title: Multi-Faceted Characterization of a Novel LuxR-Repressible Promoter Library for Escherichia coli.
abstract: The genetic elements regulating the natural quorum sensing (QS) networks of several microorganisms are widely used in synthetic biology to control the behaviour of single cells and engineered bacterial populations via ad-hoc constructed synthetic circuits. A number of novel engineering-inspired biological functions have been implemented and model systems have also been constructed to improve the knowledge on natural QS systems. Synthetic QS-based parts, such as promoters, have been reported in literature, to provide biological components with functions that are not present in nature, like modified induction logic or activation/repression by additional molecules. In this work, a library of promoters that can be repressed by the LuxR protein in presence of the QS autoinducer N-3-oxohexanoyl-L-homoserine lactone (AHL) was reported for Escherichia coli, to expand the toolkit of genetic parts that can be used to engineer novel synthetic QS-based systems. The library was constructed via polymerase chain reaction with highly constrained degenerate oligonucleotides, designed according to the consensus -35 and -10 sequences of a previously reported constitutive promoter library of graded strength, to maximize the probability of obtaining functional clones. All the promoters have a lux box between the -35 and -10 regions, to implement a LuxR-repressible behaviour. Twelve unique library members of graded strength (about 100-fold activity range) were selected to form the final library and they were characterized in several genetic contexts, such as in different plasmids, via different reporter genes, in presence of a LuxR expression cassette in different positions and in response to different AHL concentrations. The new obtained regulatory parts and corresponding data can be exploited by synthetic biologists to implement an artificial AHL-dependent repression of transcription in genetic circuits. The target transcriptional activity can be selected among the available library members to meet the design specifications of the biological system.
authors.affiliation: Dipartimento di Ingegneria Industriale e dell'Informazione, Università degli Studi di Pavia, Pavia, Italy; Centro di Ingegneria Tissutale, Università degli Studi di Pavia, Pavia, Italy.
authors.forename: Susanna
authors.initials: S
authors.lastname: Zucca
authors.affiliation: Dipartimento di Ingegneria Industriale e dell'Informazione, Università degli Studi di Pavia, Pavia, Italy; Centro di Ingegneria Tissutale, Università degli Studi di Pavia, Pavia, Italy.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Pasotti
authors.affiliation: Dipartimento di Ingegneria Industriale e dell'Informazione, Università degli Studi di Pavia, Pavia, Italy; Centro di Ingegneria Tissutale, Università degli Studi di Pavia, Pavia, Italy.
authors.forename: Nicolò
authors.initials: N
authors.lastname: Politi
authors.affiliation: Dipartimento di Ingegneria Industriale e dell'Informazione, Università degli Studi di Pavia, Pavia, Italy; Centro di Ingegneria Tissutale, Università degli Studi di Pavia, Pavia, Italy.
authors.forename: Michela
authors.initials: M
authors.lastname: Casanova
authors.affiliation: IGM-CNR, Università degli Studi di Pavia, Pavia, Italy.
authors.forename: Giuliano
authors.initials: G
authors.lastname: Mazzini
authors.affiliation: Centro di Ingegneria Tissutale, Università degli Studi di Pavia, Pavia, Italy.
authors.forename: Maria Gabriella
authors.initials: MG
authors.lastname: Cusella De Angelis
authors.affiliation: Dipartimento di Ingegneria Industriale e dell'Informazione, Università degli Studi di Pavia, Pavia, Italy; Centro di Ingegneria Tissutale, Università degli Studi di Pavia, Pavia, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Magni
mesh_terms: D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000494:pharmacology; D001483:Base Sequence; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D015723:Gene Library; D005822:Genetic Vectors / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*
chemical_list: D012097:Repressor Proteins; D015534:Trans-Activators; C064778:LuxR autoinducer binding proteins; C088386:homoserine lactone; D015107:4-Butyrolactone