Team:Caltech/Protocols/Coculture Inhibition Assay

Strains

• 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.)

Protocol

1. Grow overnight cultures of each strain in LB + Amp.
2. In the morning, back dilute cultures 1:100 into SOC + IPTG + Amp and grow to an OD600 of ~0.8.
3. 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.
4. Immediately serially dilute 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.
5. Induce the co-cultures were induced to produce hydrogen peroxide by bringing them to 10 nM AHL.
6. Incubate co-cultures for 6hrs and then plated to single colonies as before.
7. After incubation at 37C, count the CFU of each plate.