21(DE3), which produces T7 RNA polymerase in the presence of IPTG. The Fha1 protein was expressed under induced conditions.