One of the commonly employed methods in molecular biology is to utilize calcium chloride to treat Escherichia coli for the preparation of competent cells to facilitate foreign gene expression. However, the molecular mechanisms underlying Ca²⁺ mediation of competent cell formation and identification of the key genes involved in the process remain unclear. In previous studies, the combined analysis of transcriptomics and proteomics revealed bhsA as one of the crucial genes. The gene ontology functional annotation of bhsA identified it as a member of the YhcN family encoding an outer membrane protein that confers resistance to various stresses. The IPR0108542 domain found within the protein plays a significant role in stress response and biofilm formation in E. coli. Analysis of the STRING database showed that the proteins interacting with bhsA are primarily involved in biofilm formation and stress resistance. Using the RED homologous recombination method, a bhsA deletion strain was constructed to verify its role in E. coli genetic transformation. Although the mutant strain showed no significant differences in morphology or growth trend when compared to the wild-type strain, its transformation efficiency decreased by 1.14- and 1.64-fold with plasmids pUC19 and pET-32a. Furthermore, the 1-N-phenylnaphthylamine assay indicated a 1.71-fold reduction in cell membrane permeability in the mutant strain.

中文翻译：

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Ca2+处理大肠杆菌基因bhsA及其作用的生物信息学分析

分子生物学常用的方法之一是利用氯化钙处理大肠杆菌制备感受态细胞以促进外源基因表达。^{然而，Ca 2+}介导感受态细胞形成的分子机制以及参与该过程的关键基因的鉴定仍不清楚。在之前的研究中，转录组学和蛋白质组学的联合分析表明bhsA是关键基因之一。bhsA的基因本体功能注释将其鉴定为 YhcN 家族的成员，编码赋予对各种应激的抵抗力的外膜蛋白。该蛋白中的 IPR0108542 结构域在大肠杆菌的应激反应和生物膜形成中发挥着重要作用。对 STRING 数据库的分析表明，与bhsA相互作用的蛋白质主要参与生物膜形成和应激抵抗。利用RED同源重组方法构建bhsA缺失菌株，验证其在大肠杆菌遗传转化中的作用。尽管与野生型菌株相比，突变菌株在形态或生长趋势上没有显着差异，但质粒pUC19和pET-32a的转化效率下降了1.14倍和1.64倍。此外，1- N-苯基萘胺测定表明突变菌株的细胞膜通透性降低了 1.71 倍。