title: Insightful directed evolution of Escherichia coli quorum sensing promoter region of the lsrACDBFG operon: a tool for synthetic biology systems and protein expression.
abstract: Quorum sensing (QS) regulates many natural phenotypes (e.q. virulence, biofilm formation, antibiotic resistance), and its components, when incorporated into synthetic genetic circuits, enable user-directed phenotypes. We created a library of Escherichia coli lsr operon promoters using error-prone PCR (ePCR) and selected for promoters that provided E. coli with higher tetracycline resistance over the native promoter when placed upstream of the tet(C) gene. Among the fourteen clones identified, we found several mutations in the binding sites of QS repressor, LsrR. Using site-directed mutagenesis we restored all p-lsrR-box sites to the native sequence in order to maintain LsrR repression of the promoter, preserving the other mutations for analysis. Two promoter variants, EP01rec and EP14rec, were discovered exhibiting enhanced protein expression. In turn, these variants retained their ability to exhibit the LsrR-mediated QS switching activity. Their sequences suggest regulatory linkage between CytR (CRP repressor) and LsrR. These promoters improve upon the native system and exhibit advantages over synthetic QS promoters previously reported. Incorporation of these promoters will facilitate future applications of QS-regulation in synthetic biology and metabolic engineering.
authors.affiliation: Institute for Bioscience and Biotechnology Research, College Park, MD, USA.|Fischell Department of Bioengineering, University of Maryland, College Park, MD, USA.
authors.forename: Pricila
authors.initials: P
authors.lastname: Hauk
authors.affiliation: Institute for Bioscience and Biotechnology Research, College Park, MD, USA.|Fischell Department of Bioengineering, University of Maryland, College Park, MD, USA.
authors.forename: Kristina
authors.initials: K
authors.lastname: Stephens
authors.affiliation: Institute for Bioscience and Biotechnology Research, College Park, MD, USA.|Fischell Department of Bioengineering, University of Maryland, College Park, MD, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Mckay
authors.affiliation: Institute for Bioscience and Biotechnology Research, College Park, MD, USA.|Fischell Department of Bioengineering, University of Maryland, College Park, MD, USA.
authors.forename: Chelsea Ryan
authors.initials: CR
authors.lastname: Virgile
authors.affiliation: Department of Mathematics, University of Maryland, College Park, MD 20742, USA.
authors.forename: Hana
authors.initials: H
authors.lastname: Ueda
authors.affiliation: Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
authors.forename: Marc
authors.initials: M
authors.lastname: Ostermeier
authors.affiliation: Protein Structure Group, Korea Basic Science Institute, 162 Yeongudangi-Ro, Ochang-Eup, Cheongju-Si, Chungcheongbuk-Do 363-883, South Korea.
authors.forename: Kyoung-Seok
authors.initials: KS
authors.lastname: Ryu
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA.
authors.forename: Herman O
authors.initials: HO
authors.lastname: Sintim
authors.affiliation: Institute for Bioscience and Biotechnology Research, College Park, MD, USA bentley@umd.edu.|Fischell Department of Bioengineering, University of Maryland, College Park, MD, USA.
authors.forename: William E
authors.identifier: http://orcid.org/0000-0002-4855-7866
authors.initials: WE
authors.lastname: Bentley
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D019143:Evolution, Molecular; D015964:Gene Expression Regulation, Bacterial; D009154:Mutation; D059372:Nucleotide Motifs; D009876:Operon; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D053038:Quorum Sensing / Q000235:genetics*; D020218:Response Elements; D058615:Synthetic Biology; D014158:Transcription, Genetic
chemical_list: D029968:Escherichia coli Proteins