Construction of MD1

 MD1 was constructed by the suicide plamid-based, FLP recombinase-assisted method described in Pósfai et al., J Bacteriol, 1997, 179, 4426-4428.

 A 887-bp MG1655 genomic segment (coordinates: 262193-263079) was PCR-amplified and cloned into the BamHI-EcoRI sites of pSG76-C. The resulting plasmid was inserted into the genome by homologous recombination. Similarly, a 614-bp genomic segment (coordinates: 324633-325246) was cloned into the ClaI site of pST76-K, and the plasmid was recombined into the genome. The genomic segment between the frt sites of the two inserted plasmids was deleted by expressing the FLP recombinase from helper plasmid pFT-A.