title: Quorum sensing-modulated AND-gate promoters control gene expression in response to a combination of endogenous and exogenous signals.
abstract: We have constructed and characterized two synthetic AND-gate promoters that require both a quorum-sensing (QS) signal and an exogenously added inducer to turn on gene expression. The engineered promoters, LEE and TTE, contain binding sites for the QS-dependent repressor, EsaR, and either LacI or TetR, and they are induced by an acyl-homoserine lactone (AHL) signal and IPTG or aTc. Although repression of both LEE and TTE by wild-type EsaR was observed, induction of gene expression at physiologically relevant concentrations of AHL required the use of an EsaR variant with higher signal sensitivity. Gene expression from both LEE and TTE was shown to require both signal molecules, and gene expression above background levels was not observed with either signal alone. We added endogenous production of AHL to evaluate the ability of the promoters to function in a QS-dependent manner and observed that gene expression increased as a function of cell density only in the presence of exogenously added IPTG or aTc. Cell-cell communication-dependent AND-gate behaviors were demonstrated using an agar plate assay, where cells containing the engineered promoters were shown to respond to AHL produced by a second E. coli strain only in the presence of exogenously added IPTG or aTc. The promoters described in this work demonstrate that EsaR and its target DNA sequence can be used to engineer new promoters to respond to cell density or cell-cell communication. Further, the AND-gate promoters described here may serve as a template for new regulatory systems that integrate QS and the presence of key metabolites or other environmental cues to enable dynamic changes in gene expression for metabolic engineering applications.
authors.affiliation: Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute , 110 8th Street, Troy New York 12180 United States of America.
authors.forename: Jasmine
authors.initials: J
authors.lastname: Shong
authors.forename: Cynthia H
authors.initials: CH
authors.lastname: Collins
mesh_terms: D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000378:metabolism; D054742:Acyl-Butyrolactones / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000187:drug effects; D053038:Quorum Sensing / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics
chemical_list: D054742:Acyl-Butyrolactones; D001426:Bacterial Proteins; C095459:EsaR protein, Pantoea stewartii; D011994:Recombinant Proteins; D014157:Transcription Factors; D007544:Isopropyl Thiogalactoside; C030654:N-(3-oxohexanoyl)-3-aminodihydro-2(3H)-furanone; D015107:4-Butyrolactone