Generation of large libraries of random mutants in Bacillus subtilis by PCR-based plasmid multimerization
Generation of large libraries of random mutants in Bacillus subtilis by PCR-based plasmid multimerization
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DOI：
10.2144/97232rr01
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发表时间：
1997-08-01
期刊：
影响因子：
2.7
通讯作者：
Schellenberger, V
中科院分区：
文献类型：
--
作者：
Shafikhani, S;Siegel, RA;Schellenberger, V
我们描述了一种基于PCR的产生多聚体的方法，该方法可以非常高效地直接转化到枯草芽孢杆菌中。这项技术对于产生大量随机突变的基因库特别有用，这是通过定向进化优化酶所必需的。我们对编码枯草杆菌蛋白酶的基因进行了连续六轮的聚合酶链式反应，检测了三种不同的致突变水平。用我们的聚合酶链式反应多聚体方法克隆了18个群体，并通过DNA测序确定了突变频率。结果表明，通过在反应混合物中加入不同浓度的氯化锰，可以控制聚合酶链式反应过程中的突变频率。我们观察到碱基对变化的类型存在偏差，A和T的突变频率比C和G高得多。我们测定了所有群体中活跃克隆的比例，发现其自然对数与群体的平均突变频率成正比。这些数据显示，在所有可能的突变中，约有0.27个突变会导致枯草杆菌蛋白基因失活，这为其结构可塑性提供了一个衡量标准。
We describe a PCR-based method for the generation of plasmid multimers that can be directly transformed into Bacillus subtilis with very high efficiency. This technique is particularly useful for the generation of large libraries of randomly mutagenized genes, which are required for the optimization of enzymes by directed evolution. We subjected the gene coding for the protease subtilisin to six consecutive rounds of PCR at three different levels of mutagenicity. The resulting 18 populations were cloned using our PCR multimerization protocol, and the mutation frequencies were determined by DNA sequencing. The resulting data demonstrate that the mutation frequency during PCR can be controlled by adding varying concentrations of manganese chloride to the reaction mixture. We observed a bias in the type of base pair changes with A and T being mutated much more frequently than C and G. We determined the fraction of active clones in all populations and found that its natural logarithm is proportional to the average mutation frequency of the populations. These data reveal that a fraction of about 0.27 of all possible mutations leads to the inactivation of the subtilisin gene, which provides a measure for its structural plasticity.