Figure 11
Efficiency of SXT-Bet + SXT-Exo mediated recombination between a PCR-generated DNA fragment and its homologous target on the E. coli chromosome. Panel A: Schematic overview of the chromosomal targeting assay use to score exonuclease + SSAP-mediate recombination efficiency. A dsDNA 'targeting' molecule (galK<>Cmr) was synthesized by PCR using two primers sharing (ca. 20 nt) sequence homology to a chloramphenicol resistance cassette at one end; and 50 nt of sequence homology to the 5'-end (ECgalKF1) or 3'-end (ECgalKR1) of the non-essential E. coli galK gene at the other. The purified galK<>Cmr targeting cassette was electroporated into DH10B cells expressing pairs of Exo and SSAP proteins from established arabinose-inducible plasmids. The Exo and SSAP proteins mediate homologous recombination between the galK<>Cmr dsDNA cassette and the galK locus of the E. coli chromosome via 50 bp regions of flanking sequence homology. This creates a mutant E. coli strain that has its galK gene replaced with a chloramphencol resistance cassette. Panel B. Comparison of dsDNA recombination efficiencies of SXT-Bet-Exo with those of RecET and lambda-Bet-Exo. The RecET; lambda-Bet-Exo; SXT-Bet-Exo and SXT-Ssb-Bet-Exo sets of homologous recombination-promoting proteins were expressed from arabinose-inducible (PBAD/araC) plasmids established in E. coli DH10B cells (see Additional File 3). pBAD-ETγ: RecE + RecT + lambda-Gam [35]; pB1E4A: lambda-Bet + lambda Exo; pBex4b1: SXT-Bet + SXT-Exo; pBX2B: SXT-Ssb + SXT-Bet + SXT-Exo; pBAD-28MCS (negative control). Homologous recombination events were scored by plating galK<>Cmr-electroporated cells onto LB-agar with/without chloramphenicol. The dsDNA recombination efficiency was calculated by dividing the number of Cmr colonies by the total number of cells (CFUs) that survived electroporation. 8 replicates were performed; the mean recombination efficiency ± standard deviation is reported.