Functional Analysis of CRISPR-Cas9-Mediated Gene Deletion in E. coli DH5α on Membrane Permeability and Transformation Efficiency

This research utilized the CRISPR/Cas9 editing method to generate six mutant strains of <i>Escherichia coli</i> (<i>E. coli</i>) DH5α targeting specific genes. The functional characterization and phenotypic analysis confirmed the regulatory roles of these genes in modifying membrane permeability. The variations in membrane permeability among the mutant strains were assessed by measuring electrical conductivity, ortho-nitrophenyl-β-D-galactopyranoside (ONPG) hydrolysis, and propidium iodide (PI) fluorescence, with <i>E. coli</i> DH5α:<i>ompA'</i> exhibiting the most pronounced increase in membrane permeability. The function of these genes in transformation was analyzed from physicochemical and microscopic perspectives. Assays of plasmid transformation efficiency revealed a significant enhancement in the <i>E. coli</i> DH5α:<i>ompA'</i> mutant strain, underscoring the critical function of outer membrane proteins in DNA acquisition. Permeability simulations were performed utilizing the <i>E. coli</i> DH5α:<i>ompA'</i> mutant strain, grounded in a previously established model. The quantitative correlation between transformation efficiency and membrane permeability in this mutant conformed to the equation T = aP + c.