Figure 4.

Role of secondary mutations in enhanced recombination at loxK2. (A) Accelerated Cre synthesis in vivo. Escherichia coli DH5α strains expressing the indicated cre variants in pBAD33 were grown to an OD₆₀₀ of 0.5 and cre expression induced for 1 h with 0.2% l-arabinose. Cells from 1 ml of culture were harvested, lysed and 30 µl of the sonicated crude extracts subjected to SDS–PAGE, followed by western analysis with polyclonal anti-Cre serum. (B) Enhanced recombination with the FAS1 spacer. Purified mutant and wt Cre were assayed for activity in vitro on two lox² constructs, differing only by their 8 bp spacers. Reactions were as described in Materials and Methods, with termination at the indicated time points by heat inactivation of Cre. Recombination frequencies were determined by quantitation of recombination products and unprocessed substrate after gel electrophoresis.