Fig. 1
Construction of the DNA modification and conjugation transfer system tra-PRha-redαβγ in E. coli strains. (A) Electroporation transformation efficiency of four parental E. coli strains (GB2005, DH5G, GB2006 and S17-1) and the control strain ET12567 were as determined by the number of colonies grown after transfer of the plasmid pBAC-sal-phiC31-apra-oriT (116 kb). (B) Internal recombination ratio of plasmid pBAC-cm-ampF-kan-ampR-repeat in five E. coli strains. The internal recombination ratio was defined as the colony number on LB plates containing ampicillin divided by the number on LB plates containing chloramphenicol. In E. coli GB2006 and S17-1, the internal recombination ratio of the plasmid was determined to be zero. For (A, B) error bars indicate SD; n = 3; ns, p > 0.05; ****p < 0.0001. (C) Diagrams of plasmids pBAC-cm-ampF-kan-ampR-repeat and pBAC-cm-amp. Plasmid pBAC-cm-ampF-kan-ampR-repeat contains two 500-bp homologous arms (haF and haR) flanking the kan cassette. Critically, haF and haR (dark green) are truncated, non-functional fragments of the ampicillin resistance gene (amp). Homologous recombination between these arms excises the intervening kan cassette while reconstituting a functional amp through precise end-joining (pBAC-cm-amp). (D) Schematic diagram of workflow for comparing internal recombination ratios. (E) Construction strategy for engineering the E. coli strains. Firstly, the gyrA, dcm, recA, and dam genes were modified sequentially, and then, the attB1-cm-attB2 cassette was inserted into a target location (e.g., pspG) on the E. coli chromosome by Red recombination. Finally, the attB1-cm-attB2 cassette was replaced by the element tra-PRha-redαβγ.