skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor

Abstract

A whole-cell biosensor utilizing a transcription factor (TF) is an effective tool for sensitive and selective detection of specialty chemicals or anthropogenic molecules, but requires an access to an expanded repertoire of TFs. Using ligand docked homology models for binding pocket identification, assisted by conservative mutations in the pocket, we engineered a novel specificity in an Acinetobacter TF, PobR, to ‘sense’ a chemical p-nitrophenol (pNP) and measured the response via a fluorescent protein reporter expressed from a PobR promoter. Out of 10 7 variants of PobR, four were active when pNP was added as an inducer, with two mutants showing a specificity switch from the native effector 4-hydroxybenzoate (4HB). One of the mutants, pNPmut1 was then used to create a smart microbial cell responding to pNP production and detect hydrolysis of an insecticide, paraoxon, in a coupled assay involving phosphotriesterase (PTE) enzyme expressed from a separate promoter. We show that the fluorescence of the cells correlated with the catalytic efficiency of PTE variants, each cell expressed. High selectivity for similar molecules (4HB vs pNP), high sensitivity for pNP detection (~2 μM) and agreement of apo- and holo- structures of PobR scaffold with computational models are notable successes presented in thismore » work.« less

Authors:
 [1];  [1];  [2];  [2];  [2];  [3];  [1]
  1. Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
  2. Argonne National Lab. (ANL), Argonne, IL (United States)
  3. Argonne National Lab. (ANL), Argonne, IL (United States); Univ. of Chicago, IL (United States)
Publication Date:
Research Org.:
Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Sponsoring Org.:
USDOD; USDOE
OSTI Identifier:
1396115
Report Number(s):
LA-UR-16-20060
Journal ID: ISSN 0305-1048
Grant/Contract Number:
AC52-06NA25396
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Volume: 44; Journal Issue: 17; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Biological Science; whole-cell biosensor, transcription factor, protein design, high throughput screening, paraoxon, nerve agent, flow cytometry

Citation Formats

Jha, Ramesh K., Kern, Theresa L., Kim, Youngchang, Tesar, Christine, Jedrzejczak, Robert, Joachimiak, Andrzej, and Strauss, Charlie E.  M.. A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor. United States: N. p., 2016. Web. doi:10.1093/nar/gkw687.
Jha, Ramesh K., Kern, Theresa L., Kim, Youngchang, Tesar, Christine, Jedrzejczak, Robert, Joachimiak, Andrzej, & Strauss, Charlie E.  M.. A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor. United States. doi:10.1093/nar/gkw687.
Jha, Ramesh K., Kern, Theresa L., Kim, Youngchang, Tesar, Christine, Jedrzejczak, Robert, Joachimiak, Andrzej, and Strauss, Charlie E.  M.. 2016. "A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor". United States. doi:10.1093/nar/gkw687. https://www.osti.gov/servlets/purl/1396115.
@article{osti_1396115,
title = {A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor},
author = {Jha, Ramesh K. and Kern, Theresa L. and Kim, Youngchang and Tesar, Christine and Jedrzejczak, Robert and Joachimiak, Andrzej and Strauss, Charlie E.  M.},
abstractNote = {A whole-cell biosensor utilizing a transcription factor (TF) is an effective tool for sensitive and selective detection of specialty chemicals or anthropogenic molecules, but requires an access to an expanded repertoire of TFs. Using ligand docked homology models for binding pocket identification, assisted by conservative mutations in the pocket, we engineered a novel specificity in an Acinetobacter TF, PobR, to ‘sense’ a chemical p-nitrophenol (pNP) and measured the response via a fluorescent protein reporter expressed from a PobR promoter. Out of 107 variants of PobR, four were active when pNP was added as an inducer, with two mutants showing a specificity switch from the native effector 4-hydroxybenzoate (4HB). One of the mutants, pNPmut1 was then used to create a smart microbial cell responding to pNP production and detect hydrolysis of an insecticide, paraoxon, in a coupled assay involving phosphotriesterase (PTE) enzyme expressed from a separate promoter. We show that the fluorescence of the cells correlated with the catalytic efficiency of PTE variants, each cell expressed. High selectivity for similar molecules (4HB vs pNP), high sensitivity for pNP detection (~2 μM) and agreement of apo- and holo- structures of PobR scaffold with computational models are notable successes presented in this work.},
doi = {10.1093/nar/gkw687},
journal = {Nucleic Acids Research},
number = 17,
volume = 44,
place = {United States},
year = 2016,
month = 8
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 1work
Citation information provided by
Web of Science

Save / Share:
  • A whole-cell biosensor utilizing a transcription factor (TF) is an effective tool for sensitive and selective detection of specialty chemicals or anthropogenic molecules, but requires access to an expanded repertoire of TFs. Using homology modeling and ligand docking for binding pocket identification, assisted by conservative mutations in the pocket, we engineered a novel specificity in an Acinetobacter TF, PobR, to ‘sense’ a chemical p-nitrophenol (pNP) and measured the response via a fluorescent protein reporter expressed from a PobR promoter. Out of 107 variants of PobR, four were active when dosed with pNP, with two mutants showing a specificity switch frommore » the native effector 4-hydroxybenzoate (4HB). One of the mutants, pNPmut1 was then used to create a smart microbial cell responding to pNP production from hydrolysis of an insecticide, paraoxon, in a coupled assay involving phosphotriesterase (PTE) enzyme expressed from a separate promoter. We show the fluorescence of the cells correlated with the catalytic efficiency of the PTE variant expressed in each cell. High selectivity between similar molecules (4HB versus pNP), high sensitivity for pNP detection (~2 μM) and agreement of apo- and holo-structures of PobR scaffold with predetermined computational models are other significant results presented in this work.« less
  • Primary effusion lymphoma (PEL) is a rare and aggressive non-Hodgkin's lymphoma. Human telomerase reverse transcriptase (hTERT), a key component responsible for the regulation of telomerase activity, plays important roles in cellular immortalization and cancer development. Triptolide purified from Tripterygium extracts displays a broad-spectrum bioactivity profile, including immunosuppressive, anti-inflammatory, and anti-tumor. In this study, it is investigated whether triptolide reduces hTERT expression and suppresses its activity in PEL cells. The mRNA and protein levels of hTERT were examined by real time-PCR and Western blotting, respectively. The activity of hTERT promoter was determined by Dual luciferase reporter assay. Our results demonstrated thatmore » triptolide decreased expression of hTERT at both mRNA and protein levels. Further gene sequence analysis indicated that the activity of hTERT promoter was suppressed by triptolide. Triptolide also reduced the half-time of hTERT. Additionally, triptolide inhibited the expression of transcription factor specificity protein 1(Sp1) in PEL cells. Furthermore, knock-down of Sp1 by using specific shRNAs resulted in down-regulation of hTERT transcription and protein expression levels. Inhibition of Sp1 by specific shRNAs enhanced triptolide-induced cell growth inhibition and apoptosis. Collectively, our results demonstrate that the inhibitory effect of triptolide on hTERT transcription is possibly mediated by inhibition of transcription factor Sp1 in PEL cells. - Highlights: • Triptolide reduces expression of hTERT by decreasing its transcription level. • Triptolide reduces promoter activity and stability of hTERT. • Triptolide down-regulates expression of Sp1. • Special Sp1 shRNAs inhibit transcription and protein expression of hTERT. • Triptolide and Sp1 shRNA2 induce cell proliferation inhibition and apoptosis.« less
  • No abstract prepared.