Fig. 2. Isolation of the GOF Kch mutants. (A) The experimental scheme. Kch coding region together with pBluescript vector were mutagenized in vivo using XL1-Red Cells. The mutated kchs were further cloned in pB11d behind IPTG-inducible LacUV5 promoter and used to transform kch-null FRAG1 E.coli. Transformed colonies whose replicas failed to grow on any of the restrictive plates were picked, DNA sequenced and phenotyped. (B) Growth phenotype on the permissive (panel 1) and various restrictive plates (panels 2–8) with (1–4) or without (5–8) IPTG. Each row shows the growth pattern of five 5-µl drops of inoculate from cultures of stationary cells (OD₆₀₀ ∼ 3.5–4.5) diluted 10²-, 10³-, 10⁴-, 10⁵- and 10⁶-fold.