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Nat Methods. 2016 Feb;13(2):177-83. doi: 10.1038/nmeth.3696. Epub 2015 Dec 21.

Engineering an allosteric transcription factor to respond to new ligands.

Author information

1
Wyss Institute for Biologically-Inspired Engineering, Harvard University, Boston, Massachusetts, USA.
2
Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
3
Department of Biochemistry, University of Washington, Seattle, Washington, USA.
4
Howard Hughes Medical Institute, University of Washington, Seattle, Washington, USA.
5
University of California Los Angeles-Department of Energy Institute for Genomics and Proteomics, University of California Los Angeles, Los Angeles, California, USA.
6
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA.
7
Systems Biology Institute, Yale University, West Haven, Connecticut, USA.
8
Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, California, USA.
9
Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
10
Department of Medicine, University of Washington, Seattle, Washington, USA.

Abstract

Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery. We engineered an aTF, the Escherichia coli lac repressor, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol and sucralose. Using computational protein design, single-residue saturation mutagenesis or random mutagenesis, along with multiplex assembly, we identified new variants comparable in specificity and induction to wild-type LacI with its inducer, isopropyl β-D-1-thiogalactopyranoside (IPTG). The ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits.

PMID:
26689263
PMCID:
PMC4907361
DOI:
10.1038/nmeth.3696
[Indexed for MEDLINE]
Free PMC Article

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