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.
%0 Journal Article
%1 Blount2012Rational
%A Blount, Benjamin A.
%A Weenink, Tim
%A Vasylechko, Serge
%A Ellis, Tom
%D 2012
%I Public Library of Science
%J PLoS ONE
%K bt3240 gene-expression promoters regulation synthetic-biology
%N 3
%P e33279+
%R 10.1371/journal.pone.0033279
%T Rational Diversification of a Promoter Providing Fine-Tuned Expression and Orthogonal Regulation for Synthetic Biology
%U http://dx.doi.org/10.1371/journal.pone.0033279
%V 7
%X 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.
@article{Blount2012Rational,
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.},
added-at = {2018-12-02T16:09:07.000+0100},
author = {Blount, Benjamin A. and Weenink, Tim and Vasylechko, Serge and Ellis, Tom},
biburl = {https://www.bibsonomy.org/bibtex/257ac9dc0b4b52234bd9fbf7f240f10c4/karthikraman},
citeulike-article-id = {10485822},
citeulike-linkout-0 = {http://dx.doi.org/10.1371/journal.pone.0033279},
citeulike-linkout-1 = {http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3307721/},
citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/22442681},
citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=22442681},
day = 19,
doi = {10.1371/journal.pone.0033279},
interhash = {4a6229e40ea81508336f7ba61fdc0f95},
intrahash = {57ac9dc0b4b52234bd9fbf7f240f10c4},
issn = {1932-6203},
journal = {PLoS ONE},
keywords = {bt3240 gene-expression promoters regulation synthetic-biology},
month = mar,
number = 3,
pages = {e33279+},
pmcid = {PMC3307721},
pmid = {22442681},
posted-at = {2012-04-03 05:02:28},
priority = {2},
publisher = {Public Library of Science},
timestamp = {2018-12-02T16:09:07.000+0100},
title = {Rational Diversification of a Promoter Providing {Fine-Tuned} Expression and Orthogonal Regulation for Synthetic Biology},
url = {http://dx.doi.org/10.1371/journal.pone.0033279},
volume = 7,
year = 2012
}