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Team:Warsaw/Stage2/Results
From 2010.igem.org
Results
The efficiency of the kill-switch was measured by following means:
- dynamic measurement of OD
- dynamic measurement of CFU (colony-forming units)
- stationary measurement of OD
- stationary measurement of CFU (colony-forming units)
Dynamic measurement of OD and CFU
For the measurement BL21 RIL strain of E. coli (F− ompT hsdS(rB −mB −) dcm +Terr gal λ (DE3) endA Hte (argU ileY leuW Camr)) were transformed with the following plasmids:
Approximately 100 ul of over-night culture were used to inoculate 50 ml of LB medium with ampicilin and chloramphenicol (volume of inoculate was ajuted to ensure equal initial OD value). One flask was inoculated with pSB-MinC and two more with pSB-pT7-B0032-MinC. Cultures were incubated in 37 C with shaking for 30 min. Following this initial incubation, OD was measured and a small volume of culture was plated (on LA medium with amp and cm) for CFU measurement. pSB-MinC and one of the pSB-pT7-B0032-MinC cultures was induced by adding IPTG to the final concentration of 10 uM. Cultures were again placed on a shaker at 37 C. Samples for OD and CFU measurements were then taken every 30 min until the OD began to exceed 1,5.
- pSB1A2 containing MinC under T7 promoter and B0032 RBS
- pSB1A2 containing MinC without a promoter or RBS
Approximately 100 ul of over-night culture were used to inoculate 50 ml of LB medium with ampicilin and chloramphenicol (volume of inoculate was ajuted to ensure equal initial OD value). One flask was inoculated with pSB-MinC and two more with pSB-pT7-B0032-MinC. Cultures were incubated in 37 C with shaking for 30 min. Following this initial incubation, OD was measured and a small volume of culture was plated (on LA medium with amp and cm) for CFU measurement. pSB-MinC and one of the pSB-pT7-B0032-MinC cultures was induced by adding IPTG to the final concentration of 10 uM. Cultures were again placed on a shaker at 37 C. Samples for OD and CFU measurements were then taken every 30 min until the OD began to exceed 1,5.
