WO2023061222A1 - 一种细菌纤维素基生物传感器及其应用 - Google Patents

一种细菌纤维素基生物传感器及其应用 Download PDF

Info

Publication number
WO2023061222A1
WO2023061222A1 PCT/CN2022/122046 CN2022122046W WO2023061222A1 WO 2023061222 A1 WO2023061222 A1 WO 2023061222A1 CN 2022122046 W CN2022122046 W CN 2022122046W WO 2023061222 A1 WO2023061222 A1 WO 2023061222A1
Authority
WO
WIPO (PCT)
Prior art keywords
cellulose
tac
biosensor
promoter
binding domain
Prior art date
Application number
PCT/CN2022/122046
Other languages
English (en)
French (fr)
Other versions
WO2023061222A9 (zh
Inventor
龙凌凤
孙付保
胡芸
谢乐
Original Assignee
江南大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江南大学 filed Critical 江南大学
Publication of WO2023061222A1 publication Critical patent/WO2023061222A1/zh
Publication of WO2023061222A9 publication Critical patent/WO2023061222A9/zh
Priority to US18/527,123 priority Critical patent/US20240118205A1/en

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A20/00—Water conservation; Efficient water supply; Efficient water use
    • Y02A20/20—Controlling water pollution; Waste water treatment

Definitions

  • the invention relates to the fields of molecular biology, genetic engineering, nanometer materials and biosensors, in particular to a bacterial cellulose-based biosensor and its application.
  • BC Bacterial cellulose
  • Agrobacterium Bacterial cellulose
  • Rhizobium Bacterial cellulose Due to their good biocompatibility, high mechanical strength, strong water retention capacity, and high porosity, they have broad application potential as biosensor platforms.
  • BC is widely used in gas sensors, surface acoustic wave humidity sensors, electrochemical sensors, etc.
  • bacteria when used in biosensors, due to the large pores between BC fibers, bacteria cannot continue to adhere for a long time, and escape will occur during the sensing process. , limiting the application of BC as a biosensor.
  • Kim et al. developed a P(HEMA-co-HAETC) hydrogel-based bacterial sensor, which first prepared P(HEMA-co-HAETC) hydrogel beads using electrospray, and then passed a 12-hour Incubation to load cells onto hydrogel beads, the preparation process is time-consuming and requires the use of specialized equipment; Drachuk et al. method in BC materials, but this method introduces BC-producing bacteria into the biosensing system and interferes with the monitoring of analytes; in addition, Yoetz Kopelman et al.
  • the present invention discloses a bacterial cellulose-based biosensor, which enhances the adhesion of cells to bacterial cellulose by displaying CBM as an affinity tag on the cell surface.
  • the invention discloses a bacterial cellulose-based biosensor, comprising bacterial cellulose and cells displaying a cellulose-binding domain CBM on the surface, wherein the cellulose-binding domain CBM is a cellulose-binding material that can specifically bind to a cellulose crystal region domain, cells are linked to bacterial cellulose through the cellulose-binding domain CBM.
  • the cellulose-binding domain CBM is fused with an ankyrin that can be displayed on the surface to realize the surface display of the cellulose-binding domain CBM.
  • the cellulose binding domain CBM is CBM2a.
  • bacterial cellulose can be in different shapes such as spherical, flake, rod, etc., for detection in different scenarios.
  • the method for constructing the above-mentioned bacterial cellulose-based biosensor comprises the following steps: co-cultivating cells capable of displaying the cellulose-binding domain CBM on the surface with bacterial cellulose, specifically:
  • step (2) Inoculate the recombinant cells obtained in step (1) in the culture medium, add transcription inducer and bacterial cellulose when the OD600 is 0.6-0.8, and continue to cultivate for 10-12h to obtain the above-mentioned bacterial cellulose-based biosensor .
  • the cells are recombinant bacteria, Escherichia coli is used as the host, and pETDuet-tac is used as the vector, pETDuet-tac is a vector obtained by replacing the two T7 promoters on the pETDuet vector with two tac promoters, and the upstream one is The first tac promoter, the second tac promoter is located downstream, and pETDuet-tac contains a gene encoding a fluorescent protein located downstream of the first tac promoter and a gene encoding a cellulose-binding protein located downstream of the second tac promoter. Domain CBM genes.
  • the first tac promoter can be replaced with a specific promoter that affects the transcription of the downstream fluorescent protein gene in the presence of the target compound.
  • a specific promoter that affects the transcription of the downstream fluorescent protein gene in the presence of the target compound.
  • arabinose-induced promoter nucleic acid fragment containing arabinose promoter and AraC
  • nitro compound-induced promoter or heavy metal-induced promoter.
  • sequence of the arabinose-induced promoter (nucleic acid fragment containing the arabinose promoter and AraC) is shown in SEQ ID NO.3, specifically:
  • fluorescent proteins include but are not limited to green fluorescent protein, red fluorescent protein, cyan fluorescent protein, etc., such as the gene sequence shown in SEQ ID NO.2, specifically:
  • the method for constructing the above-mentioned analyte-induced fluorescent biosensor comprises the following steps:
  • step (2) reverse PCR amplification is performed with the upstream primer shown in SEQ ID NO.4 and the downstream primer shown in SEQ ID NO.5 to delete the first tac promoter.
  • the sequences of the upstream primer and the downstream primer are specifically:
  • SEQ ID NO.4 5'-CAATCGATCTCGATCCTCTACG-3';
  • SEQ ID NO.5 5'-TTTCACACAGGAAACAGTATC-3'.
  • the biosensor of the present invention is widely used in the detection of monosaccharides, explosive molecules and heavy metals. Specifically, the biosensor of the present invention is mixed with the solution to be tested, and the fluorescence intensity is detected after incubation for 3-60 hours to realize the detection of the test substance. detection.
  • the present invention has at least the following advantages:
  • the present invention can achieve efficient and specific immobilization of cells on the BC matrix without any modification of the bacterial cellulose (BC) substrate, and continuously apply After 60 h of external shear stress, the cells displaying CBM on the surface could still tightly bind to the BC carrier.
  • BC bacterial cellulose
  • the present invention adopts a synchronous display immobilization strategy to realize simple and rapid whole cell loading.
  • the bacterial cellulose (BC)-based biosensor provided by the present invention has great potential in the field of substance detection.
  • Fig. 1 is SDS-PAGE figure, and wherein, 1 is the recombinant escherichia coli E.coli BL21 (DE3) soluble protein that contains plasmid pETDuet-tac-CBM2a, 2 is original E.coli BL21 (DE3) soluble protein;
  • Figure 2 is an immunofluorescent micrograph of recombinant Escherichia coli with CBM2a displayed on the surface;
  • Fig. 3 is the SEM image of the surface morphology of the bacterial cellulose (BC) carrier loaded with CBM2a recombinant Escherichia coli displayed on the surface;
  • Fig. 4 is the fluorescence imaging diagram of the flake and spherical BC-based fluorescent biosensors that detect arabinose (320mg/L);
  • Fig. 5 is the relation of arabinose concentration and fluorescence intensity
  • Fig. 6 is a fluorescence imaging diagram of sheet-like and spherical BC-based fluorescent biosensors for detecting arabinose in soil.
  • the gene SEQ ID NO.1 which can display CBM2a on the surface of Escherichia coli E.coli BL21 (DE3), was inserted into the plasmid pETDuet-tac (with pETDuet as a template, the two T7 promoters on it were replaced with tac promoter, laboratory preservation).
  • T4 ligase was used to connect overnight at 16°C, and the SEQ ID NO.1 and the vector pETDuet-tac were ligated.
  • the ligation product was transformed into competent cells E.coli DH5 ⁇ , colony PCR and sequencing verification, and the vector pETDuet-tac-CBM2a was obtained. Transform pETDuet-tac-CBM2a into the host strain E.coli BL21(DE3) to obtain recombinant cells containing surface-displayed CBM2a.
  • BC bacterial cellulose
  • the gene that can display the CBM that specifically binds to the cellulose crystal region on the surface of Escherichia coli E. coli BL21 (DE3) was inserted into the plasmid pETDuet-tac.
  • T4 ligase was used to ligate overnight at 16°C, and the gene that can display the CBM that specifically binds to the cellulose crystal region on the surface of Escherichia coli E. coli BL21 (DE3) was ligated with the vector pETDuet-tac.
  • the ligation product was transformed into competent cells E.coli DH5 ⁇ , colony PCR and sequencing verification, and the vector pETDuet-tac-CBM2a was obtained. Transform pETDuet-tac-CBM2a into the host strain E.coli BL21(DE3) to obtain recombinant cells containing surface-displayed CBM2a.
  • Step (1) Inoculate a single colony of recombinant cell E.coli BL21(DE3) containing CBM that specifically binds to the cellulose crystal region on the surface in 5 mL of Luria-Bertani broth (LB) containing 100 ⁇ g/mL Amp culture medium at 37°C with shaking at 200rpm for 8-12h. Inoculate 1 mL of the above-mentioned bacterial solution into a 500 mL shake flask containing 100 mL of culture medium, culture at 37°C with shaking at 200 rpm, and when the OD 600 reaches 0.6-0.8, add IPTG (final concentration: 0.25 mM) and BC matrix (step 2 prepared), cultured at 25° C.
  • LB Luria-Bertani broth
  • BC bacterial cellulose
  • the gene SEQ ID NO.1 which can display CBM2a on the surface of Escherichia coli E.coli BL21 (DE3), was inserted into the plasmid pETDuet-tac (with pETDuet as a template, the two T7 promoters on it were replaced with tac promoter, laboratory preservation).
  • T4 ligase was used to connect overnight at 16°C, and the SEQ ID NO.1 and the vector pETDuet-tac were ligated.
  • the ligation product was transformed into competent cells E.coli DH5 ⁇ , colony PCR and sequencing verification, and the vector pETDuet-tac-CBM2a was obtained. Afterwards, using pETDuet-tac-CBM2a as a template, using NcoI and EcoRI enzyme cutting sites, repeat the above enzyme cutting and ligation steps to insert the green fluorescent protein gene of SEQ ID NO.2 into pETDuet-tac-CBM2a. The ligation product was transformed into competent cells E.coli DH5 ⁇ , colony PCR and sequencing verification, and the vector pETDuet-tac-EGFP-CBM2a was obtained. Then pETDuet-tac-EGFP-CBM2a was transformed into the host strain E.coli BL21(DE3) to obtain chassis fluorescent cells.
  • Step (1) Inoculate a single colony of E.coli BL21 (DE3) fluorescent cells in the chassis into 5 mL of Luria-Bertani broth (LB) medium containing 100 ⁇ g/mL Amp, culture at 37°C with shaking at 200 rpm for 8 -12h. Inoculate 1 mL of the above-mentioned bacterial solution into a 500 mL shake flask containing 100 mL of culture medium, culture at 37°C with shaking at 200 rpm, and when the OD 600 reaches 0.6-0.8, add IPTG (final concentration: 0.25 mM) and BC matrix (step 2 prepared), cultured at 25° C.
  • LB Luria-Bertani broth
  • Primer 1 5'-CAATCGATCTCGATCCTCTACG-3';
  • ClonExpress II One Step Cloning Kit purchased from Vazyme
  • the ClonExpress II One Step Cloning Kit purchased from Vazyme
  • the ligation product was transformed into competent cells E.coli DH5 ⁇ , colony PCR and sequencing verification, and the vector pETDuet-araBAD-EGFP-CBM2a was obtained.
  • the recombinant plasmid pETDuet-araBAD-EGFP-CBM2a was transformed into the host strain E.coli BL21(DE3), and connected with BC according to the method described in Example 1 to obtain an L-arabinose-inducible fluorescent biosensor.
  • L-arabinose inducible fluorescent biosensor to detect arabinose in solution.
  • the specific method is as follows: put the L-arabinose-induced fluorescent biosensors prepared above into different concentrations of arabinose solutions, culture them at room temperature for 5 hours, then measure their fluorescence intensity and perform fluorescence imaging to realize the detection of arabinose in the solution. detection.
  • the preparation method of the arabinose solution is: gradually diluting the configured arabinose stock solution with water to the final concentrations of 20mg/L, 160mg/L and 320mg/L respectively.
  • the preparation method of the 2,4-DNT solution is as follows: gradually dilute the prepared 2,4-DNT stock solution with water to the final concentrations of 5mg/L, 10mg/L and 20mg/L respectively.
  • the specific method is as follows: put the heavy metal-induced fluorescent biosensors prepared above into heavy metal solutions with different concentrations, culture them at room temperature for 24 hours, then measure their fluorescence intensity and perform fluorescence imaging to realize the detection of heavy metals in the solution.
  • Heavy metal solutions include Zn 2+ , Cd 2+ and Hg 2+ .
  • the preparation method is as follows: gradually dilute the above-mentioned Zn 2+ stock solution prepared with water to a final concentration of 20 mg/L, 100 mg/L and 300 mg/L; gradually dilute the above-mentioned Cd 2+ stock solution prepared with water to a final concentration of 0.5 mg /L, 2.0mg/L and 4.0mg/L; the above-mentioned Hg 2+ stock solutions were gradually diluted with water to a final concentration of 0.004mg/L, 0.016mg/L and 0.06mg/L, respectively.
  • Example 1 (1) Using PCR technology, the vector pETDuet-tac-EGFP-CBM2a constructed in Example 1 was used as a template to replace CBM2a with CBM44. The vector pETDuet-tac-EGFP-CBM44 was obtained.
  • CBM44 The gene sequence of CBM44 is specifically:

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Optics & Photonics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

提供了一种细菌纤维素基生物传感器，包括细菌纤维素和表面展示纤维素结合结构域CBM的细胞，细胞通过纤维素结合结构域CBM与细菌纤维素连接。所述细菌纤维素基生物传感器可实现细胞在细菌纤维素基质上的高效和特异性固定，可维持细胞生物活性，增强荧光信号输出，为检测物出入提供了足够的孔隙，显著提高了检测灵敏度。

Description

一种细菌纤维素基生物传感器及其应用 技术领域
本发明涉及分子生物学、基因工程、纳米材料及生物传感器领域，尤其涉及一种细菌纤维素基生物传感器及其应用。
背景技术
基于荧光检测的生物传感器在环境污染物检测、生化诊断和生物医学传感等领域发挥着重要作用。到目前为止，已公开报道多种工程菌株作为传感器，用以检测重金属、有机化合物和抗生素等在内的化学物质。但生物传感器在应用过程中需要将工程菌株固定到一个材料平台上以维持细胞生物活性和增强荧光信号输出。此外，理想的生物传感器平台还需具备足够的孔隙供检测物出入，同时最大限度减少对环境的污染。
细菌纤维素(BC)是指在不同条件下，由醋酸菌属(Acetobacter)、土壤杆菌属(Agrobacterium)、根瘤菌属(Rhizobium)和八叠球菌属(Sarcina)等中的某种微生物合成的纤维素的统称。因其具有生物相容性好、机械强度高、保水能力强和孔隙度高等特性，作为生物传感器平台具有广阔的应用潜力。BC在气体传感器、声表面波湿度传感器、电化学传感器等方面的应用非常广泛，然而用于生物传感器时，由于BC纤维间孔隙过大，细菌无法长时间持续附着，传感过程中会发生逃逸，限制了BC作为生物传感器的应用。
Kim等人(2019年)开发了基于P(HEMA-co-HAETC)水凝胶的细菌传感器，该传感器首先使用电喷雾制备P(HEMA-co-HAETC)水凝胶珠，然后通过12小时的孵育将细胞加载到水凝胶珠上，制备过程非常耗时，并且需要专业设备的使用；Drachuk等人(2017年)提出了重组细胞与BC生产菌(Gluconacetobacter xylinus)共培养将细胞包埋在BC材料中的方法，但该方法将BC生产菌引入生物传感系统，干扰分析物的监测；此外，Yoetz  Kopelman等人(2016年)提出了一种通过改性聚丙烯酰胺多孔微球形成正电荷，以增强带负电细菌对载体亲和力的固定化方法，尽管该方法被证明作为一种全细胞生物传感器是可行的，但会造成细胞与基质的非特异性结合。
发明内容
为解决上述技术问题，本发明公开了一种细菌纤维素基生物传感器，通过在细胞表面展示CBM作为亲和标签，增强细胞对细菌纤维素的粘附性。
本发明公开了一种细菌纤维素基生物传感器，包括细菌纤维素和表面展示纤维素结合结构域CBM的细胞，其中，纤维素结合结构域CBM为可特异性结合纤维素结晶区的纤维素结合结构域，细胞通过纤维素结合结构域CBM与细菌纤维素连接。
进一步地，将纤维素结合结构域CBM与可表面展示的锚蛋白融合，实现纤维素结合结构域CBM的表面展示。
进一步地，纤维素结合结构域CBM为CBM2a。
进一步地，编码可表面展示CBM2a的基因序列如SEQ ID NO.1所示，具体为：
5’-ATGAAGGCGACCAAACTGGTGCTGGGTGCGGTTATTCTGGGCAGCACCCTGCTGGCGGGTTGCAGCAGCAACGCGAAAATCGACCAGGGCATTAACCCGTACGTGGGTTTCGAAATGGGCTATGATTGGCTGGGTCGTATGCCGTACAAGGGTAGCGTGGAGAACGGCGCGTATAAAGCGCAGGGTGTTCAACTGACCGCGAAGCTGGGCTACCCGATCACCGACGATCTGGACATTTATACCCGTCTGGGTGGCATGGTGTGGCGTGCGGACACCAAGAGCAACGTTTACGGTAAAAACCACGATACCGGCGTGAGCCCGGTTTTTGCGGGTGGCGTGGAGTATGCGATCACCCCGGAAATTGCGACCCGTCTGGAGTATCAATGGACCAACAACATCGGTGACGCGCACACCATTGGCACCCGTCCGGATAACGGTATTCCGGGCGCTAGCTCCGGTCCGGCCGGGTGCCAGGTGCTGTGGGGCGTCAACCAGTGGAACACCGGCTTCACCGCGAACGTCACCGTGAAGAACACGTCCTCCGCTCCGGTCGACGGCTGGACGCTCACGTTCAGCTTCCCGTCCGGCCAGCAGGTCACCCAGGCGTGGAGCTCGACGGTCACGCAGTCCGGCTCGGCCGTGACGGTCCGCAACGCCCC GTGGAACGGCTCGATCCCGGCGGGCGGCACCGCGCAGTTCGGCTTCAACGGCTCGCACACGGGCACCAACGCCGCGCCGACGGCGTTCTCGCTCAACGGCACGCCCTGCACGGTCGGCCATCACCATCATCACCACTGA-3’。
进一步地，细菌纤维素可为球状、片状、杆状等不同形态，用于不同情景下的检测。
上述细菌纤维素基生物传感器的构建方法，包括以下步骤：将可在表面展示纤维素结合结构域CBM的细胞与细菌纤维素共同培养，具体地：
(1)将编码可表面展示纤维素结合结构域CBM的基因与载体连接，转化入宿主菌，得到重组细胞；
(2)将步骤(1)得到的重组细胞接种于培养基中培养，当OD 600为0.6-0.8时加入转录诱导剂和细菌纤维素，继续培养10-12h，得到上述细菌纤维素基生物传感器。
进一步地，细胞为重组菌，以大肠杆菌为宿主，以pETDuet-tac为载体，pETDuet-tac是将pETDuet载体上的两个T7启动子替换为两个tac启动子得到的载体，位于上游的为第一tac启动子，位于下游的为第二tac启动子，pETDuet-tac上含有位于第一tac启动子下游的编码荧光蛋白的基因和位于第二tac启动子下游的编码可表面展示纤维素结合结构域CBM的基因。
进一步地，为实现不同物质的检测，可将第一tac启动子替换为在靶化合物存在时，影响下游荧光蛋白基因转录的特定启动子。如阿拉伯糖诱导的启动子(含有阿拉伯糖启动子和AraC的核酸片段)、硝基化合物诱导的启动子或重金属诱导的启动子。
进一步地，阿拉伯糖诱导的启动子(含有阿拉伯糖启动子和AraC的核酸片段)序列如SEQ ID NO.3所示，具体为：
5’-TTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCACGGAACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAATACCCGCGAGAAGTAGAGTTGATCGTCAAAACCAACATTGCGACCGACGGTGGCGATAGGCATCCGGGTGGTGCT CAAAAGCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGACGCTAATCCCTAACTGCTGGCGGAAAAGATGTGACAGACGCGACGGCGACAAGCAAACATGCTGTGCGACGCTGGCGATATCAAAATTGCTGTCTGCCAGGTGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATTATCCATCGGTGGATGGAGCGACTCGTTAATCGCTTCCATGTGCCGCAGTAACAATTGCTCAAGCAGATTTATCGCCAGCAGCTCCGAATAGCGCCCTTCCCCTTGCCCGGCGTTAATGATTTGCCCAAACAGGTCGCTGAAATGCGGCTGGTGCGCTTCATCCGGGCGAAAGAACCCCGTATTGGCAAATATTGACGGCCAGTTAAGCCATTCATGCCAGTAGGCGCGCGGACGAAAGTAAACCCACTGGTGATACCATTCGCGAGCCTCCGGATGACGACCGTAGTGATGAATCTCTCCTGGCGGGAACAGCAAAATATCACCCGGTCGGCAAACAAATTCTCGTCCCTGATTTTTCACCACCCCCTGACCGCGAATGGTGAGATTGAGAATATAACCTTTCATTCCCAGCGGTCGGTCGATAAAAAAATCGAGATAACCGTTGGCCTCAATCGGCGTTAAACCCGCCACCAGATGGGCATTAAACGAGTATCCCGGCAGCAGGGGATCATTTTGCGCTTCAGCCATACTTTTCATACTCCCGCCATTCAGAGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCAT-3’。
进一步地，荧光蛋白包括但不限于绿色荧光蛋白、红色荧光蛋白、青色荧光蛋白等，如SEQ ID NO.2所示基因序列，具体为：
5’-ATGTCAAAAGGCGAAGAACTGTTTACCGGCGTTGTTCCGATTCTGGTTGAACTGGATGGTGATGTGAATGGCCATAAATTTAGCGTGTCAGGCGAAGGCGAAGGTGATGCCACCTATGGCAAACTGACCCTGAAATTTATTTGTACCACCGGCAAACTGCCGGTTCCGTGGCCGACCTTAGTGACCACCCTGACCTATGGTGTGCAGTGTTTTAGTCGCTATCCGGATCACATGAAACAGCATGATTTTTTTAAATCTGCAATGCCGGAAGGCTATGTGCAGGAACGCACCATTTTTTTTAAAGATGATGGTAATTATAAAACCCGCGCCGAAGTTAAATTTGAAGGTGATACCTTAGTTAATCGTATTGAACTGAAAGGCATTGATTT TAAAGAAGATGGCAATATTCTGGGCCATAAACTGGAATATAATTATAATAGTCATAATGTGTATATTATGGCCGATAAACAGAAAAATGGTATTAAAGTTAATTTTAAAATTCGTCATAATATTGAAGATGGCTCAGTGCAGTTAGCCGATCATTATCAGCAGAATACCCCGATTGGTGATGGTCCGGTTCTGCTGCCGGATAATCATTATCTGTCTACCCAGAGCGCCCTGAGCAAAGATCCGAATGAAAAACGCGATCACATGGTTCTGCTGGAATTTGTGACCGCAGCAGGTATTACCCTGGGCATGGATGAACTGTATAAATAA-3’。
上述待测物诱导型荧光生物传感器的构建方法，包括以下步骤：
(1)将编码可表面展示纤维素结合结构域CBM的基因和编码荧光蛋白的基因与pETDuet-tac载体连接，得到载体pETDuet-tac-EGFP-CBM；其中，编码荧光蛋白的基因位于第一tac启动子的下游，编码可表面展示纤维素结合结构域CBM的基因位于第二tac启动子的下游；
(2)以上述构建的载体pETDuet-tac-EGFP-CBM为模板，将第一tac启动子替换为待测物诱导的启动子，转化入宿主菌，按上述细菌纤维素基生物传感器的构建方法中所述方法连接细菌纤维素，得到待测物诱导型荧光生物传感器。
进一步地，在步骤(2)中，用SEQ ID NO.4所示的上游引物和SEQ ID NO.5所示的下游引物进行反向PCR扩增删除第一tac启动子。其中，上游引物和下游引物的序列具体为：
SEQ ID NO.4：5’-CAATCGATCTCGATCCTCTACG-3’；
SEQ ID NO.5：5’-TTTCACACAGGAAACAGTATC-3’。
本发明的生物传感器在检测单糖、爆炸物分子以及重金属中均有广泛应用，具体地，将本发明的生物传感器与待测溶液混合，培养3-60h后检测荧光强度，实现待测物的检测。
借由上述方案，本发明至少具有以下优点：
(1)本发明通过在细胞表面展示CBM，无需对细菌纤维素(BC)底物进行任何修饰即可实现细胞在BC基质上的高效和特异性固定，向装载有全细 胞的BC载体持续施加外部剪切力60h后，表面展示CBM的细胞仍能与BC载体紧密结合。
(2)本发明采用同步展示固定策略，实现了简单、快速的全细胞装载。
(3)本发明提供的细菌纤维素(BC)基生物传感器在物质检测领域具有巨大潜力。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
为了使本发明的内容更容易被清楚的理解，下面根据本发明的具体实施例并结合附图，对本发明作进一步详细的说明。
图1为SDS-PAGE图，其中，1是含有质粒pETDuet-tac-CBM2a的重组大肠杆菌E.coli BL21(DE3)可溶蛋白，2是原始E.coli BL21(DE3)可溶蛋白；
图2为表面展示有CBM2a的重组大肠杆菌免疫荧光显微图；
图3为装载有表面展示CBM2a重组大肠杆菌的细菌纤维素(BC)载体表面形貌SEM图；
图4为检测阿拉伯糖(320mg/L)的片状和球状BC基荧光生物传感器的荧光成像图；
图5为阿拉伯糖浓度与荧光强度的关系；
图6为检测土壤中阿拉伯糖的片状和球状BC基荧光生物传感器的荧光成像图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的 限定。
下述实施例中所使用的实验方法若无特殊说明，均为常规操作方法；所使用的材料、试剂等，若无特殊说明，均可从商业途径获得。
实施例1
(1)利用PCR技术，将可在大肠杆菌E.coli BL21(DE3)表面展示CBM2a的基因SEQ ID NO.1插入质粒pETDuet-tac(以pETDuet为模板，将其上两个T7启动子替换为tac启动子，实验室保藏)。使用内切酶NdeI和KpnI进行酶切，PCR纯化试剂盒纯化回收。后使用T4连接酶，于16℃连接过夜，将SEQ ID NO.1和载体pETDuet-tac连接。连接产物转化感受态细胞E.coli DH5α，菌落PCR及测序验证，得到载体pETDuet-tac-CBM2a。将pETDuet-tac-CBM2a转化至宿主菌E.coli BL21(DE3)，获得含有表面展示CBM2a的重组细胞。
(2)将木醋杆菌(Acetobacter xylinum ATCC)单菌落在50mL HS培养基(葡萄糖40g/L，酵母提取物5g/L，蛋白胨5g/L，Na 2HPO 42.7g/L，柠檬酸1.5g/L)中30℃静态培养2天，制备种子液。后取5mL种子液至100mL HS培养基中30℃静态培养15天，制备BC膜。
(3)挑取步骤(1)含有表面展示CBM2a的重组细胞E.coli BL21(DE3)单菌落接种于5mL含有100μg/mL Amp(氨苄西林)的Luria-Bertani broth(LB)培养基中，在37℃下，200rpm震荡培养8-12h。将上述1mL菌液接种于含100mL培养基的500mL摇瓶中，37℃下，200rpm震荡培养，当OD 600达到0.6-0.8时，同时加入IPTG(终浓度为0.25mM)和BC基质(步骤2制备得到)，25℃，150rpm培养12h，获得装载有重组大肠杆菌的BC载体。然后，用50mM磷酸钾缓冲液(pH7.0)彻底冲洗BC载体，得到细菌纤维素(BC)基生物传感器。
(4)为了确定展示CBM2a的大肠杆菌对BC的结合能力，我们通过测量孵育BC载体的溶液的荧光强度和OD 600来监测从BC载体中释放的细胞。将附载大肠杆菌的BC在4℃，50mM磷酸钾缓冲液(pH7.0)中，在旋转杂 交器中培养。然后，在不同时间点从溶液中采集样品进行荧光和OD 600测量。使用96孔细胞培养板在cytation 5成像仪上测定逃逸大肠杆菌的荧光强度和OD 600的时间历程。用λex＝480nm的激发光和λem＝520nm的发射光来表征细胞表达的EGFP的荧光。
结果显示，向装载有细胞的BC载体持续施加外部剪切力60h后，表面展示CBM的细胞仍能与BC载体紧密结合。
实施例2
(1)利用PCR技术，将可在大肠杆菌E.coli BL21(DE3)表面展示特异性结合纤维素结晶区的CBM的基因插入质粒pETDuet-tac。使用内切酶NdeI和KpnI进行酶切，PCR纯化试剂盒纯化回收。后使用T4连接酶，于16℃连接过夜，将可在大肠杆菌E.coli BL21(DE3)表面展示特异性结合纤维素结晶区的CBM的基因和载体pETDuet-tac连接。连接产物转化感受态细胞E.coli DH5α，菌落PCR及测序验证，得到载体pETDuet-tac-CBM2a。将pETDuet-tac-CBM2a转化至宿主菌E.coli BL21(DE3)，获得含有表面展示CBM2a的重组细胞。
(2)将木醋杆菌(Acetobacter xylinum ATCC)单菌落在50mL HS培养基(葡萄糖40g/L，酵母提取物5g/L，蛋白胨5g/L，Na 2HPO 42.7g/L，柠檬酸1.5g/L)中30℃静态培养2天，制备种子液。后取10mL种子液至100mL HS培养基中30℃静态培养15天，制备BC膜。
(3)挑取步骤(1)含有表面展示特异性结合纤维素结晶区的CBM的重组细胞E.coli BL21(DE3)单菌落接种于5mL含有100μg/mL Amp的Luria-Bertani broth(LB)培养基中，在37℃下，200rpm震荡培养8-12h。将上述1mL菌液接种于含100mL培养基的500mL摇瓶中，37℃下，200rpm震荡培养，当OD 600达到0.6-0.8时，同时加入IPTG(终浓度为0.25mM)和BC基质(步骤2制备得到)，25℃，150rpm培养12h，获得装载有重组大肠杆菌的BC载体。然后，用50mM磷酸钾缓冲液(pH7.0)彻底冲洗BC载体，得到细菌纤维素(BC)基生物传感器。
实施例3
(1)利用PCR技术，将可在大肠杆菌E.coli BL21(DE3)表面展示CBM2a的基因SEQ ID NO.1插入质粒pETDuet-tac(以pETDuet为模板，将其上两个T7启动子替换为tac启动子，实验室保藏)。使用内切酶NdeI和KpnI进行酶切，PCR纯化试剂盒纯化回收。后使用T4连接酶，于16℃连接过夜，将SEQ ID NO.1和载体pETDuet-tac连接。连接产物转化感受态细胞E.coli DH5α，菌落PCR及测序验证，得到载体pETDuet-tac-CBM2a。后以pETDuet-tac-CBM2a为模板，使用NcoI和EcoRI酶切位点，重复上述酶切连接步骤将SEQ ID NO.2绿色荧光蛋白基因插入pETDuet-tac-CBM2a。连接产物转化感受态细胞E.coli DH5α，菌落PCR及测序验证，获得载体pETDuet-tac-EGFP-CBM2a。后将pETDuet-tac-EGFP-CBM2a转化至宿主菌E.coli BL21(DE3)，获得底盘荧光细胞。
(2)将木醋杆菌(Acetobacter xylinum ATCC)单菌落在50mL HS培养基(葡萄糖40g/L，酵母提取物5g/L，蛋白胨5g/L，Na 2HPO 42.7g/L，柠檬酸1.5g/L)中30℃静态培养2天，制备种子液。
将10mL种子液和220mL HS培养基倒入250mL烧瓶中，30℃下150rpm震荡培养5天，制备球状BC；将10mL种子液和100mL HS培养基倒入250mL烧瓶中，30℃下静态培养15天，制备片状BC；将10mL种子液和100mL HS培养基混合，后将混合液注入硅管，30℃下将硅管静态培养10天，制备柱状BC。
(3)挑取步骤(1)底盘荧光细胞E.coli BL21(DE3)单菌落接种于5mL含有100μg/mL Amp的Luria-Bertani broth(LB)培养基中，在37℃下，200rpm震荡培养8-12h。将上述1mL菌液接种于含100mL培养基的500mL摇瓶中，37℃下，200rpm震荡培养，当OD 600达到0.6-0.8时，同时加入IPTG(终浓度为0.25mM)和BC基质(步骤2制备得到)，25℃，150rpm培养12h，根据BC基质形状，分别获得装载有重组大肠杆菌的片状和球状BC载体。然后，用50mM磷酸钾缓冲液(pH 7.0)彻底冲洗BC载体。
实施例4
以实施例1中构建载体pETDuet-tac-EGFP-CBM2a为模板，用下述引物进行反向PCR扩增，以删除含有调控绿色荧光蛋白的tac启动子(即第一tac启动子)：
引物1：5’-CAATCGATCTCGATCCTCTACG-3’；
引物2：5’-TTTCACACAGGAAACAGTATC-3’；
此外，以质粒pCAS为模板，用下述引物进行PCR扩增，以获得如SEQ ID NO.3所示的含有P araBAD和AraC的核酸片段：
引物3：
5’-TAGAGGATCGAGATCGATTGTTATGACAACTTGACGGCTACATC-3’；
引物4：
5’-GATACTGTTTCCTGTGTGAAAATGGAGAAACAGTAGAGAGTTGCG-3’；
然后，使用ClonExpress II One Step Cloning Kit(购自Vazyme)将上述片段进行连接，替换载体pETDuet-tac-EGFP-CBM2a中调控绿色荧光蛋白的tac启动子为如SEQ ID NO.3所示的含有阿拉伯糖启动子(P araBAD)和AraC的核酸片段。连接产物转化感受态细胞E.coli DH5α，菌落PCR及测序验证，获得载体pETDuet-araBAD-EGFP-CBM2a。后将重组质粒pETDuet-araBAD-EGFP-CBM2a转化至宿主菌E.coli BL21(DE3)，按实施例1所述方法与BC连接，获得L-阿拉伯糖诱导型荧光生物传感器。
实施例5
(1)利用L-阿拉伯糖诱导型荧光生物传感器检测溶液中阿拉伯糖。具体方法为：将上述制备的L-阿拉伯糖诱导型荧光生物传感器分别放入不同浓度阿拉伯糖溶液中，于室温条件下培养5h，然后测其荧光强度并进行荧光成像，实现对溶液中阿拉伯糖的检测。
阿拉伯糖溶液的制备方法为：用水逐渐稀释配置的阿拉伯糖储备液至终浓度分别为20mg/L，160mg/L和320mg/L。
(2)利用L-阿拉伯糖诱导型荧光生物传感器检测土壤中阿拉伯糖。具体方法为：将阿拉伯糖与土壤按2.4g阿拉伯糖/Kg土壤混合，制备所需土壤样品。后将荧光生物传感器放入样品中，于37℃培养24h，然后进行荧光成像，实现对土壤中阿拉伯糖的检测。
结果显示，可检测的最低浓度为15mg/L。
实施例6
(1)以实施例1中构建载体pETDuet-tac-EGFP-CBM2a为模板，替换载体pETDuet-tac-EGFP-CBM2a中调控绿色荧光蛋白的tac启动子为yqjF启动子。获得载体pETDuet-yqjF-EGFP-CBM2a。后将重组质粒pETDuet-yqjF-EGFP-CBM2a转化至宿主菌E.coli BL21(DE3)，按实施例3所述方法与BC连接，获得2,4-二硝基甲苯(2,4-DNT)诱导型荧光生物传感器。
(2)利用2,4-DNT诱导型荧光生物传感器检测溶液中2,4-DNT。具体方法为：将上述制备的2,4-DNT诱导型荧光生物传感器分别放入不同浓度2,4-DNT溶液中，于室温条件下培养12h，然后测其荧光强度并进行荧光成像，实现对溶液中2,4-DNT的检测。
2,4-DNT溶液的制备方法为：用水逐渐稀释配置的2,4-DNT储备液至终浓度分别为5mg/L，10mg/L和20mg/L。
(3)利用2,4-DNT诱导型荧光生物传感器检测土壤中2,4-DNT。具体方法为：将2,4-DNT与土壤按0.24g 2,4-DNT/Kg土壤混合，制备所需土壤样品。后将荧光生物传感器放入样品中，于37℃培养24h，然后进行荧光成像，实现对溶液中2,4-DNT的检测。
结果显示，可检测的最低浓度为4mg/L。
实施例7
(1)以实施例1中构建载体pETDuet-tac-EGFP-CBM2a为模板，替换载体pETDuet-tac-EGFP-CBM2a中调控绿色荧光蛋白的tac启动子为znt启动子(包括zntA启动子和zntR核酸序列)。获得载体pETDuet-znt-EGFP-CBM2a。后将重组质粒pETDuet-CBM2a-znt-EGFP转化至宿主菌E.coli BL21(DE3)，按实施例3所述方法与BC连接，获得重金属诱导型荧光生物传感器。
(2)利用重金属诱导型荧光生物传感器检测溶液中重金属。具体方法为：将上述制备的重金属诱导型荧光生物传感器分别放入不同浓度重金属溶液中，于室温条件下培养24h，然后测其荧光强度并进行荧光成像，实现对溶液中重金属的检测。
重金属溶液包括Zn 2+,Cd 2+和Hg 2+。制备方法为：用水逐渐稀释配置的上述Zn 2+储备液至终浓度分别为20mg/L，100mg/L和300mg/L；用水逐渐稀释配置的上述Cd 2+储备液至终浓度分别为0.5mg/L，2.0mg/L和4.0mg/L；用水逐渐稀释配置的上述Hg 2+储备液至终浓度分别为0.004mg/L，0.016mg/L和0.06mg/L。
(3)利用重金属诱导型荧光生物传感器检测土壤中重金属。具体方法为：将重金属与土壤分别按0.3gZn 2+/Kg土壤、4mgCd 2+/Kg土壤和0.06mg Hg 2+/Kg混合，制备所需土壤样品。后将荧光生物传感器放入样品中，于37℃培养24h，然后进行荧光成像，实现对溶液中重金属的检测。
结果显示，可检测的最低浓度为Zn 2+6mg/L、Cd 2+0.05mg/L、Hg 2+0.004mg/L。
对比例1
(1)利用PCR技术，以实施例1中构建载体pETDuet-tac-EGFP-CBM2a为模板，将CBM2a替换为CBM44。获得载体pETDuet-tac-EGFP-CBM44。
CBM44的基因序列具体为：
5’-AAATTCAATTTTGAAGATGGAACACTAGGGGGCTTTACCACCT CTGGCACCAATGCGACCGGTGTTGTGGTGAACACCACTGAAAAAGCGTTTAAGGGTGAACGTGGTCTGAAGTGGACCGTCACGTCCGAGGGCGAGGGCACCGCTGAGCTCAAGCTGGACGGCGGTACGATCGTGGTGCCGGGTACGACGATGACATTCCGCATTTGGATTCCGAGCGGCGCGCCAATCGCCGCAATTCAACCGTACATTATGCCGCACACCCCGGATTGGAGCGAAGTTCTGTGGAACAGCACCTGGAAAGGTTATACCATGGTCAAAACTGACGATTGGAACGAGATCACCTTGACCCTGCCGGAAGATGTTGACCCGACGTGGCCGCAGCAAATGGGTATTCAGGTTCAGACCATCGACGAAGGTGAGTTCACCATCTACGTGGATGCGATCGACTGGTGA-3’。
将pETDuet-tac-EGFP-CBM44转化至宿主菌E.coli BL21(DE3)，获得含有表面展示CBM44的重组细胞。后按照实施例1方法，得到细菌纤维素(BC)基生物传感器。
(2)为了确定展示CBM44的大肠杆菌对BC的结合能力，按照实施例1(4)中方法测量孵育BC载体的溶液的荧光强度和OD 600，以监测从BC载体中释放的细胞。
结果显示，向装载有细胞的BC载体持续施加外部剪切力60h后，表面展示CBM44的细胞发生逃逸，无法与BC载体紧密结合。
(3)以构建载体pETDuet-tac-EGFP-CBM44为模板，按照实施例4方法将tac启动子替换为含有阿拉伯糖启动子(P araBAD)和AraC的核酸片段。最终获得表面展示CBM44的L-阿拉伯糖诱导型荧光生物传感器。后按照实施例5中方法分别对溶液和土壤中阿拉伯糖进行检测。
结果显示，表面展示CBM44的L-阿拉伯糖诱导型荧光生物传感器因细胞逃逸，检测灵敏度大幅下降，如在20mg/L时无法正常检测阿拉伯糖，在320mg/L时准确度低于60％。
显然，上述实施例仅仅是为清楚地说明所作的举例，并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出 其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (10)

  1. 一种细菌纤维素基生物传感器，其特征在于：所述生物传感器包括细菌纤维素和表面展示纤维素结合结构域CBM的细胞，所述纤维素结合结构域CBM为可特异性结合纤维素结晶区的纤维素结合结构域，所述细胞通过纤维素结合结构域CBM与细菌纤维素连接；其中，所述纤维素结合结构域CBM为CBM2a。
  2. 根据权利要求1所述的生物传感器，其特征在于：编码可表面展示CBM2a的基因序列如SEQ ID NO.1所示。
  3. 根据权利要求1所述的生物传感器，其特征在于：所述细胞为重组菌，所述重组菌以pETDuet-tac为载体表达所述纤维素结合结构域CBM，所述pETDuet-tac是将pETDuet载体上的两个T7启动子替换为两个tac启动子得到的载体，位于上游的为第一tac启动子，位于下游的为第二tac启动子，所述pETDuet-tac上含有位于第一tac启动子下游的编码荧光蛋白的基因和位于第二tac启动子下游的编码可表面展示纤维素结合结构域CBM的基因。
  4. 根据权利要求3所述的生物传感器，其特征在于：将第一tac启动子替换为待测物诱导的启动子，所述待测物诱导的启动子在靶化合物存在时影响下游荧光蛋白基因的转录。
  5. 根据权利要求4所述的生物传感器，其特征在于：所述待测物诱导的启动子为阿拉伯糖诱导的启动子、硝基化合物诱导的启动子或重金属诱导的启动子。
  6. 根据权利要求5所述的生物传感器，其特征在于：所述阿拉伯糖诱导的启动子的核苷酸序列如SEQ ID NO.3所示。
  7. 权利要求1所述的生物传感器的构建方法，其特征在于，包括以下步骤：将可在表面展示纤维素结合结构域CBM的细胞与细菌纤维素共同培养，得到所述生物传感器；其中，所述纤维素结合结构域CBM为CBM2a。
  8. 权利要求1-6任一项所述的生物传感器在物质检测中的应用。
  9. 根据权利要求8所述的应用，其特征在于：所述物质包括单糖、爆炸物分子或重金属。
  10. 根据权利要求8所述的应用，其特征在于，所述的物质检测包括以下步骤：将权利要求1-6任一项所述的生物传感器与待测溶液混合培养后检测荧光强度，实现对待测物的检测。
PCT/CN2022/122046 2021-10-12 2022-09-28 一种细菌纤维素基生物传感器及其应用 WO2023061222A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US18/527,123 US20240118205A1 (en) 2021-10-12 2023-12-01 Bacterial cellulose-based biosensor and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111185375.6 2021-10-12
CN202111185375.6A CN113881616B (zh) 2021-10-12 2021-10-12 一种细菌纤维素基生物传感器及其应用

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US18/527,123 Continuation US20240118205A1 (en) 2021-10-12 2023-12-01 Bacterial cellulose-based biosensor and use thereof

Publications (2)

Publication Number Publication Date
WO2023061222A1 true WO2023061222A1 (zh) 2023-04-20
WO2023061222A9 WO2023061222A9 (zh) 2023-07-20

Family

ID=79006258

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/122046 WO2023061222A1 (zh) 2021-10-12 2022-09-28 一种细菌纤维素基生物传感器及其应用

Country Status (3)

Country Link
US (1) US20240118205A1 (zh)
CN (1) CN113881616B (zh)
WO (1) WO2023061222A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113881616B (zh) * 2021-10-12 2022-08-02 江南大学 一种细菌纤维素基生物传感器及其应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113881616A (zh) * 2021-10-12 2022-01-04 江南大学 一种细菌纤维素基生物传感器及其应用

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5624537A (en) * 1994-09-20 1997-04-29 The University Of British Columbia - University-Industry Liaison Office Biosensor and interface membrane
WO2008133464A1 (en) * 2007-04-27 2008-11-06 Labgenomics Co., Ltd Biochip for quantifying amino acid and method for analying amino acid using the same
CN102174532B (zh) * 2011-01-19 2013-05-01 中国科学技术大学 家族3纤维素结合域在真核生物中作为重组蛋白质表达和纯化的亲和标签的应用
CN105492612B (zh) * 2013-07-02 2020-11-03 韩国生命工学研究院 重组纤维素糖化酶混合物和重组酵母复合菌株及其用途
JP6348530B2 (ja) * 2015-03-26 2018-06-27 石川県公立大学法人 トリテルペンの生産方法
WO2017203281A1 (en) * 2016-05-27 2017-11-30 Customem Ltd Production of functionalised cellulose
JP2019162036A (ja) * 2016-08-05 2019-09-26 味の素株式会社 ヘミセルラーゼ
CN107446909B (zh) * 2017-09-29 2020-09-29 南京工业大学 一种大肠杆菌的固定化方法及利用固定化大肠杆菌补料发酵生产l-赖氨酸的方法
CN107741413B (zh) * 2017-11-29 2020-01-21 大连工业大学 一种预测纤维素结晶度的方法
US20220396762A1 (en) * 2019-08-21 2022-12-15 Massachusetts Institute Of Technology Growing programmable enzyme-functionalized and sense-and-response bacterial cellulose living materials with engineered microbial co-cultures
CN111139258A (zh) * 2020-01-17 2020-05-12 义乌市颂健生物科技有限公司 一种线性化DNA载体pHB-1质粒及用其制备的用于编辑细菌基因组的试剂盒
CN111304232B (zh) * 2020-02-27 2021-04-20 温州医科大学 基于膜表面融合表达策略纯化蛋白的方法及其应用

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113881616A (zh) * 2021-10-12 2022-01-04 江南大学 一种细菌纤维素基生物传感器及其应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANDRADE F K ET AL: "Improving the affinity of fibroblasts for bacterial cellulose using carbohydrate-binding modules fused to RGD", vol. 92A, no. 1, 1 January 2010 (2010-01-01), pages 9 - 17, XP002768744, DOI: 10.1002/jbm.a.32284 *
OLIVEIRA, C. ET AL.: "Recombinant CBM-fusion technology- Applications overview.", BIOTECHNOL ADV, vol. 33, no. 3-4, 14 February 2015 (2015-02-14), pages 358 - 369, XP029591428, DOI: 10.1016/j.biotechadv.2015.02.006 *
SHEMER BENJAMIN, SHARON YAGUR-KROLL, CARINA HAZAN, SHIMSHON BELKIN: "Aerobic Transformation of 2, 4-Dinitrotoluene by Escherichia coli and Its Implications for the Detection of Trace Explosives", APPL ENVIRON MICROBIOL, vol. 84, no. 4, 8 December 2017 (2017-12-08), pages e01729 - 17, XP093057058, DOI: 10.1128/AEM.01729-17 *
WANG, AIJUN ET AL.: "Specific Adhesion to Cellulose and Hydrolysis of Organophosphate Nerve Agents by a Genetically Engineered Escherichia coli Strain with a Surface-Expressed Cellulose-Binding Domain and Organophosphorus Hydrolase", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 68, no. 4, 30 April 2002 (2002-04-30), XP055152890, DOI: 10.1128/AEM.68.4.1684-1689.2002 *

Also Published As

Publication number Publication date
WO2023061222A9 (zh) 2023-07-20
CN113881616A (zh) 2022-01-04
CN113881616B (zh) 2022-08-02
US20240118205A1 (en) 2024-04-11

Similar Documents

Publication Publication Date Title
Yu et al. Aqueous sol–gel encapsulation of genetically engineered Moraxella spp. cells for the detection of organophosphates
Monošík et al. Biosensors-classification, characterization and new trends
US20240118205A1 (en) Bacterial cellulose-based biosensor and use thereof
Chen et al. Development of engineered bacteriophages for Escherichia coli detection and high-throughput antibiotic resistance determination
CN111819288B (zh) 基于杂交链式反应的免疫信号放大方法
CN111812066A (zh) 基于CRISPR/Cas12a系统的生物传感器、试剂盒及其在小分子检测中的用途
CN110066820B (zh) 一种荧光菌株E.coli C600及构建方法与应用
WO1999045143A2 (en) A fluorescence polarization screening method
Farooq et al. Principle and development of phage-based biosensors
Hong et al. Separation and colorimetric detection of Escherichia coli by phage tail fiber protein combined with nano-magnetic beads
FR2817349A1 (fr) Nouveau procede de criblage de modulateurs de la transcription bacterienne
CN108486024B (zh) 基于菌群的传感系统检测有机磷农药的方法
Xu et al. Self-assembled β-galactosidase on T4 phage capsid through affinity binding with enhanced activity and stability for rapid bacteria detection
Kim et al. A liquid-based colorimetric assay of lysine decarboxylase and its application to enzymatic assay
CN108330139B (zh) 一类可调控的腐胺生物荧光传感器及其应用
Bidmanova et al. Immobilization of Haloalkane dehalogenase LinB from Sphingobium japonicum UT26 for Biotechnological Applications. J Biocatal Biotransformation 2: 1
Rees et al. Phage for the detection of pathogenic bacteria
Liu et al. Bacillus subtilis MraY in detergent-free system of nanodiscs wrapped by styrene-maleic acid copolymers
CN113528563B (zh) 一种利用爆炸物分子降解基因合成可视化生物感应器的制备方法及其应用
Li et al. Supramolecular protein assembly in cell-free protein synthesis system
Long et al. Constructing a bacterial cellulose-based bacterial sensor platform by enhancing cell affinity via a surface-exposed carbohydrate binding module
Saxena et al. Immobilisation and biosensors
WO2022103793A1 (en) Engineered proteins that bind the sars-cov-2 nucleocapsid protein
Chen et al. Multiple amplification-based fluorometric aptasensor for highly sensitive detection of Staphylococcus aureus
CN106591347B (zh) 一种包含噬菌体溶菌酶的表达系统及其运用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22880161

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22880161

Country of ref document: EP

Kind code of ref document: A1