title: Regulation of Bacterial Gene Expression by Protease-Alleviated Spatial Sequestration (PASS).
abstract: In natural microbial systems, conditional spatial sequestration of transcription factors enables cells to respond rapidly to changes in their environment or intracellular state by releasing presynthesized regulatory proteins. Although such a mechanism may be useful for engineering synthetic biology technologies ranging from cell-based biosensors to biosynthetic platforms, to date it remains unknown how or whether such conditional spatial sequestration may be engineered. In particular, based upon seemingly contradictory reports in the literature, it is not clear whether subcellular spatial localization of a transcription factor within the cytoplasm is sufficient to preclude regulation of cognate promoters on plasmid-borne or chromosomal loci. Here, we describe a modular, orthogonal platform for investigating and implementing this mechanism using protease-alleviated spatial sequestration (PASS). In this system, expression of an exogenous protease mediates the proteolytic release of engineered transcriptional regulators from the inner face of the Escherichia coli cytoplasmic membrane. We demonstrate that PASS mediates robust, conditional regulation of either transcriptional repression, via tetR, or transcriptional activation, by the λ phage CI protein. This work provides new insights into a biologically important facet of microbial gene expression and establishes a new strategy for engineering conditional transcriptional regulation for the microbial synthetic biology toolbox.
authors.affiliation: Department of Biomedical Engineering, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Ragan A
authors.initials: RA
authors.lastname: Pitner
authors.affiliation: Interdepartmental Biosciences Graduate Program, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Andrew H
authors.initials: AH
authors.lastname: Scarpelli
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University , Evanston, Illinois 60208, United States.|Chemistry of Life Processes Institute, Northwestern University , Evanston, Illinois 60208, United States.|Robert H. Lurie Comprehensive Cancer Center, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Joshua N
authors.initials: JN
authors.lastname: Leonard
mesh_terms: D025181:ATP Synthetase Complexes / Q000235:genetics* / Q000378:metabolism; D002876:Chromosomes, Bacterial / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D056426:Genetic Loci; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D025181:ATP Synthetase Complexes; C516212:ATP synthase b subunit, E coli; D010450:Endopeptidases; C099488:TEV protease
keywords: gene regulation; microbiology; signaling; subcellular organization; transcription factor