pSim6质粒与新型反选择kil系统联用在重组工程的应用

Combination of novel counter-selection system kil and pSim6 plasmid in recombination engineering

摘要:

基因工程中，无痕修饰是一种很受欢迎的基因组操作技术。反选择系统的严谨性决定了无痕修饰的效率。近期有文献报道了一种新型的反选择系统kil，该系统与质粒pSim6联用后反选择严谨性较pKD46质粒更高，预示着kil与pSim6质粒联用可以更高效地选择出重组子。据此，对大肠杆菌菌株W3110、MG1655和DH10B中的4个不同的非必需基因位点 (lacI、dbpa、ack和glk) 进行了分析，将不同长度的外源基因片段对预先插入这些位点的tet/kil进行基因替换。结果表明kil与pSim6质粒联用较其与pKD46质粒联用，重组效率均有显著提高，并且随着外源片段的增长，效果更明显。外源片段为1 000 bp时，kil与pSim6质粒联用是其与pKD46联用的1.2–2倍；外源片段为2 000 bp时，则是pKD46的2.2–5倍。综上，kil与pSim6联用具有更高的重组效率，更有利于大肠杆菌基因组的无痕修饰操作，这为大肠杆菌重组工程提供了更多的选择。

Abstract:

Seamless modification is a popular genomic manipulation technique in genetic engineering. Selection stringency of the counter-selection system determines the efficiency of the seamless modification. Recently, a novel counter-selection system, kil, was constructed. It is reported that the selection selectivity of kil is higher in host bacteria harboring plasmid pSim6 than that harboring pKD46, indicating that recombinants could be selected out more efficiently by combining kil counter-selection system and plasmid pSim6. In order to confirm this speculation, four different loci (lacI, dbpa, ack, glk) in Escherichia coli strains W3110, MG1655 and DH10B were selected for testing: dsDNA fragments of different sizes (500 bp, 1 000 bp, and 2 000 bp) were used to substitute tet/kil. As expected, recombination efficiency was higher in host bacteria harboring plasmid pSim6 than that harboring pKD46, and the results were more obvious with the length of dsDNA increasing. Specifically, recombination efficiency was 1.2 to 2 fold higher in pSim6 harboring bacteria than in pKD46 harboring bacteria when dsDNA fragments were 1 000 bp in length. With the length of dsDNA increasing up to 2 000 bp, the gap increased to 2.2–5 fold. In conclusion, it is easier to perform seamless modification by combining kil counter-selection system and plasmid pSim6 than combining kil and pKD46. An alternative tool in genomic engineering is provided in this study.