Random Mutagenesis by Error-Prone Pol Plasmid Replication in Escherichia coli
Random Mutagenesis by Error-Prone Pol Plasmid Replication in Escherichia coli
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DOI：
10.1007/978-1-4939-1053-3_3
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发表时间：
2014-01-01
期刊：
影响因子：
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通讯作者：
Camps, Manel
中科院分区：
文献类型：
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作者：
Alexander, David L.;Lilly, Joshua;Camps, Manel
定向进化是一种在实验室中模拟自然进化的方法，目的是修改现有的酶活性或产生新的酶活性。具有所需特性的突变体的鉴定涉及遗传多样性的产生以及功能选择或筛选。遗传多样性可以通过PCR或体内方法产生，如化学诱变或在突变菌株中对所需序列进行容易出错的复制。体内诱变方法有利于迭代选择，因为它们不需要克隆，但通常产生低突变密度，突变不局限于基因内的特定基因或区域。由于这个原因，当大量的突变体可以被筛选或选择时，这种方法通常用于产生新的生化特性。在这里，我们描述了一种先进的体内诱变方法，该方法基于携带感兴趣基因的ColE1质粒的易出错复制。与其他体内诱变方法相比，这种质粒靶向方法可以增加突变负荷，并促进迭代选择方法。我们还详细描述了这种诱变方法的突变谱，并且，使用周期3 GFP作为诱变的目标，我们说明了使用我们的方法可以产生的表型多样性。总之，易于出错的Pol I复制是一种突变方法，当有功能选择时，它非常适合于新的生化活动的进化。
Directed evolution is an approach that mimics natural evolution in the laboratory with the goal of modifying existing enzymatic activities or of generating new ones. The identification of mutants with desired properties involves the generation of genetic diversity coupled with a functional selection or screen. Genetic diversity can be generated using PCR or using in vivo methods such as chemical mutagenesis or error-prone replication of the desired sequence in a mutator strain. In vivo mutagenesis methods facilitate iterative selection because they do not require cloning, but generally produce a low mutation density with mutations not restricted to specific genes or areas within a gene. For this reason, this approach is typically used to generate new biochemical properties when large numbers of mutants can be screened or selected. Here we describe protocols for an advanced in vivo mutagenesis method that is based on error-prone replication of a ColE1 plasmid bearing the gene of interest. Compared to other in vivo mutagenesis methods, this plasmid-targeted approach allows increased mutation loads and facilitates iterative selection approaches. We also describe the mutation spectrum for this mutagenesis methodology in detail, and, using cycle 3 GFP as a target for mutagenesis, we illustrate the phenotypic diversity that can be generated using our method. In sum, error-prone Pol I replication is a mutagenesis method that is ideally suited for the evolution of new biochemical activities when a functional selection is available.