Functional Analysis of CRISPR-Cas9-Mediated Gene Deletion in E. coli DH5α on Membrane Permeability and Transformation Efficiency
Feifan Leng, Xinyi Liu, Jinli He, Yubo Wang, Ning Zhu, Xiaopeng Guo, Wen Luo, Yonggang Wang
TL;DR
This study shows how deleting specific genes in E. coli using CRISPR-Cas9 increases membrane permeability and improves DNA uptake efficiency.
Contribution
The novel contribution is demonstrating a direct quantitative link between membrane permeability and transformation efficiency in CRISPR-edited E. coli.
Findings
The E. coli DH5α:ompA′ mutant showed the highest increase in membrane permeability.
Plasmid transformation efficiency was significantly enhanced in the E. coli DH5α:ompA′ mutant.
A quantitative correlation between transformation efficiency and membrane permeability was established as T = aP + c.
Abstract
This research utilized the CRISPR/Cas9 editing method to generate six mutant strains of Escherichia coli (E. coli) 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 E. coli DH5α:ompA′ 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 E. coli DH5α:ompA′ mutant strain, underscoring the critical function of outer membrane proteins in DNA…
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Taxonomy
TopicsCRISPR and Genetic Engineering · Bacterial Genetics and Biotechnology · RNA and protein synthesis mechanisms