Team:Caltech/Protocols/Coculture Inhibition Assay

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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

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 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 were induced to produce hydrogen peroxide by bringing them to 10 nM AHL.
Incubate co-cultures for 6hrs and then plated to single colonies as before.
After incubation at 37C, count the CFU of each plate.

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-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.
+===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===
+# 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 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 were induced to produce hydrogen peroxide by bringing them to 10 nM AHL.
+# Incubate co-cultures for 6hrs and then plated to single colonies as before.
+# After incubation at 37C, count the CFU of each plate.
 }}