EP1963538A2 - Differentiell fluoreszierende hefebiosensoren zur erkennung und zum biologischen abbau chemischer mittel - Google Patents

Differentiell fluoreszierende hefebiosensoren zur erkennung und zum biologischen abbau chemischer mittel

Info

Publication number
EP1963538A2
EP1963538A2 EP06851299A EP06851299A EP1963538A2 EP 1963538 A2 EP1963538 A2 EP 1963538A2 EP 06851299 A EP06851299 A EP 06851299A EP 06851299 A EP06851299 A EP 06851299A EP 1963538 A2 EP1963538 A2 EP 1963538A2
Authority
EP
European Patent Office
Prior art keywords
yeast
organophosphate
gene
paraoxon
nucleic acids
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06851299A
Other languages
English (en)
French (fr)
Inventor
David Alexander Schofield
Augustine Anthony Dinovo
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guild Associates Inc
Original Assignee
Guild Associates Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guild Associates Inc filed Critical Guild Associates Inc
Publication of EP1963538A2 publication Critical patent/EP1963538A2/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics

Definitions

  • a yeast biosensor may include a defect (e.g., mutation) in (a) cell wall synthesis, maintenance, or degradation, (b) cell membrane synthesis, maintenance, or degradation, (c) cell repair, and/or (d) cell transport (e.g., drug export pump or import pump).
  • a yeast biosensor may include one or more proteins and/or nucleic acid(s) encoding one or more proteins capable of actively importing a chemical agent into the sensor.
  • This method may further include confirming differential expression by quantitative reverse transcription PCR.
  • the present disclosure also relates to a method of identifying a yeast gene that is upregulated by or during organosphosphate hydrolysis.
  • this method may include (a) contacting a recombinant OPH+ yeast with an organophosphate under conditions that permit organophosphate hydrolysis, (b) collecting RNA from the recombinant OPH+ yeast, (c) contacting the subtracted RNA with a yeast mircoarray having feature loci that correspond to yeast genes under conditions that permit hybridization of complimentary sequences, (d) comparing a metric of the hybridization at each feature locus with the same metric of hybridization at a corresponding feature locus for RNA from yeast lacking OPH contacted with the organophosphate, (e) identifying a feature locus where the hybridization metric is higher for the recombinant OPH+ yeast contacted with the organophosphate than the yeast lacking OPH contacted with the organophosphate, and (f) correlating
  • a yeast biosensor that is sensitive to both an organophosphate and an organophosphate hydrolytic product may be prepared by (a) identifying a yeast gene that is upregulated by an organophosphate, (b) identifying at least one expression control sequence of the identified organophosphate-sensitive gene, (c) operably linking a nucleic acid comprising the organophosphate expression control sequence to a nucleic acid encoding a reporter, (d) contacting the operably linked organophosphate nucleic acids with a cell under conditions that permit uptake of the nucleic acids, (e) identifying a yeast gene that is upregulated by an organophosphate hydrolytic product and/or by a process of organophosphate hydrolysis, (f) identifying at least one expression control sequence of the identified organophosphate hydrolysis- sensitive gene, (g) operably linking a nucleic acid comprising the organophosphate hydrolytic product expression control sequence to a nucleic acid encoding a reporter, and (h) contacting the operably linked organophosphate hydro
  • FIGURE 9B is a chart illustrating the effect of 0.5 mM paraoxon on the growth (monitored at A 6 oo) of S. cerevisiae wild-type (BY4741) and the membrane mutant strain erg4;
  • the comparison between wild-type cells incubated with VX, and recombinant cells expressing OPH incubated with VX, may facilitate identification of one or more yeast genes induced by the hydrolysis of VX. Hydrolysis of VX using a yeast strain expressing OPH may result in a transcriptional profile that is distinct from cells lacking the ability to hydrolyze VX. Real-time RT-PCR may then be used on prioritized targets using different concentrations of VX to identify yeast genes that are induced at low concentrations and in a dose-dependent manner. Promoter regions of prioritized genes may be mapped using the
  • the GAPDH promoter or a similarly highly expressed constitutive promoter may be used to drive foreign gene expression in yeast.
  • the GAPDH promoter may be operably linked (e.g. , positioned adjacent to) the opd gene such that the opd gene is constitutively expressed.
  • clonal cells each harboring a specific biodegrading enzyme and specific reporter detection system for a specific chemical, may be mixed into a heterogeneous population that as a group, can detect and biodegrade different chemical agents.
  • the Flavobacterium opd gene is identical to the Pseudomonas putida opd gene (Mulbry WW & Karns JS., (1989) J Bacteriol 171, 6740-6).
  • a lkb opd fragment was PCR-amplified using a proofreading thermostable polymerase (Fig. 1).
  • the 5' primer was designed to incorporate a yeast ribosome binding site to ensure optimal translation initiation (Looman AC & Kuivenhoven JA., (1983) Nuc Acids Res 21, 4268-71).
  • the 5' and 3' primers were also designed to incorporate the restriction endonuclease sites BamHl and Hind ⁇ ll, respectively.
  • RNA was prepared using Tri reagent (Ambion) according to the manufacturers' instructions and treated with DNasel. Equal amounts of RNA (approximately 1 ⁇ g) were reverse transcribed into cDNA using the retroscript kit (Ambion). PCR was performed initially with primers designed against the S. cerevisiae housekeeping gene actin (ACTl, 547 bp product) to ensure equal amounts of cDNA were used for each sample; if required, the amount of cDNA template was then adjusted accordingly before opd analysis. PCR products were resolved and analyzed using agarose gel electrophoresis. PCR analysis was also performed with samples that lacked reverse transcriptase (RNA samples); no RT-PCR products were detected from these samples as expected.
  • RNA samples reverse transcriptase
  • Opd expression is not toxic to S. cerevisiae; however, there was a slight inhibition in the growth rate of yeast cells harboring the opd plasmid under inducing conditions.
  • Control enzyme lysates prepared from yeast cells harboring the empty plasmid, were not able to hydrolyze paraoxon.
  • yeast cells harboring the opd expression plasmid produced functional OPH protein that hydrolyzed paraoxon (Table 1).
  • Most (75%) of the enzyme activity in S. cerevisiae was membrane associated (triton extractable) with the remainder in the cytosolic fraction. This distribution is comparable to the wild-type Flavobacterium species (Mulbry WW & Karns JS., (1989) J Bacteriol 171, 6740-6).
  • Intact recombinant yeast cells were capable of hydrolyzing paraoxon and functioning as a yeast biocatalyst. Intact yeast cells displayed lower paraoxon hydrolysis compared to enzyme lysates presumably because a rate limiting step is the rate of paraoxon diffusion into the cell.
  • YeGFP is codon optimized for expression in the yeast Candida albicans, and is also highly fluorescent in S. cerevisiae (Cormack BP et al., (1997) Microbiology 143, 303-11).
  • a strain of C. albicans containing YeGFP was used.
  • the 700 bp YeGFP gene was PCR-amplified using a proofreading polymerase and C. albicans YeGFP genomic DNA as template.
  • the PCR primers were designed to contain BamRl and Spel endonuclease sites for cloning into the respective sites of pESC-HIS.
  • YGR035C and YLR346C promoters 2 putative promoter fragments for each gene containing approximately -1000 and - 500 bp upstream sequence (relative to ATG) were cloned in front of YeGFP.
  • the putative promoter regions were PCR-cloned using £. cerevisiae W3031A genomic DNA as template and a proofreading thermostable DNA polymerase.
  • the 5' and 3' primers contained SaR and Barri ⁇ l sites, respectively, for directional cloning into the same sites of p YeGFP-HIS to generate promoter-reporter gene fusions.
  • Yeast cells harboring paraoxon-inducible promoter- YeGFP fusions displayed up to 5-fold increases in fluorescence levels compared to control cells when incubated in the presence of paraoxon. Increased YeGFP levels were detected after 15 min exposure to paraoxon.
  • the YeGFP biosensor was sensitive to as low as 0.1 mM paraoxon and exhibited dose-dependent characteristics by increasing YeGFP fluorescence as the paraoxon concentration increased. The results demonstrate the ability of yeast cells to function as a biosensor and detect the presence of paraoxon.
  • the yeast codon optimized DsRed, YDsRed was also designed to contain a preferred yeast ribosome binding site and BamHI and Notl restriction endonuclease sites for cloning into the corresponding sites of pESC-LEU (Stratagene).
  • the resulting pYDsRed-LEU promoterless vector contained an adequate MCS for cloning the POXl and YGR287C promoters.
  • Cells were grown in SDgal/suc lacking leucine in the presence or absence of 3 niM paraoxon for 4.5 h at 37° C. Cells were harvested by centrifugation, washed in PBS, resuspended in 10 mM Tris-HCl pH 8.5, and duplicate samples were measured for YDsRed fluorescence (excitation and emission at 554 nm and 590 nm, respectively). All results were normalized to the number of cells present (OD 6 oo), to the 'promoterless' control vector (YDsRed-LEU) and are presented as fold-induction compared to cells lacking paraoxon.
  • Promoter Fold-induction 8 Promoter Fold-induction 1 * pYGR287C-Fl 1.41 pYGR287C-Fl 2.1 pYGR287C-F2 1.13 pYGR287C-F2 2.4 pPOXl-Fl 0.84 pPOXl-Fl 3.0
  • POXl promoter (525 cerevisiae bp upstream of ATG) driving YDsRed pYGR287C-F 1 -YDsRed-LEU E. coli/S. YGR287C promoter cerevisiae (993 bp upstream of
  • Bold indicates the BamHl or SaR restriction site for cloning into the corresponding sites of YeGFP-His or YDsRed-LEU.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
EP06851299A 2005-11-30 2006-11-29 Differentiell fluoreszierende hefebiosensoren zur erkennung und zum biologischen abbau chemischer mittel Withdrawn EP1963538A2 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US74088705P 2005-11-30 2005-11-30
PCT/US2006/045761 WO2007145661A2 (en) 2005-11-30 2006-11-29 Differentially fluorescent yeast biosensors for the detection and biodegradation of chemical agents

Publications (1)

Publication Number Publication Date
EP1963538A2 true EP1963538A2 (de) 2008-09-03

Family

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Family Applications (1)

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EP06851299A Withdrawn EP1963538A2 (de) 2005-11-30 2006-11-29 Differentiell fluoreszierende hefebiosensoren zur erkennung und zum biologischen abbau chemischer mittel

Country Status (3)

Country Link
EP (1) EP1963538A2 (de)
JP (1) JP2009518006A (de)
WO (1) WO2007145661A2 (de)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008132178A1 (de) * 2007-04-25 2008-11-06 Technische Universität Dresden Ganzzellsensor
WO2009082525A2 (en) * 2007-10-01 2009-07-02 Guild Associates, Inc. Differentially fluorescent yeast biosensors for the detection and biodegradation of chemical agents
DK3118311T3 (en) * 2014-03-12 2019-03-11 Nippon Shinyaku Co Ltd Antisense nucleic acid
US10988759B2 (en) 2016-01-15 2021-04-27 University Of Washington High throughput protein-protein interaction screening in yeast liquid culture
US20220380754A1 (en) 2020-05-11 2022-12-01 A-Alpha Bio, Inc. High-Throughput Screening Methods to Identify Small Molecule Targets
EP4158015A1 (de) 2020-06-01 2023-04-05 A-Alpha Bio, Inc. Verfahren zur charakterisierung und manipulation von protein-protein-wechselwirkungen
CN115753714B (zh) * 2022-11-21 2024-06-04 清华大学 一种生物传感器、核酸分子、表达载体及应用

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6117643A (en) 1997-11-25 2000-09-12 Ut Battelle, Llc Bioluminescent bioreporter integrated circuit
AU4660800A (en) 1999-04-26 2000-11-10 U.S. Army Medical Research And Materiel Command Immobilized enzymes as biosensors for chemical toxins
US20080044844A1 (en) 2004-01-27 2008-02-21 Shimshon Belkin Populations Of Cells And Devices And Systems Including Same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GOFFEAU A ET AL: "LIFE WITH 6000 GENES", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, WASHINGTON, DC; US LNKD- DOI:10.1126/SCIENCE.274.5287.546, vol. 274, 25 October 1996 (1996-10-25), pages 546,563 - 567, XP002070912, ISSN: 0036-8075 *
See also references of WO2007145661A2 *

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JP2009518006A (ja) 2009-05-07
WO2007145661A2 (en) 2007-12-21
WO2007145661A3 (en) 2008-04-10

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