CN102965368A - Super-sensitive yeast genotoxic carcinogen biosensor element box and its application - Google Patents
Super-sensitive yeast genotoxic carcinogen biosensor element box and its application Download PDFInfo
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Abstract
The invention discloses a super-sensitive yeast genotoxic carcinogen biosensor element box and its construction method. The invention also discloses a method for applying the element box to construct a super-sensitive yeast genotoxic carcinogen biosensor, and relates to monitoring of genotoxic carcinogens by genetically modified microorganisms. By applying the biosensor element box in a composite DNA damage super-sensitive type seven gene deleted bread yeast mutant strain, the detection sensitivity of the biosensor element box can be improved. Therefore, compared with the prior art, the biosensor element box provided in the invention has the advantages of high genotoxic carcinogen detection sensitivity, application to detection of low-dose chemical carcinogens existing in the environment, wide environmental pollutant detection range, simple operation and the like. The biosensor can promote the establishment of a real-time monitoring platform for environmentally harmful substances.
Description
Technical field
The present invention relates to biotechnology, relate in particular to the microorganism of transforming through gene to the monitoring of environmental chemical pollutants.
Background technology
Bread yeast is a kind of simple eukaryote, and unicellular, genetic modification is simple to operate, cultivates easy quick and environmental friendliness, is suitable as a kind of model animals and is applied in the life science.In the environment pollution detection technical field, bread yeast is more and more come into one's own as a kind of simple eukaryote at present, and some detection systems take bread yeast as model animals are developed.
Genotoxic carcinogens refers to and can react with DNA, cause dna damage and carcinogenic chemical carcinogen, can be with biochemical test or indirectly use the genetic toxicity test method and identify genotoxic carcinogens, comprise directly acting carcinogens, indirect acting carcinogens and some inorganic carcinogens.
Now developed and a plurality ofly transcribed the detection system of the genotoxic carcinogens of response based on dna damage, in the face of high-throughput, the actual requirement of Real-Time Monitoring, we still need continue innovation, develop highly sensitive, the biosensor of high applicability.Make up a genotoxic carcinogens biosensor, except host cell, also need possess following two primary elements: sensing element and the report element that is connected in after this.The former utilizes himself function to respond dna damage, and opens the latter's expression, thereby produces detectable signal.The present invention is called the biosensor component box with these two unit construction gene fragment together.
HUG1 (hydroxyurea and UV and gamma radiation induced) can have the significance up-regulated expression after being processed by hydroxyurea (hydroxyurea) because of cell, thereby is regarded as can playing the latent gene of transcribing response after cell is subject to dna damage and copies stagnation.Show that after deliberation the HUG1 gene is subject to the gene transcription regulation on the MEC1 path.In bread yeast, MEC1 is one of phosphatidyl-inositol 3-kinase family member, it is an important check position gene (checkpoint gene), the transcribed a large amount of regulon genes (regulon gene) of inducing of check position gene start the DNA reparation, cause cell-cycle arrest, alleviate dna damage.And the gene that the researchist finds to be present in the MEC1 check position signal path copies all transcribed response expression of inducing HUG1 of retardance and dna damage running into.It is very low to have been reported proof HUG1 expression amount under general condition, but really is subjected to significantly inducing of dna damage signal, and highly sensitive, can be used as the sensing element of preparation biosensor.
Green fluorescent protein (GFP) is isolated natural biological luminescent protein from jellyfish; Its character is extremely stable; Need not to add any substrate and cofactor, namely under the exciting of ultraviolet or blue light, send green fluorescence.It can both send fluorescence when bacterium, fungi, plant and animal cells, have live body, original position, the real-time characteristics of expressing.And yeast enhanced green fluorescence protein (yEGFP) is a kind of enhanced green fluorescence protein after codon optimized, and it can strengthen expression in yeast cell.Contrast is the reporter gene lacZ of widely used coding B-gal tilactase before this, GFP has the active somatic cell of being directly used in and detects, and can overcome and utilize enzyme (to destroy yeast cells wall as a series of shortcomings of reporter gene, affect the factor complexity of enzyme colour developing alive etc.), the series of advantages such as detection means is various, automatization, utilize green fluorescent protein as the response element of preparation biosensor, so that in the environment genetoxic compound measurement system more advantageously to high-throughput, fast, stage development easily.
Utilize SOE method (Gene Splicing by Overlap Extension) to carry out the pcr gene fragment assembly, namely realize splicing by the staggered extension of gene fragment in the PCR process.The method is to utilize round pcr to carry out the efficient gene restructuring external, and does not need restriction endonuclease digestion and ligase enzyme to process, and this utilization is simple and easy.Since primer only need to the effective combination of template, especially 5 ' terminal sequence needn't match fully with template, so 5 ' end of amplimer can add two kinds of restriction enzyme sites so that later stage clone.
Flow Cytometry is a key areas in the analysis of cells, this technology is one of cytology research means, can carry out analysis up to up to ten thousand cell individuals of per second to cell and crganelle and biomacromolecule, but and cell multiparametric analysis and sorting cells.This mode (relatively static mode) measurement cell and traditional comparing with fluoroscope detection cell to flow, it is fast to have speed, and precision is high, the characteristics that accuracy is good.
Summary of the invention
The objective of the invention is, a kind of hypersensitization type yeast genotoxic carcinogens biosensor component box HUG1-yEGFP-CYC1 and construction process thereof at first are provided, and the biosensor component box is applied in compound dna damage hypersensitization profile bag yeast seven gene deletion mutants, obtain hypersensitization type yeast genotoxic carcinogens biosensor, this biosensor has improved the susceptibility to genetoxic chemical substance in the testing environment greatly, enlarge sensing range, strengthened the handiness of detection means.
In order to achieve the above object, the present invention adopts following technical scheme:
One, the construction process of hypersensitization type yeast genotoxic carcinogens biosensor component box HUG1-yEGFP-CYC1 gene fragment is:
1, extract wild bread yeast genomic dna, by Auele Specific Primer, take wild bread yeast genomic dna as template, pcr amplification obtains HUG1 promoter gene fragment;
2, extract plasmid PUG36, by Auele Specific Primer, take the PUG36 plasmid DNA as amplification template, pcr amplification obtains respectively yEGFP coding region gene fragment;
3, extract plasmid PUG36, by Auele Specific Primer, take the PUG36 plasmid DNA as amplification template, pcr amplification obtains respectively terminator CYC1 gene fragment;
4, utilize the SOE method, pass through Auele Specific Primer, take HUG1 promoter gene fragment, yEGFP coding region gene fragment, terminator CYC1 gene fragment according to 1: 1: 1 ratio of mol ratio as amplification template, pcr amplification obtains hypersensitization type yeast genotoxic carcinogens biosensor component box HUG1-yEGFP-CYC1 gene fragment;
Two, hypersensitization type yeast genotoxic carcinogens biosensor component box changes in the compound dna damage hypersensitization profile bag yeast seven gene deletion mutants genomes, is applied to the detection of genotoxic carcinogens:
1, hypersensitization type yeast genotoxic carcinogens biosensor component box HUG1-yEGFP-CYC1 is transferred in the compound dna damage hypersensitization profile bag yeast seven gene deletion mutants genomes, forms hypersensitization profile bag yeast genotoxic carcinogens biosensor yap1 Δ cwp1 Δ cwp2 Δ snq2 Δ pdr5 Δ rad Δ mag1 Δ-HUG1-yEGFP-CYC1; Wherein compound dna damage hypersensitization profile bag yeast seven gene deletion mutants are at five genetically deficient bread yeast mutant strain (number of patent applications: on the basis 2010105941713) to the oxidative damage hypersensitivity, knock out with dna damage and repair relevant MAG1 and RAD1 gene, obtain the bread yeast mutant strain of disappearance yap1 cwp1 cwp2 snq2 pdr5 rad1 mag1 seven genes.
2, hypersensitization profile bag yeast genotoxic carcinogens biosensor can utilize low cytometric analysis, thereby by the changing conditions of measuring egfp expression genotoxic carcinogens is detected.
Advantage of the present invention and effect:
Hypersensitization profile bag yeast genotoxic carcinogens biosensor component box HUG1-yEGFP-CYC1 key advantage is, it has higher susceptibility to genotoxic carcinogens, detect easy and simple to handle, personal errors reduces, also can be used for direct-detection, have the application potential of realizing genetoxic compound Real-Time Monitoring in enormous quantities and even high-throughout.
Compare for the wild-type bread yeast with the host, the host is the hypersensitization profile bag yeast genotoxic carcinogens biosensor of compound dna damage hypersensitization profile bag yeast seven transgenation strains among the present invention, susceptibility to all types of genotoxic carcinogenses is improved, and improves more than 50 times for methyl mesylate (MMS) detection sensitivity of (25ppm) under extremely low concentration; To hydrogen peroxide (H
2O
2) improve more than 10 times in the detection sensitivity of 0.6mM concentration; Macromole is caused dna damage compound phleomycin (Phleomycin) improve 40 times in the detection sensitivity of 2.5 μ g/ml concentration; The host is the genotoxic carcinogens biosensor of wild-type bread yeast, can not detect the dna damage signal that tertbutyl peroxide (t-BHP) and dna damage agent 4-nitroquinoline 1-oxide compound (4-NQO) methyl viologen (methyl viologen) cause, cause DNA oxidative damage compound and hypersensitization profile bag yeast genotoxic carcinogens biosensor all can detect these two kinds, and higher detection sensitivity is arranged.Therefore the application of hypersensitization profile bag yeast genotoxic carcinogens biosensor can improve its detection sensitivity and enlarge its sensing range, more can promote the foundation of the Real-Time Monitoring platform of environment genotoxic carcinogens.
The comparative analysis of table 1, hypersensitization profile bag yeast genotoxic carcinogens biosensor and the sensitivity of four kinds of existing bread yeast genotoxic carcinogens biosensors,
| ? | Wild-type bread yeast RNR3-lacZ biosensor | Wild-type bread yeast RNR2-yEGF P biosensor | Wild-type bread yeast Rad54-yEG FP biosensor | Wild-type bread yeast HUG1-yEGFP-C YC1 biosensor | Compound dna damage ultra-high sensitive profile bag yeast HUG1-yEFP-CY C1 biosensor |
| The first sulfo methyl ester [25ppm) | 9.3 doubly | 4 times | 4 times | 5 times | 56 times |
| Hydrogen peroxide (0.6mM) | About 2 times | Without detecting | Without detecting | 2 times | 12 times |
| Tertbutyl peroxide (0.125mM) | Without inducement signal | Without detecting | Without detecting | Without inducement signal | 4.5 doubly |
| 4-nitroquinoline 1-oxide compound (0.005ug/ml) | Without inducement signal | Without detecting | Without detecting | Without inducement signal | 22 times |
| Phleomycin (2.5ug/ml) | Without inducement signal | 2 times | Without detecting | 20 |
40 times |
Numerical value shown in the table is the abduction delivering multiple, and expression detects the strength of signal that obtains, and the detected result after being divided by between the detection signal strength of blank; Do not detect without detecting expression.
Description of drawings
Fig. 1, genotoxic carcinogens biosensor component box HUG1-yEGFP-CYC1 are applied in the different bread yeast strains comparison to DNA alkylating agent methyl mesylate (MMS) detection sensitivity.
X-coordinate represents the concentration of DNA alkylating agent methyl mesylate (MMS) among the figure; Ordinate zou represents that biosensor detects resulting relative intensity of fluorescence to DNA alkylating agent methyl mesylate (MMS) and induces multiple;-●-curve representation genotoxic carcinogens biosensor component box HUG1-yEGFP-CYC1 is applied in the wild-type bread yeast detected result to DNA alkylating agent methyl mesylate (MMS);-s-curve representation is applied in the seven transgenation strains of compound dna damage hypersensitization profile bag yeast detected result to DNA alkylating agent methyl mesylate (MMS) in genotoxic carcinogens biosensor component box HUG1-yEGFP-CYC1.
Embodiment
One, the construction process of hypersensitization type yeast genotoxic carcinogens biosensor component box comprises the following step:
(1), from wild bread yeast, extract genomic dna:
1a, get wild bread yeast, be inoculated in the YPD nutrient solution, at 30 ℃, under 200 rev/mins the condition, shaking culture 16 hours obtains wild bread microzyme liquid;
Wherein: the formulation weight per-cent of YPD nutrient solution is: yeast extract paste 1%, and peptone 2%, glucose 2% the rest is water;
1b, get 1.5 milliliters of wild bread microzyme liquid, with 4000 rev/mins centrifugal 2 minutes, throw out is suspended from the 200 microlitre Extraction buffers;
Wherein: the prescription of Extraction buffer is: triton x-100 2%, and sodium laurylsulfonate 1%, 0.1 mole in sodium-chlor, ethylenediamine tetraacetic acid (EDTA) 1 mmole, Tri(Hydroxymethyl) Amino Methane Hydrochloride 10 mmoles the rest is water, and pH 8.0;
1c, add 0.3 gram pickling glass pearl, 100 microlitre phenol, 100 microlitre chloroforms, shake after 3 minutes, with 12000 rev/mins centrifugal 10 minutes, the absorption supernatant liquid;
1d, add the dehydrated alcohol of 2 times of the liquid volumes of getting, left standstill 30 minutes at-20 ℃, with 12000 rev/mins centrifugal 15 minutes, remove supernatant, taking precipitate;
1e, throw out is dissolved in 20 microlitre sterilized waters, is the wild yeast genomic dna;
(2), amplification HUG1 promoter gene fragment
Wild bread yeast genome is as amplification template, by upstream primer HUG1 Pro-1:5 ' AA CTG CAG GCA AAA ACT AGA GCC AAG CC 3 ' and downstream primer HUG1 Pro-2:5 ' TTA GAC ATA TAG TTT TTT TTT ATT GCT G 3 ', utilize round pcr, amplification obtains HUG1 promoter gene fragment (600bp)
(3), amplification yEGFP coding region gene fragment and terminator CYC1 gene fragment
Plasmid PUG36 (U.S. ATCC company provides) is as amplification template, by upstream primer yEGFP-1:5 ' AAA ACT ATA TGT CTA AAG GTG AAG AAT T 3 ' and downstream primer yEGFP-2:5 ' TAC ATG ACT TTG TAC AAT TCA TCC ATA C 3 ', utilize round pcr, amplification obtains yEGFP coding region gene fragment (750bp); Plasmid PUG36 is as amplification template, by upstream primer CYC1-1:5 ' TGT ACA AAG TCA TGT AAT TAG TTA TGT C 3 ' and downstream primer CYC1-2:5 ' TCCG GAA TTC GGT ACC GGC CGC AAA TTA AAG 3 ', amplification obtains terminator CYC1 gene fragment (260bp);
(4), the HUG1 promoter gene fragment that amplification hypersensitization type yeast genotoxic carcinogens biosensor component box HUG1-yEGFP-CYC1 gene fragment obtains above-mentioned amplification, yEGFP coding region gene fragment and 1: 1: 1 in molar ratio ratio of terminator CYC1 gene fragment are as amplification template, by upstream primer HUG1 Pro-1:5 ' AA CTG CAG GCA AAA ACT AGA GCC AAG CC 3 ' and downstream primer CYC1 Pro-2:5 ' TCCG GAA TTC GGT ACC GGC CGC AAA TTA AAG 3 ', amplification obtains hypersensitization type yeast genotoxic carcinogens biosensor component box, namely based on the HUG1-yEGFP-CYC1 gene fragment of cerevisiae dna damage response;
Wherein: the condition of PCR is: 94 ℃ 5 minutes; 94 ℃ 45 seconds, 55 ℃ 45 seconds, 72 ℃ 1 minute, 30 circulations; 72 ℃ 5 minutes.
Two, hypersensitization type yeast genotoxic carcinogens biosensor component box is applied to make up hypersensitization type yeast genotoxic carcinogens biosensor, the method comprises the following step:
(1), on the basis of the bread yeast to the oxidative damage hypersensitivity (number of patent application is 2010105941713) of five genetically deficients, knock out again with dna damage and repair relevant MAG1 and RAD1 gene, obtain compound dna damage hypersensitization profile bag yeast seven gene deletion mutants, step is as follows:
1a, with plasmid
ΔMag1::hisG-URA3-hisG (reference: Jin Chen, et al. (1990) Saccharomyces cerevisiae 3-methyladenine DNA glycosylase has homology to the AlkA glycosylase of E.coli and is induced in response to DNA alkylation damage.The EMBO Journal, vol.9, pp.4569-4575) carry out enzyme with restriction enzyme EcoR I and Bgal II and cut, obtain knocking out assembly mag1
Δ:: hisG-URA3-hisG;
1b, will knock out assembly and change in the bread yeast to the oxidative damage hypersensitivity with the Lithium Acetate method for transformation, homologous recombination will occur, obtain the bread yeast mutant strain of six genetically deficients by SD-Ura selectivity plate screening;
1c, with plasmid
ΔRad1::Blast (deriving from Dr.E.Perkins NIESH, NC, USA) carries out enzyme with restriction enzyme SalI and cuts, and obtains knocking out assembly rad1
Δ:: hisG-URA3-hisG, change in the bread yeast mutant strain of six genetically deficients with the Lithium Acetate method for transformation with knocking out assembly, homologous recombination occurs, obtain compound dna damage hypersensitization profile bag yeast seven gene deletion mutants by SD-Ura selectivity plate screening, this mutant strain is the bread yeast of seven genes of disappearance yap1 Δ cwp1 Δ cwp2 Δ snq2 Δ pdr5 Δ rad1 Δ mag1 Δ;
(2) with hypersensitization type yeast genotoxic carcinogens biosensor component box HUG1-yEGFP-CYC1 gene fragment clone to pBluescipt carrier (U.S. Thermo company provides), utilize the PstI restriction endonuclease to connect with the EcoR1 endonuclease digestion, obtain cloning pBluescipt-HUG1-yEGFP-CYC1;
(3), to clone pBluescipt-HUG1-yEGFP-CYC1 carries out enzyme with BamH1 restriction endonuclease and EcoR1 restriction endonuclease and cuts, obtaining the two ends restriction enzyme site is the yeast sensitive-type genotoxic carcinogens detection system sensing member UG1-yEGFP-CYC1 fragment of BamH1 and EcoR1, be cloned into again pM4366 carrier (reference: W.P.Voth, et al. (2001) Yeast vectors for integration at the HO locus.Nucleic Acids Res.Vol.29, E59-9) in, downcut fragment HO-hisG-URA3-hisG-HUG1-yEGFP-CYC1-HO with the Not1 restriction endonuclease;
(4), the HO-hisG-URA3-hisG-HUG1-yEGFP-CYC1-HO fragment is changed in compound dna damage hypersensitization profile bag yeast seven gene deletion mutants, screening obtains hypersensitization type yeast genotoxic carcinogens biosensor, and above-mentioned changing over to screening method comprises the following step:
4a, compound dna damage hypersensitization profile bag yeast seven gene deletion mutants are inoculated into 1 milliliter of YPD nutrient solution, 30 ℃, under 200 rev/mins of oscillating conditions, cultivated 16 hours;
4b, add 2 milliliters of YPD nutrient solutions again, 30 ℃, 200 rev/mins, cultivated 4 hours;
4c, get 1 milliliter of yeast among the 4b, with 2500 rev/mins centrifugal 2 minutes, taking precipitate;
4d, with throw out be suspended from 100 the milli rub/liter 400 microlitre Lithium Acetates in, with 2500 rev/mins centrifugal 2 minutes, taking precipitate;
4e, with throw out be suspended from 100 the milli rub/liter 100 microlitre Lithium Acetates in;
4f, smart single stranded DNA 1 microlitre of the salmon of 2.0 mg/ml was boiled 5 minutes, join in the Lithium Acetate of step 4e with fragment HO-hisG-URA3-hisG-HUG1-yEGFP-CYC1-HO 0.1～1 microgram behind the ice bath, room temperature was placed 5 minutes;
4g, to add 280 microlitre concentration again be 50% Macrogol 4000;
4h, vibrate to mixing, 30 ℃ left standstill 45 minutes;
4i, add 39 microlitre dimethyl sulfoxide (DMSO), 42 ℃ of heat shocks 5 minutes;
Centrifugal 2 minutes of 4j, 4000 rev/mins, taking precipitate, and throw out is suspended from the 200 microlitre sterilized waters;
Centrifugal 2 minutes of 4k, 4000 rev/mins, taking precipitate is suspended from the 100 microlitre sterilized waters, and then be applied to SD-Ura auxotroph culture medium flat plate, cultivated 3 days in 30 ℃ of incubators, grow single bacterium colony, this list bacterium colony is hypersensitization type yeast genotoxic carcinogens biosensor yeast strains;
Wherein the prescription of SD-Ura selectivity flat board is: yeast nitrogen without amino acid without ammonium sulfate 0.17%, ammonium sulfate 0.5%, glucose 2%, VITAMIN B4 0.2%, tryptophane 1%, Histidine 1%, leucine 1%, Methionin 1%, agar powder 2% the rest is water;
Three, hypersensitization type yeast genotoxic carcinogens biosensor is for detection of the method for genotoxic carcinogens, and the method comprises the following step:
1, hypersensitization type yeast genotoxic carcinogens biosensor yeast strains is inoculated in the YPD nutrient solution, 30 ℃, 200 rev/mins of lower shaking culture 16 hours;
2, the hypersensitization type yeast genotoxic carcinogens biosensor yeast strains bacterium liquid with incubated overnight is diluted to cell density OD with fresh YPD selectivity nutrient solution
600Be 0.1;
3, get 1 milliliter of hypersensitization type yeast genotoxic carcinogens biosensor yeast strains bacterium liquid after the dilution, add dna damage alkylating agent methyl methylsulfonate, cultivated 8 hours;
4, get 200 microlitre hypersensitization type yeast genotoxic carcinogens biosensor yeast strains bacterium liquid, 4000 rev/mins centrifugal 2 minutes, abandon supernatant, taking precipitate; Add 200 microlitre 0.1M PBS solution, suspended sediment; Again 4000 rev/mins centrifugal 2 minutes, abandon supernatant, taking precipitate; Add 500 microlitre 0.1M PBS solution, suspended sediment;
Wherein 0.1M PBS solution formula is: take by weighing 8 gram sodium-chlor, and 0.2 gram Repone K, 3.68 grams 12 close disodium-hydrogen, and 0.24 gram potassium primary phosphate is dissolved in the distilled water, and regulating the pH value with sodium hydroxide is 7.4, is settled to 1 liter;
5, bromination the third pyridine (PI) solution 3 microlitres that add 1 milligram every milliliter, mixing places on ice, the dark preservation until use flow cytometer to detect;
6, with flow cytometer sample is detected, set simultaneously FITC and PE two laser channelings;
7, with Flow Jo stream data analysis software output analytical data, the Mean value in the data is the average fluorescent strength numerical value of test sample;
8, measure the hypersensitization type yeast genotoxic carcinogens biosensor yeast strains average fluorescent strength numerical value of processing without methyl methylsulfonate according to step 1 to the method for step 7, as blank;
9, when showing greater than 2 the time, the ratio of the hypersensitization type yeast genotoxic carcinogens biosensor yeast strains solution average fluorescent strength numerical value of processing through methyl methylsulfonate and the average fluorescent strength numerical value of blank detects the dna damage signal.
Claims (3)
1. hypersensitization type yeast genotoxic carcinogens biosensor component box is characterized in that, this subassembly wrapper is based on the cerevisiae dna damage response
HUG1-yEGFP-CYC1Gene fragment.
2. realize the construction process of hypersensitization type yeast genotoxic carcinogens biosensor component box claimed in claim 1, it is characterized in that the method comprises the following step:
(1) from wild bread yeast, extracts genomic dna
1a, get wild bread yeast, be inoculated in the YPD nutrient solution, at 30 ℃, under 200 rev/mins the condition, shaking culture 16 hours obtains wild bread microzyme liquid;
Wherein: the formulation weight per-cent of YPD nutrient solution is: yeast extract paste 1%, and peptone 2%, glucose 2% the rest is water;
1b, get 1.5 milliliters of wild bread microzyme liquid, with 4000 rev/mins centrifugal 2 minutes, taking precipitate is suspended from the 200 microlitre Extraction buffers;
Wherein: the prescription of Extraction buffer is: triton x-100 2%, and sodium laurylsulfonate 1%, 0.1 mole in sodium-chlor, ethylenediamine tetraacetic acid (EDTA) 1 mmole, Tri(Hydroxymethyl) Amino Methane Hydrochloride 10 mmoles the rest is water, and pH 8.0;
1c, add 0.3 gram pickling glass pearl, 100 microlitre phenol, 100 microlitre chloroforms, shake after 3 minutes, with 12000 rev/mins centrifugal 10 minutes, get supernatant liquid;
1d, add the dehydrated alcohol of 2 times of the liquid volumes of getting, left standstill 30 minutes at-20 ℃, with 12000 rev/mins centrifugal 15 minutes, remove supernatant, taking precipitate;
1e, throw out is dissolved in 20 microlitre sterilized waters, is the wild yeast genomic dna;
(2), amplification
HUG1The promoter gene fragment
Wild bread yeast genome passes through upstream primer as amplification template
HUG1Pro-1:5 ' AA CTG CAG GCA AAA ACT AGA GCC AAG CC 3 ' and downstream primer
HUG1Pro-2:5 ' TTA GAC ATA TAG TTT TTT TTT ATT GCT G 3 ' utilizes round pcr, and amplification obtains
HUG1The promoter gene fragment;
(3), amplification yEGFP coding region gene fragment and terminator
CYC1Gene fragment
Plasmid PUG36 passes through upstream primer as amplification template
YEGFP-1:5 ' AAA ACT ATA TGT CTA AAG GTG AAG AAT T 3 ' and downstream primer
YEGFP-2:5 ' TAC ATG ACT TTG TAC AAT TCA TCC ATA C 3 ' utilizes round pcr, and amplification obtains
YEGFPThe coding region gene fragment;
Plasmid PUG36 passes through upstream primer as amplification template
CYC1-1:5 ' TGT ACA AAG TCA TGT AAT TAG TTA TGT C 3 ' and downstream primer
CYC1-2:5 ' TCCG GAA TTC GGT ACC GGC CGC AAA TTA AAG 3 ', amplification obtains terminator
CYC1Gene fragment;
(4), with above-mentioned
HUG1The promoter gene fragment,
YEGFPCoding region gene fragment and terminator
CYC1The gene fragment in molar ratio ratio of 1:1:1 is passed through upstream primer as amplification template
HUG1Pro-1:5 ' AA CTG CAG GCA AAA ACT AGA GCC AAG CC 3 ' and downstream primer
CYC1Pro-2:5 ' TCCG GAA TTC GGT ACC GGC CGC AAA TTA AAG 3 ', amplification obtains hypersensitization type yeast genotoxic carcinogens biosensor component box, namely based on the cerevisiae dna damage response
HUG1-yEGFP-CYC1Gene fragment;
The condition of above-mentioned PCR is: 94 ℃ 5 minutes; 94 ℃ 45 seconds, 55 ℃ 45 seconds, 72 ℃ 1 minute, 30 circulations; 72 ℃ 5 minutes.
3.
Make up hypersensitization type yeast genotoxic carcinogens biosensor with hypersensitization type yeast genotoxic carcinogens biosensor component box claimed in claim 1, it is characterized in that this construction process comprises the following step:
3a, knock out in the bread yeast to the oxidative damage hypersensitivity of five genetically deficients
MAG1With
RAD1Gene obtains compound dna damage hypersensitization profile bag yeast seven gene deletion mutants, and step is as follows:
3a1, with plasmid
Mag1::hisG-URA3-hisG restriction enzyme
The EcoR IWith
The Bgal IICarry out enzyme and cut, obtain knocking out assembly
Mag1 
:: hisG-URA3-hisG;
3a2, will knock out assembly and change over to the Lithium Acetate method for transformation in the bread yeast to the oxidative damage hypersensitivity of five genetically deficients, homologous recombination will occur, obtain the bread yeast mutant strain of six genetically deficients by SD-Ura selectivity plate screening;
3a3, with plasmid
Rad1::Blast carries out enzyme with restriction enzyme SalI and cuts, and obtains knocking out assembly rad1
:: hisG-URA3-hisG, change in the bread yeast mutant strain of six genetically deficients with the Lithium Acetate method for transformation with knocking out assembly, homologous recombination occurs, obtain compound dna damage hypersensitization profile bag yeast seven gene deletion mutants by SD-Ura selectivity plate screening, this mutant strain is disappearance
Yap1 Δ cwp1 Δ cwp2 Δ snq2 Δ pdr5 Δ rad1 Δ mag1 ΔThe bread yeast of seven genes;
3b, with in claim 2 step (4)
HUG1-yEGFP-CYC1Gene fragment clone utilizes to the pBluescipt carrier
PstIRestriction endonuclease and
EcoR1Endonuclease digestion connects, and obtains cloning pBluescipt-
HUG1-yEGFP-CYC1
3c, will clone pBluescipt-
HUG1-yEGFP-CYC1With
BamH1Restriction endonuclease and
EcoR1Restriction endonuclease carries out enzyme to be cut, and obtains the two ends restriction enzyme site and is
BamH1With
EcoR1Gene fragment, be cloned into again in the pM4366 carrier, use
Not1Restriction endonuclease downcuts fragment
HO-hisG-URA3-hisG-HUG1-yEGFP-CYC1-HO
3d, general
HO-hisG-URA3-hisG-HUG1-yEGFP-CYC1-HOFragment changes in compound dna damage hypersensitization profile bag yeast seven gene deletion mutants with the Lithium Acetate method for transformation, obtains hypersensitization type yeast genotoxic carcinogens biosensor yeast strains by SD-Ura selectivity plate screening.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103510161A (en) * | 2013-08-16 | 2014-01-15 | 中国科学院水生生物研究所 | Saccharomyces single-gene deletion library containing genotoxic biosensors |
| CN112680498A (en) * | 2020-12-28 | 2021-04-20 | 华南理工大学 | High-throughput screening method for genotoxic substances |
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| CN1824781A (en) * | 2005-12-09 | 2006-08-30 | 海南国栋药物研究所有限公司 | Method for producing high specific activity HuG CSF yeast expression carrier, high production strain and HuG-CSF |
| CN102120968A (en) * | 2010-12-18 | 2011-07-13 | 中国科学院水生生物研究所 | Baker's yeast with high sensibility on oxidative damage and preparation method thereof |
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2012
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1824781A (en) * | 2005-12-09 | 2006-08-30 | 海南国栋药物研究所有限公司 | Method for producing high specific activity HuG CSF yeast expression carrier, high production strain and HuG-CSF |
| CN102120968A (en) * | 2010-12-18 | 2011-07-13 | 中国科学院水生生物研究所 | Baker's yeast with high sensibility on oxidative damage and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| BENTON ET AL: "Deletion of MAG1 and MRE11 enhances the sensitivity of the Saccharomyces cerevisiae HUG1P-GFP promoter-reporter construct to genotoxicity", 《BIOSENSORS AND BIOELECTRONICS》 * |
| BENTON ET AL: "The utilization of a Saccharomyces cerevisiae HUG1P-GFP promoter–reporter construct for the selective detection of DNA damage", 《MUTATION RESEARCH》 * |
| SYMINGTON ET AL.: "Alteration of gene conversion tract length and associated crossing over during plasmid gap repair in nuclease-deficient strains of Saccharomyces cerevisiae", 《NUCLEIC ACIDS RESEACH》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103510161A (en) * | 2013-08-16 | 2014-01-15 | 中国科学院水生生物研究所 | Saccharomyces single-gene deletion library containing genotoxic biosensors |
| CN103510161B (en) * | 2013-08-16 | 2016-08-10 | 中国科学院水生生物研究所 | Yeast single-gene deletion libraries containing genetoxic biosensor |
| CN112680498A (en) * | 2020-12-28 | 2021-04-20 | 华南理工大学 | High-throughput screening method for genotoxic substances |
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| Publication number | Publication date |
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| CN102965368B (en) | 2014-12-24 |
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