JP4957118B2 - Improved promoter assay - Google Patents

Description

  The present invention relates to an improved promoter assay, and in particular to an improved promoter assay for assaying chemicals present in environmental samples.

  There are a huge number of chemical substances that human beings have created so far, and new chemical substances are being developed year by year. These chemical substances are used in all aspects of modern life and help improve human life. On the other hand, some chemical substances are released into the environment at various stages of production, distribution, use, disposal, etc., and remain in the environment, biological enrichment through the food chain, etc. Some of them have harmful effects on the system, and environmental pollution has become a social problem. Therefore, there is a request to evaluate the effects of chemical substances on the human body and ecosystem.

The reporter gene assay method is known as the evaluation method. The reporter gene assay method is a technique for measuring a specific gene activity as a marker for examining the function of a gene centered on transcription activity, and includes a promoter assay method and the like. The promoter assay method is a method in which a polynucleotide encoding a marker protein is operably linked to a polynucleotide sequence of a promoter of a gene to indirectly measure gene expression (Non-patent Document 1).
In a normal bioassay method, the presence of a chemical substance is evaluated by using a cellular response to a chemical substance of a microorganism, for example, changes in cell viability, proliferative capacity, respiration rate, or expression of a specific gene as an index. In the promoter assay method, a change in promoter activity, ie, a marker protein linked to a promoter, is evaluated as an index as a cellular response.

Barelle CJ, Manson CL, MacCallum DM, Odds FC, Gow Na, Brown AJ .: GFP as a quantitative reporter of gene regulation in Candida albicans.Yeast 2004 Mar; 21 (4): 333-40

  Chemicals present in environmental samples are usually at low concentrations, but in some cases may be present at high concentrations. In a bioassay method that uses the life or death of microorganisms, proliferative ability, or respiration rate as an indicator, there is a chemical substance at a level that leads to (a) a state where microorganism growth is severely inhibited, or (b) a state where microorganisms die. When present in a sample, the cellular response that serves as an indicator disappears, so that it can be evaluated that a chemical substance is present in the sample.

On the other hand, promoter assays usually use the property that the concentration of the marker protein increases in response to the presence of a chemical substance, so that there is a level of compound in the sample that can achieve the above condition (a) or (b). In this case, the promoter activity as an index does not change apparently, and it may be considered that no chemical substance is present in the sample.
That is, in the promoter assay method, there is a limit concentration at the upper limit of detection for a chemical substance in a sample, and in a sample in which a chemical substance having a concentration higher than that is present, the determination result is erroneous.
In addition, when the chemical substance to be detected is at a high concentration, monitoring the cell growth state by turbidity measurement as one of the means capable of determining the state of (a) or (b) above. However, if the sample is turbid, the turbidity of the sample and the turbidity of the cells overlap, so accurate measurement cannot be performed.
Further, when the sample is cloudy, the fluorescence intensity may be different from the case where the sample is not cloudy even if the amount of fluorescent protein is the same due to light scattering.
Furthermore, when a microorganism obtained by drying and storing microorganisms is used as a detection kit, there is also a problem that when the microorganisms are restored, the number of survivors is unknown and a reference value cannot be determined.

  Therefore, there has been a demand for a simple and highly accurate assay method that can test the presence or amount of a chemical substance even when the sample is cloudy or the number of living microorganisms is unknown.

  The present invention relates to an improved multi-indicator promoter assay for testing whether chemicals are present in the environment.

That is, the present invention
(1) A recombinant cell into which two or more reporter genes are introduced, wherein one reporter gene is linked downstream of a promoter that expresses promoter activity at a certain level regardless of the presence of a chemical substance. Recombinant cells, wherein one or more other reporter genes are linked downstream of a promoter that expresses increased promoter activity in response to a chemical substance,
(2) The recombinant cell according to (1) above, which is a recombinant yeast,
(3) The recombinant cell according to (1), wherein the two or more reporter genes are fluorescent protein genes having a fluorescence maximum wavelength of 580 nm or less,
(4) A test sample is contacted with one or more types of recombinant cells, wherein the one or more types of recombinant cells are recombinant cells into which two or more types of reporter genes have been introduced. Species reporter genes are ligated downstream of a promoter that expresses promoter activity at a constant level regardless of the presence of a chemical substance, and one or more other reporter genes are increased in response to the chemical substance. A recombinant cell characterized by being linked downstream of a promoter that expresses promoter activity;
Measuring in a test sample the promoter activity of a promoter that expresses promoter activity at a certain level regardless of the presence of a chemical substance and the promoter activity of a promoter that expresses increased promoter activity in response to a chemical substance;
Calculating the ratio of the promoter activity of a promoter that expresses increased promoter activity in response to a chemical to the promoter activity of a promoter that expresses promoter activity at a constant level regardless of the presence of the chemical;
A multi-index type promoter assay characterized by estimating the presence or abundance of a chemical substance in a test sample based on this ratio;
(5) The method according to (4) above, wherein the recombinant cell is a recombinant yeast,
(6) The method according to (4) above, wherein the promoter activity is measured by fluorescence intensity,
(7) The method according to (4) above, wherein the two or more reporter genes are fluorescent protein genes having a fluorescence maximum wavelength at 580 nm or less,
About.

According to the method of the present invention, a promoter gene that expresses a certain level of promoter activity regardless of the presence of a chemical substance downstream of a monitoring promoter gene whose promoter activity changes in response to a chemical substance in one cell. Since the control reporter gene is introduced downstream, the promoter activity for monitoring and the standard promoter activity can be measured simultaneously in the same environment.
That is, since the relative amount with respect to the standard promoter activity is used instead of the absolute amount of one type of promoter activity, one type of assay is available even when the number of viable bacteria in the test sample is unknown or cloudy. The presence of a desired chemical substance can be easily and economically determined by the method.

  In the present invention, “test sample” means environmental water and sewage such as rivers and lakes, sample water supplied from wastewater, leachate from waste treatment plants, and samples extracted from soil and agricultural products. To do. These test samples are "chemical substances" that have industrial uses such as agricultural chemicals, pharmaceuticals, dyes, paints, adhesives, etc. and are likely to be released to the environment, and are generated unintentionally by the process of disinfection and incineration. It may include “chemical substances” that are of concern for toxicity. Known toxic effects of these substances include endocrine disruptor action, mutagenicity, carcinogenicity, genotoxicity, ecotoxicity, immunotoxicity, and cell dysfunction toxicity.

  Examples of chemical substances that can be tested by the method of the present invention include (1) benzo (a) pyrene, (2) bisphenol A, (3) di (2-ethylhexyl) phthalate, and (4) 2,5-dichloro. Phenol, (5) 2,4-dichlorophenoxyacetic acid, (6) formaldehyde, (7) methylmercury chloride, (8) 4-nitroquinoline-N-oxide, (9) p-nonylphenol, (10) pentachlorophenol (11) sodium arsenite, (12) tetramethylthiuram disulfide, (13) tributyltin chloride, (14) 2,4,5-trichlorophenol, (15) Trp-P-2 (acetate), (16 ) Paraquat, (17) cadmium chloride, (18) γ-hexachlorocyclohexane, (19) marathon, (20) ethylene bisdi Ocarbamide sun manganese, (21) nickel (II) chloride, (22) potassium dichromate, (23) triphenyltin chloride, (24) phenol, (25) S-4-chlorobenzyl-N, N-diethyl Examples include thiocarbamate, (26) hexachlorophene, (27) triclosan, (28) mercury (II) chloride, (29) copper (II) sulfate, (30) potassium cyanide (31) dimethyl sulfoxide.

  In the present invention, the term “promoter assay” refers to the chemistry when a test sample is applied to a recombinant cell containing a cassette in which a reporter gene is linked downstream of a promoter screened as a gene responsive to a chemical substance. This is a bioassay method for assaying a chemical substance in a test sample, which measures the expression level of a substance-responsive gene. Specifically, a polynucleotide encoding a marker protein is operably linked to a polynucleotide sequence of a promoter of a gene, and the gene expression upon application of a test sample is measured using the transcription activity of the promoter. .

The “multi-index type promoter assay method” in the present invention is a promoter assay method using one or more, ie, one or two or more types of recombinant cells in which different marker genes are linked downstream of a plurality of promoters. At least one of the plurality of promoters in each cell is a control promoter that expresses promoter activity at a certain level regardless of the presence of a chemical substance, and the others are suspected to be present in the test sample. It is a promoter for monitoring that responds to chemical substances.
First, in the case of a promoter assay using one type of monitor promoter, one type of recombinant cell in which different marker genes are linked downstream of the control promoter and one type of monitor promoter is used. In this promoter assay method, when a chemical substance exceeding the limit concentration of the upper limit of detection is present in the test sample, the control promoter activity decreases or disappears, indicating that the limit concentration has been exceeded. Next, if the original test sample is diluted to bring the concentration of the chemical substance present in the test sample below the upper limit of detection, and the activity is measured again using recombinant cells, Since the promoter activity of the promoter increases, it can be estimated that a chemical substance to which the monitor promoter specifically responds is present in the test sample.
Next, when two or more types of monitor promoters are used, two or more types of recombinant cells in which different marker genes are linked downstream of the control promoter and one type of monitor promoter are used. In this promoter assay method, the combination of promoters for monitoring is preferably a combination of promoters of genes that specifically respond to chemical substances suspected of being present in the test sample.
For example, if the monitoring promoter 1 responds to the chemical substance A, the monitoring promoter 2 responds to the chemical substances B, C and D, and the monitoring promoter 3 responds to the chemical substances C, E, F and G, If the promoter 2 and the promoter 3 respond, the sample can be predicted to contain the chemical substance C. At this time, if the relative amount of each promoter activity is calculated based on the promoter activity of the control promoter constitutively expressed in each cell, high accuracy can be obtained even when the sample is cloudy or the number of viable bacteria is unknown. Evaluation by promoter activity becomes possible.
When two or more types of monitor promoters are used, one type of recombinant cell in which different marker genes are linked downstream of the control promoter and two or more types of monitor promoters can be used.

Based on the above concept, it is possible to estimate more types of chemical substances by creating a response database using a large number of microorganisms showing different responses to the chemical substances. A specific method for creating the database is described in detail in JP-A No. 2004-248634 (a method for identifying a toxic substance using a DNA microarray method).
Preferred combinations of microorganisms and chemicals are given in the examples below.

  The “cell” used in the present invention is a naturally occurring wild strain or wild type strain, such as animal cells derived from humans, mice and other mammals, and established strains of animal cells, fish that have been used in bioassay methods, Any of cells such as nematodes, insect cells, fungal cells such as yeast, and bacterial cells such as Escherichia coli may be used. Here, a wild strain is a cell that does not undergo natural recombination. A wild type strain is a cell in which the gene of interest has not been touched. For example, W303 has been recombined for auxotrophy and the like, but no further recombination has been performed. Established strains include those that can be subcultured with animal cells, such as those that have not undergone recombination, such as cancer cells.

As used herein, the term “promoter that expresses promoter activity at a constant level regardless of the presence of a chemical substance” refers to a promoter that constantly expresses a reporter gene linked downstream thereof regardless of the presence of a chemical substance. means.
Examples of such promoters include yeast genes selected from the group consisting of the following genes:

  The “promoter that expresses increased promoter activity in response to a chemical substance” used in the present invention means a promoter that increases the expression of a reporter gene linked downstream thereof in accordance with the amount of the chemical substance present.

  The base sequences and amino acid sequences of these yeast genes are disclosed in public databases (for example, Germany MIPS: Munich Information Center for Protein Sequence, US SGD: Saccharomyces Genome Database), and can be known via the Internet. Promoter sequences are also disclosed in public databases (SCPD: The Promoter Database of Saccharomyces cerevisiae).

  In addition, as a “promoter that expresses promoter activity at a certain level regardless of the presence of chemical substances”, the reporter gene can be used regardless of the presence of chemical substances in a state where no chemical substances are loaded by changing the growth state. It is also possible to use those that have been subjected to a treatment that constantly expresses them at a level above the detection limit of the chemical substance to be treated, for example, a treatment that changes the composition of the culture solution.

  Examples of marker proteins that can be used in the present invention include Green Fluorescence Protein (GFP) (Heim, R., Cubitt, AB and Tsien, RY (1995) Nature 373, 663-664; Heim, R , Prasher DC. And Tsien, RY (1994) Proc. Natl. Acad. Sci., 91, 12501-12504; Warg, S. and Hazerigg, T. (1994) Nature 639, 400-403; Youvan, DC and Michel-Beyerle, ME (1996) Nature Biotechnology 14 1219-1220; Chalfie, M., Tu, Y., Euskirchen, G., Ward, WW and Prasher, DC (1994) Science 263, 802-805), red fluorescence Protein (Red Fluorescent Protein: RFP), Cyan Fluorescence Protein, Blue Fluorescence Protein (BFP), Yellow Fluorescence Protein (YFP) (Gene, 111: 229-233, 1992; Nat. Biotech. 17: 969-973, 1999; Proc. Natl. Acad. Sci. USA, 98: 462-467, 2001; Annu. Rev. Biochem. 67: 509-544) . In addition, fluorescent proteins such as enhanced green fluorescent protein (EGFP) (Biochem. Biophys. Res. Commun. 227: 707-711, 1996) can also be suitably used.

In water, light is dissipated by absorption and scattering, and the degree of extinction is known to depend on the wavelength of the light (Hurburt, 1945, Oceanographic Course, Ocean Measurement Method).
FIG. 6 shows the wavelength dependence of the light absorption coefficient and scattering coefficient in distilled water. From this figure, the wavelength dependence of the scattering coefficient is not recognized so much, but a large wavelength dependence is observed in the absorption coefficient in the wavelength region exceeding 580 nm. That is, if the absorption coefficient differs depending on the emission wavelength to be measured, the observed fluorescence intensity ratio does not show a true value.

In the present invention, in order to estimate the presence or abundance of a chemical substance from the ratio of fluorescence intensities from at least two kinds of fluorescent proteins, a plurality of fluorescent proteins having a maximum fluorescence wavelength in a wavelength region having substantially the same absorption coefficient are selected. is important.
Therefore, in the present invention, it is preferable to use a fluorescent protein having a fluorescence maximum wavelength in a region of 580 nm or less, and more preferably a fluorescent protein having a fluorescence maximum wavelength in a region of 550 nm or less. In the present invention, for example, AmCyan (Clontech Laboratories, Inc.), ZsGreen (Clontech Laboratories, Inc.), ZsYellow (Clontech Laboratories, Inc.), DsRed (Clontech Laboratories, Inc.), AsRed (Clontech Laboratories, Inc.) HcRed (Clontech Laboratories, Inc.), ECFP (Clontech Laboratories, Inc.), EGFP (Clontech Laboratories, Inc.), EYFP (Clontech Laboratories, Inc.), etc. are used in combination.

One mode for carrying out the method of the present invention will be described. A "chemical substance-responsive gene recombinant cell plate" is prepared by placing recombinant cells showing different responses to chemical substances on a single plate. As a chemical substance-responsive gene recombinant cell, “a polynucleotide encoding a first fluorescent protein is operably linked downstream of a promoter that expresses a promoter activity at a certain level regardless of the presence of a chemical substance. A recombinant cell in which a polynucleotide encoding a second fluorescent protein is operably linked downstream of a promoter that expresses increased promoter activity in response is used. At that time, if the promoter for linking the polynucleotide encoding the second fluorescent protein is changed according to various chemical substances, a plurality of types of recombinant cells are prepared, and these are used in combination as appropriate. The type of substance can be determined in more detail.
A “test sample” containing the substance to be analyzed is added to this plate, and the response of each recombinant cell is measured with a dedicated device corresponding to the marker protein. Details of cell culture conditions, chemical substance identification methods, and the like at the time of measurement are described in JP-A No. 2004-248634 (toxic substance identification method using DNA microarray method).
In this embodiment, “when the promoter activity of the first promoter that expresses the promoter activity at a certain level regardless of the presence of the chemical substance is reduced or eliminated, a chemical substance in the test sample used is a microorganism ( It is considered that the sample exists at a level that leads to a) a state in which its growth is severely inhibited, or (b) a state in which microorganisms are killed. A dilution series is prepared, a sample having a dilution that does not lose the promoter activity is identified, and a reporter gene assay is performed using the diluted sample as a new test sample. For the assay method, a conventional method for assaying whether or not a chemical substance is present in a test sample can be used, for example, a change in gene expression, for example, an RNA amount Other is a method of using an index a change in mRNA levels, which includes a promoter assay.

  EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to these examples.

Example 1
A promoter gene that expresses promoter activity at a certain level regardless of the presence of chemical substances was selected.
Genes expressing GFP were selected from yeast cells regardless of the presence of chemicals. The details are as follows.

  The vector used is pYES2 (pYES2, Catno: V825-20, Invitrogen Corporation, USA), a YEp-type shuttle vector that is replicated in both E. coli and yeast (RW Old, SB Primrose) , Baifukan, pp.234-263, 2000)). The polynucleotide encoding the marker protein GFP was the GFP portion of the vector pQBI 63 (Cat no. 54-0082, Wako Pure Chemical Industries, Ltd.). First, a vector in which a GFP polynucleotide was inserted into a multiple cloning site of pYES2 was prepared. Subsequently, the GAL1 promoter portion of pYES2 was replaced with a polynucleotide containing a promoter sequence which is a yeast gene, to obtain a target plasmid vector. The operation of inserting a polynucleotide containing GFP and a promoter sequence was performed by selecting an appropriate restriction enzyme. The polynucleotide containing the promoter sequence of the yeast gene (SCPD: disclosed in The Promoter Database of Saccharomyces cerevisiae) was about 1000 bp upstream to immediately before the yeast gene.

Next, with this plasmid vector, yeast Saccharomyces cerevisiae W303 ATCC201239 (MATa / MATα leu2-3 / leu2-3 leu2-112 / leu2-112 trp1-1 / trp1-1 ura3-1 / ura3-1 his3-11 / his3- 11 his3-15 / his3-15 ade2-1 / ade2-1 can1-100 / can1-100) were transformed. The transformation procedure is shown below.
1) The yeast cell Saccharomyces cerevisiae W303 is cultured with shaking in 200 ml of YPD medium until OD660 becomes 0.5.
2) Collect and resuspend in 5 ml TE-buffer.
3) Add 250 μL of 2.5M lithium acetate.
4) Dispense 300 μl each, add 10 μl of the above plasmid vector, and incubate at 30 ° C. for 30 minutes.
5) Add 700 μl of 50% PEG4000 and shake culture at 30 ° C. for 60 minutes.
6) After heat shock (42 ° C., 5 minutes), cool rapidly.
7) Wash twice with 1M sorbitol.
8) Seed on an agar plate made with minimal nutrient medium (added with amino acids (leucine, tryptophan, uracil, histidine, adenine) necessary for SD medium).

The transformation was confirmed using a selective medium (SD medium (Yeast nitrogen base without amino acids (Difco 0919-15) + glucose + amino acids (leucine, tryptophan, histidine, adenine). Colonies grown on the agar plate of the selective medium) Furthermore, the auxotrophy of amino acids was confirmed.
The transformant thus obtained is brought into a steady state by shaking culture at 25 ° C. in SD medium (Yeast nitrogen base without amino acids (Difco 0919-15) + glucose + amino acids (leucine, tryptophan, histidine, adenine). Fluorescence intensity of cells after confirming that the absorbance at 600 nm is 0.2 to 0.5 in the logarithmic growth phase by diluting 500 times with SD medium and shaking culture at 25 ° C. for 15 hours. Was measured with a flow cytometer (FITC filter, EPICS XL-MCL, Bechmancoulter) .The flow cytometer was excited with an argon laser at an excitation wavelength of 488 nm, and 10,000 pieces in one measurement using a bandpass filter of 530 ± 30 nm. The fluorescence intensity of all cells was measured, and the average value of the fluorescence intensity of all cells was determined and used as the measurement value. A recombinant exhibiting a fluorescence intensity of 10 times or more was selected.
Thereby, it was found that the promoter of the yeast gene YNL015W can be used in the present invention.

Example 2
A preferred combination of a promoter that expresses increased promoter activity in response to a chemical substance and the chemical substance was examined.
According to the method of Example 1, in response to various chemical substances for several types of recombinants, increased promoter activity is expressed, and when the upper limit of measurement of chemical substances is exceeded, the expression level of the yeast gene decreases. Confirmed to do. This reveals a preferred combination of a yeast gene that expresses increased promoter activity in response to the chemical and the chemical.
For example, YER091C responded to mercury chloride, and YDR135C, YEL065W, YER091C, YLR303w, and YPL061W responded to ethanol.

Example 3
A recombinant yeast was prepared for measuring the promoter activity of the yeast gene YNL015W by the expression of the fluorescent protein ZsYellow (excitation maximum wavelength = 531 nm, fluorescence maximum wavelength = 540 nm YFP), and chemical susceptibility testing was performed by flow cytometry. . The details are as follows.

1) Preparation of recombinant yeast introduced with one kind of reporter gene The vector is pYES3 / CT (Cat. No. V8253-20, Invitrogen Corporation, USA), and the polynucleotide encoding ZsYellow is the vector pZsYellow (Cat. No. 632443, Clontech Laboratories, Inc.) ZsYellow part. First, a vector was prepared in which a ZsYellow polynucleotide was inserted into the multiple cloning site of pYES3 / CT. Subsequently, the GAL1 promoter part of pYES3 / CT was replaced with a polynucleotide containing a promoter sequence which is a yeast gene, to obtain a target plasmid vector. The insertion of the polynucleotide containing ZsYellow and the promoter sequence was performed by selecting an appropriate restriction enzyme. The polynucleotide containing the promoter sequence of the yeast gene (disclosed in SCPD: The Promoter Database of Saccharomyces cerevisiae) was from about 1000 bp upstream to immediately before the yeast gene.
Next, yeast Saccharomyces cerevisiae W303 ATCC201239 was transformed with this plasmid vector in the same manner as in Example 1 using a selective medium as a minimal nutrient medium (SD medium (uracil, leucine, histidine, adenine)).
Confirmation of the transformation was performed using a selective medium. Furthermore, the auxotrophy of the amino acids of the colonies grown on the agar plate of the selective medium was confirmed.

2) Chemical substance sensitivity test The obtained recombinant yeast was grown to a steady state by shaking culture at 25 ° C in an SD medium (uracil, leucine, histidine, adenine). Steady-state transformants were diluted 500-fold with SD medium and cultured with shaking at 25 ° C. for 15 hours to confirm that the absorbance at 600 nm was 0.2 to 0.5 in the logarithmic growth phase. Loaded with mercury chloride.
After loading with mercury chloride, the fluorescence of the cells cultured for a certain time (15 minutes, 4 hours) was measured using a flow cytometer (PE filter, EPICS XL-MCL, Bechmancoulter). The fluorescence intensity of 10,000 cells was measured in one measurement with a flow cytometer, and the average value of the fluorescence intensity of all cells was determined and used as the measurement value. Similarly, the fluorescence intensity of cells not loaded with mercury chloride was measured, and the difference in fluorescence intensity was determined.

3) Measurement of the number of viable bacteria After measurement with a flow cytometer, the number of viable bacteria was measured. After dilution to an appropriate concentration and seeding on an agar medium, the number of colonies was counted 3 days later. The number of colonies was multiplied by the dilution factor, and colony forming unit (CFU / ml) per ml was calculated.

4) Results The results obtained are shown in FIG. 1 and FIG. Up to 0.27 ppm, the number of viable bacteria and the fluorescence intensity are not changed by the treatment for 4 hours. A decrease in the number of viable bacteria is seen from 0.81 ppm, and at the same time the fluorescence intensity decreases.
As described above, the fluorescence intensity decreases when the cell is damaged and its growth is severely inhibited or killed. By detecting this decrease in fluorescence intensity, the upper limit of the chemical substance is exceeded. Can be determined.

Reference test example 1
An experiment was conducted to confirm whether formaldehyde directly affects GFP.
Formaldehyde was added to a GFP protein standard, and the direct influence of formaldehyde on GFP was examined, and it was confirmed that the decrease in promoter activity in the present invention was not caused by degradation of the marker protein by a chemical substance (FIG. 3).
When GFP was added to formaldehyde and measured, it was suggested that it might increase the fluorescence intensity, but it did not decrease with time.
From this, it seems that GFP and formaldehyde do not react directly.
Accordingly, in the present invention, if the promoter activity decreases, the microorganism used is exposed to a chemical substance at a level that leads to (a) a state in which its growth is severely inhibited, or (b) a state in which the microorganism is killed. Can be thought of as meaning.

Example 4
A recombinant yeast for measuring the promoter activity of the yeast gene YER091C by the ratio of the expression of the fluorescent protein GEF to the expression of the fluorescent protein ZsYellow was prepared, and a chemical sensitivity test by flow cytometry was performed. The details are as follows.

1) Preparation of recombinant yeast into which two types of reporter genes have been introduced. Promoter activity is demonstrated regardless of the presence of a chemical substance in Example 3 and the vector prepared using YER091C as a promoter that responds to chemical substances in Example 1. In both of the vectors prepared using YNL015W as a promoter expressed at a certain level, the yeast Saccharomyces cerevisiae W303 ATCC201239 was used as in Example 1 and 3, and the selection medium was a minimal nutrient medium (SD medium (leucine, histidine, adenine). ).

2) Chemical substance sensitivity test The obtained recombinant yeast was grown to a steady state by shaking culture at 25 ° C in an SD medium (leucine, histidine, adenine). Steady-state transformants were diluted 500-fold with SD medium and cultured with shaking at 25 ° C. for 15 hours to confirm that the absorbance at 600 nm was 0.2 to 0.5 in the logarithmic growth phase. Loaded with mercury chloride.
After loading with mercury chloride, fluorescence of cells cultured for a fixed time (15 minutes, 4 hours) was measured using a flow cytometer (FITC filter and PE filter, EPICS XL-MCL, Bechmancoulter). The fluorescence intensity of 10,000 cells was measured in one measurement with a flow cytometer, and the average value of the fluorescence intensity of all cells was determined and used as the measurement value. Similarly, the fluorescence intensity of cells not loaded with mercury chloride was measured, and the difference in fluorescence intensity was determined.

3) Results Changes in the fluorescence intensity derived from GFP and ZsYellow were monitored by changing the mercury chloride loading concentration, and the results are shown in FIG. FIG. 4 shows the ratio of the fluorescence intensity to the chemical substance concentration using the fluorescence intensity derived from ZsYellow and the fluorescence intensity derived from GFP. From this result, the relational expression f of the ratio of the fluorescence intensity to the chemical substance concentration can be derived. The relational expression may be a relational expression supplemented with the measured value or a complemented relational table.

  The fluorescence intensity derived from ZsYellow decreases above a certain concentration of the chemical substance, which is due to the decrease in the number of viable cells because the concentration at which the cells die is exceeded. On the other hand, although the fluorescence intensity derived from GFP begins to decrease when it exceeds a certain concentration, since the fluorescence intensity derived from ZsYellow also decreases, the chemical substance is present only below the detection limit, It can be seen that this is because the growth is severely inhibited or killed.

Example 5
A recombinant yeast for measuring the promoter activity of the yeast gene YER091C by the ratio of the expression of the fluorescent protein GEF to the expression of the fluorescent protein ZsYellow was prepared, and a chemical substance sensitivity test was performed. In this example, the obtained recombinant yeast was once dried, and the dried yeast was activated and then tested. The details are as follows.

1) Preparation of dry recombinant yeast introduced with two kinds of reporter genes From the recombinant yeast prepared in Example 4, methods well known in the art such as Reed, G. and Nagoyawithana, TW (1991) Yeast Technology, Dry yeast was prepared using the method described in 2nd edition, pp.261-314, Van Nostrand Reinhold, New York.

2) Chemical Substance Sensitivity Test In this example, a chemical substance sensitivity test was performed using the measurement container (1) shown in FIG. This container can be used alone, but a plurality of containers can be arranged to constitute a microtiter plate usually used in a promoter assay method (not shown).
In the measurement container (1), the membrane part (3) for sealing the dried yeast (2) is usually sealed so that microorganisms are not released to the outside of the container after being accommodated, and the test inside the container (4) is performed. The sample solution is made of an acceptable material. Further, the membrane part plays a role of preventing a relatively large size contaminant contained in the test sample solution from entering the container.

  In FIG. 5, the shielding part (5) is shaped to form two or more openings (6) above the membrane part (3), and at least one of the openings is attached to and detached from the tip of the syringe (20). It is configured so that it can be freely fitted. By inserting the tip of the syringe containing the test sample solution into the opening (6) of the shielding part and injecting it, the test sample solution can be press-fitted.

The container body (7) of the present invention is made of any shape and material as long as it can measure the expression of the reporter gene of the recombinant microorganism by bringing the above-mentioned recombinant microorganism into contact with the test sample solution. It may be present, but at least the bottom or side is made of a transparent material. The size of the container body (7) is a size that can be assayed sufficiently even with a smaller amount of test sample, and usually has a volume of 50 μl to 50 ml. In addition, as shown in FIG. 5, the shape of the container body is such that the volume on the opening side is larger than the volume on the bottom side so that a wide range of test sample liquids can be applied. Can do.
Furthermore, the container of the present invention may include a lid (8). Such a lid may be separated from the container body or connected to the container body by a hinge as shown.

Specifically, first, SD culture medium (leucine, histidine, adenine) is added to the measurement container (1) in which dry yeast is stored in advance to activate the dry yeast.
Thereafter, the mercury chloride solution is loaded through the membrane part (3) using the syringe (20).
After loading the mercury chloride solution, place the container in a microtiter plate reader (wavelength tunable fluorescence / absorption microtiter plate reader sapphire, TECAN), irradiate excitation light from the bottom of the container, and obtain fluorescence intensity derived from ZsYellow and GEF. Measure.

Using the obtained fluorescence intensity derived from ZsYellow and fluorescence intensity derived from GFP, the ratio of fluorescence intensity was determined. By applying the ratio of the fluorescence intensity to the relational expression f of the ratio of the fluorescence intensity to the chemical substance concentration obtained in Example 4, the concentration of mercury chloride in the container can be calculated.
Furthermore, the concentration of mercury chloride in the mercury chloride solution can be determined from the volume of the loaded mercury chloride solution and the volume of the SD medium.

3) Summary In environmental measurement kits using bioassays, it may be possible to store them for a long time by placing dry yeast in a container and sealing it. However, if dry yeast is used, it will not be damaged even if activated by the addition of a medium. The number of viable bacteria is unknown because some yeasts have received it. Therefore, conventionally, the concentration of the chemical substance could not be obtained from the fluorescence intensity per number of bacteria.
However, according to the present invention, a fluorescence intensity that does not depend on the presence of a chemical substance and a fluorescence intensity that varies depending on the concentration of a specific chemical substance, according to a relational expression of the ratio of the fluorescence intensity to the chemical substance concentration that is measured in advance Even if the number of viable bacteria is unknown, the abundance of the chemical substance can be detected.

It is a graph which shows the result of the fluorescence intensity in the mercury chloride load test with respect to the yeast containing the cassette with which the promoter of yeast gene YNL015W and YFP were connected. It is a graph which shows the result of the viable count in the mercury chloride load test with respect to the yeast containing the cassette with which the promoter of yeast gene YNL015W and YFP were connected. This is the result of examining the degradability of formaldehyde by GFP by adding various concentrations of formaldehyde to the GFP protein standard. It is a graph which shows the result of the fluorescence intensity in the mercury chloride load test with respect to the yeast containing the cassette by which the promoter of yeast gene YNL015W and YFP, and the cassette by which the promoter of yeast gene YER091C and GFP were connected. 1 is a schematic view of a container having a shape suitable for use in a promoter assay method using dried microorganisms. The figure which shows the wavelength dependence of the absorption coefficient and scattering coefficient of the light in distilled water.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Container for measurement 2 Dry yeast 3 Membrane part 4 Inside of container 5 Shielding part 6 Container opening part 7 Container body 8 Lid 20 Syringe

Claims (7)

A recombinant cell into which two or more reporter genes have been introduced, wherein one reporter gene is linked downstream of the promoter of the yeast gene YNL015W that expresses promoter activity at a constant level regardless of the presence of mercury chloride. A recombinant cell, wherein one or more other reporter genes are linked downstream of a promoter that expresses increased promoter activity in response to a chemical substance .   The recombinant cell according to claim 1, which is a recombinant yeast.   The recombinant cell according to claim 1, wherein the two or more reporter genes are fluorescent protein genes having a fluorescence maximum wavelength at 580 nm or less. Contacting the test sample to one or more recombinant cells, wherein the said one or more recombinant cells, a recombinant cell two or more reporter genes is introduced, 1 species of reporter The gene is linked downstream of the promoter of the yeast gene YNL015W, which expresses promoter activity at a constant level regardless of the presence of mercury chloride, and one or more other reporter genes are increased in response to chemicals , respectively. A recombinant cell characterized in that it is connected downstream of a promoter that expresses the promoter activity;
In the test sample, the promoter activity of the promoter of the yeast gene YNL015W that expresses promoter activity at a certain level regardless of the presence of mercury chloride and the promoter activity of the promoter that expresses increased promoter activity in response to mercury chloride are measured. And
Calculating the ratio of the promoter activity of a promoter that expresses increased promoter activity in response to a chemical to the promoter activity of the yeast gene YNL015W promoter that expresses promoter activity at a constant level regardless of the presence of mercury chloride;
A multi-index promoter assay method characterized in that the presence or amount of a chemical substance in a test sample is estimated based on this ratio.   The method according to claim 4, wherein the recombinant cell is a recombinant yeast.   The method according to claim 4, wherein the promoter activity is measured by fluorescence intensity.   The method according to claim 4, wherein the two or more reporter genes are fluorescent protein genes having a fluorescence maximum wavelength at 580 nm or less.

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