Team:Caltech/Protocols/Coculture Inhibition Assay

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Coculture Inhibition Assay

Strains

 * 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

 * 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 aliquots of the cocultures and plate to single colonies on LB+Kan plates for CFU counting.
 * 5) *Co-culture "A" should be plated at dilutions of 1:100, 1:1,000, and 1:10,000.
 * 6) *Co-culture "B" should be plated at dilutions of 1:1,000 1:10,000 and 1:100,000.
 * 7) *Co-culture "C" should be plated at dilutions of 1:10,000 1:100,000 and 1:1,000,000.
 * 8) Induce the co-cultures to produce hydrogen peroxide by bringing them to 10 nM AHL.
 * 9) Incubate co-cultures for 6hrs and then plate to single colonies as before.
 * 10) After incubation at 37C, count the CFU of each plate.

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