Multi-effect biosensor for detecting trimethylamine-n-oxide and pentaquadithionate and application thereof

Abstract

The present application belongs to the field of genetic engineering and biological detection and analysis technology, and particularly relates to a multi-effect biosensor for detecting oxidized trimethylamine and tetrathionate and application thereof. Specifically, the inventors found different physiological reaction characteristics of Vibrio parahaemolyticus to exogenous oxidized trimethylamine (TMAO) and tetrathionate, which can be used for detecting potential blood biomarker TMAO and specifically detecting intestinal inflammation biomarker tetrathionate without being interfered by other sulfur-containing compounds. The multi-effect biosensor has ultra-high sensitivity, and has the advantages of simple and rapid operation and independence on precise instruments.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and biological detection and analysis technology. Specifically, this invention relates to a multifunctional biosensor for detecting trimethylamine oxide and tetrathionine sulfate and its application. Background Technology

[0002] The gut microbiota metabolizes nutrients such as phosphatidylcholine, choline, carnitine, betaine, and creatinine from foods like red meat, eggs, and milk, producing trimethylamine. Trimethylamine can enter the liver via the portal circulation and is oxidized by flavin monooxygenase 3 (FMO3) or other flavin monooxygenases (FMOx) to produce trimethylamine-N-oxide (TMAO). TMAO is closely related to the occurrence and development of various diseases, including cardiovascular disease, myocardial infarction, stroke, diabetes, chronic kidney disease, and cancer, and may serve as a potential biomarker for the diagnosis, prognosis, and treatment intervention of these diseases. Therefore, the detection of TMAO can provide important reference for the diagnosis and prediction of related diseases, etiological and pathological research, and efficacy evaluation.

[0003] The two-component system TorSR consists of the histidine kinase sensor TorS and its homologous response regulator TorR. Its function typically also depends on the ectoplasmic binding protein TorT, which actually binds TMAO (reference available on PubMed with PMID: 17040909). TorS is a ternary hybrid sensing kinase with a histidine kinase, a receptor, and a histidine-containing phosphate transfer domain. TorT has a high affinity for TorS and can act as a core receptor, binding to TorS to form a sensor complex (TorT-TorS). Bacteria can use TorT-TorS to sense TMAO in the external environment, phosphorylating its own histidine residues and transferring the phosphate group to the response regulator TorR, which has both a receptor domain and a DNA-binding domain. This causes TorR to dimerize, further activating downstream target genes. torCAD Expression to produce TMAO metabolic enzymes. It is known in the prior art that in *E. coli*, torCAD It is located in one operator and has one promoter (the reference is available on the PubMed website via PMID: 10648521).

[0004] Furthermore, the gut microbiota of mammals can metabolize and produce large amounts of hydrogen sulfide (H2S), which is detoxified by the host intestinal mucosa, converting it into thiosulfate. Previous studies have found that after infection with enteropathogenic bacteria such as Salmonella typhimurium, which induce intestinal inflammation, thiosulfate can be oxidized to tetrathionate through a host inflammatory cytokine storm (reference available on PubMed with PMID: 20864996). Enteropathogenic bacteria have a significantly stronger ability to metabolize tetrathionate through respiration than the gut microbiota, thus gaining a colonization advantage in the gut. Therefore, the level of tetrathionate in the mammalian gut is positively correlated with intestinal inflammation. The two-component system TtrSR consists of a histidine kinase sensor TtrS and its homologous response regulator TtrR. TtrS senses exogenous tetrathionate and phosphorylates its own histidine residues, transferring the phosphate group to the response regulator TtrR, which has both a receiver domain and a DNA-binding domain. This causes TtrR to dimerize and further activate downstream target genes. ttrBCA and tsdBA Expression to participate in bacterial sulfur metabolism (references are available on PubMed via PMID: 39038066).

[0005] Currently, the detection of TMAO levels in samples is mainly based on ultra-high performance liquid chromatography-mass spectrometry (UHLC-MS), however, this method is expensive and time-consuming. Biosensors, which can capture target signals and convert them into specific outputs, have become one of the cutting-edge detection methods for disease and biomarker research. Researchers have constructed a biosensor capable of detecting TMAO levels in samples by utilizing the E. coli two-component system TorSR, the accessory protein TorT, and its target gene, combined with the green fluorescent protein (GFP) gene (see patent applications CN118773094A and CN118773095A). However, this method relies on sophisticated instruments such as fluorescence microscopy and flow cytometry, and its biosensor, built on an E. coli chassis, can only detect a single exogenous molecule, TMAO, and its detection sensitivity is generally limited. Furthermore, there are no reports of using multifunctional biosensors to detect multiple biomarkers (especially trimethylamine oxide and tetrathionine). Developing a biosensor that does not rely on sophisticated instruments and can simply and rapidly detect multiple biomarkers would be more practical. Summary of the Invention

[0006] Based on existing technology, the inventors have for the first time discovered that the two-component system TorSR in Vibrio parahaemolyticus can promote P in sensing the exogenous disease marker trimethylamine oxide (TMAO, which is usually found in the blood). torD With PtorCA (Also known as "P") torC The promoter activity of the two-component system TtrSR is inhibited when sensing sulfur-containing compounds (e.g., tetrathionate (Tet), thiosulfate (Thi), and sulfate (Sul), which are commonly found in the gut). torD With P torCA (Also known as "P") torC "Promoter activity." Based on this, the inventors incorporated the aforementioned two promoter sequences into the fluorescent reporter plasmid pBBR- lux Two recombinant fluorescent reporter plasmids P were obtained. torD - lux With P torC - lux The recombinant fluorescent reporter plasmid was introduced into Vibrio parahaemolyticus, so that the resulting modified Vibrio parahaemolyticus could detect both TMAO in the blood and sulfur-containing compounds in the intestine, thus serving as a multifunctional biosensor.

[0007] Furthermore, the inventors also discovered for the first time that in the absence of Vibrio parahaemolyticus... tsdBA Gene (Δ) tsdBA After that, it can retain both TMAO and Tet pairs for P. torD With P torCA The original influence of promoter activity can also be used to specifically detect the potential inflammatory marker Tet, without being affected by other sulfur-containing compounds besides Tet.

[0008] Specifically, the inventors discovered for the first time that, in addition to Escherichia coli and Salmonella, Vibrio parahaemolyticus also possesses the two-component system TorSR and its related signaling regulatory pathways for sensing exogenous TMAO molecules, thereby enhancing the downstream target genes. torCAD The promoter activity of this enzyme promotes the expression of downstream target genes to generate TMAO metabolic enzymes. However, in Vibrio parahaemolyticus, the downstream target genes encoding TMAO metabolic enzymes are... torD and torCA Located in different operands, they are therefore controlled by different promoters P. torD With P torCA Regulation. In other words, the two-component system TorSR in Vibrio parahaemolyticus can also sense exogenous TMAO molecules, thereby simultaneously increasing the promoter P... torD With P torCAThe activity of the compound is enhanced, and the expression of its corresponding target genes is promoted. Furthermore, the applicant has discovered for the first time that sulfur-containing compounds can inhibit the TorSR two-component system and its signaling pathway in Vibrio parahaemolyticus, particularly the intestinal inflammatory signaling factor tetrathionate (Tet). Specifically, another two-component system in Vibrio parahaemolyticus, TtrSR, can sense exogenous sulfur-containing compounds (e.g., Tet) and competitively inhibit the TorSR signaling pathway, thereby reducing the promoter P... torD With P torCA (Also known as "P") torC It can inhibit the activity of ) and suppress the expression of the corresponding target genes it regulates.

[0009] Furthermore, Vibrio parahaemolyticus can utilize the two-component system TtrSR and its target genes. ttrBAC and tsdBA Regulating sulfur metabolism promotes intestinal colonization of Vibrio parahaemolyticus (reference available on PubMed via PMID: 39038066), and the inventors have also discovered for the first time that in the absence of Vibrio parahaemolyticus... tsdBA Gene (Δ) tsdBA After the pathogenicity is weakened, it not only retains the TMAO molecule's effect on the promoter P torD With P torCA The promoting effect of activity and the effect of tetrathionate on promoter P torD With P torCA The inhibitory effect on activity, and on this basis, further exclusion of other sulfur-containing compounds besides tetrathionate from the promoter P torD With P torCA The effect on activity. Therefore, the inventors based their work on the Vibrio parahaemolyticus promoter P... torD With P torCA The different reactivity of TMAO with tetrathionine was studied using the promoter P. torD and / or P torCA By combining it with a fluorescent reporter gene, Vibrio parahaemolyticus was designed as a multifunctional biosensor that can be used to detect two disease or inflammatory markers: trimethylamine oxide and tetrathionine sulfate.

[0010] In this regard, the present invention includes, but is not limited to, the following: In one aspect, the present invention provides a multifunctional biosensor comprising genetically engineered bacteria, the genetically engineered bacteria containing a two-component system TorSR and a gene encoding the accessory protein TorT, the two-component system TorSR, and a promoter P torD and / or P torCA Regulated reporter genes.

[0011] In one aspect, the present invention provides a method for preparing a multifunctional biosensor, the method comprising: modifying bacteria to contain a two-component system TorSR and a gene encoding the accessory protein TorT, the two-component system TorSR and a gene encoded by the promoter P torD and / or P torCA Regulated reporter genes.

[0012] In one aspect, the bacteria of the present invention are selected from Salmonella, Escherichia coli and Vibrio parahaemolyticus, preferably Vibrio parahaemolyticus, more preferably Vibrio parahaemolyticus RIMD 2210633.

[0013] In one aspect, the bacteria described in this invention are Vibrio parahaemolyticus.

[0014] In one aspect, the bacteria described in this invention are Vibrio parahaemolyticus RIMD 2210633.

[0015] In one aspect, the present invention provides a multifunctional biosensor comprising genetically engineered Vibrio parahaemolyticus, the genetically engineered Vibrio parahaemolyticus containing a promoter P torD and / or P torCA Regulated reporter genes.

[0016] In one aspect, Vibrio parahaemolyticus contains an exogenous plasmid that includes the promoter P. torD and / or P torCA And the reporter genes regulated by them.

[0017] In one aspect, in the gene sequence of Vibrio parahaemolyticus, at the promoter P torD and / or P torCA The downstream of the gene and the upstream of the start codon of the gene it regulates contain reporter genes.

[0018] In one aspect, the present invention introduces a foreign plasmid into Vibrio parahaemolyticus, the plasmid containing the promoter P. torD and / or P torCA And the reporter genes regulated by them.

[0019] In one aspect, the present invention relates to the genome sequence of Vibrio parahaemolyticus, specifically to the promoter P... torD and / or P torCA A reporter gene is inserted between the downstream of the gene and the upstream of the start codon of the gene it regulates.

[0020] In one aspect, the exogenous plasmid of the present invention is a pBBR-lux plasmid, preferably, the empty plasmid sequence of which is shown in SEQ ID NO: 1.

[0021] In one aspect, in this invention, a single P torDPromoters regulate reporter genes.

[0022] In one aspect, in this invention, a single P torCA Promoters regulate reporter genes.

[0023] In one aspect, in this invention, P torD and P torCA Promoters regulate reporter genes.

[0024] In one aspect, the promoter P described in this invention torCA The nucleotide sequence is shown in SEQ ID NO: 3, and / or the promoter P is described. torD The nucleotide sequence is shown in SEQ ID NO: 4.

[0025] In one aspect, the promoter P described in this invention torCA The nucleotide sequence is shown in SEQ ID NO: 3.

[0026] In one aspect, the promoter P described in this invention torD The nucleotide sequence is shown in SEQ ID NO: 4.

[0027] In one aspect, the Vibrio parahaemolyticus of the present invention is further described as knockout. tsdBA Vibrio parahaemolyticus of the gene, preferably, the tsdBA The gene sequence is shown in SEQ ID NO: 6.

[0028] In one aspect, the reporter gene of the present invention is a fluorescent protein gene, preferably, a fluorescent protein gene as shown in SEQ ID NO: 2. And / or, the amino acid sequence of the accessory protein TorT is shown in SEQ ID NO: 7.

[0029] In one aspect, the reporter gene of the present invention is a fluorescent protein gene, preferably a fluorescent protein gene as shown in SEQ ID NO: 2.

[0030] In one aspect, the reporter gene of the present invention is a gene for a fluorescent protein as shown in SEQ ID NO: 2.

[0031] In one aspect, the amino acid sequence of the accessory protein TorT described in this invention is shown in SEQ ID NO: 7.

[0032] In one aspect, the gene encoding the accessory protein TorT described in this invention is shown in SEQ ID NO: 8.

[0033] In one aspect, the present invention provides the application of the multi-effect biosensor according to the present invention in one or more of the following: (1) Detect trimethylamine oxide or sulfur-containing compounds in the sample to be tested; (2) Prepare products for detecting trimethylamine oxide or sulfur-containing compounds; (3) Prepare a kit for diagnosing or assisting in the diagnosis of diseases related to trimethylamine oxide or sulfur-containing compounds; preferably, the sulfur-containing compound is selected from tetrathionate, thiosulfate and sulfate, more preferably, the sulfur-containing compound is tetrathionate.

[0034] In one aspect, in the application of the present invention, when detecting trimethylamine oxide, the sample to be tested is selected from blood, serum, saliva, and urine; When detecting sulfur-containing compounds, the test sample is selected from intestinal contents and feces.

[0035] In one aspect, in the application of the present invention, when detecting trimethylamine oxide, the sample to be tested is selected from blood, serum, saliva, and urine.

[0036] In one aspect, in the application of the present invention, when detecting sulfur-containing compounds, the sample to be tested is selected from intestinal contents and feces.

[0037] In one aspect, in applications of the present invention, a test sample containing trimethylamine oxide or a sulfur-containing compound is brought into contact with the multifunctional biosensor described in the present invention. For example, a test sample containing trimethylamine oxide or a sulfur-containing compound is added to a culture medium of Vibrio parahaemolyticus modified as a multifunctional biosensor according to the present invention.

[0038] In one aspect, in this invention, when the test sample contains trimethylamine oxide, the signal of the reporter gene / fluorescent protein in Vibrio parahaemolyticus is stronger compared to the blank control (e.g., Vibrio parahaemolyticus culture that does not contain the test sample / trimethylamine oxide / sulfur-containing compounds).

[0039] In one aspect, in this invention, when the test sample contains trimethylamine oxide, the signal of the reporter gene / fluorescent protein in Vibrio parahaemolyticus is weaker compared to a blank control (e.g., a culture of Vibrio parahaemolyticus that does not contain the test sample / trimethylamine oxide / sulfur-containing compounds).

[0040] In one aspect, in the application of the present invention, the minimum detectable concentration of trimethylamine oxide is 0.2 μM.

[0041] In one aspect, in the application of the present invention, the minimum detectable concentration of tetrathionate is 2 mM.

[0042] The beneficial technical effects of the present invention include at least the following: Based on the discovery of the different physiological response characteristics of Vibrio parahaemolyticus to exogenous TMAO and tetrathionine sulfate, the inventors developed a novel multifunctional biosensor (e.g., using Vibrio parahaemolyticus as the starting strain). This biosensor can be used to detect both the potential blood biomarker TMAO and the intestinal inflammatory biomarker tetrathionine sulfate, without interference from other sulfur-containing compounds. This multifunctional biosensor not only possesses extremely high sensitivity but also offers the advantages of simple and rapid operation, and independence from sophisticated instruments. Attached Figure Description

[0043] Figure 1 Displaying the pDS132 plasmid map.

[0044] Figure 2 Display pBBR- lux Plasmid map.

[0045] Figure 3 The results show that TMAO enhances P through the two-component system TorSR. torD With P torC Promoter activity.

[0046] Figure 4 The results showed that tetrathionate inhibited P through the two-component system TtrSR. torD With P torC Promoter activity.

[0047] Figure 5 The display shows Δ containing recombinant fluorescent plasmid. tsdBA The strain can sensitively detect TMAO and tetrathionine in the environment. Detailed Implementation

[0048] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings. These embodiments are implemented based on the technical solution of the present invention, providing detailed implementation methods and processes, and are intended to explain the present invention. However, these implementations do not constitute a limitation on the present invention. Some terms used herein are explained and described in detail; these explanations and descriptions are only for the convenience of those skilled in the art and should not be considered as limitations on the scope of protection of the present invention.

[0049] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0050] Example 1: Vibrio parahaemolyticus can utilize the two-component system TorSR to sense exogenous TMAO molecules and enhance promoter P. torD With P torCactivity Previous studies have reported that both *Escherichia coli* and *Salmonella* can perform anaerobic respiration via the two-component system TorSR sensing and utilize TMAO as a terminal electron acceptor; however, their biological function in *Vibrio parahaemolyticus* remains unclear. Using the genome sequence of *Vibrio parahaemolyticus* strain RIMD 2210633 as a template, Clone Manager software was used to design and construct... torR ( vp1032 )or torS ( vpa0675 Primers (P1–P4) for the gene deletion strain were synthesized by Hangzhou Youkang Biotechnology Co., Ltd. The primer sequences are as follows: P1-torR-F:GCTCTAGACTAAAAATAGGCGGTGATTATTCACCAAGA (SEQ ID NO: 9) P2-torR-UP:TTGTGGGTTCTTTCCCGCAAGCTTACTACG (SEQ ID NO: 10) P3-torR-down:AAGCTTCGGGAAAGAACCCACAAATTTTTGT (SEQ ID NO: 11) P4-torR-R:CGAGCTCGCACAAGGAAAGCAGGAATAAAGAGGGTCACCCTCATTTTTATTATCAATTGATGTTTTT (SEQ ID NO: 12) P1-torS-F:TCTTCTAGAGGTACCGCATGCACCGATACCAAACCAATATCATGG (SEQ ID NO:13) P2-torS-UP:CTCGTCCAATAATGCCATCGCGATGAATGA (SEQ ID NO: 14) P3-torS-down:ATCGCGATGGCATATTGGACGAGCAAGTC (SEQ ID NO: 15) P4-torS-R:GGAGAGCTCGATATCGCATGCTGTTTTACAAAACGGACGATCCA (SEQ ID NO: 16) PCR was performed using the complete genome of Vibrio parahaemolyticus as a template to amplify... torR or torSUpstream and downstream homologous arms of the gene were obtained, with the upstream homologous arm amplified by primers P1 and P2, and the downstream homologous arm amplified by primers P3 and P4. Fusion PCR was performed on the upstream and downstream homologous arms using primers P1 and P4 to obtain... torR or torS The missing DNA fragment was identified and purified. Specific restriction endonucleases XbaI (Catalog No.: R0145S), SacI (Catalog No.: R3156S), or SphI (Catalog No.: R3182S) from New England Biolabs (NEB) were used to isolate the DNA fragment and the pDS132 empty plasmid (plasmid map shown in figure). Figure 1 The DNA was digested overnight (as shown in the image) and then ligated using T4 DNA ligase (NEB, catalog number M0202V).

[0051] The ligation product was electroporated into *E. coli* DH5α-λpir competent cells and plated onto LB agar plates (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar powder, pH 7.4, plus 20 μg / mL chloramphenicol) and incubated overnight at 37°C. Single colonies on the plates were validated by PCR, and PCR-positive clones were sent to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing verification. Homologous recombination was performed via triparental conjugation (reference available on PubMed via PMID: 39038066), i.e., pDS132- torR or pDS132- torS Positive strains of *Escherichia coli* pRK2013-HB101 and wild-type *Vibrio parahaemolyticus* strain RIMD 2210633 (purchased from the China Medical Bacteriological Culture Collection Center) were cultured overnight at 37°C and 120 r / min in LB liquid (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.4) or MLB liquid (10 g / L tryptone, 5 g / L yeast extract, 30 g / L NaCl, pH 7.4). The cells were then collected by centrifugation at 4°C and 5000 r / min for 5 min. The three strains were mixed and conjugated on antibiotic-free LB solid medium for 6–8 h. Colonies were collected and spread onto MLB solid medium containing streptomycin (100 μg / mL) and chloramphenicol (20 μg / mL). Single colonies were streaked sequentially on MLB plates containing two antibiotics, MLB plates containing 100 μg / mL streptomycin, and LB plates containing 20% ​​sucrose to screen for corresponding deletion strains.

[0052] wild-type Vibrio parahaemolyticus strain with Δ torRThe strain was anaerobically cultured at 37°C in M9 medium (22 mM KH2PO4, 90 mM Na2HPO4, 8.5 mM NaCl, 20 mM NH4Cl, 1% casein hydrolysate, 0.2 mM MgCl2, 0.01 mM CaCl2, pH 7.4) containing 20 mM TMAO (Aladdin, catalog number T162063) to the logarithmic growth phase, and the bacterial cells were collected by centrifugation. Total RNA was extracted using TRIzol reagent. The purity and concentration of RNA were determined using a multi-well microplate reader (Agilent Technologies (China) Co., Ltd., model BioTek Synergy H1). Transcriptome libraries were prepared using the extracted RNA, sequenced on an Illumina HiSeq 2000 platform, and compared with the Vibrio parahaemolyticus reference genome.

[0053] Transcriptome sequencing revealed that, in the presence of TMAO, the response regulator in the two-component system TorSR was compared to the wild-type strain (WT). torR After gene deletion, a total of 139 genes showed significant downregulation, and 120 genes showed significant upregulation. Figure 3 A). Analysis revealed that the significantly downregulated genes included the TMAO metabolic enzymes TorA (locus tag: VP1162), TorC (locus tag: VP1161), and TorD (locus tag: VP1031). TorA and TorC are located in the same operon and are controlled by the same promoter P. torCA (Also known as "P") torC The TorD gene is regulated by the promoter P, and the TorA or TorC gene is located in two different operons. torD Regulation ( Figure 3 (A and B).

[0054] The response-regulating transcription factor TorR in the two-component system TorSR possesses a DNA-binding domain, which can bind to downstream target genes. torCAD Promoter sequence regulates target genes torCAD Transcription. To further identify the target genes of the two-component system TorSR. torCAD The inventors used the BProm program (SoftBerry) to predict the promoter sequence of the target gene online, and used the genome sequence of Vibrio parahaemolyticus strain RIMD 2210633 as a template to design a mechanism for regulating the gene. torC and torD Primers (P1–P2) for the fluorescent reporter plasmid of the gene. Primer sequences are as follows: P1-torC-F:CGCTCTAGAACTAGTGGATCCAAATGTAATTTATGTTTTGTAAATGTTGTAAG (SEQID NO: 17) P2-torC-R:GCGGCCGCAACTAGAGGATCCACATTTGCCTATTTTGTATCTAGTGTTATT (SEQ IDNO: 18) P1-torD-F:CGCTCTAGAACTAGTGGATCCTCTTCTTATTATCCAAGACGTATTCTATTC (SEQ IDNO: 19) P2-torD-R:GCGGCCGCAACTAGAGGATCCTTTTGGCTCCTGAAGGATGC (SEQ ID NO: 20) Using the complete genome of Vibrio parahaemolyticus as a template, PCR amplification was performed with primers to obtain the promoter DNA fragment of the target gene, which was then purified. The empty plasmid pBBR- was then converted to DNA using the specific restriction endonuclease BamHI (NEB, catalog number R3136S). lux (Plasmid map as shown) Figure 2 Linearization was performed, and then the target gene promoter was ligated into plasmid pBBR- using a one-step cloning kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number C112). lux (The reference for this method can be obtained from PubMed website via PMID:35508659), and the cultures were plated onto LB agar plates (containing 20 μg / mL chloramphenicol) and incubated overnight at 37°C. Single colonies on the plates were validated by PCR, and PCR-positive clones were sent to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing validation. The fluorescent reporter plasmid P was obtained. torC - lux and P torD - lux ( Figure 3 B).

[0055] The recombinant fluorescent plasmid P was introduced using the above-mentioned triparental conjugation method with the helper bacterium pRK2013-HB101. torC - lux and P torD - lux Plasmids were introduced into WT and Δ respectively. torR and Δ torSAmong the strains, well-growing strains were selected and cultured overnight at 37℃ and 120 r / min. They were then transferred at a ratio of 1:100 to MLB liquid medium supplemented with different concentrations of TMAO as exogenous molecules. After 7 hours of static incubation at 37℃, the fluorescence value (Lux) and bacterial concentration (OD) were read using a multi-functional microplate reader (Agilent Technologies (China) Co., Ltd., model BioTek Synergy H1). 600 By calculating Lux / OD 600 The promoter activity of fluorescent reporter plasmids in each strain was compared. Three replicates were set up for each strain.

[0056] The results showed that, in the absence of TMAO, P in each strain torD With P torC There was no difference in promoter activity; however, when 10 mM TMAO was present in the environment, compared to Δ torS and Δ torR Gene deletion strains, P in WT strains torD With P torC The promoter activity was significantly enhanced ( Figure 3 (C and D).

[0057] To further investigate the target genes of the two-component system TorSR torCAD The inventors used the genome sequence of Vibrio parahaemolyticus strain RIMD 2210633 as a template and designed a method for constructing the regulatory mechanism using Clone Manager software. torR Primers (P1–P2) for the prokaryotic expression plasmid of the gene. Primer sequences are as follows: P1-torR-F:GTGCCGCGCGGCAGCCATATGATGAGCTATCACGTATTAGTCGTAGAAGA (SEQ IDNO: 21) P2-torR-R:ACCAGTCATGCTAGCCATATGTTAATCACCTGCGAACATGTAGCC (SEQ ID NO:22) Using the complete genome of Vibrio parahaemolyticus as a template, PCR amplification was performed to obtain the open reading frame (OPG) DNA fragment of the target gene, which was then purified. The empty plasmid pET28a was linearized using the specific restriction endonuclease NdeI (NEB, catalog number R0111S). The OPG was then ligated into plasmid pET28a using a one-step cloning kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number C112) (the reference for this method is available on PubMed via PMID: 35508659). The plasmid was then plated on LB agar plates (containing 50 μg / mL kanamycin) and incubated overnight at 37°C. Single colonies on the plates were validated by PCR, and PCR-positive clones were sent to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing verification. The prokaryotic expression plasmid pET28a- was obtained. torR .

[0058] Extract pET28a- torR Plasmid DNA was transferred via electroporation E.coli Rosetta competent cells were transformed and plated with LB selection plates containing 50 μg / mL kanamycin. Single colonies were formed at 37°C for 16-24 h. Single colonies were then picked and inoculated into LB liquid medium containing the same antibiotic, and cultured at 37°C with shaking at 200 rpm for 12 h. 900 μL of the bacterial culture was mixed with an equal volume of 60% glycerol and stored at -80°C for later use. The activated bacterial culture was then inoculated 1:100 into 5 mL of fresh LB medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking at 200 rpm until OD (digesterone) was reached. 600 When the concentration was approximately 0.5, 1 mL was aliquoted as an uninduced control. The remaining bacterial culture was added to IPTG to a final concentration of 0.5 mM, and expression was induced at 37℃ and 200 rpm for 8 h. The induced and uninduced bacterial cultures were centrifuged at 12000 rpm for 5 min to collect the bacterial cells. After sonication, the supernatant and precipitate were separated. The protein expression difference before and after induction was compared by SDS-PAGE electrophoresis. After determining the optimal protein induction conditions, the protein was expressed and purified in prokaryotes to obtain soluble TorR protein.

[0059] The promoter sequence of the target gene and the 16S rRNA sequence of the negative control were obtained by PCR amplification. TorR protein and DNA fragments were added sequentially to an EMSA binding buffer (10 mM Tris, 1 mM EDTA, 1 mM dithiothreitol, 50 mM KCl, 50 mM MgCl2, 10% glycerol). The protein dosages for the experimental group were 0, 50, 100, and 150 nM, respectively, while those for the control group were 0 and 150 nM; the DNA fragment dosage was 30 nM. After mixing, the samples were incubated at 37°C for 30 min, and then transferred to an 8% non-denaturing gel for electrophoresis. The electrophoresis conditions were 4°C, 200 V, and 45 min. After electrophoresis, the non-denaturing gel was stained with nucleic acid dyes for 20 min, and the results were observed using a gel imaging system.

[0060] Gel retardation assays showed that the response regulator TorR protein can bind to the promoter P in a dose-dependent manner. torD and P torC The sequence, without binding to the negative control 16S rRNA sequence ( Figure 3 E).

[0061] These results fully demonstrate that Vibrio parahaemolyticus can utilize the two-component TorSR system to sense exogenous TMAO molecules and directly bind to the promoter P. torD and P torC In a sequential manner, the promoter P is regulated. torD and P torC This activity, in turn, promotes the transcriptional expression of target genes.

[0062] Example 2. Vibrio parahaemolyticus can sense exogenous sulfur-containing compounds and reduce promoter P. torD and P torCA active The applicant's previous research found that Vibrio parahaemolyticus can utilize the two-component system TtrSR to sense the intestinal signaling molecule tetrathionate (THB) to regulate sulfur metabolism and promote its colonization in the host gut (reference available on PubMed via PMID: 39038066). To investigate whether sulfur-containing compounds affect the activity of TMAO metabolic enzymes, we used the aforementioned triphilic conjugation method with the helper bacterium pRK2013-HB101 to transfer the recombinant fluorescent plasmid P... torC - lux and P torD - lux Plasmids were introduced into WT and Δ respectively. ttrR Δ ttrA and Δ tsdBAFrom the strains (which can be obtained from PMID: 39038066), well-growing strains were selected and cultured overnight at 37℃ and 120 r / min. Then, they were transferred at a ratio of 1:100 to MLB liquid medium supplemented with 10 mM tetrathionate (Tet, Sigma-Aldrich, catalog number S5758), thiosulfate (Thi, Aladdin, catalog number S433978), and sulfate (Sul, Aladdin, catalog number S433906), respectively. After static incubation at 37℃ for 7 h, the fluorescence value (Lux) and bacterial concentration (OD) were read using a multi-functional microplate reader. 600 By calculating Lux / OD 600 The promoter activity of fluorescent reporter plasmids in each strain was compared. Three replicates were set up for each strain.

[0063] The results showed that tetrathionate and thiosulfate could significantly inhibit P torD With P torC promoter activity ( Figure 4 A and E).

[0064] Vibrio parahaemolyticus utilizes the two-component system TtrSR and its target genes ttrBCA and tsdBA Regulating sulfur metabolism, the TtrBCA protein mediates the reduction of tetrathionate and thiosulfate, while the TsdBA protein mediates the oxidation of thiosulfate (references are available on PubMed via PMID: 39038066). Therefore, we utilize proteins containing P... torD - lux With P torC - lux gene deletion strain Δ ttrR (This indicates that the function of the two-component system TtrSR is disrupted, resulting in reduced pathogenicity), Δ ttrA (This indicates that the function of the TtrBCA protein is disrupted, resulting in reduced pathogenicity) and Δ tsdBA (This indicates that the TsdBA protein function is disrupted, resulting in reduced pathogenicity.) As a test strain, further analysis was conducted to determine the effect of disrupted sulfur metabolism on P. torD With P torC The effect of Δ on promoter activity. The results show that, in Δ ttrR With Δ ttrA Among the strains, tetrathionate and thiosulfate have an effect on P torD With P torC The inhibitory effect on promoter activity was significantly weakened. Figure 4 B, C, F, and G). However, in Δ tsdBA Among the strains, tetrathionate is effective against P torD With P torCThe promoter activity still exhibits a strong inhibitory effect, while other sulfur-containing compounds have an effect on P. torD With P torC The promoter showed no significant inhibitory effect. Figure 4 D and H). These results indicate that sulfur-containing compounds can reduce P. torD With P torC Promoter activity inhibits the activity of Vibrio parahaemolyticus TMAO metabolic enzyme (TorCAD), while Δ tsdBA The strain can reduce P torD With P torC Promoter activity specifically maintains the inhibition of TMAO metabolic enzymes by tetrathionine.

[0065] Example 3. Genetically engineered Vibrio parahaemolyticus (Δ) tsdBA It can be used as a multifunctional biosensor to efficiently detect TMAO and tetrathionine in samples. Using Vibrio parahaemolyticus P torD With P torC The inventors attempted to influence the different reaction characteristics of TMAO and tetrathionate with promoter activity by using Δ tsdBA :: P torD - lux With Δ tsdBA :: P torC - lux Designed as a multi-functional biosensor to detect the levels of TMAO and tetrathionate in a sample.

[0066] MLB liquid culture medium was prepared with seven concentration gradients of TMAO or Tet: 0 μM, 0.2 μM, 2 μM, 20 μM, 0.2 mM, 2 mM, and 20 mM. The recombinant fluorescent plasmid P was then conjugated using the aforementioned triphilic conjugation method. torC - lux and P torD - lux Plasmid introduction Δ tsdBA Δ was obtained from the strain. tsdBA :: P torD - lux With Δ tsdBA :: P torC - lux Two types of conjugates were selected, and well-grown strains were cultured overnight at 37°C and 120 r / min. They were then transferred at a 1:100 ratio to MLB liquid medium supplemented with different concentrations of TMAO as exogenous molecules and incubated statically at 37°C for 3 h. Fluorescence values ​​(Lux) and bacterial concentration (OD) were read every 0.5 h using a multi-functional microplate reader. 600The culture was transferred at a 1:100 ratio to MLB liquid medium containing different concentrations of Tet as exogenous molecules, and incubated at 37°C for 11 hours. The fluorescence value (Lux) and bacterial concentration (OD) were read using a multi-functional microplate reader at 3, 5, 7, 9, and 11 hours, respectively. 600 By calculating Lux / OD 600 The promoter activity of fluorescent reporter plasmids in each strain was compared. Three replicates were set up for each strain.

[0067] The results show that Δ tsdBA :: P torD - lux It can detect 2 μM levels of TMAO and 2 mM levels of tetrathionate in samples; Δ tsdBA :: P torC - lux It can detect 0.2 μM levels of TMAO and 2 mM levels of tetrathionate in samples. Figure 5 Therefore, this study developed a novel multifunctional biosensor capable of simultaneously detecting the blood disease biomarker TMAO and the intestinal inflammation biomarker tetrathionate. This multifunctional biosensor not only possesses extremely high sensitivity but also boasts the advantages of simple and rapid operation, and independence from sophisticated instruments.

[0068] sequence list SEQ ID NO: 1: pBBR- lux Plasmid sequence, where the underlined part is the fluorescent protein coding sequence. ATGACTAAAAAAATTTCATTCATTATTAACGGCCAGGTTGAAATCTTTC CCGAAAGTGATGATTTAGTGCAATCCATTAATTTTGGTGATAATAGTGTTTACCTGCCAATATTGAATGACTCTCA TGTAAAAAACATTATTGATTGTAATGGAAATAACGAATTACGGTTGCATAACATTGTCAATTTTCTCTATACGGTA GGGCAAAGATGGAAAAATGAAGAATACTCAAGACGCAGGACATACATTCGTGACTTAAAAAAATATATGGGATATT CAGAAGAAATGGCTAAGCTAGAGGCCAATTGGATATCTATGATTTTATGTTCTAAAGGCGGCCTTTATGATGTTGT AGAAAATGAACTTGGTTCTCGCCATATCATGGATGAATGGCTACCTCAGGATGAAAGTTATGTTCGGGCTTTTCCG AAAGGTAAATCTGTACATCTGTTGGCAGGTAATGTTCCATTATCTGGGATCATGTCTATATTACGCGCAATTTTAA CTAAGAATCAGTGTATTATAAAAACATCGTCAACCGATCCTTTTACCGCTAATGCATTAGCGTTAAGTTTTATTGA TGTAGACCCTAATCATCCGATAACGCGCTCTTTATCTGTTATATATTGGCCCCCACCAAGGTGATACATCACTCGCA AAAGAAATTATGCGACATGCGGATGTTATTGTCGCTTGGGGAGGGCCAGATGCGATTAATTGGGCGGTAGAGCATG CGCCATCTTATGCTGATGTGATTAAAATTTGGTTCTAAAAAGAGTCTTTGCATTATCGATAATCCTGTTGATTTGAC GTCCGCAGCGACAGGTGCGGCTCATGATGTTTGTTTTTACGATCAGCGAGCTTGTTTTTCTGCCCAAAACATATAT TACATGGGAAATCATTATGAGGAATTTAAGTTAGCGTTGATAGAAAAACTTAATCTATATGCGCATATATTACCGA ATGCCAAAAAAGATTTTGATGAAAAGGCGGCCTATTCTTTAGTTCAAAAAGAAAGCTTGTTTGCTGGATTAAAAGT AGAGGTGGATATTCATCAACGTTGGATGATTATTGAGTCAAATGCAGGTGTGGAATTTAATCAACCACTTGGCAGA TGTGTGTACCTTCATCACGTCGATAATATTGAGCAAATATTGCCTTATGTTCAAAAAAATAAGACGCAAACCATAT CTATTTTTCCTTGGGAGTCATCATTTAAATATCGAGATGCGTTAGCATTAAAAGGTGCGGAAAGGATTGTAGAAGC AGGAATGAATAACATATTTCGAGTTGGTGGATCTCATGACGGAATGAGACCGTTGCAACGATTAGTGACATATATT TCTCATGAAAGGCCATCTAACTATACGGCTAAGGATGTTGCGGTTGAAATAGAACAGACTCGATTCCTGGAAGAAG ATAAGTTCCTTGTATTTGTCCCATAATAGGTAAAAGTAGGAAAATGAATCAAAATATAAAACCATCGACCACGTT ATTGTGTTGAAGGAAATAAAAAAATTCATGTTTGGGAAACGCTGCCAGAAGAAAACAGCCCAAAGAGAAAGAATG CCATTATTATTGCGTCTGGTTTTGCCCGCAGGATGGATCATTTTGCTGGTCTGGCGGAATATTTATCGCGGAATGG ATTTCATGTGATCCGCTATGATTCGCTTCACCACGTTGGATTGAGTTCAGGGACAATTGATGAATTTACAATGTCT ATAGGAAAGCAGAGCTTGTTAGCAGTGGTTGATTGGTTAACTACACGAAAAATAAATAACTTCGGTATGTTGGCTT CAAGCTTATCTGCGCGGATAGCTTATGCAAGCCTATCTGAAATCAATGCTTCGTTTTTAATCACCGCAGTCGGTGT TGTTAACTTAAGATATTCTCTTGAAAGAGCTTTAGGGTTTGATTATCTCAGTCTACCCATTAATGAATTGCCGGAT AATCTAGATTTTGAAGGCCATAAATTGGGTGCTGAAGTCTTTGCGAGAGATTGTCTTGATTTTGGTTGGGAAGATT TAGCTTCTACAATTAATAACATGATGTATCTTGATATACCGTTTATTGCTTTTACTGCAAATAACGATAATTGGGT CAAGCAAGATGAAGTTATCACATTGTTATCAAATATTCGTAGTAATCGATGCAAGATATATTCTTTGTTAGGAAGT TCGCATGACTTGAGTGAAAATTTAGTGGTCCTGCGCAATTTTTATCAATCGGTTACGAAAGCCGCTATCGCGATGG ATAATGATCATCTGGATATTGATGTTGATATTACTGAACCGTCATTTGAACATTTAACTATTGCGACAGTCAATGA ACGCCGAATGAGAATTGAGATTGAAAATCAAGCAATTTCTCTGTCTTAAAATCTATTGAGATATTCTATCACTCAA ATAGCAATATAAGGACTCTCTATGAAATTTGGAAACTTTTTGCTTACATACCAACCTCCCCAATTTTCTCAAACAG AGGTAATGAAACGTTTGGTTAAATTAGGTCGCATCTCTGAGGAGTGTGGTTTTGATACCGTATGGTTACTGGAGCA TCATTTCACGGAGTTTGGTTTGCTTGGTAACCCTTATGTCGCTGCTGCATATTTACTTGGCGCGACTAAAAAATTG AATGTAGGAACTGCCGCTATTGTTCTTCCCACAGCCCATCCAGTACGCCAACTTGAAGATGTGAATTTATTGGATC AAATGTCAAAAGGACGATTTCGGTTTGGTATTTGCCGAGGGCTTTACAACAAGGACTTTCGCGTATTCGGCACAGA TATGAATAACAGTCGCGCCTTAGCGGAATGCTGGTACGGGCTGATAAAGAATGGCATGACAGAGGGATATATGGAA GCTGATAATGAACATATCAAGTTCCATAAGGTAAAAGTAAACCCCGCGGCGTATAGCAGAGGTGGCGCACCGGTTT ATGTGGTGGCTGAATCAGCTTCGACGACTGAGTGGGCTGCTCAATTTGGCCTACCGATGATATTAAGTTGGATTAT AAATACTAACGAAAAGAAAGCACAACTTGAGCTTTATAATGAAGTGGCTCAAGAATATGGGCACGATATTCATAAT ATCGACCATTGCTTATCATATATAACATCTGTAGATCATGACTCAATTAAAGCGAAAGAGATTTGCCGGAAATTTC TGGGGCATTGGTATGATTCTTATGTGAATGCTACGACTATTTTTGATGATTCAGACCAAACAAGAGGTTATGATTT CAATAAAGGGCAGTGGCGTGACTTTGTATTAAAAGGACATAAAGATACTAATCGCCGTATTGATTACAGTTACGAA ATCAATCCCGTGGGAACGCCGCAGGAATGTATTGACATAATTCAAAAAGACATTGATGCTACAGGAATATCAAATA TTTGTTGTGGATTTGAAGCTAATGGAACAGTAGACGAAATTATTGCTTCCATGAAGCTCTTCCAGTCTGATGTCAT GCCATTTCTTAAAGAAAAACAACGTTCGCTATTATATTAGCTAAGGAGAAAGAAATGAAATTTGGATTGTTCTTCC TTAACTTCATCAATTCAACAACTGTTCAAGAACAAAGTAGTTCGCATGCAGGAAATAACGGAGTATGTTGATAA GTTGAATTTTGAACAGATTTTAGTGTATGAAAATCATTTTTCAGATAATGGTGTTGTCGGCGCTCCTCTGACTGTT TCTGGTTTTCTGCTCGGTTTAACAGAGAAAATTAAAATTGGTTCATTAATCACATCATTACAACTCATCATCCTG TCGCCATAGCGGAGGAAGCTTGCTTATTGGATCAGTTAAGTGAAGGGAGATTTATTTAGGTTTAGTGATTGCGA AAAAAAGATGAAATGCATTTTTTAATCGCCCGGTTGAATATCAACAGCAACTATTTGAAGAGTGTTATGAAATC ATTAACGATGCTTTAACAACAGGCTATTGTAATCCAGATAACGATTTTTATAGCTTCCCTAAAATATCTGTAAATC CCCATGCTTATACGCCAGGCGGACCTCGGAAATATGTAACAGCAACCAGTCATCATATTGTTGAGTGGGCGGCCAA AAAAGGTATTCCTCTCATCTTTAAGTGGGATGATTCTAATGTTAGATATGAATATGCTGAAAGATATAAAGCC GTTGCGGATAAATATGACGTTGACCTATCAGAGATAGACCATCAGTTAATGATATTAGTTAACTATAACGAAGATA GTAATAAAGCTAAACAAGACGCGTGCATTTTAGTGATTATGTTCTTGAAATGCACCCTAATGAAAATTTTCGA AAATAAACTTGAAGAAATATTGCAGAAAACGCTGTCGGAAATTATACGGAGTGTATAACTGCGGCTAAGTTGGCA ATTGAAAAGTGTGGTGCGAAAGTGTATTGCTGTCCTTTGAA SEQ ID NO: 2: lux Fluorescent protein coding sequence SEQ ID NO: 3: P torC AAATGTAATTTATGTTTTGTAAATGTTGTAAGTTTAACAAGGTTTTAAAAGACTTATTTTTTCATATCTGTTCATGTTTTTTAATAAATAATATCAATCCCACACAACTCCGGATGAGCTCAAAAAAGTGTTGAATTACAAGGCTTGAAGGGAGTTATTTGATTGAACCTTACTATTAGTATGGTCTAACATTTTCAATTAGAGATTGTAAAAACTTAAGCTTGGTTACACGGGCTTTTCAATAACACTAGATACAAAATAGGCAAATGT; SEQ ID NO: 4: P torD TCTTCTTATTATCCAAGACGTATTCTATTCATAACTAGTAACATTTCTAGTCATAGATACATTAAAAAGCAAGATTTAGCGTTTTTATTGATTCAAAACAAGCTTGCCCATCACTGTTAATAAGTGCACACAATTCAACATAATCAACAATAATTATAGTTACTGCTATTCATCTTATTTGATTACACTTAGGGGATATCCCTAATTGACTTTATGATGCAATACTCGAAGCATCCTTCAGGAGCCAAAA; SEQ ID NO: 5: pDS132 plasmid sequence SEQ ID NO: 6: tsdBA gene sequence SEQ ID NO: 7: Accessory protein TorT (VPA0674) MVINQRSIRTIFKHSVLAASLCASLPSVAAEKICAIYPHLKDSYWLSVNYGMVSEAEKQGVNLRVLEAGGYPNKSRQEQQLALCTQWGANAIILGTVDPHAYEHNLKSWVGNTPVFATVNQLDLDEEQSTLLKGEVGVDWYWMGYEAGKYLAERHPKGSGKTNIALLLGPRTRGGTKPVTTGFYEAIKNSDIHIVDSFWADNDKELQRNLVQRVIDMGNIDYIVGSAVAIEAAISELRSADKTHDIGLVSVYLSHGVYRGLLRNKVLFAPTDKMVQQGRLSVMQAAHYLRHQPYEKQASPIIKPLTPKTLHDDTIEESLSPSEYRPTFSIKAVD; SEQ ID NO: 8: Accessory protein TorT coding sequence

Claims

1. A multi-functional biosensor, characterized in that, The multifunctional biosensor comprises genetically engineered bacteria, which contain a two-component system TorSR and a gene encoding the accessory protein TorT, the two-component system TorSR, and a promoter P torD and / or P torCA The reporter gene for regulation is Vibrio parahaemolyticus RIMD 2210633, which is further described as a knockout strain. tsdBA Vibrio parahaemolyticus gene, the promoter P torCA The nucleotide sequence is shown in SEQ ID NO: 3, and the promoter P torD The nucleotide sequence is shown in SEQ ID NO:

4.

2. A method for preparing a multi-functional biosensor, characterized in that, The method includes: modifying bacteria to contain a two-component system TorSR and a gene encoding the accessory protein TorT, the two-component system TorSR, and a gene encoded by the promoter P. torD and / or P torCA The reporter gene for regulation is Vibrio parahaemolyticus RIMD 2210633, which is further described as a knockout strain. tsdBA Vibrio parahaemolyticus gene, the promoter P torCA The nucleotide sequence is shown in SEQ ID NO: 3, and the promoter P torD The nucleotide sequence is shown in SEQ ID NO:

4.

3. The method according to claim 2, characterized in that, An exogenous plasmid containing the promoter P was introduced into Vibrio parahaemolyticus. torD and / or P torCA And the reporter genes regulated by them.

4. The method according to claim 2, characterized in that, In the genome sequence of Vibrio parahaemolyticus, at the promoter P torD and / or P torCA A reporter gene is inserted between the downstream of the gene and the upstream of the start codon of the gene it regulates.

5. The multi-functional biosensor according to claim 1 or the method according to claim 2, characterized in that, The tsdBA The gene sequence is shown in SEQ ID NO:

6.

6. The multi-functional biosensor according to claim 1 or the method according to claim 2, characterized in that, The reporter gene is a fluorescent protein gene; and / or, the amino acid sequence of the accessory protein TorT is shown in SEQ ID NO:

7.

7. The multi-functional biosensor or method according to claim 6, characterized in that, The reporter gene is the gene for the fluorescent protein shown in SEQ ID NO:

2.

8. The use of the multifunctional biosensor according to any one of claims 1, 5, 6 and 7 in the preparation of a kit for detecting trimethylamine oxide or a sulfur-containing compound in a sample to be tested, wherein the sulfur-containing compound is tetrathionate.

9. The use of the multifunctional biosensor according to any one of claims 1, 5, 6 and 7 in the preparation of products for detecting trimethylamine oxide or sulfur-containing compounds, wherein the sulfur-containing compound is tetrathionate.

10. The application according to claim 8 or 9, characterized in that, When detecting trimethylamine oxide, the test samples are selected from blood, serum, saliva, and urine; When testing for sulfur-containing compounds, the samples to be tested are selected from intestinal contents and feces.

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