Engineering an allosteric transcription factor to respond to new ligands
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. In this paper, 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). Finally, the ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits.
- Authors:
-
[5];
- Harvard Univ., Boston, MA (United States). Wyss Inst. for Biologically-Inspired Engineering; Harvard Medical School, Boston, MA (United States). Dept. of Genetics
- Univ. of Washington, Seattle, WA (United States). Dept. of Biochemistry. Howard Hughes Medical Inst.
- Univ. of California, Los Angeles, CA (United States). UCLA-USDOE Inst. for Genomics and Proteomics
- Yale Univ., New Haven, CT (United States). Dept. of Molecular, Cellular and Developmental Biology; Yale Univ., West Haven, CT (United States). Systems Biology Inst.
- Univ. of California, Los Angeles, CA (United States). Dept. of Chemistry and Biochemistry
- Univ. of Washington, Seattle, WA (United States). Dept. of Genome Sciences. Dept. of Medicine. Howard Hughes Medical Inst.
- Publication Date:
- Research Org.:
- Harvard Univ., Cambridge, MA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER); National Inst. of Health (NIH) (United States)
- Contributing Org.:
- Yale Univ., New Haven, CT (United States); Univ. of California, Los Angeles, CA (United States); Univ. of Washington, Seattle, WA (United States)
- OSTI Identifier:
- 1239401
- Grant/Contract Number:
- FG02-02ER63445; FC02-02ER63421; AC02-06CH11357; 1P41 GM103533; P41 RR015301; P41 GM103403
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Nature Methods
- Additional Journal Information:
- Journal Volume: 13; Journal Issue: 2; Journal ID: ISSN 1548-7091
- Publisher:
- Nature Publishing Group
- Country of Publication:
- United States
- Language:
- ENGLISH
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; Genetic engineering; Protein design
Citation Formats
Taylor, Noah D., Garruss, Alexander S., Moretti, Rocco, Chan, Sum, Arbing, Mark A., Cascio, Duilio, Rogers, Jameson K., Isaacs, Farren J., Kosuri, Sriram, Baker, David, Fields, Stanley, Church, George M., and Raman, Srivatsan. Engineering an allosteric transcription factor to respond to new ligands. United States: N. p., 2015.
Web. doi:10.1038/nmeth.3696.
Taylor, Noah D., Garruss, Alexander S., Moretti, Rocco, Chan, Sum, Arbing, Mark A., Cascio, Duilio, Rogers, Jameson K., Isaacs, Farren J., Kosuri, Sriram, Baker, David, Fields, Stanley, Church, George M., & Raman, Srivatsan. Engineering an allosteric transcription factor to respond to new ligands. United States. https://doi.org/10.1038/nmeth.3696
Taylor, Noah D., Garruss, Alexander S., Moretti, Rocco, Chan, Sum, Arbing, Mark A., Cascio, Duilio, Rogers, Jameson K., Isaacs, Farren J., Kosuri, Sriram, Baker, David, Fields, Stanley, Church, George M., and Raman, Srivatsan. Mon .
"Engineering an allosteric transcription factor to respond to new ligands". United States. https://doi.org/10.1038/nmeth.3696. https://www.osti.gov/servlets/purl/1239401.
@article{osti_1239401,
title = {Engineering an allosteric transcription factor to respond to new ligands},
author = {Taylor, Noah D. and Garruss, Alexander S. and Moretti, Rocco and Chan, Sum and Arbing, Mark A. and Cascio, Duilio and Rogers, Jameson K. and Isaacs, Farren J. and Kosuri, Sriram and Baker, David and Fields, Stanley and Church, George M. and Raman, Srivatsan},
abstractNote = {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. In this paper, 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). Finally, the ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits.},
doi = {10.1038/nmeth.3696},
journal = {Nature Methods},
number = 2,
volume = 13,
place = {United States},
year = {2015},
month = {12}
}
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