Physiological effects of Hin-mediated recombination/knotting. (A) Assay for the effect of knotting on the function of a gene. The ovals represent E. coli cells. The Hin expression vector, pHIN, and plasmid substrates pBR, pREC and pKNOT containing the bla gene (encoding β-lactamase) are depicted. Wild-type recombination sites are depicted as black arrows. The mutant hix site is shown as a grey arrow. (B) Effect of DNA knotting on ampicillin sensitivity of E. coli strain W3110 containing pHIN and either pBR, pREC or pKNOT. Single colonies were streaked from left to right across LB-agar that contained an ampicillin gradient and constant IPTG (1 mM) and spectinomycin (50 μg/ml) for Hin overexpression and maintenance. The experiment was repeated five times in either strain C600 or W3110, and was carried out either from high to low or from low to high ampicillin concentration with identical results. (C) Ampicillin sensitivity (MIC50) was quantified using the plate dilution method.