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Yet, the field of membrane protein structural biology is in a "log" phase.
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Briefly, the cells were grown to a log phase in liquid EMM as previously described and washed to remove thiamine.
Total RNA was extracted when cells were in log phase growth and had been cultured for approximately the same number of passages (approx. 4 6).
After checking the reactions to be in the log phase, thirty PCR cycles were performed with sets at 92°C for 30 sec., 55°C for 30 sec., and 72°C for 90 sec., using a Mastercycler 5330 (Eppendorf).
This relationship had been determined earlier by sampling, diluting, plating, and counting colony-forming units from fermentations that were in the log phase of growth.
The quantification of each amplicon was determined as the cycle at which the PCR amplification was in log phase in fluorescent signal (Ct), relative to β-globin as the internal control.
Small changes in the starting cell density should not affect the kinds and levels of damage inflicted as long as the cells are in log phase and thus in a similar metabolic state.
This procedure resulted in a culture in which the bacteria were in the early log phase.
The plasmid was then transformed into E. coli strain TOP10 (Invitrogen BV, Leek, The Netherlands) and urease B expression was induced with 1 mM IPTG when the cells were in log phase growth at 37°C.
Cells were harvested with a solution of 0.25% trypsin 0.03% EDTA when they were in log phase of growth, and maintained at the above-described culture conditions for all experiments.
The threshold cycle value (CT) was obtained for each well as the cycle number at which the measured fluorescence crossed the arbitrary threshold of 150 units (all values are in log phase).
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