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Title: Sort-Seq Approach to Engineering a Formaldehyde-Inducible Promoter for Dynamically Regulated Escherichia coli Growth on Methanol

Abstract

Tight and tunable control of gene expression is a highly desirable goal in synthetic biology for constructing predictable gene circuits and achieving preferred phenotypes. Elucidating the sequence–function relationship of promoters is crucial for manipulating gene expression at the transcriptional level, particularly for inducible systems dependent on transcriptional regulators. Sort-seq methods employing fluorescence-activated cell sorting (FACS) and high-throughput sequencing allow for the quantitative analysis of sequence–function relationships in a robust and rapid way. Here we utilized a massively parallel sort-seq approach to analyze the formaldehyde-inducible Escherichia coli promoter (Pfrm) with single-nucleotide resolution. A library of mutated formaldehyde-inducible promoters was cloned upstream of gfp on a plasmid. The library was partitioned into bins via FACS on the basis of green fluorescent protein (GFP) expression level, and mutated promoters falling into each expression bin were identified with high-throughput sequencing. The resulting analysis identified two 19 base pair repressor binding sites, one upstream of the -35 RNA polymerase (RNAP) binding site and one overlapping with the -10 site, and assessed the relative importance of each position and base therein. Key mutations were identified for tuning expression levels and were used to engineer formaldehyde-inducible promoters with predictable activities. Engineered variants demonstrated up to 14-fold lowermore » basal expression, 13-fold higher induced expression, and a 3.6-fold stronger response as indicated by relative dynamic range. Finally, an engineered formaldehyde-inducible promoter was employed to drive the expression of heterologous methanol assimilation genes and achieved increased biomass levels on methanol, a non-native substrate of E. coli.« less

Authors:
ORCiD logo; ORCiD logo;
Publication Date:
Research Org.:
Univ. of Delaware, Newark, DE (United States); Tulane Univ., New Orleans, LA (United States)
Sponsoring Org.:
USDOE Advanced Research Projects Agency - Energy (ARPA-E); National Inst. of Health (NIH) (United States)
OSTI Identifier:
1355946
Alternate Identifier(s):
OSTI ID: 1508258
Grant/Contract Number:  
AR0000432; R01GM085232; F32GM109617-01A1
Resource Type:
Published Article
Journal Name:
ACS Synthetic Biology
Additional Journal Information:
Journal Name: ACS Synthetic Biology; Journal ID: ISSN 2161-5063
Publisher:
American Chemical Society
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; biosensor; promoter engineering; sort-seq; transcription factor binding; transcriptional fine-tuning

Citation Formats

Rohlhill, Julia, Sandoval, Nicholas R., and Papoutsakis, Eleftherios T. Sort-Seq Approach to Engineering a Formaldehyde-Inducible Promoter for Dynamically Regulated Escherichia coli Growth on Methanol. United States: N. p., 2017. Web. doi:10.1021/acssynbio.7b00114.
Rohlhill, Julia, Sandoval, Nicholas R., & Papoutsakis, Eleftherios T. Sort-Seq Approach to Engineering a Formaldehyde-Inducible Promoter for Dynamically Regulated Escherichia coli Growth on Methanol. United States. doi:10.1021/acssynbio.7b00114.
Rohlhill, Julia, Sandoval, Nicholas R., and Papoutsakis, Eleftherios T. Tue . "Sort-Seq Approach to Engineering a Formaldehyde-Inducible Promoter for Dynamically Regulated Escherichia coli Growth on Methanol". United States. doi:10.1021/acssynbio.7b00114.
@article{osti_1355946,
title = {Sort-Seq Approach to Engineering a Formaldehyde-Inducible Promoter for Dynamically Regulated Escherichia coli Growth on Methanol},
author = {Rohlhill, Julia and Sandoval, Nicholas R. and Papoutsakis, Eleftherios T.},
abstractNote = {Tight and tunable control of gene expression is a highly desirable goal in synthetic biology for constructing predictable gene circuits and achieving preferred phenotypes. Elucidating the sequence–function relationship of promoters is crucial for manipulating gene expression at the transcriptional level, particularly for inducible systems dependent on transcriptional regulators. Sort-seq methods employing fluorescence-activated cell sorting (FACS) and high-throughput sequencing allow for the quantitative analysis of sequence–function relationships in a robust and rapid way. Here we utilized a massively parallel sort-seq approach to analyze the formaldehyde-inducible Escherichia coli promoter (Pfrm) with single-nucleotide resolution. A library of mutated formaldehyde-inducible promoters was cloned upstream of gfp on a plasmid. The library was partitioned into bins via FACS on the basis of green fluorescent protein (GFP) expression level, and mutated promoters falling into each expression bin were identified with high-throughput sequencing. The resulting analysis identified two 19 base pair repressor binding sites, one upstream of the -35 RNA polymerase (RNAP) binding site and one overlapping with the -10 site, and assessed the relative importance of each position and base therein. Key mutations were identified for tuning expression levels and were used to engineer formaldehyde-inducible promoters with predictable activities. Engineered variants demonstrated up to 14-fold lower basal expression, 13-fold higher induced expression, and a 3.6-fold stronger response as indicated by relative dynamic range. Finally, an engineered formaldehyde-inducible promoter was employed to drive the expression of heterologous methanol assimilation genes and achieved increased biomass levels on methanol, a non-native substrate of E. coli.},
doi = {10.1021/acssynbio.7b00114},
journal = {ACS Synthetic Biology},
number = ,
volume = ,
place = {United States},
year = {2017},
month = {5}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1021/acssynbio.7b00114

Citation Metrics:
Cited by: 14 works
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Works referencing / citing this record:

A Strongly Fluorescing Anaerobic Reporter and Protein-Tagging System for Clostridium Organisms Based on the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST)
journal, May 2019

  • Streett, Hannah E.; Kalis, Katie M.; Papoutsakis, Eleftherios T.
  • Applied and Environmental Microbiology, Vol. 85, Issue 14
  • DOI: 10.1128/aem.00622-19

A Strongly Fluorescing Anaerobic Reporter and Protein-Tagging System for Clostridium Organisms Based on the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST)
journal, May 2019

  • Streett, Hannah E.; Kalis, Katie M.; Papoutsakis, Eleftherios T.
  • Applied and Environmental Microbiology, Vol. 85, Issue 14
  • DOI: 10.1128/aem.00622-19