WO2002053772A1 - Nouveau systeme de biocapteurs - Google Patents

Nouveau systeme de biocapteurs Download PDF

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Publication number
WO2002053772A1
WO2002053772A1 PCT/EP2001/013231 EP0113231W WO02053772A1 WO 2002053772 A1 WO2002053772 A1 WO 2002053772A1 EP 0113231 W EP0113231 W EP 0113231W WO 02053772 A1 WO02053772 A1 WO 02053772A1
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promoter
biosensor system
hpd
cell
organism
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PCT/EP2001/013231
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Michael Schledz
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Greenovation Biotech Gmbh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • 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
    • C12Q1/001—Enzyme electrodes
    • 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
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • the present invention relates to the field of qualitative and/or quantitative analyses and provides a new biosensor system for the detection of analytes which is based on a reporter gene that can also be used as a reporter system in molecularbiological approaches and pharmacological screenings.
  • the present invention provides a biosensor system for the evaluation of the presence and quantity of chemical substances desired to be analyzed as well as processes using said biosensor system.
  • the biosensor system according to the present invention uses metabolic/catabolic pathways of living cells which have been transformed to display specific characteristics that can be used to identify the presence and quantity of a broad range of analytes.
  • test systems which enable detection of certain analytes.
  • these test sytems are mostly cost and labour intensive and only a small number of them are based on living cells or organisms.
  • the present invention is primarily based on the use of j?-hydroxyphenyl-pyruvic acid dioxygenase (HPD) cDNA as a reporter gene.
  • HPD j?-hydroxyphenyl-pyruvic acid dioxygenase
  • cDNAs coding for acid dioxygenase from different sources were cloned under the control of inducible promoters in appropriate vectors, which, upon transformation, ensure inducible expression of the reporter gene in selected model organisms such as E. coli and Saccharomyces cerevisiae.
  • the enzymatic activity of the translation product HPD catalyzes the metabolization of p- hydroxyphenyl-pyruvic acid and the formation of homogentisic acid which is secreted from the transformed host cell into the extracellular space.
  • Spontaneous oxidation of homogentisic acid by e.g. oxygen and/or NaOH forms ochronotic acid, a brown coloured pigment.
  • hpd-de ⁇ ved expression vectors can be used as reporter and biosensor systems.
  • the emergence and quantity of the ochronotic pigment are time-dependent and saturable by an excess of substrate.
  • the experiments conducted have shown, that the ochronotic pigment can most easily be measured quantitatively over a spectral range from 350 to 600 nm.
  • hpd cDNA can be used as a versatile reporter, eg.
  • the present invention provides a novel biosensor system comprising a cell or organism harbouring a DNA molecule enabling said cell or organism to express 7-hydroxyphenyl-pyruvic acid dioxygenase under the control of a promotor which is inducible by the presence of substances or conditions to be analyzed.
  • the substances to be analyzed are selected from the group consisting of air, water and soil contaminants, toxins and toxic compounds, whereas said conditions are selected from the group consisting of salt, osmotic and oxidative stress.
  • procaryotic promoter zntA and the eucaryotic promoter Cupl-2 a vast quantity of specific procaryotic and eucaryotic promoters can be applied in the biosensor system according to the principles of the present invention in dependence of the specific analytes or conditions to be detected and/or quantified.
  • promotors can easily be identified by the skilled artisan and are preferably selected from the following also indicating the respective analytes to be detected with the aid of the selected promotor:
  • zntA-ptomoter from E.coli and ca ⁇ A-operon from Staphylococcus aureus, respectively, can suitably be selected.
  • mercury (Hg) and lead (Pb) the mer-4-promoter from E.coli will be appropriate.
  • Ni nickel
  • Cr chrome
  • the ⁇ ra-operon from Staphylococcus aureus can be selected for the detection of arsenic, whereas other metals (Fe, Cu, Al, Ni) can be detected by using the ⁇ t ' C-promoter from E.coli.
  • the metals manganese (Mn) and cobalt (Co) are preferably detected by using the cor A -promoter from E.coli.
  • the zrt- promoter members of the zip-family from yeast (Zn), the ycfl -promoter from yeast (Cd), and the cupl -promoters from yeast (Cu), respectively, can suitably be selected.
  • Zn the zip-family from yeast
  • Cd the ycfl -promoter from yeast
  • Cu cupl -promoters from yeast
  • other metals such as iron (Fe)
  • use of the gefl-mdftrl -promoters from yeast are preferred.
  • the promoters hall (salt), grel (osmotic), and aad4 (oxidative) from yeast are preferably selected.
  • promoters exemplified above can be used in the native or modified form and can be substituted by selection of homologues from other species or organisms without departing from the principles underlying the present invention.
  • present invention can be carried out by using novel promoter sequences which will be identified in the future to be inducible by certain chemical compounds, compositions or conditions to be detected.
  • this soluble reporter system can be integrated into molecularbiological techniques for the detection and analysis of cw-acting elements (promoters, enhancers), signal transduction pathways in eucaryotes, and DNA-protein interactions. In pharmaco- logical approaches it can be applied in affinity screenings for membrane receptors and ligands.
  • a method is provided to study and monitor the activity of a promoter by use of a vector which comprises hpd cloned 3'- downstream of a multiple cloning site (MCS), followed by a transcriptional terminator.
  • MCS multiple cloning site
  • the turnover rate of HPD protein can be influenced by cloning a PEST-domain (Pro-Glu-Ser-Thr; (19)) into the coding sequence of hpd.
  • a functional expression cassette for hpd is generated.
  • Transient transformation of host cells with such a vector and subsequent selection leads to the generation of transformants.
  • the activity of the promoter can then be monitored in the dependence of agents and/or environmental factors (such as, e.g., light, salt, water, temperature). Due to the PEST-domain, which is preferably used in studies of eucaryotic promoters, the response to the exogenous agent and/or transcription factor can be diminished, i.e. HPD protein will undergo degradation within a few hours.
  • cis-acting elements i.e. promoters, enhancer sequences
  • trans-acting factors i.e. transcription factors
  • insertion of a known or putative promoter element into the multiple cloning site of an appropriate vector will promote the transcription translation (expression) of hpd gene products after transformation of suitable host cells.
  • the expression can subsequently be analyzed by extracellularly detecting the quantity of ochronotic pigment accumulated in the culture medium.
  • the minimally required promoter region By insertion of different lengths or parts of the overall promoter sequence and quantification of the ochronotic pigment synthesis (s.a.), e.g. the minimally required promoter region can be characterized. Furthermore, mutagenization of the promoter sequence allows the identification of functionally relevant sequence stretches. With the knowledge of the promoter sequence, a putative enhancer sequence 5'-upstream of the promoter can be identified, cloned and analyzed. The dependence of the promoter activity from the presence of a transcriptional activator, e.g. transcription factor, can be quantified by using the same vector for the transformation of two different cell cultures, one of them lacking or being deficient in the transcriptional activator. A comparable system using a different reporter
  • SEAP reporter system 3 secretory alkaline phosphatase
  • the screening system according to the invention allows direct analysis without the necessity of additionally performing and evaluating an enzymatic assay.
  • the quantification of ochronotic pigment is in proportion to cellular RNA levels of hpd and can easily be performed in the cell culture medium. A measurement of promoter activity is thus achieved without lysing transformed cells.
  • transformed cells can be further studied using other methods, e.g. Northern blots, RNAse protection assays or Western Blots.
  • Sample collection can be automated by using cultures grown in e.g. 96-well plates. Therefore, the system provided herein enables high-throughput screenings.
  • appropriate vectors comprise expression cassettes which may be e.g. composed from 5' to 3' of a specific cw-acting enhancer element, the TATA-like region from the thymidine kinase promoter or only its TATA-Box, and the hpd-reporter with transcriptional terminator.
  • cw-acting elements which can be utilized according to the invention for the detection of transcription factors such as API (PKC-pathway), GRE (steroidhormone pathway), NFkB (NFkB-pathway) and SRE (MAPK-pathway).
  • a screening method for isolating potential interaction partners of specific DNA-sequences such as e.g. transcriptional activators, from a One-Hybrid library by preferably using yeast cells transformed with a 2 reporter-system comprising both the his3 -reporter and the hpd-reporter. Positive interactions are indicated by growth of transformed yeast cells on histidin-deficient agar plates. After this selection, false positive clones can be eliminated by growing them in normal yeast medium. The medium of positive clones turn ochre, due to the expression of HPD, whereas the medium of false positive clones does not change colour.
  • specific DNA-sequences such as e.g. transcriptional activators
  • the present invention provides a superior method, because the time consuming control-assay by another reporter (e.g. ⁇ -galactosidase-filter assay) can be omitted and positive colonies can directly be subjected to other analytical procedures (e.g. plasmid-rescue).
  • another reporter e.g. ⁇ -galactosidase-filter assay
  • positive colonies can directly be subjected to other analytical procedures (e.g. plasmid-rescue).
  • Receptor based ligand-affinitv-screening there is provided a method to screen for agonistic/antagonistic ligands having a specific affinity for a given receptor or extracellular domain or part thereof.
  • a method to perform studies on extracellular receptor domains desired to be characterized The principle of these methods is based on a known signal transduction cascade, which is triggered by binding of a ligand to an extracellular domain or part of a transmembrane receptor. According to the invention, this binding can be measured by the accumulation of ochronotic pigment in the medium.
  • the cascade will generally comprise a membrane receptor, being a chimeric protein composed of the cytoplasmatic and transmembrane domains of an already known receptor, and the extracellular domain or part of a receptor to be analyzed.
  • An expression cassette for this chimeric receptor is part of a vector used to transform suitable host cells which are either transgene with respect to a hpd expression cassette according to the invention or which are co-transformed with such a genetic construct enabling expression profiling on the same or different vector. After ligand binding to this transmembrane receptor, a transcription factor will be released which activates the transcription of the hpd expression cassette.
  • the translated gene product then catalyzes the synthesis of homogentisic acid, which is released into the medium where it spontaneously oxydizes to ochronotic pigment.
  • the accumulation of ochronotic pigment is quantitatively dependent on the affinity of the ligand to the receptor.
  • EGFR epidermal growth factor receptor
  • cytoplasmic region In the cytoplasmic region three conserved sequence domains can be recognized: a region for feedback attenuation by protein kinase C (PKC), the tyrosine kinase domain and a C-terminal domain, which contains motifs for internalization and degradation of the receptor.
  • PKC protein kinase C
  • EGF-receptor is activated by ligand-binding to the monomeric form and dimerizes before internalization.
  • the chimeric receptor will consist of two forms, where the extracellular domain will be changed and the tyrosine kinase domain completely removed.
  • the split ubiquitin system may be used as a sensor of protein interactions in vivo (17; US Patent US 5,503,977).
  • this system can be used to analyze the interactions of integral membrane proteins via a short signal transduction cascade (35).
  • the system will be used for interactions between two different monomeric forms of EGF-receptor.
  • Ubiquitin is a conserved protein of 76 amino acids, which is usually attached to the N terminus of proteins as a signal for their degradation (39). The ubiquitin moiety is recognized by ubiquitin-specific proteases, resulting in the cleavage of the attached protein.
  • the cleavage can be visualized with a transcription factor attached to the C-terminus of ubiquitin, which after release leads to the activation of reporter gene expression (35).
  • the ubiquitin-fusion can be divided and expressed in two parts, a N-terminal part (Nub/, amino acids 1-34, with / being isoleucin at position 13) and a C-terminal part of ubiquitin (Cub, amino acids 35-76 of ubiquitin), fused to a transcription factor.
  • Nub/ and Cwb-reporter assemble in the cell and fo ⁇ n split-ubiquitin.
  • the split-ubiquitin is recognized by ubiquitin- specific proteases, resulting in the cleavage of the transcription factor attached to Cub.
  • the protein interaction can be provided by two forms of monomeric EGF-receptor after ligand- binding, one fused to Nub and the other fused to Cwb-transcription factor.
  • the released transcription factor might be proteinA-LexA-VP16 (PLV), as already described in Stagljar et al. (35). It can activate the hpd reporter gene and by this allows the analysis of ligand-receptor binding via extracellular soluble ochronotic pigment.
  • PV proteinA-LexA-VP16
  • the quality of the end product of many biotechnological processes is dependent on the vitality of the organism, of whom it has been synthesized.
  • the expression of hpd under the control of a specific promoter in a transgenic organism can be an indicator for this.
  • changes in pH, temperature, osmolarity or vitality can be observed in a change of colour in the medium, being an indicator of stress conditions for the organism.
  • the present invention provides a simple means to monitor the vitality of an organism during cultivation.
  • Figure 1 Biochemical pathway for the degradation of L-tyrosine to ochronotic pigment.
  • Figure 2 Formation of ochronotic pigment in over night-cultures of E. coli.
  • E.coli strain JM109 was transformed with pQE60 or pHPDQE, respectively. Cultures were induced with 500 ⁇ M IPTG and grown for 24 hours in LB/ampicillin (100 ⁇ g ml). Due to the presence of HPD in the culture of pHPDQE, homogentisic acid was formed which was oxidized on air to ochronotic pigment. This colour change was not observed in the control culture of pQE60.
  • Figure 3 Identification of homogentisic acid in bacterial lysates of pHPDQE - cultures. Reversed phase Cig - HPLC was performed as indicated in the example section.
  • Figure 4 Spectrum of ochron
  • JM109 was transformed/induced with: 1: pQE60/500 ⁇ M IPTG; 2:pHPDQE/0 ⁇ M IPTG; 3: pHPDQE/10 ⁇ M IPTG 4:pHPDQE/100 ⁇ M IPTG; 5: pHPDQE/500 ⁇ M IPTG; 6: pHPDQE/1 mM IPTG; 7: pHPDQE/10 mM IPTG.
  • M broad range protein marker (No. P7702S, New England Biolabs,
  • Figure 6 Arabinose-induction of HPD expression in E.coli transformed with pBADHPD. 25 ⁇ g protein of lysates from Top 10 transformed with pBADHPD or the anti- sense construct pBADDPH and induced with different concentrations of arabinose were analyzed by (A) 10 % SDS-PAGE/coo assie or (B) immuno blot with affinity-purified mouse anti-HPD-antibodies. Whereas in (A) the induction of HPD was not evident, immuno blot analysis (B) revealed a steep increase of HPD expression dependent on the arabinose-concentration.
  • Top 10 transformed with pBADHPD was induced with 2 x 10 "2 % arabinose and the formation of ochronotic pigment in the cleared lysate was spectroscopically measured against induced (2 x 10 "2 % arabinose) Top
  • Figure 10 Time dependence of enzymatic activity.
  • a culture of Top 10 transformed with pzntHPD was cultivated in Zn (II)- minimal medium and induced with 20 ⁇ M ZnCl 2 .
  • the formation of ochronotic pigment in the cleared lysate was measured spectroscopically at 400 nm against an induced (20 ⁇ M Zn(II)) Top 10 control lysate within time.
  • Figure 11 Substrate dependence of enzymatic activity.
  • a culture of Top 10 transformed with pzntHPD was cultivated in Zn (II)- minimal medium and induced with 20 ⁇ M ZnCl 2 .
  • Figure 12 Inducibility of enzymatic activity is dependent on log (Zn(II)).
  • Top 10 transformed with pzntHPD were cultivated in Zn (I ⁇ )-minimal medium, including 500 ⁇ M L-tyrosine and induced with different concentrations of ZnCl 2 .
  • B visible colour change in the clear lysates in 24 well-plates.
  • ochronotic pigment in the cleared lysate was dependent on the concentration of the inducer and revealed a linear kinetic within the range 1 ⁇ M to 10 ⁇ M Zn (II).
  • M9, M9-medium without cells BL, induced (20 ⁇ M Zn(II)), non-transformed BL21DE3(RIL); -T, non-induced, transformed TOP10 in M9-medium without L-tyrosine.
  • Figure 13 The induction of a bacterial hpd by Zn(II) is similar to that of hpd from Arabidopsis.
  • BL21DE3(RIL) transformed with pzntSYN were cultivated in Zn (II)-minimal medium, including 500 ⁇ M L-tyrosine and induced with different concentrations of ZnCl 2 .
  • Absorbance was measured spectroscopically at 400 nm against an induced (20 ⁇ M Zn(II)) Top 10 control lysate after 24 hours.
  • a linear increase of absorption can be observed within the range of 1 to 10 ⁇ M Zn (II) in lysates.
  • Figure 14 Inducibility of CUPl-2 promoter and enzymatic activity of HPD dependent on logC (Cu(II)) in transformed cells with an eucaryotic background (yeast).
  • Saccharomyces cerevisiae strain INVScl transformed with pYCA were induced for 48 hours with different concentrations of CuSO .
  • the absorbance of cell lysates were measured spectroscopically at 400 nm against an induced (500 ⁇ M Cu(II)) yeast control and plotted versus logC (Cu(II)).
  • the formation of ochronotic pigment in the cleared lysate was dependent on the concentration of the inducer and revealed a linear kinetic within the range of 5 ⁇ M to 500 ⁇ M Cu (II).
  • Figure 15 Comparison of ochronotic pigment formation in yeast supernatants and lysates.
  • Top 10 bacterial cells were transfomed with pzntGal, a vector with a ⁇ - galactosidase expression cassette in the same genetical background as pzntHPD and induced with different concentrations of Zn(II) for 24 hours.
  • lysates were measured at 420 nm and at 550 nm as a reference.
  • the cell concentration in harvested culture suspensions was constant independent of the concentration of the inducer as indicated by the absorbance at 600 nm.
  • Figure 18 Standardization of ochronotic pigment formation.
  • ochronotic pigment has been linked to the metabolism of aromatic amino acids.
  • Figure 1 In the oxidative degradation of L-tyrosine ( Figure 1), it is first converted to p- hydroxyphenyl-pyruvate by transamination with alpha-ketoglutarate, in a reaction catalyzed by aromatic transaminase (20). ;?-hydroxyphenyl-pyruvic acid dioxygenase (HPD) then catalyzes its oxidation to homogentisic acid (HGA) in a complex reaction, involving hydroxylation of the phenyl ring, decarboxylation, oxidation and migration of the side chain (14).
  • HPD homogentisic acid
  • the enzyme appears to be ubiquitary in living organisms and has been purified from vertebrates (22, 28, 40), procaryotes (21) and plants (9).
  • cDNAs and encoding genes have been identified from numerous sources, such as mammals, fungi, bacteria and plants (9, 6, 29, 26). They show between 25 % to 95 % identity at the amino acid level (26).
  • HPDs from Arabidopsis thaliana and Synechocystis sp. PCC6803
  • HGA the product of the reaction, is further catabolized by the next enzyme in the degradation pathway, homogentisic acid 1,2-dioxygenase ( Figure 1).
  • This enzyme has also been purified to homogeneity from different sources (32, 37) and the respective genes have been identified (33, 11, 23, 7).
  • Hpd from Arabidopsis (26, 1) was amplified from an Arabidopsis thaliana Matchmaker cDNA library (Clontech, Heidelberg, Germany) by proof-reading PCR using the primers hpdfor (5'-TGAAATCCATGGGCCACCAAAACGCCGCCGTT-3'; SEQ ID No.l) and hpdback (5'- TCTTCTTGTGGATCCCACTAACTGTTTGGC-3*; SEQ ID No.2).
  • the 1355 bp-PCR-fragment was digested with Ncol and Bam ⁇ l and inserted into pQE60 (Qiagen, Hilden, Germany) which had been digested with the same enzymes.
  • the resulting plasmid pHPDQE was transformed into E.coli strain JMl 09.
  • hpd5 5'-ACCATGGGCCACCAAAACGCCGCC-3*; SEQ ID No.3
  • hpd3 5'- TCCCACTAACTGTTTGGCTTCAAG-3'; SEQ ID No.4
  • pBADTopo TA Invitrogen, Groningen, Netherlands
  • E.coli strain Top 10 was transformed with the resulting plasmid pBADHPD and cultures were used for the induction experiments.
  • znt5 5'-TCCGTGCGGATATCGCGATTGCTGCGG-3': SEQ ID No.5
  • znt3 5'-CAGGAGTCGCCATGGCATCCTCCGGTT-3*; SEQ ID No.6
  • the 163 bp fragment was digested with Ncol and EcoRY and cloned into pBADHPD which had been digested with the same enzymes.
  • the ⁇ r ⁇ -promoter, the enterokinase-domain and most of the ⁇ raC-regulator were deleted.
  • the distance and sequence between zHtA-promoter and hpd-coding sequence were the same as between zntA- promoter and z «tA in the native context of the E.coli chromosome.
  • E.coli strain Top 10 was transformed with the resulting plasmid pzntHPD and respective cultures were used for induction experiments.
  • znt5 5'-TCCGTGCGGATATCGCGATTGCTGCGG-3'; SEQ ID No.5
  • zn ⁇ mod 5'-GTCGGGATCCCATCCTCCGGTTAAGTTTTTTCT-3'; SEQ ID No.9
  • the zflt-4-promoter was amplified from pzntHPD by proof-reading-PCR as above (denatiiration at 94°C for 2 min; 30 Cycles: 30s at 94°C/45s at 60°C/60s at 68°C; extension at 68°C for 5 min).
  • the 138 bp fragment was digested with BamHl and EcoRV and cloned into pSYN which had been cut with the same enzymes, h the resulting vector pzntSYN the distance and sequence between z «t ⁇ -promoter and bpd-coding sequence were the same as between z «t_4-promoter and zntA in the native context of the E.coli genome.
  • E.coli strain B121(DE3)R1L was transformed with pzntSYN and respective cultures were used for induction experiments.
  • PCR was performed by denaturation for 2 min at 94°C, 30 cycles (30s at 94°C/45s at 60°C/60s at68°C) and an extension of 5 min at 68°C, using primers CUP5 (5'-TTAGGAGCTCGATCCCATTACCG- ACATTTGGGCG-3'; SEQ ID No.10) and CUP3 (5'-TATCGGATCCTACAGTTTG- TTTTTCTTAATATCTATTTCG-3'; SEQ ID No.11).
  • CUP5 5'-TTAGGAGCTCGATCCCATTACCG- ACATTTGGGCG-3'; SEQ ID No.10
  • CUP3 5'-TATCGGATCCTACAGTTTG- TTTTTCTTAATATCTATTTCG-3'; SEQ ID No.11
  • the achieved 430 bp fragment was digested with Sacl and BamHl and ligated into the equally digested vector p426ADH (23), resulting in plasmid pYCUP
  • hpd was amplified by proof-reading-PCR as above from pBADHPD, using the primers YHPD5 (5'-TATCGGATCCATGGGCCACCAAAACGCCGCC-3'; SEQ ID No.12) and YHPD3 (5'-TTAGAAGCTTTCATCCGACTAACTGTTTGGCTTC-3'; SEQ ID No.13). After denaturation for 2 min at 94°C, 30 cycles ( 30s at 94°C/45s at 60°C/60s at 68°C) and an extension of 5 min. at 68°C were performed.
  • Hpd of 1300 bp was digested with BamHl and Hindlll and ligated into pYCUP which had been cut with the same enzymes.
  • the resulting vector pYCA was transformed into chemocompetent Saccharomyces cerevisiae strain JNNScl (Invitrogen, Groningen, Netherlands) and plated on selective plates without uracile. Incubation of transformed yeast clones of pYCA and induction with Cu (II) was performed in the same selective liquid medium.
  • 3'-dA-overhangs were generated by incubating the purified PCR-product with Taq-polymerase and an excess of dATP in the appropriate buffer for 10 min (72°C). To facilitate digestion with Pmll and Ncol the fragment was cloned into pCR4.1Topo (Invitrogen, Groningen, Netherlands). Nector pzntHPD was digested with Eel 13611 and Ncol and ligated with the respective, repurified fragment of ⁇ -galactosidase. Transformants of pzntGal in ToplO were used for induction assays with Zn (II).
  • Competent Yeast Cells Saccharomyces cerevisiae cells strain INVScl (Invitrogen, Groningen, Netherlands) were grown over night in YPD-medium at 30°C. For inoculating the starter culture the next day 1,5 ml over night-culture were used in 100 ml of YPD-medium. Cells were grown at 30°C until the OD 6 oo was 0,6. They were harvested by a 5 min. centifugation at 1600 x g and resuspended in 50 ml solution A (15 g ethylene glycole/5 ml 1 M bicine pH 8.35/91,1 g sorbitol, diluted in water and filtrated in a final volume of 500 ml). The suspension was pelleted again as above, resuspended in 2 ml solution A before adding 110 ml dimethyl- sulfoxide and aliquotated in 200 ⁇ l portions. Until transformation they were stored at -80°C.
  • Yeast transformation Saccharomyces cerevisiae strain INVScl (Invitrogen, Groningen, Netherlands) was transformed by adding 1 ⁇ g DNA to 200 ⁇ l of frozen competent cells, followed by a 5 min. incubation at 37°C, thereby carefully mixing every minute of incubation. Afterwards, 1,5 ml of solution B (60 g poly ethylene glycole 1000/30 ml 1M bicine pH 8,35 diluted in H 2 0 de st. and filtrated in a final volume of 150 ml) was added and the mixture was incubated for one hour at 37°C. Then cells were harvested by a 3 min.
  • solution B 60 g poly ethylene glycole 1000/30 ml 1M bicine pH 8,35 diluted in H 2 0 de st. and filtrated in a final volume of 150 ml
  • Protein-analytical methods Affinity-purification ofantisera Purified recombinant HPD was used for immunization in mice. The antiserum was affinify- purified by binding to the electro-transferred overexpressed antigen onto nitrocellulose according to the method described by Smith and Fisher (34). For immunoblot analysis this antibody preparation was used in a 1:5 (v/v) dilution, corresponding to a 1:200 (v/v) dilution with respect to the serum.
  • inducer induction assays defined concentrations of inducer were added to the transformed cultures after aliquoting in 10 ml to sterile 50 ml-tubes (Greiner, Frickenhausen, Germany). The Top 10 control cultures were induced by 1 mM IPTG, 2 x 10 "2 % (w/v) arabinose or 50 ⁇ M ZnCl 2 .
  • Cultures were then grown at 22°C with shaking (180 ⁇ m) for 24 hours (promoter induction assays) or for a different time interval. Afterwards, 20 ⁇ l 5 N NaOH were added to 1 ml of culture to give a final concentration of 0,1 N NaOH, vortexed and incubated at room temperature for 10 min. Supernatants were cleared by a short centrifugation (5 min., 21.000 x g) at room temperature and wavelength scanned in a 2-beam Uvikon 941plus spectrophotometer (Kontron Instruments, Milan, Italy) against the induced ToplO control.
  • Zn (II) -minimal medium was a sterile filtrated M9 minimal medium (30), containing 2
  • % (w/v) glucose/ 1 mM MgSO 4 x7H 2 O/ 15 mM thiamine pyrophosphate and a dropout supplement (30 ⁇ g/ml L-isoleucin, 150 ⁇ g/ml L-valin, 20 ⁇ g/ml L-adenine hemisulfate, 20 ⁇ g/ml L-arginine HC1, 20 ⁇ g/ml L-histidin monohydrate, 100 ⁇ g/ml L-leucin, 30 ⁇ g/ml L- lysine HC1, 20 ⁇ g/ml L-methionine, 50 ⁇ g/ml L-phenylalanine, 200 ⁇ g/ml L-threonine, 20 ⁇ g/ml L-tryptophane, 30 ⁇ g/ml L-tyrosine, 20 ⁇ g/ml L-uracil).
  • the culture was induced with 20 ⁇ M ZnCl 2 , in the induction course with different concentrations of Zn (II).
  • different amounts of L-tyrosine were added at the time point of induction together with 20 ⁇ M Zn (II).
  • ⁇ -Galactosidase-assay ofprocaryotes after Zn (II) induction For assays using the pzntGal-vector construct, the detection of ⁇ -Galactosidase activity was performed as recommended in most laboratory manuals (30): 1 ml of the previously induced transformed Top 10 culture was pelleted by a short centrifugation (5 min., 21.000 x g) and resuspended in 800 ⁇ l Z-Buffer. The cell mix was diluted 1/100 (v/v) with Z-Buffer, then 40 ⁇ l of 0,1% (w/v) SDS and 60 ⁇ l of chloroforme were added on ice and the mixture was vortexed for 15 sec.
  • Cu (II)-minimal medium was a sterile filtrated YNB (Yeast Nitrogen Base)-medium, containing 2% (w/v) glucose, supplemented with 20 mg/ml L-histidine-HCl, 30 mg/ml L- leucine and 20 mg/ml L-tryptophan (1,5 % agar was added for plates) to ensure selection for His " , Leu " , T ⁇ " and Ura + (selective marker on p426ADH).
  • YNB Yeast Nitrogen Base
  • the recovered supernatants were diluted threefold with 10 mM acetic acid and filtered with a Millipore HV filter (0,2 ⁇ m pore size). 20 ⁇ l of the probes were applied to a HPLC system consisting of a Nucleosil C ⁇ 8 column (5 ⁇ m; 200 mm; Macherey -Nagel, D ⁇ ren, Germany) and a linear gradient system using 10 mM acetic acid/methanol (85:15, by vol.) at a flow rate of 0,8 ml/min.
  • UV/VIS spectra were monitored by a photodiode array detector (Waters 986, Eschborn, Germany) and chromatograms analyzed at 290 nm using the Millennium software package PDA software (Waters, Eschborn, Gennany). Products were identified by chromatographic comparison to a 500 ⁇ M HGA-Standard (Sigma-Aldrich, Germany), diluted in LB-brotiVlOmM acetic acid. The spectrum of HGA had a maximal abso ⁇ tion at 290 nm.
  • the foe-promoter in pHPDQE is an inducible promoter, it is not tightly regulated.
  • Example for a biosensor system for environmental pollution The sensor system based on hpd-cDNA and an ⁇ r ⁇ -promoter might appear quite artificial.
  • the transcriptional control protein MerR in E.coli is a metalloregulatory switch, activating transcription of a mercury resistance operon in the presence of mercuric ions and repressing transcription in their absence (4). It is a direct Hg(II) sensor that catalyzes transcriptional activation of a Hg(II) efflux gene. Since working with Hg(II) is harmful, we chose a Zn(II)-responsive MerR homologue in E.coli, ZntR.
  • ZntR is a trans activator of zntA transcription and binds to the z «tA-promoter.
  • the induction of this promoter by Zn(II) via ZntR is well characterized on a transcriptional level by run-off transcription assays (27).
  • the zrctA-promoter was amplified from E.coli K12 - DNA by proof-reading-PCR and cloned into pBADHPD, thereby deleting the tfr ⁇ -promoter, the enterokinase-domain and most of the r ⁇ C-regulator. Cloning of the zntR-gene in the resulting vector pzntHPD was unnecessary, since it is already an endogeneous feature of E.coli genome. Nevertheless, it was tried to generate a vector consisting of a z/.tR-gene under the constitutive control of the nptll- promoter. Only expression cassettes with zntR in the wrong orientation were obtained indicating, that a constitutive overexpression of the regulator might be harmful in E.coli.
  • Vector pzntHPD was transformed into E. coli, cultures grown in LB-broth and induced with different concentrations of Zn (II) ranging from 0 to 300 ⁇ M.
  • Zn Zn
  • Figure 9A Western Blot -analysis revealed a clear Zn(II)-dependent induction of HPD ( Figure 9B).
  • Figure 9B Western Blot -analysis revealed a clear Zn(II)-dependent induction of HPD.
  • a steady increase of HPD protein was detectable from 1/ 2,5/5/10 to 25 ⁇ M Zn (II), reaching a plateau of expression ( Figure 9B; lanes 3 to 9).
  • ochronotic pigment is not only a feature of plant HPDs (eg. from Arabidopsis; see above) or eucaryotic HPDs. It could be shown that bacterial HPDs, cloned under the same genetical background, reveal the same kinetics of time, substrate dependency and induction. For this pu ⁇ ose, hpd from Synechocystis sp. PCC6803 was cloned under the control of the Zn (ID-promoter (see above).
  • the hpd reporter system according to the invention is as sensitive as other reporter systems already commercially available.
  • ⁇ -galactosidase from E.coli the most common reporter system, was chosen.
  • This reporter was cloned under the same genetical background as the hpd reporter and analyzed under the same conditions. Measurements of ⁇ - galactosidase activity were performed 2 and 24 hours after induction. Kinetics of this reporter system were equivalent to the hpd reporter showing a linear increase between 1 to 10 ⁇ M ( Figure 17). In both systems the amount of accumulated end product is solely a feature of transcriptional promoter activity and can be calibrated by exogeneous standards (Figure 18). This also demonstrates that the spontaneous oxidation process leading to ochronotic pigment formation is comparable to the enzymatic reaction of ⁇ -galactosidase.
  • the ⁇ -galactosidase technique requires cell lysis prior to perform the enzymatic assay.
  • This enzymatic assay which is not necessary in the case of hpd reporter, is time-consuming, needs personal trained in laboratory practice and far more equipment than the hpd system provided herein. Additionally, this ⁇ -galactosidase assay cannot be performed as a high troughput assay like the hpd biosensor.
  • the use of organic solvents that might be harmful to the technicians represents an additional drawback inherent in comparable assay techniques.
  • the non-enzymatic reaction is faster than the enzymatic reaction. Pigment formation therefore behaves in a quantifiable manner. Its formation can be standardized in equivalents to the enzymatic reaction.
  • the enzymatic activity of the gene product i.e. hydroxyphenyl pyruvate dioxygenase
  • the enzymatic activity of the gene product is very high, so that a minimal induction of the expression cassette in procaryotes as in eucaryotes already leads to discrete amounts of homogentisic acid.
  • promoter- analyses and/or biosensor systems can be provided.
  • Homogentisic acid is soluble. Unexpectedly, it is secreted into the extracellular space in procaryotes as in eucaryotes and is not retained in cells. For analysis, the fast non- enzymatic reaction to the soluble and quantifiable ochronotic pigment has to be performed. This happens spontaneously in the medium and can be increased to a maximum by the addition of basic agents, such as addition of NaOH to a final concentration of 0,1 N.
  • the colour change can be measured over a wide spectral range and is not restricted to a specific wavelength, so that there are several possibilities of quantification dependent on the abso ⁇ tion of the inducer.
  • Time for quantitative measurements is in the range of 24 hours post induction; variabilities of cell cultures will diminish within this time, as E.coli will already arrest to the fog-phase.
  • the substrate of the enzymatic reaction or its biochemical precursors don't need to be components of the medium. They also don't need to be added exogenously before, within or after induction or for the development" of the system. Synthesis of the substrate is an endogenous feature of the amino acid-metabolism in all living organisms. Su ⁇ risingly, the amount of fonned substrate in the assayed transformed organisms is high enough to allow quantification after induction.
  • the FtsQ protein of Escherichia coli membrane topology, abundance and cell division phenotypes due to ove ⁇ roduction and insertion mutations. J.Bacteriol. 173, 2187-2195. 4. Comess K.M., Shewchuk L.M., Ivanetich K. and Walsh C.T. (1994). Construction of a synthetic gene for the metalloregulatory protein MerR and analysis of regionally mutated proteins for transcriptional regulation. Biochemistry 33, 4175-4186.

Abstract

L'invention relève de la détection qualitative et/ou quantitative d'analytes. Elle concerne notamment un système de biocapteurs servant à évaluer la présence et la quantité de substances chimiques à analyser ainsi que des procédés utilisant ce système de capteurs biologiques. Le système de capteurs biologiques de l'invention utilise des voies métaboliques/cataboliques des cellules vivantes, transformées pour manifester des caractéristiques spécifiques pouvant être utilisées pour identifier la présence et la quantité d'une vaste gamme d'analytes.
PCT/EP2001/013231 2000-12-28 2001-11-15 Nouveau systeme de biocapteurs WO2002053772A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104845998A (zh) * 2015-02-13 2015-08-19 温州医科大学 一种检测水体中重金属铜的微生物学方法
CN104845996A (zh) * 2015-02-13 2015-08-19 温州医科大学 一种检测水体金属汞的微生物学方法
CN104946682A (zh) * 2014-12-24 2015-09-30 温州医科大学 一种检测水体重金属铅的微生物学方法
CN104946681A (zh) * 2014-12-24 2015-09-30 温州医科大学 一种检测水体重金属镉的微生物学方法
CN105132346A (zh) * 2014-12-24 2015-12-09 温州医科大学 一株检测镉的大肠埃希氏菌
CN112899209A (zh) * 2021-02-20 2021-06-04 温州医科大学 一种锌离子诱导重组蛋白表达体系、其诱导方法和应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992015687A1 (fr) * 1991-03-01 1992-09-17 Vito Genes fusionnes et utilisation de ces genes pour determiner la presence de metaux ou de composes xenobiotiques
US5571722A (en) * 1991-07-30 1996-11-05 Bio-Technical Resources L.P. Plasmid for detecting mercury in water
WO1997027285A1 (fr) * 1996-01-29 1997-07-31 Arizona Board Of Regents, On Behalf Of University Of Arizona Dioxygenase vegetale clonee de l'acide p-hydroxyphenylpyruvique
WO1998004685A1 (fr) * 1996-07-25 1998-02-05 American Cyanamid Company Gene et inhibiteurs de 4-hydroxyphenylpyruvatedioxygenase
WO1998004716A1 (fr) * 1996-07-29 1998-02-05 Crc For Waste Management And Pollution Control Limited Biocapteurs

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992015687A1 (fr) * 1991-03-01 1992-09-17 Vito Genes fusionnes et utilisation de ces genes pour determiner la presence de metaux ou de composes xenobiotiques
US5571722A (en) * 1991-07-30 1996-11-05 Bio-Technical Resources L.P. Plasmid for detecting mercury in water
WO1997027285A1 (fr) * 1996-01-29 1997-07-31 Arizona Board Of Regents, On Behalf Of University Of Arizona Dioxygenase vegetale clonee de l'acide p-hydroxyphenylpyruvique
WO1998004685A1 (fr) * 1996-07-25 1998-02-05 American Cyanamid Company Gene et inhibiteurs de 4-hydroxyphenylpyruvatedioxygenase
WO1998004716A1 (fr) * 1996-07-29 1998-02-05 Crc For Waste Management And Pollution Control Limited Biocapteurs

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ERBE J L ET AL: "Cyanobacteria carrying an smt-tux transcriptional fusion as biosensors for the detection of heavy metal cations.", JOURNAL OF INDUSTRIAL MICROBIOLOGY, vol. 17, no. 2, 1996, pages 80 - 83, XP001070093, ISSN: 0169-4146 *
HESTBJERG HANSEN LARS ET AL: "Versatile biosensor vectors for detection and quantification of mercury.", FEMS MICROBIOLOGY LETTERS, vol. 193, no. 1, 2000, pages 123 - 127, XP001069410, ISSN: 0378-1097 *
WILLARDSON BARRY M ET AL: "Development and testing of a bacterial biosensor for toluene-based environmental contaminants.", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 64, no. 3, March 1998 (1998-03-01), pages 1006 - 1012, XP001069414, ISSN: 0099-2240 *

Cited By (10)

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Publication number Priority date Publication date Assignee Title
CN104946682A (zh) * 2014-12-24 2015-09-30 温州医科大学 一种检测水体重金属铅的微生物学方法
CN104946681A (zh) * 2014-12-24 2015-09-30 温州医科大学 一种检测水体重金属镉的微生物学方法
CN105132346A (zh) * 2014-12-24 2015-12-09 温州医科大学 一株检测镉的大肠埃希氏菌
CN105132346B (zh) * 2014-12-24 2019-01-25 温州医科大学 一株检测镉的大肠埃希氏菌
CN104946681B (zh) * 2014-12-24 2019-05-07 温州医科大学 一种检测水体重金属镉的微生物学方法
CN104946682B (zh) * 2014-12-24 2019-05-10 温州医科大学 一种检测水体重金属铅的微生物学方法
CN104845998A (zh) * 2015-02-13 2015-08-19 温州医科大学 一种检测水体中重金属铜的微生物学方法
CN104845996A (zh) * 2015-02-13 2015-08-19 温州医科大学 一种检测水体金属汞的微生物学方法
CN104845998B (zh) * 2015-02-13 2019-05-07 温州医科大学 一种检测水体中重金属铜的微生物学方法
CN112899209A (zh) * 2021-02-20 2021-06-04 温州医科大学 一种锌离子诱导重组蛋白表达体系、其诱导方法和应用

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