Use of Mutagenesis and Functional Screens to Characterize Essential Genes Involved in Lipopolysaccharide Transport.
Use of Mutagenesis and Functional Screens to Characterize Essential Genes Involved in Lipopolysaccharide Transport.
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DOI：
10.1007/978-1-0716-2581-1_1
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发表时间：
2022
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结构-功能分析是表征特定残基对蛋白质的生物发生和功能贡献的一种强有力的策略。这种方法需要鉴定在没有野生型蛋白的情况下表达突变等位基因的菌株。在研究非必需细菌基因时，通过将感兴趣的质粒编码的等位基因引入到已经缺乏野生型基因的菌株中，可以很容易地构建突变株集合。然而，这种高通量的方法不适用于研究必需基因，因为它们各自的空菌株是不可行的。虽然目前有几种工具可以修改基本基因，但它们可能会受到构建和分析每个突变株所需工作量的极大限制。在这里，我们描述了一个高通量的系统，用于快速分析参与大肠杆菌内毒素运输的必要基因的结构和功能。这种方法可以用于研究任何必要的基因，依赖于单个细菌菌株的初始构建，该菌株可以在不到24小时的时间内产生多个质粒编码的等位基因并对其进行功能鉴定。我们将讨论与其他常用方法相比，我们的协议的优点和可能的缺点。
Structure-function analysis is a powerful strategy to characterize the contribution of specific residues to the biogenesis and function of a protein. This approach requires the characterization of strains that express mutant alleles in the absence of the wild-type protein. When studying non-essential bacterial genes, collections of mutants can be easily constructed by introducing plasmid-encoded alleles of interest into a strain that already lacks the wild-type gene. However, this high-throughput approach is not applicable to studying essential genes since their respective null strains are not viable. While there are several tools currently available to modify essential genes, they can be greatly limited by the amount of effort it takes to build and analyze each mutant strain. Here, we describe a high-throughput system for the rapid structure-function analysis of essential genes involved in lipopolysaccharide transport in Escherichia coli. This method, which can be applied to study any essential gene, relies on the initial construction of a single bacterial strain that can be used to generate and functionally characterize multiple plasmid-encoded alleles in under 24 hours. We will discuss the advantages and possible shortcomings of our protocol in comparison to other commonly used methods.