Bacterium for detecting genotoxic substances and application thereof

Abstract

The invention provides a high-sensitivity bacterium for genotoxic substance detection and application thereof, the bacterium comprises a recombinant vector, and the recombinant vector comprises a sulA promoter or a mutant thereof. The bacterium constructed by the invention shows a huge application prospect in the aspect of detecting genetic toxic substances in the environment, and has the advantages of high specificity, quick response, low cost, simplicity and convenience in operation and the like.

Description

Technical Field

[0001] The present application belongs to the field of bioengineering technology, and specifically relates to bacteria for detecting genotoxic substances and applications thereof. Background Art

[0002] Genotoxic substances are chemicals or physical agents that can directly or indirectly cause genetic damage. These damages may result in DNA strand breaks, base modifications, chromosomal aberrations, and DNA replication errors. Exposure to genotoxic substances may cause cell death, mutations, and even cancer. Therefore, the research and assessment of genotoxic substances is of great significance in the fields of environmental protection, occupational safety, and public health.

[0003] Genotoxic substances are widely present in the environment, including water, soil, and pesticides, and may enter the human body directly or indirectly through various pathways, increasing the risk of diseases such as cancer. Genotoxic substances in the natural environment have attracted considerable attention due to their potential health hazards. If reproductive cells are exposed to genotoxic substances, the resulting genetic mutations may be passed on to future generations, even if these offspring have not been directly exposed to the genotoxic agent.

[0004] Currently, the International Organization for Standardization has established the SOS / umu test as a standard method specifically for detecting environmental genotoxicity. However, a standardized testing system has not yet been established for this method when addressing emerging pollutants. In addition, the chassis cells relied on by the SOS / umu test method are conditionally pathogenic Salmonella typhimurium, and access to its recombinant bacterial system is relatively difficult, making it difficult to widely promote in the domestic industry. Therefore, the development of a new method that is efficient, convenient, and suitable for screening genotoxic substances is particularly urgent. Summary of the Invention

[0005] In response to the problems existing in the prior art, the purpose of this application is to provide a highly sensitive bacterium for detecting genotoxic substances in the environment and its application.

[0006] Specifically, this application involves the following aspects:

[0007] 1. Application of the sulA promoter or its mutants in the detection of genotoxic substances. Preferably, the genotoxic substances include any one or more of H2O2, MMC, MNNG, NA, CH2O, DEN, EMS, DMS, furazolidone, and 9-aminoacridine.

[0008] 2. The use according to item 1, wherein the nucleotide sequence of the sulA promoter is shown as SEQ ID NO: 1, or the nucleotide sequence of the sulA promoter mutant is shown as SEQ ID NO: 2.

[0009] 3. A sulA promoter mutant, the nucleotide sequence of which is shown in SEQ ID NO: 2.

[0010] 4. A recombinant vector comprising a sulA promoter or a mutant thereof.

[0011] 5. The recombinant vector according to item 4, wherein the nucleotide sequence of the sulA promoter is shown as SEQ ID NO: 1, or the nucleotide sequence of the sulA promoter mutant is shown as SEQ ID NO: 2.

[0012] 6. The recombinant vector according to item 4 or 5, wherein the recombinant vector comprises a reporter gene, and expression of the reporter gene is driven by the sulA promoter or a mutant thereof;

[0013] Preferably, the reporter gene is a fluorescent protein;

[0014] Further preferably, the fluorescent protein is green fluorescent protein, yellow fluorescent protein, red fluorescent protein or blue fluorescent protein.

[0015] 7. The recombinant vector according to any one of items 4 to 6, wherein the starting vector used to construct the recombinant vector is pBR322, ColE1, pSC101, p15A or R6K vector.

[0016] 8. A bacterium for detecting genotoxic substances, comprising the vector according to any one of items 4 to 7;

[0017] Preferably, the bacteria is Escherichia coli;

[0018] Further preferably, the genotoxic substance includes any one or more of H2O2, MMC, MNNG, NA, CH2O, DEN, EMS, DMS, furazolidone, and 9-aminoacridine.

[0019] 9. Use of the recombinant vector according to any one of items 4 to 7 or the bacterium according to item 8 in the preparation of a kit for detecting genotoxic substances.

[0020] 10. A kit for detecting genotoxic substances, comprising the recombinant vector according to any one of items 4 to 7 or the bacterium according to item 8.

[0021] 11. Use of the recombinant vector according to any one of items 4 to 7, the bacterium according to item 8, or the kit according to item 10 in the detection of genotoxic substances.

[0022] 12. A method for detecting a genotoxic substance, comprising:

[0023] mixing the bacteria described in item 8 with a sample to be tested to detect genotoxic substances in the sample;

[0024] Preferably, the genotoxic substance includes any one or more of H2O2, MMC, MNNG, NA, CH2O, DEN, EMS, DMS, furazolidone, and 9-aminoacridine.

[0025] Beneficial effects:

[0026] This application is based on the SOS stress repair principle, focuses on the screening of genotoxic substances, and successfully constructs a highly sensitive bacterium. The bacterium provides a more efficient and reliable solution for the detection of genotoxic substances in the environment, and is expected to show significant application potential in the field of drinking water safety assessment.

[0027] Among the bacteria, this application selected a highly safe model organism as the host strain, which has low laboratory grade requirements and will not cause harm to the human body. It has the advantages of high sensitivity, low cost, and short test cycle. It has great application prospects in the detection of genotoxic substances and provides a powerful platform for the detection of genotoxic substances in future environments.

[0028] In the bacteria, the present application uses the sulA promoter, which is highly sensitive to DNA damage. This promoter has the advantages of rapid response, high specificity, and convenient detection, enabling real-time monitoring of the degree of DNA damage. Furthermore, the present application also discovered that by mutating the sulA promoter, the sensitivity of detection can be significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The results are shown in Figure 5. PCR amplification results of sulA, recA, cda and umu promoter genes.

[0030] Figure 2 The results are those of PCR amplification of the backbone gene.

[0031] Figures 3A-3D These are the sequencing results of four biosensors. Figure 3A p15A-P sulA -sfGFP sequencing results; Figure 3B p15A-P recA -sfGFP sequencing results; Figure 3C p15A-P cda -sfGFP sequencing results; Figure 3D p15A-P umu -sfGFP sequencing results.

[0032] Figures 4A-4DResults of testing four biosensors for 0.5 mM H2O2. Figure 4A To test p15A-P sulA - Results of the sfGFP sensor; Figure 4B To test p15A-P recA - Results of the sfGFP sensor; Figure 4C To test p15A-P cda - Results of the sfGFP sensor; Figure 4D To test p15A-P umu - Results for the sfGFP sensor.

[0033] Figures 5A-5D Results of testing four biosensors for 50 μM MNNG. Figure 5A To test p15A-P sulA - Results of the sfGFP sensor; Figure 5B To test p15A-P recA - Results of the sfGFP sensor; Figure 5C To test p15A-P cda - Results of the sfGFP sensor; Figure 5D To test p15A-P umu - Results for the sfGFP sensor.

[0034] Figures 6A-6E To test the p15A-P of H2O2, MMC, MNNG, NA, CH2O genotoxic substances at different concentrations sulA -sfGFP sensor results. Among them, Figure 6A This is the result of the H2O2 test; Figure 6B The result of the MMC test; Figure 6C The results of the MNNG test; Figure 6D The result of the NA test; Figure 6E This is the result of the CH2O test.

[0035] Figures 7A-7E To test the p15A-P of H2O2, MMC, MNNG, NA, CH2O genotoxic substances at different times sulA -sfGFP sensor results. Among them, Figure 7A This is the result of the H2O2 test; Figure 7B The result of the MMC test; Figure 7C The results of the MNNG test; Figure 7D The result of the NA test; Figure 7E This is the result of the CH2O test.

[0036] Figures 8A-8ETo test the p15A-P of DEN, EMS, DMS, furazolidone and 9-aminoacridine genotoxic substances at different times sulA -sfGFP sensor results. Among them, Figure 8A The results of the DEN test; Figure 8B The results of the EMS test; Figure 8C The result of the DMS test; Figure 8D The results of the furazolidone test; Figure 8E This is the result of the 9-aminoacridine test.

[0037] Figures 9A-9J is the standard curve of ten genotoxic substances. Among them, Figure 9A is the standard curve of H2O2; Figure 9B is the standard curve of MMC; Figure 9C is the standard curve of MNNG; Figure 9D is the standard curve of NA; Figure 9E is the standard curve of CH2O; Figure 9F is the standard curve of DEN; Figure 9G is the standard curve of EMS; Figure 9H is the standard curve of DMS; Figure 9I is the standard curve of furazolidone; Figure 9J is the standard curve of 9-aminoacridine.

[0038] Figure 10 For mutant No. 55 and p15A-P sulA - Results of comparison with the sfGFP sensor (sulA).

[0039] Figure 11 This is the sequencing result of mutant strain 55.

[0040] Figures 12A-12J The results of testing mutant strain 55 with ten genotoxic substances at different concentrations are shown. Figure 12A This is the result of the H2O2 test; Figure 12B The result of the MMC test; Figure 12C The results of the MNNG test; Figure 12D The result of the NA test; Figure 12E is the result of CH2O test; Figure 12F The result of the DEN test; Figure 12G The results of the EMS test; Figure 12H The result of the DMS test; Figure 12I The results of the furazolidone test; Figure 12J This is the result of the 9-aminoacridine test.

[0041] Figures 13A-13J is the standard curve of ten genotoxic substances. Among them, Figure 13Ais the standard curve of H2O2; Figure 13B is the standard curve of MMC; Figure 13C is the standard curve of MNNG; Figure 13D is the standard curve of NA; Figure 13E is the standard curve of CH2O; Figure 13F is the standard curve of DEN; Figure 13G is the standard curve of EMS; Figure 13H is the standard curve of DMS; Figure 13I is the standard curve of furazolidone; Figure 13J is the standard curve of 9-aminoacridine. DETAILED DESCRIPTION

[0042] The present application is further described below with reference to examples. It should be understood that the examples are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0043] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art. Although methods and materials similar or identical to those described herein may be used in experiments or practical applications, the materials and methods are described herein below. In the event of a conflict, the present specification, including definitions, will prevail. Additionally, the materials, methods, and examples are provided for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific examples, which are not intended to limit the scope of this application.

[0044] definition

[0045] As used herein, the term "about" refers to a variation of no more than 10% of the associated figure. In certain embodiments, the term "about" refers to a variation of no more than 5% of the associated figure.

[0046] As used herein, the terms "comprise," "include," and "contain" are used interchangeably to include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of," "consisting essentially of."

[0047] As used herein, the term "variant" or "mutant" refers to a polynucleotide or polypeptide comprising an alteration (i.e., substitution, insertion, and / or deletion) at one or more (e.g., several) positions relative to a wild-type or comparable polynucleotide or polypeptide, wherein a substitution refers to replacing the nucleotide or amino acid occupying a position with a different nucleotide or amino acid. A deletion refers to the removal of a nucleotide or amino acid occupying a position. An insertion refers to the addition of a nucleotide or amino acid adjacent to and immediately following the nucleotide or amino acid occupying the position.

[0048] As used herein, the term "mutation" generally refers to any type of change or modification to a sequence (nucleic acid or amino acid sequence), including deletion, truncation, inactivation, disruption, substitution, translocation, or insertion of amino acids or nucleotides.

[0049] As used herein, the terms "nucleic acid," "nucleic acid sequence," "nucleotide sequence," "polynucleotide," "polynucleotide sequence," "RNA sequence," or "DNA sequence" refer to oligonucleotides, nucleotides, or polynucleotides, and fragments and portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded and represent the sense or antisense strand. The sequence may be a non-coding sequence, a coding sequence, or a mixture of the two. The nucleic acid sequences of the present application can be prepared using standard techniques well known to those skilled in the art.

[0050] As used herein, the term "recombinant vector" generally refers to a recombinant DNA molecule constructed by connecting an exogenous target gene to a vector in vitro. It can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous target gene into the recipient cell and provide the exogenous target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0051] As used herein, the term "biosensor" is intended to be used for detecting genotoxic substances in the environment, and refers to a detection system with bacteria as the core sensitive element, wherein the bacteria are designed to be able to sense the presence of genotoxic substances and to achieve rapid and accurate detection of genotoxic substances. In some embodiments, the bacteria can produce a measurable biological signal (such as fluorescence intensity) by expressing a specific reporter gene (such as a fluorescent protein), thereby achieving detection of genotoxic substances. In some embodiments, the bacteria is Escherichia coli.

[0052] As used herein, the term "promoter" has a meaning well known to those skilled in the art, and refers to a non-coding nucleotide sequence located upstream of a gene that can initiate expression of downstream genes. In this article, the term "sulA promoter" can be used interchangeably with "P sulA ” is used interchangeably, the term “recA promoter” can be used with “P recA ” is used interchangeably, the term “cda promoter” can be used with “P cda ” are used interchangeably, the term “umu promoter” can be used with “P umu ” are used interchangeably.

[0053] sulA promoter or its mutants

[0054] The sulA promoter is a key promoter in the SOS stress response system, responsible for regulating the expression of the sulA gene; under normal circumstances, the sulA promoter is inhibited by the LexA protein, but when the SOS response is activated, the LexA protein will be degraded, thereby relieving the inhibition of the sulA promoter, allowing it to initiate transcription and express the sulA protein.

[0055] In this application, the inventors discovered that the sulA promoter can be used to construct a highly sensitive bacterium that has the advantages of strong specificity, rapid response, low cost and easy operation in detecting genotoxic substances in the environment, and can effectively meet the needs of environmental monitoring and safety assessment.

[0056] Furthermore, after extensive experimental verification, the inventors discovered that modifying the LexA binding site in the sulA promoter can optimize its response characteristics, thereby significantly improving the sensitivity of genotoxic substance detection. Specifically, the inventors constructed two mutant libraries by randomly mutating the LexA binding site and randomly inserting 4bp bases into the LexA binding site to extend its length. After screening, the inventors ultimately determined that the insertion of the 4bp base TAAA in the LexA binding site was the key factor in determining detection sensitivity.

[0057] Based on this, the present application provides the application of sulA promoter in the detection of genotoxic substances.

[0058] The nucleotide sequence of the sulA promoter is shown in SEQ ID NO: 1.

[0059] The sequence of SEQ ID NO: 1 is as follows:

[0060]

[0061] The sulA promoter is derived from Escherichia coli. Those skilled in the art will appreciate that the sequence of the sulA promoter is not limited to the specific sequences listed above. The sulA promoter sequence should include sequences that contain one, two, or three or more nucleotide mutations compared to the sequence of SEQ ID NO: 1, but are still substantially functionally identical thereto; sequences that are 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 1; and sequences that have one or more nucleotides deleted, one or more nucleotides added, or one or more nucleotides substituted from the sequence shown in SEQ ID NO: 1 but are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence shown in SEQ ID NO: 1.

[0062] The present application is not intended to limit the source of the sulA promoter. Specifically, as long as the selected E. coli has a sequence identical to the above promoter sequence, it meets the requirements of the present application. In some embodiments, the sulA promoter is derived from E. coli MG1655.

[0063] The present application also provides a sulA promoter mutant, which is obtained by mutating the sulA promoter as shown in the nucleotide sequence of SEQ ID NO: 1, wherein the mutation is to insert TAAA into the sequence shown in SEQ ID NO: 1. The nucleotide sequence of the mutant is shown in SEQ ID NO: 2.

[0064] The sequence of SEQ ID NO: 2 is as follows:

[0065]

[0066] Those skilled in the art will also understand that the sequence of the sulA promoter mutant is not limited to the specific sequences listed above. The sequence of the sulA promoter mutant should include sequences that contain one, two, or three or more nucleotide mutations compared to the sequence of SEQ ID NO: 2, but are still substantially functionally identical thereto, sequences that are 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 2, and sequences that delete one or more nucleotides, add one or more nucleotides, or replace one or more nucleotides based on the nucleotide sequence shown in SEQ ID NO: 2 but are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence shown in SEQ ID NO: 2.

[0067] The present application also provides the use of the above-mentioned sulA promoter mutant in the detection of genotoxic substances.

[0068] Those skilled in the art will appreciate that this application is not intended to limit the specific type or substance of the genotoxic substance. The genotoxic substance can be any substance known in the art, as long as it can induce a response in the sulA promoter or a mutant thereof. In some embodiments, the genotoxic substance includes any one or more of H2O2, MMC, MNNG, NA, CHO, DEN, EMS, DMS, furazolidone, and 9-aminoacridine, for example, any two, three, four, five, six, seven, eight, nine, or all ten of the genotoxic substance.

[0069] Recombinant vectors, bacteria and kits

[0070] The present application provides a recombinant vector comprising the above-mentioned sulA promoter or a mutant thereof.

[0071] In some embodiments, the recombinant vector further comprises a reporter gene, and the expression of the reporter gene is driven by the sulA promoter or a mutant thereof.

[0072] Wherein, the reporter gene can be, for example, a luminescent gene (such as luxCDABE), a color gene, an enzyme, or a fluorescent protein. In some embodiments, the reporter gene is a fluorescent protein, and the fluorescent protein is, for example, green fluorescent protein, yellow fluorescent protein, red fluorescent protein, or blue fluorescent protein. In some embodiments, the reporter gene is green fluorescent protein.

[0073] In some embodiments, the starting vector used to construct the recombinant vector is pBR322, ColE1, pSC101, p15A or R6K vector.

[0074] The present application is not intended to limit the type of starting vector, and the starting vector can be any suitable vector. In some embodiments, the starting vector is a p15A vector.

[0075] In some embodiments, the recombinant vector comprises a sulA promoter and a fluorescent protein. The nucleotide sequence of the sulA promoter is shown in SEQ ID NO: 1; the starting vector used to construct the recombinant vector is p15A; and the fluorescent protein is green fluorescent protein, expression of which is driven by the sulA promoter. In some embodiments, the nucleotide sequence of the recombinant vector is shown in SEQ ID NO: 28.

[0076] The sequence of SEQ ID NO:28 is as follows:

[0077]

[0078]

[0079] In some embodiments, the recombinant vector comprises a sulA promoter mutant and a fluorescent protein. The nucleotide sequence of the sulA promoter mutant is shown in SEQ ID NO:2; the starting vector used to construct the recombinant vector is p15A; and the fluorescent protein is green fluorescent protein, expression of which is driven by the sulA promoter mutant. In some embodiments, the nucleotide sequence of the recombinant vector is shown in SEQ ID NO:29.

[0080] The sequence of SEQ ID NO:29 is as follows:

[0081]

[0082]

[0083]

[0084] In some embodiments, the recombinant vector is a recombinant plasmid.

[0085] The construction of any of the above recombinant vectors can be carried out by any method known in the art. For example, the recombinant vector can be obtained by restriction endonuclease method, homologous recombination method, TOPO cloning method, Gateway cloning method, CRISPR / Cas9 technology, Gibson assembly method, and / or synthetic biology method.

[0086] The present application provides a bacterium for detecting genotoxic substances, wherein the bacterium comprises any one of the above-mentioned recombinant vectors.

[0087] Wherein, the bacteria is Escherichia coli. In some embodiments, the bacteria is Escherichia coli MG1655. The Escherichia coli MG1655 is commercially available.

[0088] The recombinant vector can be introduced into the bacteria according to conventional methods in the art. For example, transformation methods (including heat shock transformation or electroporation), transduction, calcium ion-mediated methods, or gene gun methods can be used. In the present application, heat shock transformation is used for introduction.

[0089] In some embodiments, the bacteria used for genotoxic substance detection is Escherichia coli MG1655, which includes a recombinant vector containing a sulA promoter and a fluorescent protein. The nucleotide sequence of the sulA promoter is shown in SEQ ID NO: 1; the starting vector used to construct the recombinant vector is p15A; and the fluorescent protein is green fluorescent protein, the expression of which is driven by the sulA promoter. In some embodiments, the nucleotide sequence of the recombinant vector is shown in SEQ ID NO: 28. When the bacteria are exposed to genotoxic substances or radiation with DNA-damaging properties, the SOS repair system is activated, releasing the inhibition of the sulA promoter and activating the expression of the downstream fluorescent protein. The presence and intensity of the genotoxic substance can be characterized by measuring the fluorescence value of the fluorescent protein.

[0090] In some embodiments, the bacteria used for genotoxic substance detection is Escherichia coli MG1655, which includes a recombinant vector containing a sulA promoter mutant and a fluorescent protein. The nucleotide sequence of the sulA promoter mutant is shown in SEQ ID NO:2; the starting vector used to construct the recombinant vector is p15A; and the fluorescent protein is green fluorescent protein, the expression of which is driven by the sulA promoter mutant. In some embodiments, the nucleotide sequence of the recombinant vector is shown in SEQ ID NO:29. When the bacteria are exposed to genotoxic substances or DNA-damaging radiation, the SOS repair system is activated, releasing the inhibition of the sulA promoter mutant and activating the expression of the downstream fluorescent protein. The presence and intensity of the genotoxic substance can be characterized by measuring the fluorescence value of the fluorescent protein.

[0091] The present application provides a kit for detecting genotoxic substances, which comprises any of the above-mentioned recombinant vectors or any of the above-mentioned bacteria.

[0092] The present application also provides the use of any of the above-mentioned recombinant vectors, any of the above-mentioned bacteria, or any of the above-mentioned kits in the detection of genotoxic substances.

[0093] Those skilled in the art will appreciate that this application is not intended to limit the specific types or specific substances of genotoxic substances. The above-mentioned genotoxic substances can be any substances known in the art, as long as they can induce the response of the sulA promoter or its mutants. In some embodiments, the genotoxic substances include any one or more of H2O2, MMC, MNNG, NA, CHO, DEN, EMS, DMS, furazolidone, and 9-aminoacridine, for example, any two, three, four, five, six, seven, eight, nine, or all ten of the genotoxic substances.

[0094] Methods for detecting genotoxic substances

[0095] The present application also provides a method for detecting genotoxic substances, which comprises: mixing any of the above-mentioned bacteria with a sample to be tested to detect the genotoxic substances in the sample.

[0096] Among them, the method of detecting genotoxic substances is routine in the art.

[0097] In some embodiments, a method for detecting genotoxic substances includes: mixing bacteria with a sample to be tested, culturing the mixed bacteria, and detecting the genotoxic substances in the sample to be tested by measuring fluorescence values. The bacteria are Escherichia coli MG1655, and the Escherichia coli MG1655 includes a recombinant vector containing a sulA promoter and a fluorescent protein; the nucleotide sequence of the sulA promoter is shown in SEQ ID NO:1; the starting vector used to construct the recombinant vector is p15A; the fluorescent protein is green fluorescent protein, and the expression of the green fluorescent protein is driven by the sulA promoter. In some embodiments, the nucleotide sequence of the recombinant vector is shown in SEQ ID NO:28. Specifically, after the bacteria are mixed with the sample to be tested, the fluorescence value of the fluorescent protein is measured to determine whether the sample to be tested is genotoxic.

[0098] In some embodiments, a method for detecting genotoxic substances includes: mixing bacteria with a sample to be tested, culturing the mixed bacteria, and detecting the genotoxic substances in the sample to be tested by measuring fluorescence. The bacteria is Escherichia coli MG1655, which includes a recombinant vector containing a sulA promoter mutant and a fluorescent protein; the nucleotide sequence of the sulA promoter mutant is shown in SEQ ID NO:2; the starting vector used to construct the recombinant vector is p15A; the fluorescent protein is green fluorescent protein, and expression of the green fluorescent protein is driven by the sulA promoter mutant. In some embodiments, the nucleotide sequence of the recombinant vector is shown in SEQ ID NO:29. Specifically, after the bacteria are mixed with the sample to be tested, the fluorescence value of the fluorescent protein is measured to determine whether the sample to be tested is genotoxic.

[0099] In some embodiments, the culture temperature is 37°C.

[0100] Similarly, those skilled in the art will appreciate that this application is not intended to limit the specific types or specific substances of genotoxic substances. The above-mentioned genotoxic substances can be any substances known in the art, as long as they can induce the response of the sulA promoter or its mutants. In some embodiments, the genotoxic substances include any one or more of H2O2, MMC, MNNG, NA, CHO, DEN, EMS, DMS, furazolidone, and 9-aminoacridine, for example, any two, three, four, five, six, seven, eight, nine, or all ten of the genotoxic substances.

[0101] This application successfully addresses the challenges of prior art SOS / umu assays, such as demanding host strain culture conditions, cumbersome detection procedures, and low sensitivity. By selecting a highly safe model organism as the host strain and employing the sulA promoter, which is highly sensitive to DNA damage, this application successfully constructs a highly sensitive bacterium. This bacterium demonstrates promising application in the detection of genotoxic substances in the environment, with advantages such as high specificity, rapid response, low cost, and ease of operation.

[0102] Example

[0103] The present application will be described below in conjunction with specific examples, but the scope of the present application is not limited thereto. Unless otherwise specified, the reagents and instruments used in the following examples are conventional reagents and instruments in this area and can be obtained commercially. The methods used are all conventional test methods, and those skilled in the art can undoubtedly implement the scheme and obtain corresponding results based on the examples.

[0104] Example 1 Construction of a Detection Vector and Biosensor for Genotoxic Substances

[0105] 1.1 Strain culture

[0106] In Example 1, all bacterial cultures were inoculated into LB medium (containing 1 / 1000 chloramphenicol) at a ratio of 1:100 and cultured at 37° C. and 220 rpm with shaking.

[0107] 1.2 Promoter amplification

[0108] Escherichia coli MG1655 (derived from W1485, a derivative strain of K12, preserved in our laboratory) was used as a template and p15A-P sulA -F / R, p15A-P recA -F / R, p15A-P cda -F / R and p15A-P umu -F / R primers (the specific sequences of which are shown in Table 1 below) were used for PCR to amplify the sulA, recA, cda and umu promoters. The target band size was approximately 200-300 bp. Agarose gel electrophoresis showed that each promoter was successfully amplified ( Figure 1 ).

[0109] The nucleotide sequence of the sulA promoter is as follows (SEQ ID NO: 1):

[0110]

[0111] The nucleotide sequence of the recA promoter is as follows (SEQ ID NO: 3):

[0112]

[0113] The nucleotide sequence of the cda promoter is as follows (SEQ ID NO: 4):

[0114]

[0115] The nucleotide sequence of the umu promoter is as follows (SEQ ID NO: 5):

[0116] Table 1 PCR amplification primer sequences in Example 1

[0117]

[0118]

[0119] 1.3 Construction of genotoxic substance carriers

[0120] Using p15A as a vector, the p15A linear fragment was connected with the superfolder green fluorescent protein (sfGFP) fragment by homologous recombination to obtain the p15A-sfGFP recombinant vector; sulA -bF / R, p15A-P recA -bF / R, p15A-P cda -bF / R and p15A-P umu -bF / R primers (the specific sequences are shown in Table 1) were used to linearize p15A-sfGFP and amplify the corresponding promoter backbone. The target band size was approximately 2900 bp. Agarose gel electrophoresis showed that each backbone was successfully amplified ( Figure 2 ).

[0121] Furthermore, the four promoters (sulA, recA, cda, and umu) were assembled with the corresponding p15A-sfGFP fragments using seamless cloning technology to obtain p15A-P. sulA -sfGFP, p15A-P recA -sfGFP, p15A-P cda -sfGFP and p15A-P umu -sfGFP four recombinant vectors. Among them, p15A-P sulA -sfGFP nucleotide sequence is shown in SEQ ID NO: 28, and p15A-P recA -sfGFP, p15A-Pcda -sfGFP and p15A-P umu The nucleotide sequence of -sfGFP can be obtained by replacing the sulA promoter region in SEQ ID NO: 28 with the corresponding recA, cda and umu promoter sequences.

[0122] 1.3 Screening and identification of biosensors

[0123] The obtained p15A-P with promoter and green fluorescent protein sulA -sfGFP, p15A-P recA -sfGFP, p15A-P cda -sfGFP and p15A-P umu The four recombinant vectors of -sfGFP were transformed into MG1655 competent cells by heat shock transformation to obtain biosensors, which were verified by Sanger sequencing. Figures 3A-3D As shown, four biosensors have been successfully constructed.

[0124] Example 2 Testing of Biosensors for Genotoxic Substances

[0125] 2.1 Strain culture

[0126] In Example 2, all bacterial cultures were inoculated into LB medium (containing 1 / 1000 chloramphenicol) at a ratio of 1:100 and cultured at 37° C. and 220 rpm with shaking.

[0127] 2.2 H2O2 test of biosensor performance

[0128] The working performance of the biosensor was tested using 0.5 mM H2O2. The four biosensors after recovery were added to 20 mL of fresh LB medium (containing 1 / 1000 chloramphenicol) and cultured at 37 °C and 220 rpm until the OD 600 =0.2-0.3, add 100 μL H2O2 respectively, and measure the fluorescence value and OD every 1 h 600 , and calculate the relative fluorescence value (fluorescence value / OD 600 ); the corresponding biosensor with equal amount of DMSO added was used as blank control.

[0129] The results are as follows Figures 4A-4D As shown, after adding 0.5 mM H2O2, p15A-P sulA -sfGFP sensor and p15A-P cda -sfGFP sensor downstream of sfGFP expression, and p15A-P sulA -sfGFP sensor showed an increasing trend over time; p15A-P cda-sfGFP sensor has a high background fluorescence, and although it shows an upward trend with time, it is basically no different from the control strain; while p15A-P recA -sfGFP sensor and p15A-P umu -sfGFP sensor downstream sfGFP was not activated, suggesting that the constructed p15A-P recA -sfGFP sensor and p15A-P umu - The sfGFP sensor may not respond to H2O2.

[0130] 2.3MNNG test of biosensor performance

[0131] The working performance of the biosensor was tested using 50 μM MNNG. The four biosensors after recovery were added to 20 mL of fresh LB medium (containing 1 / 1000 chloramphenicol) and cultured at 37 °C and 220 rpm until the OD 600 =0.2-0.3, add 100 μL MNNG respectively, and measure the fluorescence value and OD every 1 h 600 , and calculate the relative fluorescence value (fluorescence value / OD 600 ); the corresponding biosensor with equal amount of DMSO added was used as blank control.

[0132] The results are as follows Figures 5A-5D As shown, the four biosensors showed consistent results with the H2O2 test, activating p15A-P sulA -sfGFP sensor and p15A-P cda -sfGFP sensor downstream of sfGFP expression, and p15A-P sulA -sfGFP sensor showed an increasing trend with time; while p15A-P recA -sfGFP sensor and p15A-P umu -sfGFP sensor downstream of sfGFP is not activated.

[0133] The p15A-P was determined by H2O2 test and MNNG test. sulA -sfGFP sensor has the highest sensitivity.

[0134] Example 3 Response detection of genotoxic substances by biosensor

[0135] 3.1 Determination of different genotoxic substances by biosensors

[0136] According to the method in Example 2, different concentrations of H2O2, MMC, MNNG, NA, and CH2O were used to test p15A-P sulA -sfGFP sensor, the results are as follows Figures 6A-6EAs shown in the figure, in the tests of five genotoxic substances, the genotoxic substances at different concentrations showed an upward trend as time increased, and at the same time, a good gradient was shown as the concentration increased.

[0137] In order to determine the optimal detection time, curves of different times with increasing concentration were drawn. The results are shown in Figure 2. Figures 7A-7E As shown in the figure, the five genotoxic substances showed a good trend at about 2 h, so 2 h was selected as the detection time.

[0138] In environmental samples, due to the diversity of genotoxic substances, p15A-P sulA -sfGFP sensor has a broad spectrum of activity to respond to various genotoxic substances. sulA Based on the broad spectrum characteristics of the sfGFP sensor, the inventors of this application selected more genotoxic substances, including EMS, DMS, DEN, furazolidone, and 9-aminoacridine, and conducted further tests. The results are as follows: Figures 8A-8E As shown, in the test of five genotoxic substances, p15A-P sulA The -sfGFP sensor also responded well.

[0139] 3.2 Calculation of detection limits for genotoxic substances

[0140] In order to more accurately evaluate the relationship between genotoxic substances and their SOS response to strains, the relationship between genotoxic substances and p15A-P sulA -sfGFP sensor fluorescence value / OD 600 The detection method is used to determine the relationship between the two.

[0141] The detection time was selected as 2 h, and the standard curves of different genotoxic substances (H2O2, MMC, MNNG, NA, CH2O, DEN, EMS, DMS, furazolidone, 9-aminoacridine) were established according to the data of 3.1 in Example 3. The results are as follows: Figures 9A-9J As shown, the X-axis represents the different concentrations of genotoxic substances, the Y-axis represents the corresponding induction ratio (IR), and the R of each curve is 2 The values are all around 0.99, indicating a good fit and can accurately reflect the relationship between the two.

[0142] Furthermore, according to the EN ISO standard (ISO, 2000), IR = 1.5 is defined as the threshold value of genotoxic effects. When IR> 1.5, it indicates potential genotoxicity. In this application, IR = 1.5 is used as the evaluation index and inserted into the standard curve to obtain the detection limit of the biosensor for genotoxic substances. The results are shown in Table 2 below. It was found that p15A-P sulA The detection limits of the -sfGFP sensor for MMC, MNNG, CH2O, and DMS are lower than those of conventional SOS / umu.

[0143] Table 2p15A-P sulA -Detection limits of sfGFP sensors for different genotoxic substances

[0144]

[0145] Example 4 Optimization of biosensor detection sensitivity

[0146] 4.1 lexA binding site modification

[0147] The LexA binding site on the sulA promoter was randomly mutated using different primers (the specific sequences of which are shown in Table 3 below). RM-R was a universal primer, and PCR was performed using InPbp-F, RM-F, RM-1-F, RM-2-F, RM-3-F, and RM-R, respectively. sulA The -sfGFP recombinant vector was linearized and then subjected to Gibson assembly, and then transformed into MG1655 competent cells by heat shock transformation to obtain mutant strains.

[0148] Table 3 PCR amplification primer sequences in Example 4

[0149]

[0150]

[0151] Two mutant libraries were constructed by randomly mutating the LexA binding site or randomly inserting a 4bp extension of the binding site. Screening of the mutant library revealed that the response multiple of mutant 55 was 1.7 times that of the wild type at 2h, and the background was 1.4 times that of the wild type ( Figure 10 ); Sanger sequencing revealed the mutation to be TAAA (SEQ ID NO: 2), indicating that TAAA is the key to determining the sensitivity of the sulA promoter ( Figure 11 ).

[0152] The nucleotide sequence of the sulA promoter of mutant strain 55 is as follows (SEQ ID NO: 2):

[0153]

[0154] 4.2 Determination of different genotoxic substances by mutant strain 55

[0155] According to the method in Example 2, different concentrations of H2O2, MMC, MNNG, NA, CH2O, DEN, EMS, DMS, furazolidone, and 9-aminoacridine were used to test mutant No. 55. The results were as follows. Figures 12A-12J As shown in the figure, in the tests of ten genotoxic substances, the genotoxic substances at different concentrations showed an upward trend as time increased, and at the same time, a good gradient was shown as the concentration increased.

[0156] 4.3 Calculation of detection limits for genotoxic substances

[0157] The data of 4.2 in Example 4 when the detection time is 2h are selected to draw a standard curve. The results are as follows: Figures 13A-13J As shown, the R of each curve 2 The values were all around 0.99, indicating a good fit and accurately reflecting the relationship between genotoxic substances and their SOS response to mutant strain 55.

[0158] Furthermore, the detection limit was calculated based on IR=1.5, and the results are shown in Table 4 below. It was found that the detection limit of mutant strain 55 for H2O2, NA, CHO, DEN, furazolidone and 9-aminoacridine was lower than that of the wild type, and the detection limit of mutant strain 55 for H2O2, MMC, MNNG, CHO, DEN and DMS was lower than that of the traditional SOS / umu, indicating that the TAAA mutation can significantly improve the sensitivity of the sulA promoter.

[0159] Table 4 Detection limits of mutant strain 55 for different genotoxic substances

[0160]

[0161] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application in any other manner. Any person skilled in the art may utilize the above disclosed technical content to modify or modify the present application into equivalent embodiments with equivalent variations. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present application and are based on the technical essence of the present application shall still fall within the scope of protection of the present application.

Claims

1. Application of the sulA promoter or its mutants in the detection of genotoxic substances. Preferably, the genotoxic substances include any one or more of H2O2, MMC, MNNG, NA, CH2O, DEN, EMS, DMS, furazolidone, and 9-aminoacridine.

2. The use according to claim 1, wherein the nucleotide sequence of the sulA promoter is shown as SEQ ID NO: 1, or the nucleotide sequence of the sulA promoter mutant is shown as SEQ ID NO:

2.

3. A sulA promoter mutant, the nucleotide sequence of which is shown in SEQ ID NO:

2.

4. A recombinant vector comprising a sulA promoter or a mutant thereof. 5 . The recombinant vector according to claim 4 , wherein the nucleotide sequence of the sulA promoter is shown as SEQ ID NO: 1, or the nucleotide sequence of the sulA promoter mutant is shown as SEQ ID NO:

2.

6. The recombinant vector according to claim 4 or 5, wherein the recombinant vector comprises a reporter gene, and expression of the reporter gene is driven by the sulA promoter or a mutant thereof; Preferably, the reporter gene is a fluorescent protein; Further preferably, the fluorescent protein is green fluorescent protein, yellow fluorescent protein, red fluorescent protein or blue fluorescent protein.

7. The recombinant vector according to any one of claims 4 to 6, wherein the starting vector for constructing the recombinant vector is pBR322, Col E1, pSC101, p15A or R6K vector.

8. A bacterium for detecting genotoxic substances, comprising the recombinant vector according to any one of claims 4 to 7; Preferably, the bacteria is Escherichia coli; Further preferably, the genotoxic substance includes any one or more of H2O2, MMC, MNNG, NA, CH2O, DEN, EMS, DMS, furazolidone, and 9-aminoacridine.

9. Use of the recombinant vector according to any one of claims 4 to 7 or the bacterium according to claim 8 in the preparation of a kit for detecting genotoxic substances.

10. A kit for detecting genotoxic substances, comprising the recombinant vector according to any one of claims 4 to 7 or the bacterium according to claim 8.

11. Use of the recombinant vector according to any one of claims 4 to 7, the bacterium according to claim 8, or the kit according to claim 10 in the detection of genotoxic substances.

12. A method for detecting a genotoxic substance, comprising: mixing the bacteria according to claim 8 with a sample to be tested to detect genotoxic substances in the sample; Preferably, the genotoxic substance includes any one or more of H2O2, MMC, MNNG, NA, CH2O, DEN, EMS, DMS, furazolidone, and 9-aminoacridine.

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