Whole Genome Sequencing and CRISPR/Cas9 Gene Editing of Enterotoxigenic Escherichia coli BE311 for Fluorescence Labeling and Enterotoxin Analyses

Abstract

Some prevention strategies, including vaccines and antibiotic alternatives, have been developed to reduce enterotoxigenic Escherichia coli proliferation in animal production. In this study, a wild-type strain of BE311 with a virulent heat-stable enterotoxin gene identical to E. coli K99 was isolated for its high potential for gene expression ability. The whole genome of E. coli BE311 was sequenced for gene analyses and editing. Subsequently, the fluorescent gene mCherry was successfully knocked into the genome of E. coli BE311 by CRISPR/Cas9. The E. coli BE311–mCherry strain was precisely quantified through the fluorescence intensity and red colony counting. The inflammatory factors in different intestinal tissues all increased significantly after an E. coli BE311–mCherry challenge in Sprague–Dawley rats (p < 0.05). The heat-stable enterotoxin gene of E. coli BE311 was knocked out, and an attenuated vaccine host E. coli BE311-STKO was constructed. Flow cytometry showed apoptotic cell numbers were lower following a challenge of IPEC-J2 cells with E. coli BE311-STKO than with E. coli BE311. Therefore, the E. coli BE311–mCherry and E. coli BE311-STKO strains that were successfully constructed based on the gene knock-in and knock-out technology could be used as ideal candidates in ETEC challenge models and for the development of attenuated vaccines.

Exopolysaccharide production by Lactobacillus plantarum T10 is responsible for the probiotic activity in enhancing intestinal barrier function in vitro and in vivo

“…As a modified ETEC strain, E. coli BE311-mCherry attaches to the IPEC-J2 cells via adhesins and subsequently releases enterotoxins, causing cellular barrier damage and inflammation. 15 Our study results are in line with earlier findings, demonstrating that L. plantarum T10 and L. plantarum T13 can decrease the production of IL-8 and TNF-α in IPEC-J2 cells following infection with E. coli BE311-mCherry. A previous study showed that Lactobacillus spp.…”

“…The enterotoxigenic Escherichia coli (ETEC) strain BE311-mCherry with red-fluorescing mCherry protein was constructed in the previous study. 15 The strain was cultivated in Luria-Bertani (LB) broth in shake culture at 37 °C and was used in constructing a cellular infection model. Porcine intestinal epithelial cells (IPEC-J2) were obtained from Hunan Fenghui Biotechnology Co., Ltd (Hunan, China).…”

Strategies to Enhance l-Isoleucine Synthesis by Modifying the Threonine Metabolism Pathway in Escherichia coli

“…l -isoleucine is referred to as a branched chain amino acid together with l -valine and l -leucine. It is a high-value-added amino acid with muscle repair function, which is also often used to treat cirrhosis, obesity, coma, and other conditions. − l -isoleucine has received much attention in recent years as a synthetic precursor for the antiglycolytic drug 4-hydroxyisoleucine. , Fermentation is the primary method for industrial production of l -isoleucine; meanwhile, chemical synthesis and extraction from hair also play a key role in its production. − Additionally, metabolic engineering of the l -isoleucine biosynthesis pathway in Corynebacterium glutamicum and E. coli has also been widely used for the industrial production of this amino acid.…”

“…It is a high-value-added amino acid with muscle repair function, which is also often used to treat cirrhosis, obesity, coma, and other conditions. 1 − 4 l -isoleucine has received much attention in recent years as a synthetic precursor for the antiglycolytic drug 4-hydroxyisoleucine. 5 , 6 Fermentation is the primary method for industrial production of l -isoleucine; meanwhile, chemical synthesis and extraction from hair also play a key role in its production.…”