Construction and use of a biosensor selection system for lactose or analogue thereof in bacillus subtilis

Abstract

Provided are construction and use of a biosensor selection system for lactose or an analogue thereof in Bacillus subtilis. A lactose operon-based "dual-selection" genetic circuit used for selecting repressor proteins for biosensors and applicable to Bacillus subtilis is designed; the selection of repressor protein DNA binding activity is realized by means of a negative-selection lethal circuit; the allosteric activation-based activity screening is realized by means of a positive selection fluorescence signal; and finally, a biosensor having target lactose analogue recognition activity is obtained. A novel biosensor responding to substances such as lactose, 2'-fucosyllactose, and 3-fucosyllactose can be obtained by using the selection platform, and upon verification, the biosensor constructed on the basis of the selection result has high detection sensitivity and specificity.

Description

Construction and application of a screening system for lactose or its analogs biosensor in bacillus subtilis TECHNICAL FIELD

[0001] The present application relates to a screening system for lactose or its analogs biosensor in bacillus subtilis and application, belonging to the field of synthetic biology and metabolic engineering. BACKGROUND

[0002] In 1961, Monord and Jacob of Pasteur Institute in France found that E. coli could determine whether to produce lactose metabolism related enzymes according to the presence or absence of glucose and lactose in the environment, and through the study of the above phenomenon, they proposed the concept of operon and operator. After the concept of lactose operon was proposed, researchers have successively discovered tryptophan operon, histidine operon, arabinose operon and other various operons. The regulation of operon on genes enables microorganisms to turn on or off the expression of certain genes according to the changes of external environment, so as to rapidly synthesize the required enzymes and metabolites when specific metabolites are needed, and to stop the synthesis of other types of metabolites. In the face of rapidly changing complex external environment, this regulation mechanism is indispensable for organisms, and the existence of operon enables organisms to have stronger adaptability in the changing external environment.

[0003] Researchers have developed various metabolite biosensors based on various operon elements. When the target metabolite exists, the metabolite biosensor can realize the response characteristics by changing the protein conformation and can convert the target metabolite concentration signal into fluorescence signal, growth rate signal, metabolic pathway signal, etc. In recent years, metabolite biosensors have played a great role in the construction of microbial cell factories. At the same time, with the expansion of application range, complex application conditions have put forward higher requirements for the metabolite recognition range, molecular response level and signal output intensity of the biosensor. However, most of the biosensors used at present are natural metabolite biosensors developed based on biological elements in nature that respond to a certain metabolite. The above situation leads to the fact that natural metabolite biosensors cannot adapt to the requirements of responding to multiple new metabolites.

[0004] To expand the application range of metabolite biosensors, it is necessary to change the response target of natural metabolite sensing elements. Generally, it is difficult to change the specificity of the inducing agent of these biosensor response proteins, because changing the specificity of the inducing agent may destroy its original allosteric characteristics, ultimately affecting its binding effect with specific gene elements, causing the failure of the whole system and making it unable to be used. Therefore, in order to increase the response range of the biosensor and make it respond to different metabolites or inducing agent types, so as to be used in a more extensive range, it is necessary to develop a rapid, accurate and efficient screening platform for the screening of response target substance biosensors. SUMMARY

[0005] To solve the above problems, the application provides a construction method of a lactose or its analog biological sensor screening platform in Bacillus subtilis and application thereof. The screening platform is composed of a DNA binding transcriptional repressor LacI expression frame, a screening system gene circuit expression frame (an IPTG inducible promoter, a reporter gene and a suicide gene), and a gene expression element required for expression of the above genes such as a promoter, an RBS and a terminator. The screening platform can be constructed in a plasmid vector, transformed into the cytoplasm of Bacillus subtilis cells or integrated into the genome of Bacillus subtilis for use, so as to realize screening of biological sensors or their components.

[0006] A first object of the application is to provide a screening method of a lactose or its analog biological sensor, comprising the following steps:

[0007] S1, constructing a DNA binding transcriptional repressor LacI mutant library;

[0008] S2, constructing a recombinant cell library containing a DNA binding transcriptional repressor LacI mutant gene expression frame and a screening system gene circuit expression frame; the screening system gene circuit expression frame contains an inducible promoter, a reporter gene and a suicide gene;

[0009] S3, negative screening: culturing the recombinant cell library obtained in S2 to make the DNA binding transcriptional repressor LacI mutant and the genes in the screening system gene circuit expression frame express, and then transferring the recombinant cell library to a first culture medium for culture to screen out surviving cells; the first culture medium contains a non-cytotoxic prodrug, and the protein encoded by the suicide gene can convert the non-cytotoxic prodrug into a cytotoxic active drug;

[0010] S4, positive screening: transferring the surviving cells in S3 to a second culture medium for culture, screening the cells according to the signal intensity of the reporter gene to obtain a target biological sensor; the second culture medium contains lactose or its analog.

[0011] Further, the host cells of the recombinant cell library can be selected according to the type of biological sensor, such as common Bacillus subtilis, Escherichia coli and the like.

[0012] Further, in step S1, the mutant library construction method includes saturation mutation and / or random mutation.

[0013] Further, in step S2, the DNA-binding transcription repressor LacI mutant gene expression cassette is any DNA sequence that can transcribe and translate a DNA-binding transcription repressor LacI mutant, and contains not only the LacI mutant coding gene, but also the promoter, RBS, terminator and other expression elements required for the expression of the gene, such as the sequence of the expression cassette sequence includes but is not limited to SEQ ID NO. 9.

[0014] Further, the promoter that initiates the expression of the DNA-binding transcription repressor LacI mutant gene is preferably a constitutive promoter, and the specific selection can be selected according to the host cell, and it is appropriate to have lower expression or no leakage expression in the target host (no leakage is the best).

[0015] Further, in step S2, the screening system gene circuit expression cassette is integrated expression or free expression, and the DNA-binding transcription repressor LacI mutant gene expression cassette is free expression.

[0016] Further, in step S2, the reporter gene can be any gene that can directly or indirectly detect the signal of its encoded product, and the types of reporter genes include but are not limited to substrate-free fluorescent emission (such as fluorescent protein genes), interaction with radioactive or fluorescent substrates (such as luciferase), etc.

[0017] Further, the fluorescent protein gene includes: green fluorescent protein gene, yellow fluorescent protein gene, red fluorescent protein gene, deep red fluorescent protein gene, near-infrared fluorescent protein gene, orange fluorescent protein gene, cyan fluorescent protein gene, blue fluorescent protein gene, deep blue fluorescent protein gene, etc., and the fluorescent protein gene derived from the mutant gene or modification. Such as sfGFP with an amino acid sequence as shown in SEQ ID NO. 3 or eGFP with an amino acid sequence as shown in SEQ ID NO. 4.

[0018] Further, in step S2, the suicide gene can be thymidine kinase gene (hsvTK), cytosine deaminase gene (CD), etc., and correspondingly, the non-cytotoxic prodrug selected in step S3 is 5-fluoro-2'-deoxyuridine (5FdU), 5-fluorocytosine (5-FC), etc. Among them, 5FdU is converted into 5-fluoro-2'-deoxyuridine-5'-monophosphate (5FdUMP) by thymidine kinase; 5-FC is metabolized by CD into 5-fluorouracil in microorganisms, causing cell death.

[0019] Further, in step S2, the fluorescent protein gene is connected with a herpes simplex virus thymidine kinase (hsvTK) gene through a flexible linker, that is, the screening platform gene circuit expression frame is composed of an inducible promoter, a reporter gene, a linker, and a suicide gene in series.

[0020] Further, in step S3, the non-toxic prodrug is added in the first culture medium at a concentration of 1 nM to 1000 mM.

[0021] Further, in step S4, the lactose analogs include, but are not limited to, 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), and the like.

[0022] Further, in step S4, the lactose or its analogs are added in the second culture medium at a concentration of 1 nM to 1000 mM.

[0023] Further, in step S4, the method for screening cells includes, but is not limited to, flow cytometry screening, microfluidic screening, plate coating screening, liquid medium culture screening, and the like.

[0024] Further, in step S4, the method for screening cells includes, but is not limited to, flow cytometry screening, microfluidic screening, plate coating screening, liquid medium culture screening, and the like.

[0025] Further, after step S4, if necessary, the method for sequencing the LacI mutant in the target cell is also included, as a component for preparing a biosensor.

[0026] A second object of the present application is to provide a screening system for a lactose or its analog biosensor, which comprises:

[0027] A recombinant cell library containing a DNA-binding transcriptional repressor LacI mutant library gene expression frame and a screening system gene circuit expression frame; the screening system gene circuit expression frame contains an inducible promoter, a reporter gene, and a suicide gene.

[0028] The first culture medium contains a non-cytotoxic prodrug, and the protein encoded by the suicide gene can convert the non-cytotoxic prodrug into a cytotoxic active drug. Of course, the person skilled in the art knows that when the expression frame of the screening system gene circuit is expressed independently, the first culture medium also contains the antibiotic corresponding to the resistance gene of the plasmid vector used.

[0029] The second culture medium contains lactose or its analogs. Similarly, when the expression frame of the screening system gene circuit is expressed independently, the second culture medium also contains the antibiotic corresponding to the resistance gene of the plasmid vector used.

[0030] A third object of the present application is to provide the use of the above-mentioned screening system in screening a lactose or its analogs biosensor or a component for the biosensor.

[0031] A fourth object of the present application is to provide a Bacillus subtilis biosensor for lactose or its analogs, which comprises a recombinant Bacillus subtilis containing a DNA-binding transcriptional repressor LacI mutant gene expression frame; the DNA-binding transcriptional repressor LacI mutant has the following amino acid residue substitution at any one of the following positions based on the sequence shown in SEQ ID NO. 10:

[0032] Valine at position 150 is replaced by alanine;

[0033] Serine at position 193 is replaced by glycine;

[0034] Glutamine at position 291 is replaced by isoleucine;

[0035] Glutamine at position 291 is replaced by valine;

[0036] Isoleucine at position 79 is replaced by histidine;

[0037] Glutamine at position 291 is replaced by phenylalanine.

[0038] Further, the Bacillus subtilis biosensor further comprises an inducible promoter and a reporter gene expressed by the promoter, so as to detect the presence or absence of lactose or its analogs according to the expression of the reporter gene.

[0039] Further, in step S2, the inducible promoter is derived from P lac promoter, including but not limited to P hy-spank promoter, P grac100 promoter, etc.; the P hy-spank promoter, P grac100The nucleotide sequence of the promoter is shown in SEQ ID NO. 1 and SEQ ID NO. 2 respectively.

[0040] A fifth object of the present application is to provide a DNA-binding transcription repressor LacI mutant, which is based on the sequence shown in SEQ ID NO. 10 and has substitution of amino acid residues at any of the following positions:

[0041] substituting valine at position 150 with alanine;

[0042] substituting serine at position 193 with glycine;

[0043] substituting glutamine at position 291 with isoleucine;

[0044] substituting glutamine at position 291 with valine;

[0045] substituting isoleucine at position 79 with histidine;

[0046] substituting glutamine at position 291 with phenylalanine.

[0047] A sixth object of the present application is to provide a nucleic acid molecule encoding the DNA-binding transcription repressor LacI mutant.

[0048] A seventh object of the present application is to provide an expression vector containing the nucleic acid molecule.

[0049] An eighth object of the present application is to provide a recombinant cell containing the nucleic acid molecule. Preferably, the host cell is a microbial cell, most preferably a Bacillus subtilis cell.

[0050] A ninth object of the present application is to provide use of the Bacillus subtilis biosensor, DNA-binding transcription repressor LacI mutant, nucleic acid molecule, expression vector or recombinant cell in detecting lactose or its analogs.

[0051] The beneficial effects of the present application are:

[0052] (1) The application provides a construction method of a lactose and its analogs biosensor screening platform, specifically, a DNA binding transcription inhibitor LacI mutant library and a screening system gene circuit expression frame containing an inducible promoter, a reporter gene and a suicide gene are transformed into cells, first, 5-fluoro-2'-deoxyuridine (5FdU) is added in the culture medium, and the screening of the DNA binding transcription inhibitor LacI mutant with promoter sequence binding activity is realized by whether the cells survive or not; second, specific lactose analogs (lactose, 2'-FL, 3-FL, etc.) are added in the culture medium, and the screening of the DNA transcription inhibitor LacI mutant with specific lactose analog binding ability is realized by the reporter gene signal. The screening platform is simple to construct, and high-throughput screening of the DNA binding transcription inhibitor LacI mutant library is realized, and the DNA binding transcription inhibitor LacI mutant responding to specific lactose analogs is obtained.

[0053] (2) The application screens out LacI mutants with strong inducible promoter binding activity and sensitive response to lactose and its analogs, such as V150A, S193G, I79H, Q291I, Q291V, Q291F, etc., through the self-constructed screening platform. These mutants have strong binding force with the promoter in the absence of lactose and its analogs, thereby inhibiting transcription, and in the presence of the test substance, they respond sensitively and are separated from the promoter, the reporter gene is started to express, and detection is realized. It is verified that the detection effect of these mutants is much better than that of the wild type LacI, and the mutants have specificity. BRIEF DESCRIPTION OF DRAWINGS

[0054] Fig. 1 is a gene circuit schematic diagram of the lactose analog biosensor screening platform in Bacillus subtilis and a lactose analog biosensor mutant screening flowchart.

[0055] Fig. 2 is a mutant lactose response curve. DETAILED DESCRIPTION

[0056] The application will be further described below in combination with the drawings and specific embodiments, so that those skilled in the art can better understand the application and implement it, but the embodiments are not limiting to the application.

[0057] The materials involved in the following embodiments are as follows:

[0058] Liquid LB medium (g / L): peptone 10, yeast powder 5, NaCl 10.

[0059] The primer sequences involved in the following embodiments are as follows:

[0060] Table 1 primer sequence

[0061] Construction of Bacillus subtilis lactose biosensor screening platform in Example 1

[0062] The Bacillus subtilis lactose biosensor plasmid screening platform in this example is composed of a DNA-binding transcriptional repressor LacI expression frame, an IPTG inducible promoter, an sfGFP fluorescent protein expression gene, a fusion protein linker Linker1, a herpes simplex virus thymidine kinase gene, and gene expression elements required for expression of the above genes, such as promoters, RBSs and terminators.

[0063] (1) Construct the screening system gene circuit expression frame and the DNA-binding transcriptional repressor LacI expression frame to obtain the biosensor screening platform plasmid:

[0064] The P hy-spank The primer F1 and R1 were designed using the promoter and its RBS sequence (nucleotide sequence as shown in SEQ ID NO. 1) as a template, and the P hy-spank The primer F2 and R2 were designed using the sfGFP fluorescent protein DNA sequence (amino acid sequence as shown in SEQ ID NO. 3) as a template, and the sfGFP fluorescent protein DNA fragment was obtained by PCR amplification; the primer F3 and R3 were designed using the herpes simplex virus thymidine kinase sequence (amino acid sequence as shown in SEQ ID NO. 8) as a template, and the herpes simplex virus thymidine kinase DNA fragment was obtained by PCR amplification; the fusion protein linker Linker1 DNA sequence is shown in SEQ ID NO. 5, and since the sequence is short, it is added to the primer R2 and F3; the primer F4 and R4 were designed using the pHT-LacI plasmid vector sequence as a template, and the plasmid vector fragment containing the DNA-binding transcriptional repressor LacI expression frame sequence was obtained by PCR amplification;

[0065] The above DNA fragments were fused by PCR and transformed into E. coli DH5α to construct the pHT-GLH lactose biosensor screening platform plasmid.

[0066] (2) Construction of LacI mutant library (containing saturated mutant library and random mutant library):

[0067] The pHT-GLH plasmid is extracted, and a DNA-binding transcription inhibitor LacI saturation mutation primer is designed; the pHT-GLH plasmid is used as a template to obtain a pHT-GLH plasmid saturation mutation fragment by PCR, and the pHT-GLH plasmid saturation mutation fragment is transformed into Escherichia coli DH5α to construct a DNA-binding transcription inhibitor LacI saturation mutation library; the Escherichia coli DH5α containing the DNA-binding transcription inhibitor LacI saturation mutation library is cultured, and the DNA-binding transcription inhibitor LacI saturation mutation library is extracted; similarly, random mutation primers F5 and R5 are designed, and a DNA-binding transcription inhibitor LacI random mutant library is constructed using a random mutation kit, the Escherichia coli DH5α containing the DNA-binding transcription inhibitor LacI random mutant library is cultured, and the DNA-binding transcription inhibitor LacI random mutant library is extracted.

[0068] (3) Construction of genetically engineered strains:

[0069] The DNA-binding transcription inhibitor LacI saturation mutation library or the random mutant library is transformed into the B. subtilis competent cell to obtain a B. subtilis lactose biosensor screening platform cell library.

[0070] The screening system gene circuit expression frame and the DNA-binding transcription inhibitor LacI expression frame are constructed on the same plasmid and are expressed freely in the host cell, a screening cell library is constructed, and the screening cell library is used for subsequent experiments. Of course, a person skilled in the art can also choose a genomic integration method to construct a lactose biosensor screening platform. The specific steps are as follows:

[0071] The B. subtilis genomic integration type lactose biosensor screening platform is composed of a DNA-binding transcription inhibitor LacI expression plasmid (pHT-lacI plasmid), an IPTG inducible promoter, a fluorescent protein expression gene, a fusion protein linker Linker, a herpes simplex virus thymidine kinase gene, and a promoter, an RBS and a terminator required for expression of the above genes and other gene expression elements.

[0072] The PCR primers F6, R6, F7 and R7 for gene integration are designed using the B. subtilis amyE integration site DNA sequence as a template, and the upstream homologous arm and the downstream homologous arm fragments for gene integration are obtained by PCR amplification. hy-spank The primers F1 and R1 are designed using the promoter and its RBS sequence (as shown in SEQ ID NO. 1) as a template, and the P hy-spankPromoter and its RBS sequence DNA fragment; the sfGFP fluorescent protein DNA sequence (as shown in SEQ ID NO. 3) was used as a template to design primers F2 and R2, and the herpes simplex virus thymidine kinase sequence (as shown in SEQ ID NO. 8) was used as a template to design primers F3 and R3, and a herpes simplex virus thymidine kinase DNA fragment was obtained by PCR amplification; the fusion protein linker Linker1 DNA sequence is shown in SEQ ID NO. 5, and since the sequence is short, the sequence is added to primers R2 and F3, respectively; the above DNA fragments are subjected to fusion PCR to construct a gene integration fragment; the above gene integration fragment is transformed into Bacillus subtilis 168 for gene integration, and after the colonies grow, colony PCR verification is performed to select Bacillus subtilis successfully integrated with the fusion fragment; and the above successfully integrated Bacillus subtilis is prepared into a competent state for standby.

[0073] Using pHT-lacI plasmid (donated by Dr. Li Yang of Jiangnan University) as a template, a pHT-lacI plasmid saturation mutation fragment is obtained by PCR using saturation mutation primers and is transformed into E. coli DH5α to construct a DNA binding transcriptional repressor LacI saturation mutant library; the E. coli DH5α containing the DNA binding transcriptional repressor LacI saturation mutant library is cultured to scale, and the DNA binding transcriptional repressor LacI saturation mutant library is extracted; similarly, random mutation primers F5 and R5 are designed, and a DNA binding transcriptional repressor LacI random mutant library is constructed using a random mutation kit, the E. coli DH5α containing the DNA binding transcriptional repressor LacI random mutant library is cultured to scale, and the DNA binding transcriptional repressor LacI random mutant library is extracted.

[0074] The DNA binding transcriptional repressor LacI saturation mutant library or the random mutant library is transformed into the competent state of the Bacillus subtilis successfully integrated with the fusion fragment to obtain a Bacillus subtilis lactose biosensor screening platform cell library.

[0075] Example 2 Screening of lactose-responsive LacI mutants using a lactose biosensor screening platform

[0076] This example aims to screen LacI mutants with strong promoter binding activity and sensitive response to lactose, and the screening includes negative screening and positive screening: LacI binding activity screening is performed by negative screening lethal circuit, and allosteric activation activity screening is performed by positive screening fluorescent signal.

[0077] A screening plate containing 5FdU is prepared, and Bacillus subtilis containing the lactose biosensor screening platform cell library is inoculated on the screening plate for culture. LacI mutants in Bacillus subtilis are constitutively expressed, and different mutants have different promoter P hy-spankwith different binding ability, thus playing different degrees of inhibition expression. LacI mutant and promoter P hy-spank binding will inhibit transcription, herpes simplex virus thymidine kinase lethal gene is not expressed, cell survival (when herpes simplex virus thymidine kinase gene is expressed, the engineered bacteria will convert the 5FdU precursor in the culture medium which is not toxic to cells into 5FdUMP which is cytotoxic), so the single colony grown on the plate is the negative screening of the LacI mutant containing DNA binding ability, and the single colony is eluted to prepare the bacterial solution after the single colony is grown;

[0078] The cells are eluted with liquid LB medium, and liquid shaking culture is carried out (37℃, 220rpm, 10-12h) by adding lactose with a final concentration of 1mM. The LacI mutant strain containing lactose response ability loses the ability to bind to the promoter P hy-spank because the protein conformation of the LacI mutant changes after binding to lactose, and the promoter P hy-spank triggering sfGFP expression to produce fluorescence signal. Therefore, after the bacterial solution is turbid, flow cytometry is used to screen cells with sfGFP fluorescence signal (about 1 / 10000-1 / 100000 cells with fluorescence signal), and the obtained cells contain DNA binding transcriptional repressor LacI mutant which can respond to lactose. DNA sequencing of the obtained single colony can obtain the DNA sequence information of the DNA binding transcriptional repressor which can respond to lactose (the amino acid sequence of the wild type is shown in SEQ ID NO. 10).

[0079] Example 3 Verification of lactose response

[0080] The mutant strain screened in Example 2 is inoculated into liquid LB medium containing chloramphenicol antibiotic, and cultured at 37℃, 220rpm for 10-12h. Fresh seed liquid is inoculated into a 96-well plate containing 1mM lactose and chloramphenicol antibiotic (inoculation amount 1%). The 96-well plate is placed in a hole plate shaker for culture (37℃, 700rpm). After 6h of shaking culture, the fluorescence intensity of the hole plate is measured every 3h using an enzyme marker instrument, and a fluorescence intensity change curve is drawn (as shown in Figure 2, note: Blank in Figure 2 represents the control strain containing the original lacI repressor protein).

[0081] Obviously, the above examples are only examples for the sake of clarity, and are not limited to the embodiments. Based on the above description, other different forms of changes or variations can be made by those of ordinary skill in the art. Here, it is not necessary and impossible to exhaust all embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A method of screening for a lactose or analogue thereof biosensor, characterized by: The method comprises the following steps: S1, constructing a DNA-binding transcription repressor LacI mutant library; S2, constructing a recombinant cell library containing a DNA-binding transcription repressor LacI mutant gene expression frame and a screening system gene circuit expression frame; the screening system gene circuit expression frame contains an inducible promoter, a reporter gene and a suicide gene; S3, culturing the recombinant cell library obtained in S2 to express the DNA-binding transcription repressor LacI mutant and the genes in the screening system gene circuit expression frame, and then transferring the recombinant cell library to a first culture medium for culture to screen out surviving cells; the first culture medium contains a non-cytotoxic prodrug, and the protein encoded by the suicide gene can convert the non-cytotoxic prodrug into a cytotoxic active drug; S4, transferring the surviving cells in S3 to a second culture medium for culture, screening the cells according to the signal intensity of the reporter gene, and obtaining a target biosensor; the second culture medium contains lactose or its analogs.

2. The screening method according to claim 1, characterized in that, The screening system gene circuit expression frame is integrated expression or free expression, and the DNA-binding transcription repressor LacI mutant gene expression frame is free expression.

3. The screening method according to claim 1, characterized by, The suicide gene is selected from a thymidine kinase gene or a cytosine deaminase gene, and the non-cytotoxic prodrug is selected from 5-fluorocytosine or 5-fluoro-2'-deoxyuridine.

4. The screening method according to claim 1, characterized by, The lactose analogs at least include 2'-fucosyllactose and 3-fucosyllactose.

5. A screening system for a lactose or analogue thereof biosensor, characterized in that, The screening system comprises: a recombinant cell library containing a DNA-binding transcription repressor LacI mutant library gene expression frame and a screening system gene circuit expression frame; the screening system gene circuit expression frame contains an inducible promoter, a reporter gene and a suicide gene; a first culture medium; the first culture medium contains a non-cytotoxic prodrug, and the protein encoded by the suicide gene can convert the non-cytotoxic prodrug into a cytotoxic active drug; a second culture medium; the second culture medium contains lactose or its analogs.

6. The screening system of claim 5 is used in a biosensor or a biosensor element for screening lactose or its analogs.

7. A Bacillus subtilis biosensor for lactose or its analogs, characterized by, The Bacillus subtilis biosensor comprises: a recombinant Bacillus subtilis containing a DNA-binding transcription repressor LacI mutant gene expression frame; the DNA-binding transcription repressor LacI mutant has an amino acid residue substitution at any one of the following positions based on the sequence shown in SEQ ID NO. 10: valine at position 150 is replaced by alanine; serine at position 193 is replaced by glycine; glutamine at position 291 is replaced by isoleucine; glutamine at position 291 is replaced by valine; isoleucine at position 79 is replaced by histidine; glutamine at position 291 is replaced by phenylalanine.

8. The B. subtilis biosensor of claim 7, wherein, The Bacillus subtilis biosensor further comprises: an inducible promoter, and a reporter gene and a suicide gene expressed by the promoter.

9. The B. subtilis biosensor of claim 8, wherein, The inducible promoter includes P hy-spank Promoter or P grac100 Promoter.

10. A mutant of the DNA-binding transcriptional repressor LacI, characterized in that, The DNA-binding transcription repressor LacI mutant has an amino acid residue substitution at any one of the following positions based on the sequence shown in SEQ ID NO. 10: substituting alanine for valine at position 150; substituting glycine for serine at position 193; substituting isoleucine for glutamine at position 291; substituting valine for glutamine at position 291; substituting histidine for isoleucine at position 79; substituting phenylalanine for glutamine at position 291.

11. A nucleic acid molecule encoding the DNA-binding transcriptional repressor LacI mutant of claim 10.

12. An expression vector comprising the nucleic acid molecule of claim 11.

13. A recombinant cell comprising the nucleic acid molecule of claim 11.

14. Use of the B. subtilis biosensor of any one of claims 7-9, the DNA-binding transcriptional repressor LacI mutant of claim 10, the nucleic acid molecule of claim 11, the expression vector of claim 12, or the recombinant cell of claim 13 for detecting lactose or analogs thereof.

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