CL111 was first transformed with the plasmids containing BCFA-producing pathway. The straight-chain-specific fabH was then eliminated by phage P1 transduction with lysates grown on strain CAG12094. The transduction mixtures were plated on LB-agar medium supplemented with appropriate anti-biotics (tetracycline, 15 mg/L; kanamycin, 50 mg/L; ampicillin,100 mg/L; chloramphenicol, 30 mg/L). Colonies were screened for sensitivity to a mixture of spectinomycin and streptomycin(17.5 mg/L each). Deletion of fabH was confirmed by colony PCR. Correct colonies were then screened for BCFA production.
DH1, DH1(tesA) & BC33 – E. coli strains for producing branched-chain fatty acids
Tech ID: T-017808