title: A series of template plasmids for Escherichia coli genome engineering.
abstract: Metabolic engineering strategies often employ multi-copy episomal vectors to overexpress genes. However, chromosome-based overexpression is preferred as it avoids the use of selective pressure and reduces metabolic burden on the cell. We have constructed a series of template plasmids for λ Red-mediated Escherichia coli genome engineering. The template plasmids allow construction of genome integrating cassettes that can be used to integrate single copies of DNA sequences at predetermined sites or replace promoter regions. The constructed cassettes provide flexibility in terms of expression levels achieved and antibiotics used for selection, as well as allowing construction of marker-free strains. The modular design of the template plasmids allows replacement of genetic parts to construct new templates. Gene integration and promoter replacement using the template plasmids are illustrated.
authors.affiliation: DBT-ICT Centre for Energy Biosciences, Institute of Chemical Technology, Mumbai 400019, Maharashtra, India.
authors.forename: Shalini S
authors.initials: SS
authors.lastname: Deb
authors.affiliation: DBT-ICT Centre for Energy Biosciences, Institute of Chemical Technology, Mumbai 400019, Maharashtra, India. Electronic address: ss.reshamwala@ictmumbai.edu.in.
authors.forename: Shamlan M S
authors.initials: SM
authors.lastname: Reshamwala
authors.affiliation: DBT-ICT Centre for Energy Biosciences, Institute of Chemical Technology, Mumbai 400019, Maharashtra, India; Department of Chemical Engineering, Institute of Chemical Technology, Mumbai 400019, Maharashtra, India.
authors.forename: Arvind M
authors.initials: AM
authors.lastname: Lali
mesh_terms: D002875:Chromosomes; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D016680:Genome, Bacterial; D008164:Luminescent Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein
keywords: E. coli; Gene integration; Genome engineering; Promoter replacement; Template plasmids