Recombineering-mediated construction of E . coli strain MW005. Panel A. An rpsL-tetA(C) counter-selection cassette, PCR-amplified from an NcoI fragment of pBAC-RT using ODNs 6090/6091 (Table 1) and flanked with 50-nt homology arms, was used to replace, by recombineering, the chromosomal copy of cre in E. coli strain EL350 to give strain MW004. Panel B. A trfA replacement cassette, PCR-amplified from pMW010 using ODNs 6088/6089 (Table 1) and flanked with the same 50-nt homology arms as the RT-cassette, was used to replace the RT-cassette in MW004 with the trfA gene sequence to give MW005.