title: Rational diversification of a promoter providing fine-tuned expression and orthogonal regulation for synthetic biology.
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.
authors.affiliation: Centre for Synthetic Biology and Innovation, Imperial College London, London, United Kingdom.
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Blount
authors.forename: Tim
authors.initials: T
authors.lastname: Weenink
authors.forename: Serge
authors.initials: S
authors.lastname: Vasylechko
authors.forename: Tom
authors.initials: T
authors.lastname: Ellis
mesh_terms: D015966:Gene Expression Regulation, Fungal / Q000502:physiology*; D015723:Gene Library; D051304:Profilins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D058615:Synthetic Biology / Q000379:methods*
chemical_list: C547108:PFY1 protein, S cerevisiae; D051304:Profilins