一种用于高效筛选极端酶突变基因的质粒营救法

Plasmid Rescue Method for Highly Efficient Screening of Extremozyme Mutant Gene

为简化定向进化筛选突变耐高温和耐酸碱酶等极端酶的繁复工作，建立了一种快速质粒营救法，可直接从加热处理的平板中回收携带有突变基因的质粒，用于进一步的随机突变基因的筛选和基因测序等。研究了在不同的热处理条件下的质粒营救效率，获得了质粒营救转化效率最高的处理条件。结果表明，本方法适于质粒大小在2-10kb范围的载体，可用于平板筛选最适酶活性温度在60-100℃、pH4-10的突变体酶，并可将一轮的筛选过程缩短1-2d。

A plasmid rescue method to release plasmid DNA from Escherichia coli by heat treatment was used to screen the extremozyme mutants from directed evolution mutagenesis library. Plasmid rescue efficiency was measured under different heat treatment conditions. The effects of optimal process parameters on the efficiency of plasmid rescue and scope of applications were defined. The whole preparation requires 2-3 hours only. To screen recombinant clones rapidly, this method is better than traditional methods. It is very suitable for screening 2-10 kb plasmids sizes,optimal pH 4-10 and temperature scope of 60-100 ℃ enzyme mutants. The thermolysis procedure is quick to perform and, therefore, especially suitable for screening large numbers of Escherichia coli transformants for directed evolution process.