Because the copy number of each plasmid is different, we performed reciprocal assays in which we switched the protein fusions (i.e. from the low copy to the high copy plasmid, and vice versa) as internal controls. Both fused plasmid sets (pDD866 and pDD868, or pDD867 and pDD859) or the unfused vectors (pSR658 and pSR659) were co-transformed and co-expressed in the reporter strain
SU202. This strain has a chromosomal construct that consists of a lacZ reporter gene controlled by the strong sulA promoter, which contains an engineered LexA operator sequence. When there is no fusion to the LexA DBD, the strain constitutively expresses a high level of β-galactosidase. However, if a protein fused to the LexA DBD in pSR658 and another protein fused to the LexA DBD Selleckchem STA-9090 in pSR659 buy BAY 80-6946 can heterodimerize, a competent LexA dimer is formed that can bind to the engineered LexA operator and repress transcription of lacZ in the reporter strain SU202. Homodimers, if formed, cannot bind to the engineered operator site. Expression of the LexA fusion in pSR658 and pSR659 is induced by IPTG, and since β-galactosidase is a very stable enzyme, the reporter strain is routinely grown overnight with IPTG, so that any enzyme that was transcribed prior to induction of the LexA chimera has the opportunity to degrade. This strategy resulted in a more reliable and accurate quantitation of heterodimerization.
Following overnight incubation in LB broth with 1 mM IPTG, the reporter strain carrying pSR658 and pSR659, or the LexA DBD fusions, was diluted and grown to log phase in LB broth isothipendyl with 1 mM IPTG. The amount of heterodimerization was quantitated by the repression of lacZ activity as indicated
by β-galactosidase activity assays and compared to the activity of the reporter strain carrying pSR658 plus pSR659 (no fusion). The algorithm for determining β-galactosidase activity is: [OD420-(1.75*OD550)/t*v*OD600*1000, where t=time of reaction development in minutes, v=volume of sample in milliliters, and OD600 is the optical density of the culture at 600 nm . This equation allows normalization of different culture densities for comparison purposes. VapX and VapD: for these assays, vapX was fused to the LexA DBD in pSR658, resulting in pDD882, and to the LexA DBD in pSR659, resulting in pDD883. Likewise, vapD was fused to the LexA DBD in pSR659, resulting in pDD884, and the LexA DBD in pSR658, resulting in pDD885. Heterodimerization assays measuring β-galactosidase activity were carried out and quantitated as above. Each pair was analyzed at least three times in triplicate. Cloning and purification of VapD, Cat, and VapX To perform ribonuclease (RNase) activity assays, the cat (chloramphenicol acetyltransferase) gene was PCR-amplified from pACYC184 by high-fidelity polymerase and ligated to the SacI/XhoI-cut pET24b expression vector, resulting in Cat with a C-terminal polyhistidine tag in pDD689.