大肠杆菌hfq和rne-710基因的双质粒无痕敲除技术

Two-plasmid scarless genetic modification in Escherichia coli hfq and rne-710

摘要:

基因敲除技术是了解基因功能的重要技术手段。以大肠杆菌K-12 MG1655基因组hfq (309 bp) 和rne-710 (1056 bp) 基因为模型，首先构建Δhfq∷Spe和Δrne-710∷Spe菌株，通过融合PCR方法分别构建缺失hfq (309 bp) 和rne-710 (1056 bp) 的融合片段并连接至辅助质粒，缺失hfq和rne-710的片段经重组分别替换壮观霉素抗性盒，得到无痕敲除株Δhfq和Δrne-710。双质粒无痕敲除和融合PCR方法相结合为大片段基因缺失开辟了新的途径。

Abstract:

Gene modification is an important technique to understand gene function. We firstly constructed Δhfq::Spe and Δrne-710::Spe mutant strains of Escherichia coli MG1655. The fragment lacking of hfq and rne-710 was ligated to the auxiliary plasmid and separately replace the spectinomycin box by homologous recombinase system to obtain the Δhfq and Δrne-710 mutant strains. The combination of two-plasmid scarless genetic modification and fusion PCR led to a new way for the long DNA fragment gene deletions.