Rational diversification of a promoter providing fine-tuned expression and orthogonal regulation for synthetic biology

Blount, Benjamin A.; Weenink, Tim; Vasylechko, Serge; Ellis, Tom

Abstract

Yeast is an ideal organism for the development and application of synthetic biology, yet there remain relatively few well-characterised biological parts suitable for precise engineering of this chassis. In order to address this current need, we present here a strategy that takes a single biological part, a promoter, and re-engineers it to produce a fine-graded output range promoter library and new regulated promoters desirable for orthogonal synthetic biology applications. A highly constitutive Saccharomyces cerevisiae promoter, PFY1p, was identified by bioinformatic approaches, characterised in vivo and diversified at its core sequence to create a 36-member promoter library. TetR regulation was introduced into PFY1p to create a synthetic inducible promoter (iPFY1p) that functions in an inverter device. Orthogonal and scalable regulation of synthetic promoters was then demonstrated for the first time using customisable Transcription Activator-Like Effectors (TALEs) modified and designed to act as orthogonal repressors for specific PFY1-based promoters. The ability to diversify a promoter at its core sequences and then independently target Transcription Activator-Like Orthogonal Repressors (TALORs) to virtually any of these sequences shows great promise toward the design and construction of future synthetic gene networks that encode complex "multi-wire" logic functions. © 2012 Blount et al.

Citation

Blount, B. A., Weenink, T., Vasylechko, S., & Ellis, T. (2012). Rational diversification of a promoter providing fine-tuned expression and orthogonal regulation for synthetic biology. PLoS ONE, 7(3), Article e33279. https://doi.org/10.1371/journal.pone.0033279

• Journal Article Type: Article
• Acceptance Date: Feb 13, 2012
• Publication Date: Mar 19, 2012
• Deposit Date: Aug 3, 2022
• Publicly Available Date: Aug 5, 2022
• Journal: PLoS ONE
• Electronic ISSN: 1932-6203
• Publisher: Public Library of Science
• Peer Reviewed: Peer Reviewed
• Volume: 7
• Issue: 3
• Article Number: e33279
• DOI: https://doi.org/10.1371/journal.pone.0033279
• Public URL: https://nottingham-repository.worktribe.com/output/4183565
• Publisher URL: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033279