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Team:Caltech/Protocols/Coculture Inhibition Assay
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===Strains=== | ===Strains=== | ||
| - | * The engineered strain | + | * The engineered strain was JI377 transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K137076 K137076] (spxB) in pSB1A2 (AmpR). |
| - | * The target strain was JI377 transformed with | + | * The target strain was JI377 transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 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.) | + | * The negative control was JI377 transformed with a modified pUC18 vector (AmpR) containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K137017 galactose oxidase]. (Kindly provided by Professor Arnold at Caltech.) |
===Protocol=== | ===Protocol=== | ||
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# In the morning, back dilute cultures 1:100 into SOC + IPTG + Amp and grow to an OD600 of ~0.8. | # 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. | # 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 cocultures and plate to single colonies on LB+Kan plates for CFU counting. | + | # 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 "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 "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. | #*Co-culture "C" should be plated at dilutions of 1:10,000 1:100,000 and 1:1,000,000. | ||
| - | # Induce the co-cultures | + | # Induce the co-cultures to produce hydrogen peroxide by bringing them to 10 nM AHL. |
| - | # Incubate co-cultures for 6hrs and then | + | # Incubate co-cultures for 6hrs and then plate to single colonies as before. |
# After incubation at 37C, count the CFU of each plate. | # After incubation at 37C, count the CFU of each plate. | ||
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Latest revision as of 01:06, 30 October 2008
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Coculture Inhibition Assay
Strains
Protocol
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