Whole-cell biosensor construction based on yersinia quorum sensing system and application thereof

Abstract

The invention belongs to the field of life science, and particularly relates to a yersinia quorum sensing system research method, whole-cell biosensor construction based on the method and application of the whole-cell biosensor. The method comprises the following steps: (1) constructing knockout bacteria and YpsR back-supplementing bacteria of a yersinia pseudotuberculosis quorum sensing system YpsR / YpsI; (2) analysis and screening of the affinity of an N-acyl homoserine lactone (AHL) signal small molecule and a YpsR (Yttrium limacinum) regulatory protein, and (3) analysis and screening of the affinity of the N-acyl homoserine lactone (AHL) signal small molecule and the YpsR regulatory protein; (3) construction of a gene line based on AHL and YpsR regulatory protein; and (4) functional verification of the Y.pstb whole-cell sensor based on AHL detection. The biosensor capable of detecting the AHL generated by the yersinia pseudotuberculosis is constructed by researching a YpsR / YpsI quorum sensing system and a molecular signal recognition and combination mechanism, and more theoretical basis and technical support are provided for prevention, control and treatment of the yersinia pseudotuberculosis.

Description

Technical Field

[0001] The invention belongs to the field of life sciences, and in particular relates to a biosensor and an application thereof in detecting Yersinia pseudotuberculosis. Background Art

[0002] Yersinia pseudotuberculosis (abbreviated as pseudotuberculosis) is a human intestinal pathogenic bacterium. It is a Gram-negative bacterium with a rod-shaped flagella system and motility [1-3]. The bacterium has a wide host range and can be transmitted through contaminated water and food, causing intestinal diseases in mammals and rodents. It is an important zoonotic pathogen. Symptoms of human infection include fever, headache, abdominal pain, and vomiting, which can cause gastrointestinal diseases such as enteritis and mesenteric lymphadenitis in the host. Yersinia pseudotuberculosis is an imported pathogen and poses a potential threat to human health. In Finland and Japan, there have been many cases of Yersinia pseudotuberculosis infection [4-6].

[0003] Bacteria use quorum sensing systems (QS) to regulate the expression of genes related to virulence, invasion, and symbiosis, which is crucial in the interaction between bacteria and hosts, improving their own survival and pathogenicity [7,8]. Therefore, the quorum sensing system is a key factor affecting the pathogenicity of pathogens, and it is of great significance to analyze the cell signal selection and transmission mechanism of the quorum sensing system of Yersinia [9-12]. There are two luxR / luxI type QS systems in Yersinia pseudotuberculosis, namely ytbI / ytbR and ypsI / ypsR [8]. YtbI and YpsI encoded by the ytbI and ypsI genes of Yersinia pseudotuberculosis are responsible for the synthesis of signal molecules as LuxI-type synthetases, and YtbR and YpsR are transcriptional regulatory proteins that can respond to N-acyl homoserine lactones (Acyl-homoserine lactones, AHL) signal molecules, thereby regulating the transcription of downstream target genes. Among them, YpeI and YspI synthetases are responsible for the synthesis of at least 24 AHLs, including N-3-oxo-hexanoyl homoserine lactone (3-oxo-C6-HSL, 3OC6) (molecular formula: C10H15NO4), and YspI synthetase is responsible for the synthesis of hexanoyl-L-homoserine lactone (C6-HSL) (molecular formula: C10H17NO3), octanoyl-L-homoserine lactone (C8-HSL) (molecular formula: C12H21NO3), N-3-oxo-octanoyl homoserine lactone (3-oxo-C8-HSL, 3OC8) (molecular formula: C12H19NO4), etc. [13-15]. YpsR, a regulatory protein of the LuxR family of the Yersinia pseudotuberculosis quorum sensing system, is a signal transduction hub protein of the quorum sensing system, regulating the expression of downstream proteins, thereby affecting the entire quorum sensing process.

[0004] Biosensors have been widely used in the detection of a variety of special signal molecules, and then for efficient screening of specific strains. By coupling high-throughput screening devices to establish corresponding small molecule metabolite screening technology, it can provide important guarantees for the rapid detection and screening of pathogens. However, there are currently no reports on AHL biosensors for detecting the production of Yersinia pseudotuberculosis. Due to the lack of simple and fast detection methods, the prevention ability of Yersinia pseudotuberculosis is greatly limited. The present invention analyzes the molecular signal recognition and binding mechanism of YpsR, designs and constructs an AHL whole-cell biosensor with sensitivity and specificity based on YpsR / I and its downstream genes, which is used to detect pathogens such as Yersinia pseudotuberculosis, and provides more theoretical basis and technical support for the prevention, control and treatment of Yersinia.

[0005] References

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[0015]

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[0019]

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[0020]

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[0021]

[18] Zhang W, Xu S, Li J, et al. Modulation of a thermoregulated type VIsecretion system by AHL-dependent quorum sensing in Yersiniapseudotuberculosis. Arch Microbiol, 2011, 193(5): 351-363. Summary of the invention

[0022] The present invention aims to provide an N-acyl homoserine lactone (AHL) biosensor produced by Yersinia pseudotuberculosis, and use the biosensor to detect Yersinia pseudotuberculosis.

[0023] Based on the tuberculosis characteristics of LuxR-type quorum sensing regulatory protein YpsR and AHL, the present invention verifies the binding strength of YpsR with 10 different AHL small molecules through a series of in vitro and in vivo experiments. The invention provides a method for determining the binding strength of YpsR and AHL in vitro and in vivo, and screens out 3OC6 with the strongest binding strength to YpsR protein through in vitro and in vivo experiments.

[0024] The present invention provides an in vitro determination method, which uses surface plasmon resonance (SPR) technology to determine the equilibrium dissociation constants of different YpsR-AHL protein complexes. The binding force between AHL and YpsR is negatively correlated with the equilibrium dissociation constant.

[0025] The present invention provides a method for determining the binding force between YpsR and AHL in vivo. The specific steps are as follows: by constructing a quorum sensing system ypsI / ypsR knockout bacteria and ypsR complementing bacteria, culturing the complementing strain, adding AHL molecules of different concentration gradients during the culturing process, and measuring the OD after a certain period of time. 600 The values ​​were compared with the OD of the control group without AHL. 600 Compare and calculate the bacterial OD 600 Down-regulation rate. Effects of different AHLs on bacterial growth status (OD 600 ) is proportional to the binding strength of YpsR and AHL, and is directly proportional to the bacterial OD 600 The downward adjustment rate is inversely proportional.

[0026] In a specific embodiment, gene editing technology is used to construct a quorum sensing system ypsI / ypsR knockout bacteria and ypsR complement bacteria, and after obtaining the culture suspension, a set of YpsR-AHL interaction research methods without leakage interference is formed.

[0027] According to the binding characteristics of YpsR and AHL, the present invention selects transcriptional regulatory factors that can respond to AHLs with similar structures as candidate elements, and first provides a method for constructing an AHL biosensor based on the transcriptional regulatory factor YpsR, which can convert the concentration of AHLs produced by Yersinia pseudotuberculosis into a fluorescence intensity signal. The biosensor is composed of a transcriptional regulatory factor YpsR encoding gene derived from Yersinia pseudotuberculosis, a Pyps6 promoter region, a gfp marker gene, and a plasmid backbone. The biosensor provided by the present invention can specifically respond to AHLs produced by Yersinia pseudotuberculosis, and exhibits good sensitivity under the condition of a ratio of biosensor recombinant bacteria: Yersinia pseudotuberculosis = 1000:1.

[0028] The present invention provides an AHL biosensor based on the transcriptional regulatory factor YpsR, which includes a LuxR-type quorum sensing regulatory protein (such as a transcriptional regulatory factor YpsR protein encoding gene or a homologous gene thereof, such as a homologous gene derived from a gene encoding a YpeR protein with the same amino acid sequence in Yersinia), a promoter region (such as a Pyps2 or Pyps6 promoter region or a homologous promoter), a marker gene and a plasmid backbone to form an expression vector.

[0029] Specifically, it contains a constitutive expression promoter such as J23105 promoter and a YpsR protein encoding gene operably linked thereto, and a Pyps2 or Pyps6 promoter and a fluorescent protein GFP gene operably linked thereto.

[0030] Preferably, the gene encoding the transcriptional regulatory factor YpsR is derived from Yersinia pseudotuberculosis, and the promoter region of Pyps2 or Pyps6 is derived from Corynebacterium glutamicum.

[0031] In a specific embodiment, the marker gene is a fluorescent marker gene, preferably a green fluorescent protein GFP gene.

[0032] In another specific embodiment, the expression vector is an expression vector suitable for bacteria, such as an expression vector suitable for expression in Escherichia coli or Yersinia.

[0033] Preferably, the nucleotide sequence of the gene encoding the transcriptional regulatory factor YpsR is as shown in SEQ ID No: 1 or has a sequence identity of more than 90% thereto; the nucleotide sequence of the Pyps6 promoter is as shown in SEQ ID No: 7; and the nucleotide sequence of GFP is as shown in SEQ ID No: 3.

[0034] The present invention provides a recombinant bacterium containing the biosensor, preferably a bacterium, such as the following bacteria:

[0035]

[0036]

[0037] The present invention provides the use of the biosensor or the recombinant bacteria in preparing a reagent for detecting AHL produced by Yersinia. The Yersinia is Yersinia pseudotuberculosis, Yersinia pestis or Yersinia enterocolitica

[0038] The present invention further provides a method for detecting AHL produced by Yersinia pseudotuberculosis, which comprises the following steps: mixing the biosensor or the recombinant bacteria with a sample to be detected, the intensity of the labeling signal such as the fluorescence signal represents the concentration of AHL produced by the sample to be detected, and the fluorescence intensity is higher than the fluorescence intensity of the control group, then it is determined that the target strain is detected; preferably, the AHL is 3OC6. Preferably, the control group is a recombinant bacterium containing a biosensor, such as Escherichia coli; the Yersinia is Yersinia pseudotuberculosis, Yersinia pestis or Yersinia enterocolitica.

[0039] Specifically, the sample is human or animal body fluid, urine, blood, feces; food, feed sample; or sample from the environment, such as water, soil, waste; or culture or bacterial liquid of the bacteria to be detected.

[0040] The present invention has the following advantages: the protein translated by the transcriptional regulatory factor utilized in the present invention can form a protein complex with AHL, the protein complex activates the expression of downstream genes by binding to promoter regions such as the Pyps6 promoter, and the expression of AHL and the intensity of the fluorescence signal are combined to realize sensitive detection of Yersinia pseudotuberculosis, thus providing a theoretical basis and technical support for the prevention, control and treatment of Yersinia. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 .YpsR protein expression and purification verification. Among them, (A) is the SDS-PAGE verification result of YpsR (1-179); (B) is the verification result of size exclusion, and (C) is the verification result of liquid chromatography-mass spectrometry.

[0042] Figure 2 In vitro experiments verified the affinity of YpsR to 10 AHL small molecules. Among them; (AJ) is the surface plasmon resonance characterization of the binding of 10 AHL small molecules to YpsR 1-179 residues; (K) is the correlation between the experimental and predicted binding affinities calculated using the Vina program.

[0043] Figure 3 .Schematic diagram of the structures of 10 AHL small molecules.

[0044] Figure 4 .Interaction and signal transduction ability of YpsR / YpsI with AHL small molecules. Among them, (A) is a schematic diagram of YpsR / YpsI gene knockout; (B) is the use of primers to verify the knockout of ypsR / ypsI gene and ytbR / ytbI gene. In the figure, WT represents the amplification product of Y.pseudotuberculosis wild-type strain, and WC represents the amplification product of water control; (C) is the wild-type, ΔYpsR / YpsI and ΔYpsR / YpsI +ypsR (D) shows the growth curve of ypsR complementation bacteria (ΔYpsR / YpsI) under different concentrations of 3OC6. +ypsR ) growth; (E) under the conditions of 10 AHL small molecules, ypsR complementation bacteria (ΔYpsR / YpsI +ypsR ) growth reduction rate; (F) is the inhibitory effect on three bacteria under the condition of 10-5M concentration of 3OC6, 6 hours; (G, H) is the inhibitory effect on ypsR complementary bacteria (ΔYpsR / YpsI) under the condition of 10-5M concentration of 3OC6. +ypsR ) plate count experiment. (n=3**P=<0.001,***P=<0.01).

[0045] Figure 5.Effects of different concentrations of ten AHLs on the growth of ypsR-rescued bacteria (ΔYpsR / YpsI+ypsR).

[0046] Figure 6 .Gene circuit design and mutation site simulation based on YpsR. (A) is the design and synthesis of gene circuits using components from the biological standard component library, with YpsR as the sensor protein and GFP as the reporter protein; (B) is the activation effect of 10-5M 3OC6 on 10 whole-cell biosensors; (C) is the activation effect of 10 AHLs on whole-cell biosensors based on Pyps6.

[0047] Figure 7 .Application of biosensors based on YpsR-AHL. Wherein, (A) is a schematic diagram of the co-culture of the biosensor based on YpsR-AHL and Y.PSTB; (B) is the relative fluorescence intensity of the biosensor after co-culture with Y.PSTB and ΔyspR / I for 6 hours; (C) is the relative fluorescence intensity of the biosensor after co-culture with Yersinia enterocolitica, Salmonella typhimurium and Klebsiellapneumoniae for 6 hours; (D) is the relative fluorescence intensity of the biosensor after co-culture with Y.PSTB and ΔYpsR / YpsI for 9 hours; (E) is the relative fluorescence intensity of the biosensor after co-culture for 9 hours. DETAILED DESCRIPTION

[0048] The present invention is described in more detail below in conjunction with specific embodiments, which is for a better understanding of the present invention but does not constitute a limitation of the present invention.

[0049] Example 1: In vitro experiments to verify the affinity of YpsR to AHL

[0050] In this example, the YpsR gene was obtained by gene synthesis, and an expression vector was constructed using pMAT9s. The MBP tag protein at the N-terminus of the vector was used for affinity purification, and the expression was optimized in E. coli, and stable proteins were separated and purified. In this example, the 1-179 amino acid sequence of YpsR was selected, and its N-terminus was connected to the MBP tag and the C-terminus was connected to the His tag to obtain a stable YpsR protein. In addition, we analyzed the purified protein based on LC-MS for the natural intact protein mass (LC-MS Intact Mass Analysis) and peptide mass fingerprinting (Peptide mass fingerprinting), and further confirmed that we had obtained a complete YpsR 1-179 high-purity protein ( Figure 1 ).

[0051] Among them, the YpsR protein sequence is as follows (SEQ ID NO: 1):

[0052] MIINFFDNESINEDIKNYIQRRIKAYGNIRYSYLLMNKKVPLHPAIISNYPLDWVKKYKKNSYHLIDPVILTAKGK VAPFAWDDNSVINIKSTDSAVFNLAREYNIVNGYTFVLHDNNNNMATLNVSSGDDDSIFFDESIEVNKEKIQMLLIFIHD KMLGLYNKSHHENNTLNKKENKR。

[0053] Among them, the sequence of the YpsR protein with an MBP tag linked to the N-terminus and a His tag linked to the C-terminus is as follows (SEQ ID NO: 2):

[0054] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTMIINFFDNESINEDIKNYIQRRIKAYGNIRYSYLLMNKKVPLHPAIISNYPLDWVKKYKKNSYHLIDPVILTAKGKVAPFAWDDNSVINIKSTDSAVFNLAREYNIVNGYTFVLHDNNNNMATLNVSSGDDDSIFFDESIEVNKEKIQMLLIFIHDKMLGLYNKSHHENNTLNKKENKRHHHHHHHH。

[0055] In this study, the rigid maltose binding protein (MBP) technology was used to produce a stable, soluble Y. pseudotuberculosis MBP-YpsR (residues 1–179, N-terminal ligand binding domain) and to measure the activity of 10 AHL small molecules (whose structures are shown in Figure 2 ) The equilibrium dissociation constant of YpsR fused to MBP. The equilibrium dissociation constant is negatively correlated with the affinity of AHL small molecules.

[0056] Specifically, in this example, a rigid helix was attached to the C-terminus of MBP. This helix limits the flexibility of the attached recombinant protein, thereby improving its solubility. The equilibrium dissociation constants of the 10 AHLs are shown below, among which 3OC6 has the lowest equilibrium dissociation constant (1.3M), indicating that 3OC6 has the strongest affinity for the YpsR regulatory protein, which is consistent with the results of the previous in vivo experiment that 3OC6 has the best antibacterial effect ( Figure 3 ).

[0057] Table 1

[0058]

[0059] Example 2: YpsR / YpsI quorum sensing system regulates bacterial growth

[0060] In this example, the CRISPR / Cas9 system was used to knock out the quorum sensing systems YpsR / YpsI and YtbR / YtbI in Yersinia pseudotuberculosis. However, YtbR / YtbI was not found in colony PCR identification. Therefore, a wild-type strain and a YpsR / YpsI knockout strain (ΔYpsR / YpsI) were constructed, and on this basis, a YpsR complement strain (ΔYpsR / YpsI) was constructed. +ypsR ). The growth curves of the wild type, knockout bacteria, and complemented bacteria were tested. All strains were able to grow in order to observe the regulatory effect of the quorum sensing system on bacterial growth. The growth conditions of the knockout strain and the complemented strain were similar. Both reached the logarithmic growth phase in 4 hours and entered the growth plateau phase after 7.5 hours, while the wild type reached the logarithmic growth phase in 5.5 hours and entered the growth plateau phase after 8.5 hours. This shows that the knockout of the YpsR / YpsI gene can promote the growth activity of the pseudoconjugated bacteria themselves, confirming that the YpsR / YpsI system does affect the growth process of Y.pstb ( Figure 4 Middle C).

[0061] On this basis, this example will complement the strain (ΔYpsR / YpsI +ypsR) were added to LB liquid medium containing 10 common AHLs small molecules (3OC6, 3OC8, 3OC10, 3OC12, 3OC14, C6, C8, C10, C12 and C14), and the mixture was transferred to a 96-well microplate. The microplate reader was used to culture and observe the complementation bacteria (ΔYpsR / YpsI +ypsR ) at different concentrations of each small molecule (10 -5 ,10 -6 ,10 -7 ,10 -8 ,10 -9 ,10 -10 ,10 -11 ,10 -12 M) in the growth Figure 5 ). The inhibition degree of 10 different AHL small molecules on bacterial growth was determined. -5 The OD of the supplemented bacteria in the 3OC6-added group of M was compared with that in the group without 3OC6. 600 It can reduce 57.13% (P<0.001) ( Figure 4 By comparing the 10 AHLs at the same concentration (10 - 5 The results of the antibacterial effect of 3OC6 after culturing for 6 hours showed that the antibacterial effect of 3OC6 was the highest and its inhibitory effect on the growth of tuberculosis bacteria was significantly better than that of other small molecules ( Figure 4 (E). Further comparison of wild type, ΔYpsR / YpsI and ΔYpsR / YpsI +ypsR In 10 -5 The growth status of the wild type and complementing bacteria at 6 h after culture under the condition of M concentration 3OC6 was found to be more obvious inhibitory effect, while there was no significant difference in the knockout strain ( Figure 4 F), indicating that YpsR / YpsI may regulate bacterial growth activity through its binding with AHLs. To further determine the antibacterial effect of 3OC6, we observed the number of bacteria growing after adding 3OC6 by CFU plate counting experiment. The results are shown in Figure 4 As shown in G and H, at 10 11 and 10 12 Under the dilution multiple, the experimental group with 3OC6 addition was significantly downregulated compared with the control group without 3OC6, with the downregulation rates of 21.99% and 29.55% respectively (P<0.01), further verifying that 3OC6 has a significant inhibitory effect on Yersinia pseudotuberculosis through the quorum sensing system. The difference in the degree of bacterial growth inhibition also reflects the interaction strength between different AHL molecules and YpsR, which is consistent with the previous in vitro biochemical analysis results.

[0062] In this example, the knockout bacteria ΔYpsR / YpsI and ΔYtbR / YtbI were identified by four pairs of primers designed and synthesized, and the wild bacteria and knockout bacteria were amplified by primers YtbR-F1 and YtbR-R1, YtbI-F1 and YtbI-R1, YpsR-F1 and YpsR-R1, YpsI-F1 and YpsI-R1, and the target gene was detected by electrophoresis after colony PCR ( Figure 4 A and B). The primer sequences used in the identification process are as follows:

[0063] YtbR-F1:CATTGTCACTAGCTACCCAAATGAATGGGC,

[0064] YtbR-R1: CCTATGTGATATTTTACTGTTCTGGGGGTG.

[0065] YtbI-F1:CATTGTCACTAGCTACCCAAATGAATGGGC,

[0066] YtbI-R1:CTTAATACTGAACATGGATCCTGAAACGC.

[0067] YpsR-F1:GTACCGCTACATCCGGCGATCATATCGAAC,

[0068] YpsR-R1:GCATGTTTTGGCATTGAGAACACCTAAC.

[0069] YpsI-F1:CGTTTTGGACTGGAAAGTAACCTGTATTG,

[0070] YpsI-R1:CTGCTGTTGGCTATTACTGTCAATGCTC.

[0071] PCR amplification system for plasmid construction: 5uL 2×HieffHotStart PCR Genotyping MasterMix (With Dye), 1uL 10uM upstream primer, 1uL 10uM downstream primer, 1.5uLDMSO, 1uL template, 0.5uLPhusion polymerase, add ddH2O to a final volume of 10ml. PCR amplification conditions: 95℃ pre-denaturation for 5min; 95℃ denaturation for 15s, 60℃ annealing for 15s, 72℃ extension for 30s, 30 cycles; 72℃ extension for 5min.

[0072] The steps of competent culture preparation and transformation are as follows: Pick a monoclonal bacterium from the plate and inoculate it into 5 mL of antibiotic-free LB medium in a 50 mL shake flask, and culture it overnight at 37°C and 220 rpm. The next morning, take 1 ml of the overnight cultured bacterial solution and inoculate it into 50 mL of LB medium. After culturing it at 37°C and 220 rpm for 2 hours, measure the OD 600 Until OD 600 = about 0.6. Collect the cells by centrifugation at 5000rpm, and wash the collected cells with 10ml pre-cooled sterile water for 3 times. Centrifuge at 4℃, 3000g for 2 minutes, remove the supernatant, add 1ml pre-cooled 10% glycerol to resuspend the cells, and dispense the competent cells into 1.5ml sterile EP tubes, 100μL per tube, and store in a -80℃ refrigerator for later use. Add 100ng pBAD-ypeR and pBAD-yspR plasmids to the Yersinia pseudotuberculosis wild type and knockout bacteria ΔYpsR / YpsI competent cells, mix gently, and transfer them to the bottom of the electroporation cup with a pipette, immediately cover the lid and place on ice for 5min, then add 1ml LB medium after electroporation by electroporator, mix gently, and recover at 37℃, 220rpm for 3h, then apply the bacterial solution to the selection plate containing ampicillin antibiotics, and invert and culture at 37℃ until colonies grow. The grown single clones were identified by colony PCR using primers araBAD-F1 and GFP-R1 to screen positive clones.

[0073] The single clones grown after electroporation were identified by colony PCR using primers araBAD-F1 and GFP-R1 to screen positive clones. The results showed that YtbR and Ytbl did not exist in the strain and could not be knocked out. The YspR and YspI genes could not be detected in the knockout strain, indicating that ΔYpsR / YpsI was successfully constructed.

[0074] Example 3: Construction of a biosensor plasmid based on YpsR-AHL

[0075] In this embodiment, each component module of the biosensor based on YpsR-AHL is amplified by PCR to obtain nucleotide fragments, and each fragment is connected in a certain order by molecular cloning technology to complete the construction of the biosensor plasmid based on YpsR-AHL. Among them, the biosensor is composed of the transcriptional regulatory factor YpsR encoding gene derived from Yersinia pseudotuberculosis (Genebank: YP 071012.1 for the complete amino acid sequence, Genebank NC_006155.1 for the nucleotide sequence), the PypsX promoter region, the marker gene and the plasmid backbone. In this gene circuit, the conventional promoter J23105 controls the expression of ypsR, PypsX is regulated by YpsR, and the gfp gene is driven by the PypsX promoter. The fluorescence level of the FP product indicates the activity of this regulated promoter ( Figure 6(A).

[0076] The nucleotide sequence of the ypsR encoding gene can be referenced to Genebank NC_006155.1, and the amino acid sequence is shown in SEQ ID NO:1.

[0077] The nucleotide sequences of the 10 PypsX promoters are as follows:

[0078]

[0079]

[0080]

[0081] The specific steps are as follows: Plasmid construction was assembled according to the BioBrick RCF standard using a variety of molecular cloning techniques. Biological components were obtained from the standard biological component registry or by chemical synthesis. As the YpsR transmitter module, the orthostatic promoter PCon (BBa_J23105) and the ribosome binding site (RBS; BBa_B0034) were inserted upstream of the synthetic Yersinia pseudotuberculosis ypsR gene. As the YpsR receiver module, the RBS (BBa_B0034)–GFP (BBa_E0040)–terminator (BBa_B0015) was inserted downstream of PypsR [a YpsR-HSL-regulated promoter (BBa_C0062)]. These modules were assembled into the pSB1A3 vector.

[0082] In this example, the sensitivity of 10 PypsX promoter-based gene circuits to 3OC6-AHL was tested. The specific method was as follows: 10 PypsX promoter-based gene circuits were assembled into plasmid pSB1A3, and the plasmid was transformed into Yersinia pseudotuberculosis by electroporation (Voltage = 2500v, Capacity = 25μF, External resistance = 200Ω, Time = 5ms), 37°C, 220rpm shaking culture for 1h, 4500rpm centrifugation for 5min to obtain colony precipitation, and 200μL fresh LB medium (tryptone 10g / L; yeast extract 5g / L; sodium chloride 10g / L) was resuspended and coated on an LB solid plate containing a final concentration of 100μg / ml ampicillin, and incubated at 37°C for 14h to prepare 10 recombinant bacteria containing PypsX promoter gene circuits. Pick 10 monoclonal colonies of recombinant bacteria and inoculate them into LB liquid medium (tryptone 10 g / L; yeast extract 5 g / L; sodium chloride 10 g / L) containing a final concentration of 100 μg / mL ampicillin, and culture overnight at 37°C, 220 rpm for 12 h. Inoculate the above overnight culture solution into fresh LB liquid medium without antibiotics at a ratio of 1:100, and add a final concentration of 10 -5 M 3OC6, under the culture conditions with and without 3OC6, 37°C, 220rpm shaking culture for 6h, the relative fluorescence intensity of 10 recombinant bacteria was tested, thereby determining the effectiveness of 10 PypsX promoter-based sensors. Among them, the sensor based on the Pyps6 promoter showed the highest fluorescence intensity in the presence of AHL molecules, although the baseline fluorescence was slightly higher than that of other promoters in the absence of AHL. In addition to Pyps6, the sensor based on the Pyps2 promoter also showed significant AHL sensing ability, but its absolute fluorescence intensity was lower than that of Pyps6 ( Figure 6 (middle B).

[0083] Therefore, we further used the Pyps6-based sensor to detect different types of AHL. Only 3OC6 could significantly induce GFP expression, increasing the fluorescence intensity by about 50% compared to the absence of AHL. Other types of AHL increased the fluorescence intensity by less than 20% at most, and the average increase was less than 10% (C6, Figure 6 Therefore, we used a plasmid containing the Pyps6 promoter gene circuit to transform into the engineering model strain E. coli DH5α by heat transformation to construct an AHL biosensor.

[0084] Example 4: Functional verification of Y.pstb whole-cell sensor based on AHL detection

[0085] In order to verify the sensitivity and specificity of the biosensor constructed in Example 3, the Pyps6 plasmid described in Example 3 was transferred into E. coli DH5α to obtain recombinant bacteria containing AHLs biosensors. Single clone colonies were picked and inoculated into LB liquid culture medium (tryptone 10 g / L; yeast extract 5 g / L; sodium chloride 10 g / L) containing a final concentration of 100 μg / mL ampicillin, and cultured overnight at 37°C and 220 rpm for 12 h. At the same time, single colonies of wild-type Yersinia pseudotuberculosis, knockout bacteria ΔYpsR / YpsI, Yersinia enterocolitica, Salmonella typhimurium and Klebsiella pneumoniae were picked and inoculated into fresh LB liquid culture medium without antibiotics, and cultured overnight at 37°C and 220 rpm for 12 h. The above five overnight culture solutions were inoculated into fresh LB liquid medium without antibiotics at a ratio of 1:100, and cultured at 37°C, 220 rpm, with shaking until OD 600 =1. The recombinant bacteria were mixed with wild-type Yersinia pseudotuberculosis, knockout bacteria ΔYpsR / YpsI, Yersinia enterocolitica, Salmonella typhimurium and Klebsiella pneumoniae at a ratio of 1000:1, and mixed cultured at 37°C and 220rpm. The mixed bacterial liquids at 6h and 9h were collected and diluted with 1x PBS buffer. The recombinant bacteria 1000:1 LB culture medium was used as a control. The relative fluorescence intensity of each group of mixed bacterial liquids was measured by flow cytometry. The fluorescence intensity curve was drawn with the relative fluorescence intensity as the horizontal axis and the number of bacteria as the vertical axis, and the fluorescence intensity bar graph was drawn with the number of bacteria as the horizontal axis and the relative fluorescence intensity as the vertical axis.

[0086] The results showed that the biosensor could detect the AHLs signal molecules produced by Yersinia pseudotuberculosis at a concentration ratio of 1000:1, and the recombinant strain containing the biosensor had a correspondingly increased fluorescence intensity. At the same time, the functional verification determined the response of the biosensor or the recombinant strain containing the biosensor to other intestinal microorganisms. At a concentration ratio of 1000:1, Yersinia enterocolitica, Salmonella typhimurium, and Klebsiella pneumoniae did not cause significant changes in the fluorescence intensity of the host bacteria, indicating that the biosensor has good specificity ( Figure 7 ).

Claims

1. An AHL biosensor based on the transcriptional regulatory factor YpsR, characterized in that: It includes (1) LuxR-type quorum sensing regulatory protein (such as transcriptional regulatory factor YpsR protein encoding gene or its homologous gene), (2) promoter region (such as Pyps2 or Pyps6 The expression vector is composed of (1) a promoter region or a homologous promoter), (2) a marker gene and (3) a plasmid backbone.

2. The biosensor according to claim 1, wherein It contains a constitutive expression promoter such as J23105 A promoter and a YpsR protein encoding gene or a homologous gene operably linked thereto, and Pyps2 or Pyps6 The promoter is operably linked to a fluorescent protein GFP gene.

3. The biosensor according to claim 1, wherein The transcriptional regulatory factor YpsR encoding gene is derived from Yersinia pseudotuberculosis, Pyps2 or Pyps6 The promoter region is derived from Corynebacterium glutamicum.

4. The biosensor according to claim 1, wherein The marker gene is a fluorescent marker gene, preferably a green fluorescent protein GFP gene.

5. The biosensor according to any one of claims 1 to 4, characterized in that: The expression vector is an expression vector suitable for expression in Escherichia coli or Yersinia pseudotuberculosis.

6. The biosensor according to claim 5, characterized in that The nucleotide sequence of the gene encoding the transcriptional regulatory factor YpsR is as shown in SEQ ID No: 1 or has a sequence identity of more than 90% thereto; Pyps6 The nucleotide sequence of the promoter is shown in SEQ ID No: 7; the nucleotide sequence of GFP is shown in SEQ ID No:

3.

7. The recombinant bacteria containing the biosensor according to any one of claims 1 to 6, preferably bacteria, for example, Yersinia pseudotuberculosis, Yersinia pestis, Yersinia enterocolitica, Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, Salmonella enterica, Vibrio fischeri, Vibrio harveyi, Pseudomonas aeruginosa, Pseudomonas putida, Aeromonas hydrophila, Vibrio anguillarum, Erwinia spp., Sinorhizobium alfalfa, Burkholderia glanders, Chromobacterium violaceum, Citrobacter rodentium, Pectobacterium carotovora or Rhizobium leguminosarum.

8. Use of the biosensor according to any one of claims 1 to 6 or the recombinant bacteria containing the biosensor according to claim 7 in the preparation of a reagent for detecting AHL produced by bacteria; The bacterium is, for example, a bacterium, such as Yersinia pseudotuberculosis, Yersinia pestis, Yersinia enterocolitica, Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, Salmonella enterica, Vibrio fischeri, Vibrio harveyi, Pseudomonas aeruginosa, Pseudomonas putida, Aeromonas hydrophila, Vibrio anguillarum, Erwinia spp., Sinorhizobium meliloti, Burkholderia glanders, Chromobacterium violaceum, Citrobacter rodentium, Pectobacterium carotovora or Rhizobium leguminosarum; preferably, the Yersinia is Yersinia pseudotuberculosis, Yersinia pestis or Yersinia enterocolitica.

9. A method for detecting AHL produced by Yersinia, characterized in that: The method comprises the following steps: mixing the biosensor according to any one of claims 1 to 6 or the recombinant bacteria containing the biosensor according to claim 7 with a sample to be detected, wherein the intensity of a marker signal such as a fluorescent signal represents the concentration of AHL produced by the test sample, and if the fluorescent intensity is higher than the fluorescent intensity of a control group, it is determined that the target strain is detected; Preferably, the control is a recombinant bacterium containing a biosensor, and the recombinant bacterium is Escherichia coli; Preferably, the AHL is 3OC6; and the Yersinia is Yersinia pseudotuberculosis, Yersinia pestis or Yersinia enterocolitica.

10. The method according to claim 9, characterized in that The sample is human or animal body fluid, urine, blood, feces; food, feed sample; or sample from the environment, such as water, soil, waste; or culture or bacterial liquid of the bacteria to be detected.

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