Rapid generation of CRISPR/dCas9-regulated, orthogonally repressible hybrid T7-lac promoters for modular, tuneable control of metabolic pathway fluxes in E. coli
AuthorCress, B.; Jones, J.; Daniel, K.; Leitz, Q.; Englaender, J.; Collins, S.; Linhardt, Robert J.; Koffas, M.
SubjectBiology; Chemistry and chemical biology; Chemical and biological engineering; Biomedical engineering
Full CitationRapid generation of CRISPR/dCas9-regulated, orthogonally repressible hybrid T7-lac promoters for modular, tuneable control of metabolic pathway fluxes in E. coli, B. Cress, J. Jones, K. Daniel, Q. Leitz, J. Englaender, S. Collins, R. J. Linhardt, M. Koffas, Nucleic Acids Research, 44, 4472–4485, 2016.
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AbstractRobust gene circuit construction requires use of promoters exhibiting low crosstalk. Orthogonal promoters have been engineered utilizing an assortment of natural and synthetic transcription factors, but design of large orthogonal promoter-repressor sets is complicated, labor-intensive, and often results in unanticipated crosstalk. The specificity and ease of targeting the RNA-guided DNA-binding protein dCas9 to any 20 bp user-defined DNA sequence makes it a promising candidate for orthogonal promoter regulation. Here, we rapidly construct orthogonal variants of the classic T7-lac promoter using site-directed mutagenesis, generating a panel of inducible hybrid promoters regulated by both LacI and dCas9. Remarkably, orthogonality is mediated by only two to three nucleotide mismatches in a narrow window of the RNA:DNA hybrid, neighboring the protospacer adjacent motif. We demonstrate that, contrary to many reports, one PAM-proximal mismatch is insufficient to abolish dCas9-mediated repression, and we show for the first time that mismatch tolerance is a function of target copy number. Finally, these promoters were incorporated into the branched violacein biosynthetic pathway as dCas9-dependent switches capable of throttling and selectively redirecting carbon flux in Escherichia coli. We anticipate this strategy is relevant for any promoter and will be adopted for many applications at the interface of synthetic biology and metabolic engineering.;
DescriptionNucleic Acids Research, 44, 4472–4485; Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.
DepartmentThe Linhardt Research Labs.; The Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS);
PublisherOxford University Press
RelationshipsThe Linhardt Research Labs Online Collection; Rensselaer Polytechnic Institute, Troy, NY; https://harc.rpi.edu/;
AccessCC BY — Creative Commons Attribution; A full text version is available in DSpace@RPI; Open Access;
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