DNA damage inducible promoter and application thereof

Abstract

The invention discloses a DNA damage inducible promoter and application thereof. The promoter is combined with a cracking gene SRRz and a terminator rrnB to form a recombinant vector, and the recombinant vector is transferred into escherichia coli to obtain an escherichia coli detection strain. The detection strain is applied to detection of genotoxic substances, and compared with an original SOS / umu system, the system is more sensitive and convenient. Compared with a natural umuDC promoter, the promoter provided by the invention has lower detection limit and higher responsivity, and integrally shows higher sensitivity. By combining the characteristics of the detection system, the genetic toxicity can be more intuitively detected, and under the action of genetic toxicity substances with the same concentration, the promoter induces the expression of more SRRz cleavage proteins compared with a natural umuDC promoter, so that the cleavage degree of thalli is larger, the survival rate of the thalli is lower, and the bacterial liquid is clearer and more transparent from the appearance. The system can provide technical support for genetic toxicity preliminary screening of samples such as water and food.

Description

Technical Field

[0001] The present invention relates to the technical field of high-throughput screening of genotoxic substances, in particular to a DNA damage-inducible promoter and application thereof, and specifically to detecting genotoxic substances using recombinant Escherichia coli carrying a modified promoter. Background Art

[0002] Whole-cell biosensors have received increasing attention in recent years due to the rise of synthetic biology. Sensors based on bacterial cells have been studied for various applications, such as environmental monitoring, disease diagnosis and biotherapy, bioproduction, mineral measurement, and mine clearance. However, these performances are insufficient to meet real-world detection requirements, especially in terms of low sensitivity and limit of detection (LOD), and limited input / output dynamic range.

[0003] The SOS / umu test is a detection method that uses whole-cell biosensors and has been continuously developed over the past four decades. The original umu::lacZ system uses the ability of the SOS response to induce the expression of downstream genes to detect genetic toxicity by fusing the umuDC promoter with the lacZ reporter gene. After that, the sensitivity was improved by changing the reporter gene, such as GFP, adding exogenous metabolic enzyme genes to increase the response range of chemicals, knocking out host cell genes to increase sensitivity, and changing the host cell to improve its detection performance. However, the SOS promoter used is generally a natural SOS promoter, which is a promoter obtained from the SOS system of one bacterium and replaced in another bacterium. The performance of these natural promoters has certain limitations in terms of sensitivity, detection limit, output range, etc.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide a DNA damage-inducible promoter and its application. The promoter is a highly sensitive Escherichia coli DNA damage-induced specific promoter with higher sensitivity, lower detection limit and higher response intensity.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A DNA damage-inducible promoter, whose sequence is shown in SEQ ID No. 1 or its reverse complementary sequence.

[0008] An expression cassette comprising the above-mentioned DNA damage-inducible promoter.

[0009] Furthermore, the expression cassette also contains a target gene and a terminator.

[0010] The target gene is a lytic gene or a toxic protein gene that affects the growth of bacteria (preferably Escherichia coli) strains, but is not limited thereto.

[0011] Furthermore, the lytic gene is a bacteriophage lytic gene, etc.;

[0012] Furthermore, the phage lysis gene can be any phage lysis gene, preferably the lysis gene SRRz of lambda phage, whose gene sequence is SEQ ID No.4.

[0013] The terminator is an E. coli terminator, further comprising an rrnB T1 terminator and an rrnB T2 terminator, etc.;

[0014] Furthermore, the nucleotide sequence of the rrnB T1 terminator is shown as SEQ ID No.5; the nucleotide sequence of the rrnBT2 terminator is shown as SEQ ID No.6.

[0015] A recombinant vector comprising the above-mentioned DNA damage-inducible promoter or the above-mentioned expression cassette;

[0016] The starting vector of the recombinant vector can be any E. coli vector, such as pUC series vectors (such as pUC18, pUC19), pET series vectors (such as pET30a(+)), pRSF series vectors, etc.;

[0017] Furthermore, in the embodiment of the present invention, pUC18 was used as a starting vector to construct a recombinant vector pUC-ATA-SR.

[0018] A recombinant strain contains the DNA damage-inducible promoter or the expression box or the recombinant vector.

[0019] Furthermore, the recombinant strain is obtained by integrating the above expression cassette into a host bacterium.

[0020] Furthermore, the recombinant strain is obtained by transforming the above recombinant vector into a host bacterium.

[0021] Furthermore, the host bacteria is Escherichia coli; furthermore, it is Escherichia coli containing T7 RNA polymerase; furthermore, it is Escherichia coli BL21 (DE3) or E. coli BL21 (DE3)PLysS, etc.

[0022] In the embodiment of the present invention, E. coli BL21 (DE3) is used as a host bacterium to construct a recombinant E. coli BL21 (DE3) / pUC-ATA-SR, which can be used for the detection of genotoxic substances.

[0023] Application of the above DNA damage inducible promoter, expression cassette, recombinant vector or recombinant strain in detecting genetic toxic substances.

[0024] Furthermore, the above-mentioned DNA damage-inducible promoter, expression cassette, recombinant vector or recombinant strain is used in high-throughput detection of genotoxic substances.

[0025] A high-throughput detection method for genotoxic substances comprises the following steps:

[0026] The recombinant strain detection solution was prepared using the above recombinant strain, and the recombinant strain detection solution was incubated with the sample to be tested in a microplate; at the same time, the recombinant strain detection solution with the same volume of pure solvent was set as a negative control, and the absorbance OD was measured every hour. 600 , the bacterial survival rate is calculated according to the absorbance to achieve high-throughput detection.

[0027] The sample to be tested is a genetic toxic substance, including but not limited to at least one of cumene hydroperoxide (CHP), nalidixic acid (NAL), 5-fluorouracil (5-FU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and dimethyl sulfate (DMS).

[0028] Furthermore, the microplate can be selected from a 96-well plate, a 48-well plate, a 24-well plate, etc. A 96-well plate is used in the embodiment of the present invention.

[0029] Furthermore, the incubation time is 4±1h.

[0030] Compared with the prior art, the present invention has the following advantages and effects:

[0031] The DNA damage inducible promoter of the present invention is a SOS inducible promoter specific to Escherichia coli (such as E.coli BL21 (DE3)). The promoter is newly constructed by using the binding site (SOS box) of the repressor protein LexA of the T7 promoter and the SOS inducible promoter. The newly constructed promoter ATA is combined with the lysis gene SRRz and the terminator rrnB to form a recombinant vector, and the recombinant vector is transferred into Escherichia coli to obtain an Escherichia coli detection strain. The detection strain is applied to the detection of genotoxic substances, 5 direct genotoxic substances are detected and the effect of the promoter is evaluated. Compared with the original SOS / umu system, this system is more sensitive and convenient. In this system comparison, this promoter has a lower detection limit and a higher responsiveness than the natural umuDC promoter, and shows a higher sensitivity as a whole. Combined with the characteristics of this detection system, genetic toxicity can be detected more intuitively. Under the same concentration of genetic toxic substances, this promoter induces more SRRz lysis protein expression than the natural umuDC promoter, resulting in a greater degree of bacterial lysis and a lower bacterial survival rate. From the appearance point of view, the bacterial solution is clearer and more transparent. This system can provide technical support for the initial screening of genetic toxicity of water, food and other samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a schematic diagram of the structure of the DNA damage-inducible promoter ATA in Example 1 and a schematic diagram of the structure of the genotoxicity detection vector.

[0033] Figure 2 This is a graph showing the change in relative bacterial survival rate over time when using the detection strain to detect chemicals at specific concentrations in Example 2.

[0034] Figure 3 This is the dose-effect curve of the detection of the chemical by the detection strain in Example 2. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0036] The experimental methods in the following examples without specific experimental conditions are usually carried out under conventional experimental conditions or the experimental conditions recommended by the manufacturer. The materials and reagents used are commercially available unless otherwise specified.

[0037] Example 1: DNA damage inducible promoter P ATA Design and construction of genotoxicity detection vectors and strains

[0038] The T7 promoter (the gene sequence is SEQ ID No. 2) is used as the basic design, and a LexA protein binding site is added (the preferred gene sequence is SEQ ID No. 3), and finally the promoter ATA is obtained, which is denoted as P ATA (The gene sequence is SEQ ID No. 1).

[0039] The LexA protein binding site, i.e., the SOS box, is a repressor protein induced by the SOS signal. When DNA damage activates the SOS signal, the LexA protein will be cleaved to remove the repressive effect, ultimately initiating the expression of downstream genes. An SOS box sequence was added upstream and downstream of the T7 promoter to enhance the repressive effect of the LexA protein on the T7 promoter. Finally, an ATA promoter with a length of 57 bp was obtained.

[0040] By using homologous recombination, SRRz (sequence is SEQ ID No.4), rrnB T1 terminator (sequence is SEQ ID No.5), rrnB T2 terminator (sequence is SEQ ID No.6) and umuDC promoter (sequence is SEQID No.7) or ATA promoter (each element is connected in the order of umuDC promoter or ATA promoter-SRRz-rrnB T1terminator-rrnB T2 terminator) are integrated into the multiple cloning site of pUC18 plasmid. The recombinant vector containing umuDC promoter is denoted as pUC-USR, and the recombinant vector containing ATA promoter is denoted as pUC-ATA-SR. The difference between the two lies only in the promoter sequence. The structural schematic diagram of DNA damage inducible promoter ATA and the structural schematic diagram of genotoxicity detection vector are shown in FIG. Figure 1 shown.

[0041] The vectors pUC-USR and pUC-ATA-SR were respectively transferred into E. coli BL21 (DE3) competent cells to obtain recombinant E. coli BL21 (DE3) / pUC-USR or E. coli BL21 (DE3) / pUC-ATA-SR for detecting genotoxic substances.

[0042] Example 2: Detection of five genotoxic substances

[0043] In this example, 5 kinds of direct genotoxic substances were tested for genotoxicity, and the relative bacterial survival rate was used as a standard indicator. The genotoxicity degree of the corresponding genotoxic substances can be reflected in the size of the bacterial survival rate.

[0044] Calculation of relative survival rate:

[0045] After the E. coli detection solution contacts the sample to be tested, the light absorption value (OD) of the bacterial solution at 600 nm is measured at a specific time point.

[0046] Relative bacterial survival rate (%) = OD of the sample well to be tested 600 / Pure solvent pore OD 600 *100%.

[0047] The genotoxicity test method is as follows:

[0048] 1. Resuscitation and activation of recombinant E. coli

[0049] Take out the recombinant E. coli BL21 (DE3) / pUC-USR or E. coli BL21 (DE3) / pUC-ATA-SR from the -80 ° C refrigerator, streak it on the LB plate culture medium, and resume culture at 37 ° C for 16 hours. Pick a single colony and inoculate it into LB culture medium. Culture it at 37 ° C and 220 rpm for 12 to 16 hours to obtain a saturated bacterial solution.

[0050] 2. Preparation of E. coli detection solution

[0051] The saturated bacterial solution was inoculated into fresh LB medium at a volume ratio of 1:50-1:100 and cultured at 37°C and 220 rpm until OD 600 to 0.2-0.4, add IPTG to a final concentration of 0.5 mM, take 198 μL and add it to each well of a 96-well plate to obtain an E. coli detection solution.

[0052] 3. Contact with the chemical to be tested

[0053] A solution containing the chemicals to be tested (CHP, NAL, 5-FU, MNNG, DMS) with a certain concentration gradient was prepared using DMSO as a solvent as the test sample. The concentration gradient of CHP in the solution containing CHP was 8, 4, 2.5, 2, 1.5, 1, 0.75, 0.5, 0.25, 0.125, 0.1, 0.0625, 0.05, 0.0125, 0.01 mg / mL, and the concentration gradient of NAL in the solution containing NAL was 10, 5, 2.5, 1.92, 1.25, 0.96, 0.625, 0.48, 0.3125, 0.25, 0.15625, 0.12, 0.078125, 0.06, 0.03, 0.015625, 0.015, 0.003 mg / mL. g / mL, the concentration gradient of 5-FU in the solution containing 5-FU was 40, 20, 10, 8, 6, 4, 2, 1, and 0.2 mg / mL, the concentration gradient of MNNG in the solution containing MNNG was 2, 1.5, 1, 0.5, 0.25, 0.125, 0.03125, 0.015625, and 0.003125 mg / mL, and the concentration gradient of DMS in the solution containing DMS was 271.6, 128, 64, 32, 27.16, 16, 10, 8, 5.9, 4, 3.5, 2, 1.3, 1, 0.35, and 0.25 mg / mL.

[0054] Add 2 μL of the sample to be tested into the above 96-well plate and set up 3 parallel groups. At the same time, add the same volume of pure solvent (dimethyl sulfoxide) as a negative control and measure the OD per hour. 600 .

[0055] 4. Calculation of relative survival rate, dose-effect curve and detection limit

[0056] Calculate the relative bacterial survival rate test results as follows Figure 2 Shown (one of the concentrations is shown).

[0057] The dose-effect curve at 4 hours was drawn based on the relative bacterial survival rate and the detection limit was calculated. Figure 3 and Table 1.

[0058] Table 1 Detection limits of five chemicals

[0059]

[0060] Note: Ref.TA1535 / pSK1002 is the reference strain Salmonella typhimurium TA1535 / pSK1002 (commonly available in the market), which can be purchased from Huizhi Heyuan Biotechnology (Suzhou) Co., Ltd.

[0061] [1]: Nakamura SI, Oda Y, Shimada T, et al. SOS-inducing activity ofchemical carci nogens and mutagens in Salmonella typhimurium TA1535 / pSK1002:examination with 151chemicals.[J].Mutation Research Letters,1987,192(4):239-246.DOI:10.1016 / 0165-7992(87)90063-7.

[0062] In the test results of this embodiment, the results of the recombinant E. coli BL21 (DE3) / pUC-USR or E. coli BL21 (DE3) / pUC-ATA-SR on 5 chemicals are as follows: Figure 3 As shown, the dose-effect curves correspond to:

[0063] (1)E.coli BL21(DE3) / pUC-USR:

[0064] CHP:

[0065] R 2 =0.9442(-0.2041≤x≤1.903)

[0066] NAL:

[0067] R 2 =0.9625(-0.1072≤x≤2.000)

[0068] 5-FU:

[0069] R 2 =0.9814(0.3010≤x≤2.602)

[0070] MNNG:

[0071] R 2 =0.9671(-0.8062≤x≤1.301)

[0072] DMS:

[0073] R 2 =0.8481(1.102≤x≤3.433)

[0074] (2)E.coli BL21(DE3) / pUC-ATA-SR:

[0075] CHP:

[0076] R 2 =0.9819(-0.3010≤x≤1.602)

[0077] NAL:

[0078] R 2 =0.9809(-0.8239≤x≤1.283)

[0079] 5-FU:

[0080] R 2 =0.9116(0.3010≤x≤2.602)

[0081] MNNG:

[0082] R 2 =0.9816(-0.8062≤x≤1.301)

[0083] DMS:

[0084] R 2 =0.9524(1.000≤x≤3.107)

[0085] Wherein, X represents the logarithm of the concentration of the genotoxic substance with base 10, Y represents the relative bacterial survival rate (%), and R represents the curve fitting correlation coefficient.

[0086] Figure 2 and Figure 3 The results in Table 1 show that the promoter ATA designed by the present invention has a higher responsiveness than the wild-type promoter umuDC under the same conditions, which is manifested in that at the same concentration of the same chemical, the recombinant strain E. coli BL21 (DE3) / pUC-ATA-SR has a higher degree of lysis, that is, a lower relative bacterial survival rate; it has a lower detection limit, and the five chemicals detected all show a lower detection limit, ranging from 2 times to several dozen times. Compared with the strain Salmonella typhimurium TA1535 / pSK1002 (commonly available on the market) constructed by Oda, the founder of SOS / umu, its detection limit is also equivalent to or better than that.

[0087] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A DNA damage-inducible promoter, characterized in that: Its sequence is shown as SEQ ID No. 1 or its reverse complementary sequence.

2. A biomaterial related to a DNA damage inducible promoter, characterized in that: Any one or more combinations of the following biological materials: (1) An expression cassette containing the DNA damage-inducible promoter according to claim 1; (2) A recombinant vector containing the DNA damage-inducible promoter according to claim 1; (3) a recombinant vector containing the expression cassette described in (1); (4) A recombinant strain containing the DNA damage-inducible promoter according to claim 1; (5) a recombinant strain containing the expression cassette described in (1); (6) A recombinant strain containing the recombinant vector described in (2) or (3).

3. The biomaterial according to claim 2, characterized in that: The expression cassette described in (1) further contains a target gene and a terminator.

4. The biomaterial according to claim 3, characterized in that: The target gene is a lytic gene or a toxic protein gene that affects the growth of bacterial strains; The terminator is an Escherichia coli terminator.

5. The biomaterial according to claim 4, characterized in that: The lysis gene is a bacteriophage lysis gene; The terminators are rrnB T1 terminator and rrnB T2 terminator.

6. The biomaterial according to any one of claims 2 to 5, characterized in that: The starting vector of the recombinant vector described in (2) and (3) is an Escherichia coli vector; The host bacteria corresponding to the recombinant strains described in (4), (5) and (6) is Escherichia coli.

7. The biomaterial according to claim 6, characterized in that: The starting vector of the recombinant vector described in (2) and (3) is a pUC series vector, a pET series vector or a pRSF series vector; The host bacteria corresponding to the recombinant strains described in (4), (5) and (6) are Escherichia coli containing T7 RNA polymerase.

8. Use of the DNA damage inducible promoter according to claim 1 or the biomaterial according to any one of claims 2 to 7, characterized in that: Application in the detection of genotoxic substances; further, application in high-throughput detection of genotoxic substances.

9. A high-throughput detection method for genotoxic substances, characterized in that: The steps include: A recombinant strain detection liquid is prepared using the recombinant strain in the biological material according to any one of claims 2 to 7, and the recombinant strain detection liquid is incubated with the sample to be tested in a microplate; at the same time, a recombinant strain detection liquid with the same volume of pure solvent added is set as a negative control, and the absorbance OD per hour is measured. 600 , the bacterial survival rate is calculated according to the absorbance to achieve high-throughput detection.

10. The high-throughput detection method for genotoxic substances according to claim 9, characterized in that: The sample to be tested is a genotoxic substance, including but not limited to at least one of cumene hydroperoxide, nalidixic acid, 5-fluorouracil, N-methyl-N'-nitro-N-nitrosoguanidine and dimethyl sulfate.

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