CN106906209A - A kind of DNA damage detects response element and its application - Google Patents

A kind of DNA damage detects response element and its application Download PDF

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Publication number
CN106906209A
CN106906209A CN201710138812.6A CN201710138812A CN106906209A CN 106906209 A CN106906209 A CN 106906209A CN 201710138812 A CN201710138812 A CN 201710138812A CN 106906209 A CN106906209 A CN 106906209A
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coli
detection
genotoxic
application
dna damage
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卓敏
李爽
袁鹏飞
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South China University of Technology SCUT
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract

The invention discloses a kind of DNA damage detection response element and its application, its gene order is as shown in SEQ ID 1.Application of the described DNA damage detection response element in terms of Genotoxic quick detection, it is that the element is inserted into coli expression carrier, and the recombinant vector is transferred to Escherichia coli, recombination bacillus coli is obtained, then the recombination bacillus coli is applied to Genotoxic quick detection.Detection method operation is convenient, it is time-consuming it is short, disturbed without additionally addition chemical reagent, not by pigment, it is easy to accomplish high flux sample detection.

Description

A kind of DNA damage detects response element and its application
Technical field
The present invention relates to the technical field to the Genotoxic detection in environment, specially using reporter gene Recombination bacillus coli detection environment in Genotoxic.
Background technology
In recent years, genetoxic pollutant is distributed more widely in China's environment, and species and quantity are larger, in environmental contaminants In have certain representativeness, it is larger to harm.Factor in food, medicine, environment etc. cause to DNA damage, chromosome The cancerations such as distortion are more and more, and the detection to these stress factors is also increasingly necessary.DNA damage typically passes through genetoxic Testing inspection, with the fast development of Protocols in Molecular Biology, various genotoxicity testing methods are also constantly being improved.
Genetoxic detection refer to for detect can directly or indirectly inducing cell development genetics damage experiment, and The damage of DNA is one of link of malignant progression process.Some short-term quickly external genetoxics are established in recent years Test method detects the damage of DNA.The genetoxic short term detection method set up at present is more than 200 kinds.Wherein research and There are Sister chromatid exohange (sister chromatid exchange, SCE), program to synthesize for NDA using more method (unscheduled DNA synthesis), comet (coment assay), also known as single cell gel electrophoresis test (single cell gel electrophoresis), ames test (reverse mutation Test of salmonella typhi munine, also known as Salmonella reversion test), SOS colour tests, Prophage induction experiment side Method etc..Generally there is these detection methods sterile working that trivial operations, detection time are long, needs are strict etc. to be unfavorable for what is promoted Factor.And due to reasons such as patent protections, indivedual detection methods are simultaneously unfavorable for Spread in China, limit it and widely use.
The content of the invention
The present invention be directed to the deficiencies in the prior art, there is provided a kind of gene that can cause DNA damage in quick response environment Element, and the element is applied to the quick detection of environment Genotoxic.Detection method operation is convenient, take short, nothing Chemical reagent need to additionally be added, do not disturbed by pigment, it is easy to accomplish high flux sample detection.
Technical scheme is as follows：
A kind of DNA damage detects response element, and its gene order is as shown in SEQ ID 1.
Application of the described DNA damage detection response element in terms of Genotoxic quick detection.
The element is inserted into coli expression carrier, constructed dna damage check recombinant vector, and by the recombinant vector Escherichia coli are transferred to, recombination bacillus coli is obtained, then the recombination bacillus coli are applied to Genotoxic quick detection, Will recombinant bacterium and Genotoxic incubation, Bacillus coli cells cracking.
The recombinant vector, from 5 ' to 3 ' ends are followed successively by DNA damage detection response element, phage splitting gene and termination Son.
Described phage splitting gene, can be any one phage splitting gene, preferably lambda bacteriophages Lysis genes SRRz, its gene order is SEQ ID2.
Described Escherichia coli terminator can be any one Escherichia coli terminator, preferably T7 terminators.
The method of Genotoxic quick detection provided by the present invention, comprises the following steps：
(1) E. coli detection liquid is prepared using described recombination bacillus coli；
(2) testing sample is mixed with E. coli detection liquid, while adding the E. coli detection of same volume neat solvent Liquid is compared；Two groups of samples continue to cultivate 15-60min；
(3) the E. coli detection liquid that will mix with testing sample is measured respectively with the E. coli detection liquid of control group OD600；
(4) lysis efficiency is calculated, according to Genotoxic concentration in lysis efficiency standard curve calculating testing sample.
The preparation of the E. coli detection liquid：
(1) recombination bacillus coli reserve is cultivated with LB solid mediums, is made its recovery activation；
(2) the activation Escherichia coli single bacterium colony that will be obtained carries out concussion and cultivate to logarithm life in being linked into LB fluid nutrient mediums The long-term later stage；
(3) the saturation bacterium solution that will be obtained is with 1:100 volume ratios are inoculated into fresh LB culture mediums, culture to bacterium solution OD600 It is 0.15-0.25, obtains E. coli detection liquid.
The coli expression carrier can be any one escherichia coli vector, such as pBluescript, pUC18, PUC19, pET serial carrier (such as pET30a).It is the carrier that sets out with pUC18, the Escherichia coli cracking carrier of structure is pRST.
Described Escherichia coli, are E.coli BL21, E.coli DH5a, E.coli XL1-blue or E.coli HB101。
Described Genotoxic can be Cr6+, methyl mesylate (MMS) and 4- nitroquinoline 1- oxides (4- NQO)。
Recombination bacillus coli containing Escherichia coli Genotoxic response carrier falls within protection of the invention Scope.
Compared with prior art, beneficial effects of the present invention are as follows：
(1) operation object is Escherichia coli, easy to operation without risk of causing a disease.
(2) test period is short, be can detect in 2h and completed.
(3) detection process need not add additional agents (such as zymolyte), with low cost.
(4) detection sensitivity is high, to Cr6+, 4-NQO, MMS sensitive concentration scope be respectively in 0.01-0.1,1-5,40- It is sensitive under the conditions of 100mg/L.
Brief description of the drawings
Fig. 1 vector construction schematic diagrames.
Fig. 2 recombination bacillus colis (E.coli BL21/pRST) are to Cr6+(a), 4-NQO (b), MMS (c) difference genetoxics The cracking response diagram of material.
Specific embodiment
The present invention is described in further detail with reference to specific embodiment.
Embodiment 1
Genetoxic responds the structure of carrier
It is the carrier that sets out with pUC18, builds genetoxic response carrier pRST.Specific construction method is as follows：
1. synthetic DNA damage response element Prec(Seq ID 1) and T7 terminator sequences, respectively in PrecWith T7 terminators Upstream addition EcoRI and XbaI sites, downstream addition SpeI and PstI restriction enzyme sites.
2., with lambda phage genomes DNA as template, SRRz genes are expanded.Also distinguish in the upstream and downstream of SRRz genes Addition EcoRI and XbaI, SpeI and PstI restriction enzyme sites.
3. using biological building blocks (Biobricks) method, using digestion, connection method by each element according to promoter-split Solution gene-terminator is linked in sequence, and is inserted into pUC18 carriers, obtains genetoxic response carrier pRST.
4. carrier pRST is transferred to E.coli BL21 competent cells, obtains the restructuring of Genotoxic detection E. coli BL21/pRST.
Embodiment 2
The calculating of lysis efficiency
20min after E. coli detection liquid contact measured sample, determines light absorbs of each test bacterium solution at 600nm respectively Value (OD600)。
Cleavage rate (%)=(A-B)/A*100%
Wherein, the bacterium solution OD of A-addition methyl alcohol600
The bacterium solution OD of B-addition test specimens600。
Embodiment 3
Genotoxic is detected
1. the recovery activation of recombination bacillus coli
During recombination bacillus coli to line from -80 ° of refrigerators LB plating mediums, the renewal cultivation under the conditions of 37 ° 14h.Picking single bacterium colony, is seeded in LB culture mediums, and 12-16h is cultivated under the conditions of 37 °, 250rpm.
2. prepared by E. coli detection liquid
By the bacterium solution of activation of recovering with 1:100 volume ratios are seeded to fresh LB, are added to 190 μ l volumes In 96 well culture plates, cultivated at 35-38 DEG C, under the conditions of 600-1000rpm to OD600To 0.15-0.2 (used time about 1-1.5h).
3. contacted with given the test agent
The μ l of given the test agent 10 are added in 96 orifice plates, continue to cultivate 20 minutes at 35-38 DEG C, under the conditions of 800rpm, if Put 3 parallel groups.The neat solvent (methyl alcohol) of addition same volume, and addition saturated concentration (Cr are set simultaneously6+, 4-NQO, MMS Saturated concentration is respectively 0.1,100,25mg/L) to wild type E.coli BL21 bacterial strains as negative control.
4. test result indicate that, under the conditions of the saturated concentration, without genetoxic respond carrier wild type E.coli BL21 strain growths are normal, and cellular lysate does not occur.Testing result in the present embodiment is as shown in Fig. 2 each Genotoxic is examined The standard curve of survey is corresponded to：
Cr6+, Y=3.685+601.6X, R2=0.99 (0.01<X<0.1)
4-NQO, Y=-4.532+12.58X, R2=0.99 (1<X<5)
MMS, Y=-31.14+0.9355X, R2=0.97 (40<X<100)
Wherein, X represents the concentration (mg/L) of Genotoxic, and Y represents cellular lysate efficiency (%), R2Represent that curve is intended Close coefficient correlation.
The result shows, in the range of detectable concentration, sample concentration is linear to cellular lysate efficiency related.Therefore, may be used Whether sample is detected containing Genotoxic with bacterial strain E.coli BL21/pRST.
Embodiment 4
Different water sample genetoxic detections
The present embodiment is intended carrying out Genotoxic detection for the water sample of separate sources, with Cr6+It is standard toxicant, The Genotoxic concentration of corresponding detection sample, Cr is scaled according to lysis efficiency6+Equivalent concentration.
1. E.coli BL21/pRST detection bacterium solutions are prepared according to the step 1-2 of embodiment 3.
2. water sample to be measured is taken, with the resin adsorption that the speed of 40mL/min is activated, then ethyl acetate wash-out.Eluent Using centrifugation freeze drying process removal ethyl acetate, then with the distilled water sample dissolution of certain volume to required volume.By water to be measured Sample mixes with E. coli detection liquid, while the E. coli detection liquid for adding same volume neat solvent is compareed；Two groups of samples are equal Continue to cultivate 20min.The water sample to be measured take respectively daily urban water (releasing of laboratory tap), urban water plant water source, Certain chemical plant wastewater and Zhujiang River water inlet water sample B.
3. the E. coli detection liquid that will mix with water sample to be measured is measured respectively with the E. coli detection liquid of control group OD600。
4. lysis efficiency is calculated, according to Genotoxic Cr in lysis efficiency standard curve calculating testing sample6+Equivalent Concentration.Acquired results are as shown in table 1.
The result of table 1 shows, the Genotoxic Cr at daily urban water and urban water plant water source6+Equivalent concentration be less than 2.5ng/L, and chemical plant wastewater water sample Cr6+Equivalent concentration be about 9.3ng/L, Zhujiang River water inlet water sample Cr6+Equivalent it is dense Degree is about 4.8ng/L.
Above-mentioned testing result shows, includes the genetic engineering of Genotoxic quick response element of the present invention Bacterium, detects, finally with Cr in response concentration range to determinand6+Equivalent concentration represents that the genetoxic of testing sample contains Amount.The method is without pathogenic risk, and simple to operate, detection sensitivity is high, time-consuming short, easy to spread.
SEQUENCE LISTING
<110>South China Science ＆ Engineering University
<120>A kind of DNA damage detects response element and its application
<130> 1
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> 1
<400> 1
gctacttgat actgtatgag catacagtat aattg 35
<210> 2
<211> 1320
<212> DNA
<213> Artificial Sequence
<220>
<223> 1
<400> 2
gaccttattc ctaattaaat agagcaaatc cccttattgg gggtaagaca tgaagatgcc 60
agaaaaacat gacctgttgg ccgccattct cgcggcaaag gaacaaggca tcggggcaat 120
ccttgcgttt gcaatggcgt accttcgcgg cagatataat ggcggtgcgt ttacaaaaac 180
agtaatcgac gcaacgatgt gcgccattat cgcctggttc attcgtgacc ttctcgactt 240
cgccggacta agtagcaatc tcgcttatat aacgagcgtg tttatcggct acatcggtac 300
tgactcgatt ggttcgctta tcaaacgctt cgctgctaaa aaagccggag tagaagatgg 360
tagaaatcaa taatcaacgt aaggcgttcc tcgatatgct ggcgtggtcg gagggaactg 420
ataacggacg tcagaaaacc agaaatcatg gttatgacgt cattgtaggc ggagagctat 480
ttactgatta ctccgatcac cctcgcaaac ttgtcacgct aaacccaaaa ctcaaatcaa 540
caggcgccgg acgctaccag cttctttccc gttggtggga tgcctaccgc aagcagcttg 600
gcctgaaaga cttctctccg aaaagtcagg acgctgtggc attgcagcag attaaggagc 660
gtggcgcttt acctatgatt gatcgtggtg atatccgtca ggcaatcgac cgttgcagca 720
atatctgggc ttcactgccg ggcgctggtt atggtcagtt cgagcataag gctgacagcc 780
tgattgcaaa attcaaagaa gcgggcggaa cggtcagaga gattgatgta tgagcagagt 840
caccgcgatt atctccgctc tggttatctg catcatcgtc tgcctgtcat gggctgttaa 900
tcattaccgt gataacgcca ttacctacaa agcccagcgc gacaaaaatg ccagagaact 960
gaagctggcg aacgcggcaa ttactgacat gcagatgcgt cagcgtgatg ttgctgcgct 1020
cgatgcaaaa tacacgaagg agttagctga tgctaaagct gaaaatgatg ctctgcgtga 1080
tgatgttgcc gctggtcgtc gtcggttgca catcaaagca gtctgtcagt cagtgcgtga 1140
agccaccacc gcctccggcg tggataatgc agcctccccc cgactggcag acaccgctga 1200
acgggattat ttcaccctca gagagaggct gatcactatg caaaaacaac tggaaggaac 1260
ccagaagtat attaatgagc agtgcagata gagttgccca tatcgatggg caactcatgc 1320

Claims (10)

1. a kind of DNA damage detects response element, it is characterised in that its gene order is as shown in SEQ ID 1.
2. application of the DNA damage detection response element described in claim 1 in terms of Genotoxic quick detection.
3. application according to claim 1, it is characterised in that the element is inserted into coli expression carrier, and will The recombinant vector is transferred to Escherichia coli, obtains recombination bacillus coli, then the recombination bacillus coli is applied into genetoxic thing Matter quick detection.
4. application according to claim 3, it is characterised in that the recombinant vector, from 5 ' to 3 ' ends are followed successively by DNA damage Detection response element, phage splitting gene and terminator.
5. application according to claim 4, it is characterised in that the phage splitting gene is base sequence such as SEQ ID Shown in 2；Described terminator is T7 terminator sequences.
6. the application described in claim 3~5 any one, it is characterised in that comprise the following steps：
(1) E. coli detection liquid is prepared using described recombination bacillus coli；
(2) testing sample is mixed with E. coli detection liquid, while the E. coli detection liquid for adding same volume neat solvent is made Control；Two groups of samples continue to cultivate 10-60min；
(3) the E. coli detection liquid that will mix with testing sample measures OD respectively with the E. coli detection liquid of control group600；
(4) lysis efficiency is calculated, according to Genotoxic concentration in lysis efficiency standard curve calculating testing sample.
7. application according to claim 6, it is characterised in that the preparation of the E. coli detection liquid：
(1) recombination bacillus coli reserve is cultivated with LB solid mediums, is made its recovery activation；
(2) the activation Escherichia coli single bacterium colony that will be obtained carries out concussion and cultivate to exponential phase in being linked into LB fluid nutrient mediums Later stage；
(3) the saturation bacterium solution that will be obtained is with 1:100 volume ratios are inoculated into fresh LB culture mediums, culture to bacterium solution OD600For 0.15-0.25, obtains E. coli detection liquid.
8. the application according to claim 3~5 any one, it is characterised in that the coli expression carrier is PUC18, pUC19, pBluscript or pET30a；Described Escherichia coli, are E.coliBL21, E.coli DH5a, E.coli XL1-blue or E.coli HB101.
9. the application according to claim 2~6 any one, it is characterised in that described Genotoxic includes Cr6 +, 4- nitroquinoline 1- oxides (4-NQO), methyl mesylate (MMS).
10. the recombination bacillus coli described in the application of claim 3~5 any one.
CN201710138812.6A 2017-03-09 2017-03-09 A kind of DNA damage detects response element and its application Pending CN106906209A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018133513A1 (en) * 2017-01-18 2018-07-26 华南理工大学 Genotoxic substance detection vector and detection method thereof
CN110873790A (en) * 2018-09-03 2020-03-10 华南理工大学 Whole-cell biosensor for detecting heavy metal ions in water-soluble sample and construction and application thereof
CN112680498A (en) * 2020-12-28 2021-04-20 华南理工大学 High-throughput screening method for genotoxic substances

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20040026253A (en) * 2002-09-23 2004-03-31 광주과학기술원 Recombinant bioluminescent/fluorescent bacteria for the detection oxidative damage and DNA damage simultaneously
CN1880461A (en) * 2006-04-28 2006-12-20 清华大学 Escherichia coli self-cracking method and its dedicated carrier and application
CN102605036A (en) * 2012-04-01 2012-07-25 清华大学 In-situ detection method for genetic toxicity of contaminated soil

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20040026253A (en) * 2002-09-23 2004-03-31 광주과학기술원 Recombinant bioluminescent/fluorescent bacteria for the detection oxidative damage and DNA damage simultaneously
CN1880461A (en) * 2006-04-28 2006-12-20 清华大学 Escherichia coli self-cracking method and its dedicated carrier and application
CN102605036A (en) * 2012-04-01 2012-07-25 清华大学 In-situ detection method for genetic toxicity of contaminated soil

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018133513A1 (en) * 2017-01-18 2018-07-26 华南理工大学 Genotoxic substance detection vector and detection method thereof
CN110873790A (en) * 2018-09-03 2020-03-10 华南理工大学 Whole-cell biosensor for detecting heavy metal ions in water-soluble sample and construction and application thereof
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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Inventor after: Zhuo Min

Inventor after: Yuan Pengfei

Inventor after: Li Shuang

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Inventor before: Yuan Pengfei

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Application publication date: 20170630