title: Developing an extended genomic engineering approach based on recombineering to knock-in heterologous genes to Escherichia coli genome.
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.
authors.affiliation: Department of Chemical Engineering, University of Waterloo, Waterloo, ON N2L 3G1, Canada.
authors.forename: Karan
authors.initials: K
authors.lastname: Sukhija
authors.forename: Michael
authors.initials: M
authors.lastname: Pyne
authors.forename: Saad
authors.initials: S
authors.lastname: Ali
authors.forename: Valerie
authors.initials: V
authors.lastname: Orr
authors.forename: Daryoush
authors.initials: D
authors.lastname: Abedi
authors.forename: Murray
authors.initials: M
authors.lastname: Moo-Young
authors.forename: C Perry
authors.initials: CP
authors.lastname: Chou
mesh_terms: D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D055879:Gene Knock-In Techniques / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D016680:Genome, Bacterial; D008049:Lipase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D010399:Penicillin Amidase / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid
chemical_list: D029968:Escherichia coli Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D008049:Lipase; D010399:Penicillin Amidase