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Title: Construction of genetic logic gates based on the T7 RNA polymerase expression system in Rhodococcus opacus PD630

Abstract

Rhodococcus opacus PD630 (R. opacus) is a non-model, gram-positive bacterium which holds promise as a biological catalyst for the conversion of lignocellulosic biomass into value-added products. In particular, it demonstrates both a high tolerance for and an ability to consume inhibitory lignin-derived aromatics, generates large quantities of lipids, exhibits a relatively rapid growth rate, and has a growing genetic toolbox for engineering. Yet, the availability of genetic parts for tunable, high-activity gene expression is still limited in R. opacus. Moreover, genetic logic circuits for sophisticated gene regulation have never been demonstrated in Rhodococcus spp. To address these shortcomings, two inducible T7 RNA polymerase-based expression systems were implemented for the first time in R. opacus and applied to constructing AND and NAND genetic logic gates. Additionally, three IPTG-inducible promoters were created by inserting LacI binding sites into newly-characterized constitutive promoters. Furthermore, four novel aromatic sensors for 4-hydroxybenzoic acid, vanillic acid, sodium benzoate, and guaiacol were developed, expanding the gene expression toolbox. Lastly, the T7 RNA polymerase platform was combined with a synthetic IPTG-inducible promoter to create an IMPLY logic gate. Overall, this work represents the first demonstration of a heterologous RNA polymerase system and synthetic genetic logic in R. opacus, enablingmore » complex and tunable gene regulation in this promising non-model host for bioproduction.« less

Authors:
 [1];  [1]
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  1. Washington Univ., St. Louis, MO (United States)
Publication Date:
Research Org.:
Washington Univ., St. Louis, MO (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1545618
Grant/Contract Number:  
SC0018324
Resource Type:
Accepted Manuscript
Journal Name:
ACS Synthetic Biology
Additional Journal Information:
Journal Volume: 8; Journal Issue: 8; Journal ID: ISSN 2161-5063
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; T7 RNA polymerase; genetic circuit; Boolean logic; non-model organism; aromatic sensors

Citation Formats

DeLorenzo, Drew, and Moon, Tae Seok. Construction of genetic logic gates based on the T7 RNA polymerase expression system in Rhodococcus opacus PD630. United States: N. p., 2019. Web. doi:10.1021/acssynbio.9b00213.
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DeLorenzo, Drew, & Moon, Tae Seok. Construction of genetic logic gates based on the T7 RNA polymerase expression system in Rhodococcus opacus PD630. United States. https://doi.org/10.1021/acssynbio.9b00213
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DeLorenzo, Drew, and Moon, Tae Seok. Tue . "Construction of genetic logic gates based on the T7 RNA polymerase expression system in Rhodococcus opacus PD630". United States. https://doi.org/10.1021/acssynbio.9b00213. https://www.osti.gov/servlets/purl/1545618.
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@article{osti_1545618,
title = {Construction of genetic logic gates based on the T7 RNA polymerase expression system in Rhodococcus opacus PD630},
author = {DeLorenzo, Drew and Moon, Tae Seok},
abstractNote = {Rhodococcus opacus PD630 (R. opacus) is a non-model, gram-positive bacterium which holds promise as a biological catalyst for the conversion of lignocellulosic biomass into value-added products. In particular, it demonstrates both a high tolerance for and an ability to consume inhibitory lignin-derived aromatics, generates large quantities of lipids, exhibits a relatively rapid growth rate, and has a growing genetic toolbox for engineering. Yet, the availability of genetic parts for tunable, high-activity gene expression is still limited in R. opacus. Moreover, genetic logic circuits for sophisticated gene regulation have never been demonstrated in Rhodococcus spp. To address these shortcomings, two inducible T7 RNA polymerase-based expression systems were implemented for the first time in R. opacus and applied to constructing AND and NAND genetic logic gates. Additionally, three IPTG-inducible promoters were created by inserting LacI binding sites into newly-characterized constitutive promoters. Furthermore, four novel aromatic sensors for 4-hydroxybenzoic acid, vanillic acid, sodium benzoate, and guaiacol were developed, expanding the gene expression toolbox. Lastly, the T7 RNA polymerase platform was combined with a synthetic IPTG-inducible promoter to create an IMPLY logic gate. Overall, this work represents the first demonstration of a heterologous RNA polymerase system and synthetic genetic logic in R. opacus, enabling complex and tunable gene regulation in this promising non-model host for bioproduction.},
doi = {10.1021/acssynbio.9b00213},
journal = {ACS Synthetic Biology},
number = 8,
volume = 8,
place = {United States},
year = {Tue Jul 30 00:00:00 EDT 2019},
month = {Tue Jul 30 00:00:00 EDT 2019}
}
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Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1021/acssynbio.9b00213
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Citation Metrics:
Cited by: 15 works
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Figures / Tables:

Figure 1 Figure 1: Inducible T7 RNAP system in R. opacus. A) Schematic of the phenol- or aTc-inducible T7 RNA polymerase (T7 RNAP) expression platform. A phenol-inducible promoter (pLPD06575; referred to as pPhenol) or an optimized pTet promoter was placed upstream of the T7 RNAP gene and integrated into the R. opacusmore » genome at a previously determined neutral site (ROCI3). The T7 promoter (pT7) was placed upstream of eGFP on the pAL5000(S) plasmid backbone. B) Normalized fluorescence of a strain containing pPhenol-T7 RNAP in response to 0, 0.01, 0.025, 0.05, 0.1, 0.2, 0.3, 0.5, or 0.6 g/L phenol. The increase in fluorescent output from 0 to 0.5 g/L phenol was 55-fold. C) Normalized fluorescence of a strain containing pTet-T7 RNAP in response to 0, 0.01, 0.025, 0.1, 0.5, 1, 5, or 10 ng/mL aTc. The increase in fluorescent output from 0 to 1 ng/mL aTc was 5.3-fold. Values are averages of three replicates, and error bars represent one standard deviation. The solid black line in each case represents a fitted curve (see Supplementary Methods and Supplementary Table 4).« less
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