Western blotting for proper Tse2 expression

In order to determine that Tse2 and Tsi2 were only being produced in the presence of HSL, E. coli MG1655 containing the F2620-Tse2-Tsi2 construct was cultured in liquid LB containing either 10µM HSL, or no HSL. the cultures were pelleted, and western blotted for Tse2. The cultures grown in HSL+ media showed bands on the western blot (Figure x) indicative of Tse2 being produced when HSL is present. The cultures grown in media without HSL showed no bands ( Figure X), meaning that Tse2 is not being expressed unless HSL is present. This is exactly the behavior that was expected if the F2620-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.

Figure X: Western Blot Showing Proper Expression of Tse2

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.