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. 2024 Aug;64(8):e2400113.
doi: 10.1002/jobm.202400113. Epub 2024 Jun 25.

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

Xiaona Chen  1 , Ning Zhu  1   2 , Guangrui Yang  3   4 , Xiaopeng Guo  1 , Shangchen Sun  1   2 , Feifan Leng  1 , Yonggang Wang  1
Affiliations

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

Xiaona Chen et al. J Basic Microbiol. 2024 Aug.

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.

Keywords: CaCl2; competent cells; cspA; transformation efficiency.

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