Knocking out Analysis of the CpxP gene using Crispr/Cas9 in Escherichia coli MG1655

Xiaoliang He,Xinran Yu,Yuwen Ren,Xiaohui Zhou,Wanli Meng

doi:10.1186/s13568-020-01099-z

Abstract

Based on the analysis of cpxP genes among Escherichia coli strains, cpxP gene-targeting short guide RNA (sgRNA) was designed and inserted into the pGL3-MGP-RNA. The donor sequences (MG-HR) for homologous repair were designed and cloned by PCR. MG-HR and pGL3-MGP-RNA were transformed into E. coli MG1655 (pCas9). The cpxP gene expression cassette was amplified by PCR and subcloned into pBBR1MCS-2. Then the pBBR-cpxP was independently transformed into E. coli MG1655. The results of motility experiment suggest that cpxP gene had a significant effect on the movement ability of E. coli strain. The CpxP protein had a significant inhibition of bacterial activity. The lastest 81 CpxP proteins sequences were selected and analyzed by multi-sequence alignment and molecular cluster. The CpxP proteins were roughly divided into three categories. Our results suggest that the CpxP protein was involved in bacterial motility, infection and pathogenicity.

Highlights

The Cpx system is one of the most common two-component regulatory systems in Gram-negative bacteria
Identification of the knockout of cpxP gene The knockout of cpxP gene was identified by PCR using MGP-RNA. The donor sequences (MG-HR)-S-F and MG-HR-X-R as primers, the genomic DNA of E. coli MG1655 as a template
The results showed that the diffusion diameters of E. coli MG1655ΔcpxP is significantly greater than E. coli MG1655 (Student’s t-test, P < 0.05) (Fig. 1)

Summary

Introduction

The Cpx system is one of the most common two-component regulatory systems in Gram-negative bacteria. It consists of the membrane-anchored sensor kinase CpxA, the cytosolic response regulator CpxR, and the peripheral spatial helper protein CpxP (Dong et al 1993; Ruiz and Silhavy 2005). The CRISPR-Cas system was used recently as efficient genome engineering technology in several prokaryotes and eukaryotes, including (but not limited to) Escherichia coli (Jiang et al 2013), Saccharomyces cerevisiae (DiCarlo et al 2013), yeast (DiCarlo et al 2015), Streptomyces spp. The CRISPR/Cas system was used to remove plasmid harbouring mcr-1 from Escherichia coli (Dong et al 2019)

Methods

Results

Conclusion