Figure 3

Terminator elements up-stream of the I-SceI cleavage site enhance the efficiency of I-SceI cleavage. One or two terminator elements were introduced ahead of the I-SceI cleavage site in mutation cassettes designed to introduce a sequence encoding a C-terminal 3 × FLAG tag on GapA in E. coli K-12 MG1655 and S. enterica Typhimurium SL1344. The efficiency of double-strand break formation by I-SceI was determined by spreading cells on agar plates containing LB Miller medium and 100 μg/mL ampicillin in the presence or absence of 100 ng/ml anhydrotetracycline.