一种新的基于Red重组及I-Sec I体内切割的大肠杆菌基因组无痕删减方法

Markerless DNA deletion based on Red recombination and in vivo I-Sec I endonuclease cleavage in Escherichia coli chromosome

摘要:

目前常用的基因修饰方法是在Red同源重组介导下，电转线性PCR片段替换染色体上指定序列。因PCR过程错误掺入，该方法常常会在同源序列部位产生一些突变。为了避免此类突变，我们建立了一种新的无痕删除方法。首先将含有抗性标记 (两侧带有I-Sec I识别位点) 的线性DNA电转到Red重组感受态细胞内，用抗性基因替换基因组上指定序列；然后，将携带融合同源臂 (两侧带有I-Sec I位点) 的供体质粒导入上述细胞，诱导表达I-Sec I内切酶切割供体质粒释放同源片段，同时切除染色体上抗性基因产生双链断裂，通过分子间同源重组实现无痕删除。我们应用该方法连续删除了大肠杆菌DH1基因组上11个非必需区，使基因组减小10.59%。PCR测序证明所有删减区域同源臂未发生突变，基因组重测序证明指定区域被删除。删减菌的生长变化不大，但耐酸能力有所改变，并对番茄红素合成有不同影响。

Abstract:

Red-based recombineering has been widely used in Escherichia coli genome modification through electroporating PCR fragments into electrocompetent cells to replace target sequences. Some mutations in the PCR fragments may be brought into the homologous regions near the target. To solve this problem in markeless gene deletion we developed a novel method characterized with two-step recombination and a donor plasmid. First, generated by PCR a linear DNA cassette which comprises a I-Sec I site-containing marker gene and homologous arms was electroporated into cells for marker-substitution deletion of the target sequence. Second, after a donor plasmid carrying the I-Sec I site-containing fusion homologous arm was chemically transformed into the marker-containing cells, the fusion arms and the marker was simultaneously cleaved by I-Sec I endonuclease and the marker-free deletion was stimulated by double-strand break-mediated intermolecular recombination. Eleven nonessential regions in E. coli DH1 genome were sequentially deleted by our method, resulting in a 10.59% reduced genome size. These precise deletions were also verified by PCR sequencing and genome resequencing. Though no change in the growth rate on the minimal medium, we found the genome-reduced strains have some alteration in the acid resistance and for the synthesis of lycopene.