Role of cspA on the Preparation of Escherichia coli Competent Cells by Calcium Chloride Method

IF 3.5 4区 生物学 Q2 MICROBIOLOGY
Xiaona Chen, Ning Zhu, Guangrui Yang, Xiaopeng Guo, Shangchen Sun, Feifan Leng, Yonggang Wang
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引用次数: 0

Abstract

One of the fundamental techniques in genetic engineering is the creation of Escherichia coli competent cells using the CaCl2 method. However, little is known about the mechanism of E. coli competence formation. We have previously found that the cspA gene may play an indispensable role in the preparation of E. coli DH5α competent cells through multiomics analysis. In the present study, the cellular localization, physicochemical properties, and function of the protein expressed by the cspA gene were analyzed. To investigate the role of the cspA gene in E. coli transformation, cspA-deficient mutant was constructed by red homologous recombination. The growth, transformation efficiency, and cell morphology of the cspA-deficient strain and E. coli were compared. It was found that there were no noticeable differences in growth and morphology between E. coli and the cspA-deficient strain cultured at 37°C, but the mutant exhibited increased transformation efficiencies compared to E. coli DH5α for plasmids pUC19, pET-32a, and p1304, with enhancements of 2.23, 2.24, and 3.46 times, respectively. It was proved that cspA gene is an important negative regulatory gene in the CaCl2 preparation of competent cells.

cspA 对用氯化钙法制备大肠杆菌合格细胞的作用
基因工程的基本技术之一是使用 CaCl2 方法创建大肠杆菌能力细胞。然而,人们对大肠杆菌能力形成的机制知之甚少。我们之前通过多组学分析发现,cspA 基因可能在制备大肠杆菌 DH5α 能力细胞的过程中发挥着不可或缺的作用。本研究分析了 cspA 基因表达的蛋白质的细胞定位、理化性质和功能。为了研究 cspA 基因在大肠杆菌转化中的作用,研究人员通过红色同源重组构建了 cspA 基因缺失突变体。比较了 cspA 基因缺陷株和大肠杆菌的生长、转化效率和细胞形态。结果发现,37℃培养的大肠杆菌与 cspA 基因缺陷突变株在生长和形态上没有明显差异,但突变株对质粒 pUC19、pET-32a 和 p1304 的转化效率比大肠杆菌 DH5α 有所提高,分别提高了 2.23 倍、2.24 倍和 3.46 倍。实验证明,cspA 基因是能育细胞 CaCl2 制备过程中一个重要的负调控基因。
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来源期刊
Journal of Basic Microbiology
Journal of Basic Microbiology 生物-微生物学
CiteScore
6.10
自引率
0.00%
发文量
134
审稿时长
1.8 months
期刊介绍: The Journal of Basic Microbiology (JBM) publishes primary research papers on both procaryotic and eucaryotic microorganisms, including bacteria, archaea, fungi, algae, protozoans, phages, viruses, viroids and prions. Papers published deal with: microbial interactions (pathogenic, mutualistic, environmental), ecology, physiology, genetics and cell biology/development, new methodologies, i.e., new imaging technologies (e.g. video-fluorescence microscopy, modern TEM applications) novel molecular biology methods (e.g. PCR-based gene targeting or cassettes for cloning of GFP constructs).
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