And its homology Trichostatin A in vivo regions were quite long, meaning several rounds of PCR amplification and more manipulation steps were needed.

As previously reported, multi-copy Red plasmid pTP223 failed to promote gene replacement using the PCR-generated substrates with short homology extensions in E. coli, since the linear multimers of this plasmid generated through high dosage of lambda Gam protein drove the plasmid replication in rolling circle mode may be toxic to E. coli host or compete with the recombination substrates [27–30]. Based on these observations, we learn more constructed plasmid pRKaraRed derived from RK2, low-copy and broad-host-range expression. As expected, plasmid pRKaraRed was able to promote efficient homologous recombination with short homology extension in E. coli, in P. aeruginosa PAO1, and also in Pseudomonas sp. M18 (data not shown). In E. coli, PCR cassettes flanked by only

35 bp homology region could induce the homologous recombination and efficient recombination happened when the PCR fragments flanked by 40 bp homology regions were used (data not shown). But in Pseudomonas PAO1 and M18, almost no transformant could be obtained using the PCR fragments with 35 bp or 40 bp homology extension, and at least 50 bp homology regions were required for efficient recombination (30~80 transformants). This is consistent with previous results that the minimum length of homology LCZ696 chemical structure extension required for efficient recombination may be different when the lambda Red system is used in different organisms,

which may have relevance to the characteristics of the organisms, such as the difference in GC content and so on [22–25]. Although the efficiency of recombination in Pseudomonas was lower than that in E. coli, plasmid pRKaraRed was still suitable for the gene modification in Pseudomonas. Differences in the expression of Red proteins, DNA uptake, sequence contexts and the species-specific restriction may result in the variations of recombination efficiency [27]. The scarless modification strategy based on plasmid pRKaraRed was efficient and rapid. Single-point mutation, deletion of large operons and consecutive next deletion of multiple genes could be achieved easily. One plasmid and PCR cassette flanked by 50 bp homology regions were enough to induce efficient recombination, meaning only one step PCR amplification was needed. And as the marker cassettes could be used repeatedly, only the homology regions should be changed to perform the modifications of different genes, which may alleviate the workload of primer design. Furthermore, the expression of the lambda Red proteins were driven by the tightly regulated promoter P BAD , of which the basal expression level was very low in the absence of its inducer. This will minimize the unwanted recombination and increase the efficiency of homologous recombination.