Team:Caltech/Protocols/Coculture Inhibition Assay

The engineered strain JI377 was transformed with pSB1A2 (AmpR) harboring luxR-spxB. The target strain was JI377 transformed with pSB1AK3 (AmpR KanR) harboring only a transcriptional terminator (part B0015). The negative control was JI377 transformed with a modified pUC18 vector (AmpR) containing galactose oxidase. (Kindly provided by Professor Arnold at Caltech.) Overnight cultures in LB were backdiluted 1:100 into SOC +IPTG +Amp, and grown to an OD600 of ~0.8. To begin the assay, the target strain was inoculated into 2.5 ml cultures of the engineered or control strains in amounts of (A) 1:1,000 (B) 1:10,000 and (C) 1:100,000. Co-cultures were immediately serially diluted and plated to single colonies on LB+Kan plates for CFU counting. (Co-culture “A” plated at dilutions of 1:100, 1:1,000, and 1:10,000. Co-culture “B” plated at dilutions of 1:1,000 1:10,000 and 1:100,000. Co-culture “C” plated at dilutions of 1:10,000 1:100,000 and 1:1,000,000.) The co-cultures were induced to produce hydrogen peroxide by bringing them to 10 nM AHL. Co-cultures were incubated for 6hrs and then plated. After incubation at 37C, the CFU of each plate was counted.