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Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells
Published on: April 2, 2016
Functional Analysis of CRISPR-Cas9-Mediated Gene Deletion in E. coli DH5α on Membrane Permeability and Transformation
Feifan Leng1, Xinyi Liu1, Jinli He1
1School of Life Science and Engineering, Lanzhou University of Technology, Pengjiaping Road 36, Qilihe District, Lanzhou 730050, China.
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 acquisition. Permeability simulations were performed utilizing the E. coli DH5α:ompA' 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.
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