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Developing an Extended Genomic Engineering Approach Based on Recombineering to Knock-in Heterologous Genes to <Emphasis Type="Italic">Escherichia coli</Emphasis> Genome
Authors:Karan Sukhija  Michael Pyne  Saad Ali  Valerie Orr  Daryoush Abedi  Murray Moo-Young  C Perry Chou
Institution:(1) Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, Waterloo, ON, N2L 3G1, Canada;(2) Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy, Isfahan University of Medical Sciences, 81745-359 Isfahan, Iran;
Abstract:Most existing genomic engineering protocols for manipulation of Escherichia coli are primarily focused on chromosomal gene knockout. In this study, a simple but systematic chromosomal gene knock-in method was proposed based on a previously developed protocol using bacteriophage λ (λ Red) and flippase–flippase recognition targets (FLP–FRT) recombinations. For demonstration purposes, DNA operons containing heterologous genes (i.e., pac encoding E. coli penicillin acylase and palB2 encoding Pseudozyma antarctica lipase B mutant) engineered with regulatory elements, such as strong/inducible promoters (i.e., P trc and P araB ), operators, and ribosomal binding sites, were integrated into the E. coli genome at designated locations (i.e., lacZYA, dbpA, and lacI-mhpR loci) either as a gene replacement or gene insertion using various antibiotic selection markers (i.e., kanamycin and chloramphenicol) under various genetic backgrounds (i.e., HB101 and DH5α). The expression of the inserted foreign genes was subjected to regulation using appropriate inducers isopropyl β-d-1-thiogalactopyranoside (IPTG) and arabinose] at tunable concentrations. The developed approach not only enables more extensive genomic engineering of E. coli, but also paves an effective way to “tailor” plasmid-free E. coli strains with desired genotypes suitable for various biotechnological applications, such as biomanufacturing and metabolic engineering.
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