When using a temperatureinducible system

a. Inoculate 1-ml cultures of LB medium containing the appropriate antibiotics with 1 or 2 colonies of E. coli (carrying the cIts857 allele) containing the empty expression vector, and 1 or 2 colonies containing the recombinant expression vector. Incubate the cultures overnight at 30°C.

b. Inoculate 10 ml of LB medium containing antibiotic in a 50-ml flask with 50 pl of an overnight culture. Grow the culture to the mid-log phase of growth (A550 = 0.5-1.0) at 30°C.

c. Transfer 1 ml of each uninduced culture (zero-time aliquot) to microfuge tubes. Immediately process the zero-time aliquots as described in Steps 6 and 7.

d. Induce the remainder of each culture by shifting the incubation temperature to 40°C. Proceed to Step 6.

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