The engineered strain was JI377 transformed with K137076 (spxB) in pSB1A2 (AmpR).
The target strain was JI377 transformed with B0015 (a transcriptional terminator) in pSB1AK3 (AmpR KanR).
The negative control was JI377 transformed with a modified pUC18 vector (AmpR) containing galactose oxidase. (Kindly provided by Professor Arnold at Caltech.)
Protocol
Grow overnight cultures of each strain in LB + Amp.
In the morning, back dilute cultures 1:100 into SOC + IPTG + Amp and grow to an OD600 of ~0.8.
To begin the assay, inoculate the target strain 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.
Immediately serially dilute aliquots of the cocultures and plate to single colonies on LB+Kan plates for CFU counting.
Co-culture "A" should be plated at dilutions of 1:100, 1:1,000, and 1:10,000.
Co-culture "B" should be plated at dilutions of 1:1,000 1:10,000 and 1:100,000.
Co-culture "C" should be plated at dilutions of 1:10,000 1:100,000 and 1:1,000,000.
Induce the co-cultures to produce hydrogen peroxide by bringing them to 10 nM AHL.
Incubate co-cultures for 6hrs and then plate to single colonies as before.
After incubation at 37C, count the CFU of each plate.