WO2019076262A1 - Biocapteur permettant la détection d'un composé moléculaire cible et système comprenant un biocapteur - Google Patents

Biocapteur permettant la détection d'un composé moléculaire cible et système comprenant un biocapteur Download PDF

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WO2019076262A1
WO2019076262A1 PCT/CN2018/110245 CN2018110245W WO2019076262A1 WO 2019076262 A1 WO2019076262 A1 WO 2019076262A1 CN 2018110245 W CN2018110245 W CN 2018110245W WO 2019076262 A1 WO2019076262 A1 WO 2019076262A1
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biosensor
sequence
target molecule
nucleotide sequence
reporter gene
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PCT/CN2018/110245
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Chinese (zh)
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刘龙英
陈泰
沈玥
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深圳华大生命科学研究院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to the field of compound detection technology, and in particular to a biosensor for detecting a target molecular compound and a system comprising the biosensor.
  • Natural products are components of organisms or their metabolites. They usually have special biological activities and functions. They are the main pathway for the discovery of new drugs and are also a hot spot in current metabolic research. Since the natural product usually has a complicated structure due to containing more active groups, the chemical synthesis step is cumbersome and the yield is extremely low, the main acquisition route is extraction from plants, etc. However, the plant has a long growth cycle and a large number of extraction processes. Loss, as well as the effects of the process, make the acquisition of active natural products far from satisfactory. Therefore, the biosynthesis of natural products has become the main way to achieve industrial production.
  • the three-step reaction catalyzed by CPR1, CYB5, ADH1, ALDH1) reconstituted the biosynthetic pathway of artemisinic acid in yeast, which greatly increased the yield of artemisinic acid.
  • Stephanopoulos et al. introduced the synthetic pathway of taxadiene into E. coli and optimized the whole metabolic pathway to obtain high-yield taxadiene-producing Escherichia coli cells, which is a biosynthesis study of the new anticancer drug paclitaxel. Significant progress.
  • Zhang Xueli and others cooperated with Huang Qiqi and others to jointly carry out research on the synthesis of medicinal terpenoids by artificial cells, which significantly improved the ability of artificial yeast cells to synthesize diterpenoids and triterpenoids, and synthesized tanshinone IIA into prodrugs.
  • the yield of miltiradiene increased to 488 mg/L
  • the yield of squalene was increased to 852 mg/L
  • the yield of lycopene was increased to 21.17 mg/L.
  • Liu Tianqi and others have used synthetic biology methods to construct efficient metabolic pathways in model organisms, effectively producing rare-source drugs and prodrugs, including various high-value-added natural substances such as lycopene, astaxanthin, and paclitaxel. Compound.
  • biosensors of small molecule compounds have been widely used in the study of metabolic pathways.
  • Gil et al. designed biosensors to monitor environmental toxins;
  • Paige et al. designed a biosensor capable of detecting the solubility of the corresponding metabolite.
  • many researchers have made relevant research on biosensors of various metabolites and made some progress, but the current method does not propose a simple strategy that can be designed to be very effective and broad. Applied biosensors.
  • LBD ligand-binding domain
  • steroid-binding proteins as an example to locate the amino acid side chains around the optimal orientation ligand by computer calculations, and to map possible binding sites on the scaffold protein structure, according to the nature and structure of these side chains.
  • This design is based on these typical natural complexes or protein structures with low Boltzmann-weighted values in the free state and side chain interactions already present. Based on the high complementarity of these methods and phenotypes, 17 proteins with potential binding capabilities were designed for experimental screening.
  • the three fusion proteins have been shown to significantly increase the activity of the target molecule digoxin (10 times, DBD mutation). The body is 60 times).
  • the fusion protein is used as a biosensor, and finally an LBD capable of highly recognizing the target molecule digoxin is obtained.
  • the design of the biosensor still needs to rely on the high specificity of the computer designed, constructed and optimized to combine the LBD of the target small molecule, and the whole excavation process is very cumbersome.
  • the target small molecule changes, it is necessary to perform complicated calculations such as computer calculation, construction, and screening. Therefore, current biosensor design strategies are designed and constructed only for a certain metabolite, and do not achieve a very effective and broadly applicable biosensor.
  • the invention provides a biosensor for detecting a target molecular compound and a system comprising the biosensor, which can conveniently construct a biosensor of various molecular compounds with high specificity and effective recognition capability, so that various molecular metabolites Achieving accurate, high-throughput screening greatly reduces cumbersome manual operations and reduces the cost of using high-precision instrumentation for a wide range of applications.
  • a biosensor for detecting a target molecular compound being a fusion protein, the biosensor comprising a target molecule synthetase, and two synthase enzymes respectively linked to the target molecule a DNA binding domain, a transcriptional activation domain, and a degradation determinant; when the precursor of the target molecule is absent, the degradation determinant degrades the biosensor; when the precursor of the target molecule is present, the target The molecular synthetase recognizes and binds to the precursor substance to stabilize the biosensor, such that the degradation determinant fails to automatically detach, and the DNA binding domain binds to the promoter region of the reporter gene, and the transcription activation domain activates the reporter gene. expression.
  • the biosensor described above sequentially connects a degradation determinant, a DNA binding domain, a target molecule synthetase, and a transcriptional activation domain from a nitrogen terminal to a carbon terminal.
  • the target molecule synthetase is an enzyme that synthesizes the natural product.
  • natural product is a chemical substance which is usually produced by living organisms in nature and which is usually pharmacologically or biologically active, and mainly includes proteins, polypeptides, amino acids, nucleic acids, various enzymes, monosaccharides, Oligosaccharides, polysaccharides, glycoproteins, resins, colloids, lignin, vitamins, fats, oils, waxes, alkaloids, volatile oils, flavonoids, glycosides, terpenoids, phenylpropanoids, organic acids, phenols, anthraquinones Naturally occurring chemical constituents such as lactones, steroids, tannins, and antibiotics are commonly used in pharmaceutical drug discovery and drug design.
  • the above-mentioned target molecule synthetase is a wild type enzyme which synthesizes the above natural product.
  • the above-mentioned target molecular compound is selected from a drug and/or a prodrug compound, and accordingly, the above-mentioned target molecule synthetase is an enzyme which synthesizes the above-mentioned drug and/or prodrug compound.
  • the target molecule is lycopene
  • the target molecule synthetase is lycopene synthase; preferably, the lycopene synthase is encoded by a CrtI gene; preferably, the precursor of the target molecule is octahydrogen Lycopene.
  • the target molecule is resveratrol
  • the target molecule synthetase is a p-diphenylene synthase; preferably, the p-diphenylene synthase is encoded by an STS gene; preferably, the precursor substance of the target molecule It is p-coumaroyl-CoA.
  • the above degradation determinant is the degradation determinant MAT ⁇ 2 in yeast.
  • the above reporter gene is a resistance screening gene and/or a fluorescent marker gene.
  • the above resistance screening gene is selected from the group consisting of ampicillin, kanamycin, spectinomycin, and/or tetracycline; or the fluorescent marker gene is selected from the group consisting of a green fluorescent protein GFP gene, a red fluorescent protein RFP gene, and/or yellow Fluorescent protein YFP gene.
  • an embodiment provides a method of constructing a biosensor of the first aspect, comprising: first, a nucleic acid fragment encoding a target molecule synthetase, and a DNA respectively ligated to both ends of the target molecule synthetase
  • the nucleic acid fragment of the binding domain, the nucleic acid fragment of the transcriptional activation domain, and the nucleic acid fragment of the degradation determinant are assembled; and the assembled nucleic acid fragment is then ligated into the plasmid vector.
  • the above method further comprises: constructing an expression plasmid of the reporter gene, wherein the DNA binding domain binds to a promoter region of the reporter gene, and the transcription activation domain activates the expression of the reporter gene.
  • an embodiment provides a nucleotide sequence encoding the biosensor of the first aspect.
  • nucleotide sequence includes the sequence shown as SEQ ID NO: 134.
  • nucleotide sequence includes the sequence shown as SEQ ID NO: 171.
  • nucleotide sequence further includes a first vector sequence.
  • the first vector sequence described above is the sequence of the vector backbone pRS415.
  • the above reporter gene expression sequence comprises the sequence shown as SEQ ID NO:133. Further, the above reporter gene expression sequence further comprises a second vector sequence.
  • the second vector sequence described above is the sequence of the vector backbone pRS413.
  • an embodiment provides a method for detecting a target molecule compound, comprising: introducing the biosensor of the first aspect or the nucleotide sequence of the third aspect into a host cell for expression together with a reporter gene; Qualitative and/or quantitative analysis of the target molecule compound by detecting the expression of the above reporter gene.
  • an embodiment provides a biosensing system for detecting a target molecule compound, comprising a nucleotide sequence encoding the biosensor of the first aspect, a reporter gene expression sequence, and optionally, further comprising The nucleotide sequence of the biosensor and the host cell in which the reporter gene expression sequence is expressed.
  • nucleotide sequence of the above biosensor comprises the sequence shown as SEQ ID NO: 134
  • the above reporter gene expression sequence comprises the sequence shown as SEQ ID NO: 133.
  • nucleotide sequence of the above biosensor comprises the sequence shown as SEQ ID NO: 171
  • reporter gene expression sequence comprises the sequence shown as SEQ ID NO: 133.
  • nucleotide sequence of the above biosensor further includes a first vector sequence
  • the reporter gene expression sequence further includes a second vector sequence
  • the host cell is a yeast cell.
  • the first vector sequence is the sequence of the vector backbone pRS415
  • the second vector sequence is the sequence of the vector backbone pRS413.
  • an embodiment provides the use of the biosensor of the first aspect or the nucleotide sequence of the third aspect or the biosensing system of the fifth aspect for detecting a target molecule compound.
  • the above uses include relatively qualitative, quantitative analysis, and/or screening of host cells producing the above-described target molecular compound.
  • the design of the biosensor for detecting a target molecular compound of the present invention if only for the screening of the final product, selecting the reporter gene as a resistance screening gene, will greatly reduce the costly batch screening process, if it is necessary to obtain a strain bank of different yields.
  • the reporter gene can be designed into various fluorescent marker genes, and the metabolic products can be qualitatively and quantitatively analyzed by detecting the intensity of fluorescence.
  • the biosensor for detecting a target molecular compound of the present invention is designed according to a naturally occurring target metabolic pathway, and various target molecular synthetases are used as recognition groups, and the above-mentioned target molecule synthetase is naturally occurring and is front of the target molecule.
  • the bulk material has a high degree of specificity and effective recognition ability, omitting the cumbersome excavation process for computer design, construction and optimization screening of the target molecule LBD, which significantly reduces the time cost.
  • the biosensor of the present invention selects a target molecular synthetase as a group for identifying a precursor substance of a target molecule, and the components of the designed fusion biosensor are relatively independent, the single element replacement is relatively simple, and all natural in nature Compound molecules can find groups that can recognize their precursors. Even if the target molecules are replaced, they can quickly assemble their fusion biosensors, and have a relatively wide range of applications.
  • FIG. 1 is a schematic diagram showing a model structure and a function of a biosensor according to an embodiment of the present invention
  • FIG. 2 is a schematic view showing the synthesis of a lycopene body according to an embodiment of the present invention
  • FIG. 3 is a schematic view showing the synthesis of a resveratrol body according to an embodiment of the present invention.
  • 5 is an electropherogram showing the construction of an integrated plasmid in an embodiment of the present invention.
  • FIG. 6 is a diagram showing fluorescence results observed on a fluorescent display device after transformation of a yeast strain by a lycopene biosensor according to an embodiment of the present invention
  • FIG. 7 is a diagram showing fluorescence results of a lycopene biosensor after detecting a yeast strain on a microplate reader according to an embodiment of the present invention
  • FIG. 8 is a diagram showing fluorescence results observed on a fluorescent display device after a yeast strain is transformed by a resveratrol biosensor according to an embodiment of the present invention
  • Figure 9 is a graph showing the fluorescence results of a resveratrol biosensor after detection of a yeast strain on a microplate reader in accordance with an embodiment of the present invention.
  • the whole process of identifying the target molecular group in the current biosensor is mainly the technology of protein engineering, and the binding relationship between the protein and the ligand molecule is calculated by a computer operation model to predict the binding ability between the two. It is necessary to construct a universal and simple biosensor, and the purpose is to eliminate the cumbersome work of computer calculation, construction, screening, etc., along with the change of the target molecule.
  • the interaction of many enzymes is required in the metabolic pathway to achieve the expression of the target molecule.
  • the formation of the target molecule is the result of the action of the enzyme responsible for the step of identifying the substrate of the previous step. Then, the synthetase of this step should have a high recognition ability or binding ability to the precursor substance.
  • the biosensor is a fusion protein including a target molecule synthetase (as a "recognition group"), and respectively A DNA binding domain (DBD), a transcriptional activation domain (TAD), and a degradation determinant (eg, ligated upstream of the DBD) attached to both ends of the target molecule synthetase.
  • a target molecule synthetase as a "recognition group”
  • DBD DNA binding domain
  • TAD transcriptional activation domain
  • degradation determinant eg, ligated upstream of the DBD
  • the reporter gene can select a resistance screening marker or a fluorescent display marker, and finally introduce the constructed fusion protein (ie, a biosensor) into the cell.
  • the recognition group cannot bind to the premise substance of the target molecule, thereby causing the biosensor to be unstable, and the degradation determinant degrades the unstable biosensor; when the precursor substance of the target molecule When generated in the body, the target molecule synthetase highly recognizes the precursor substance and binds it effectively.
  • the biosensor becomes more stable due to the effective binding of the substrate, and the degradation determinant is thus automatically detached.
  • the DBD will be able to effectively combine the downstream report.
  • TAD will effectively activate the reporter gene for expression. Therefore, the host cell producing the target molecule can be finally screened by the resistance screening marker, or the target molecule can be subjected to relative qualitative/quantitative analysis by detecting the expression level of the reporter gene.
  • lycopene is formed by synthesizing phytoene into tomato red under the action of lycopene synthase CrtI. Prime. Therefore, CrtI can be used as a recognition group for a biosensor.
  • the optimal mutant of DBD is Gal4, and the two mutants L77F and R60S obtained in the Gal4 dimer can improve the recognition ability of the recognition group. Therefore, G L77F or G R60S can be used as a mutant of DBD to construct a biosensor of lycopene, and in this embodiment, G L77F is selected.
  • the optimal mutant of TAD is VP16 or VP64, both of which can drive the expression of yEGFP by controlling the downstream GAL1 promoter. Therefore, VP16 or VP64 can be used as a mutant of TAD to construct a biosensor of lycopene, and VP16 is selected in this embodiment.
  • the N-terminal of DBD is linked to the degradation determinant MAT ⁇ 2 commonly found in the yeast system, and the downstream reporter gene selects the fluorescent protein yEGFP in yeast, which can quantitatively quantify the target metabolite by detecting the expression level of the fluorescent protein.
  • sequence fragments of the fusion fragments were synthesized by gene synthesis, and then the fusion fragments were inserted into the plasmid by Gibson assembly, and the plasmids having the fusion protein were assembled into a yeast strain capable of metabolizing lycopene, and the expression of the fluorescent protein was observed and detected.
  • the experimental process was mainly divided into two major modules: reporter plasmid construction (pRS413+GYC) and GIV (Gal4-crtI-VP16) plasmid construction (pRS415+MGIV).
  • the reporter plasmid contains yEGFP and selects the GAL1 promoter as the promoter, the CYC1 terminator as the terminator, and the vector backbone pRS413;
  • the GIV (Gal4-crtI-VP16) plasmid contains the degradation determinant MAT ⁇ 2, the DBD optimal mutant G L77F , tomato The erythrin synthase CrtI and the TAD-optimal mutant VP16, as well as the vector backbone pRS415, are shown in Figure 4.
  • the 5'-GAL1 promoter + yEGFP + CYC1-3' terminator fragment was synthesized by DNA column (1467 bp, cut into 3 segments: GYC-F1 581 bp; GYC-F2 491 bp; GYC-F3 565 bp) and MAT ⁇ 2-G L77F- IV Fragment (2610 bp, split into 5 segments: MGIV-F1 567 bp; MGIV-F2 583 bp; MGIV-F3 558 bp; MGIV-F4 577 bp; MGIV-F5 580 bp), oligonucleotides resolved by each fragment (see Table 1 below)
  • the PCA was assembled separately to obtain sub-fragments, for example, GYC-F1-1 to GYC-F1-16 were assembled by PCA to obtain sub-fragment GYC-F1 (SEQ ID NO: 125), and the others were deduced to obtain sub-fragments G
  • Lycopene yeast engineering bacteria from the National Gene Bank of Shenzhen Huada Gene Life Science Research Institute, which is based on the Saccharomyces cerevisiae strain SynYII (Accession No. CTCCC NO: M 2014434) as the chassis cell, the lycopene biosynthesis pathway
  • the three foreign genes (CrtE, CrtB, CrtI) were spliced to form a recombinant plasmid, which was introduced into the chassis cells for metabolite detection.
  • the strain capable of expressing lycopene was lycopene yeast engineering bacteria.
  • Shenzhen Huada Gene Technology Service Co., Ltd. was commissioned to synthesize all the oligonucleotides in Table 1, and the synthesized oligonucleotides were diluted to 10 ⁇ M, respectively, and 2.5 ⁇ L of each was taken out, and 2 ⁇ L was taken as a template for the first assembly of SOE PCA.
  • 0.2 ⁇ L of Q5 DNA polymerase, 4 ⁇ L of 5 ⁇ buffer, 1.6 ⁇ L of 2.5 mM dNTPs, and 20 ⁇ L of water were added.
  • Step 2 Configure the following reaction system, Ex Taq DNA polymerase (TAKARA) 0.5 ⁇ L, 10 ⁇ buffer 5 ⁇ L, 2.5 mM dNTPs 4 ⁇ L, first step PCR product 10 ⁇ L, first and last oligos of each subfragment Nucleotides were used as primers, each taking 2 ⁇ L, and ddH 2 O was supplemented with 50 ⁇ L.
  • the reaction procedure was 94 ° C for 5 min, 94 ° C for 30 sec, 55 ° C for 30 sec, 72 ° C for 30 sec, 29 cycles, 72 ° C for 2 min, and 12 ° C.
  • Electrophoretic detection Prepare 1% agarose gel, take 5 ⁇ L PCR product for electrophoresis detection, 3 ⁇ L DL 2000 DNA ladder, 180V voltage for 30min, electropherogram shown in Figure 5(a), and obtain the final spliced sub-fragment of about 500bp. .
  • the PCR product in step 1 was purified by gel purification using a gel purification kit, and the product was purified by PCR using a TA cloning kit (TAKARA).
  • TAKARA TA cloning kit
  • the monoclonal clones were selected from the TA cloning plate in step 2.
  • 2 ⁇ L of the bacterial liquid was taken as a template for PCR, 0.5 ⁇ L of M13-F and M13-R, 5 ⁇ L of 2 ⁇ PCR Mix, and 10 ⁇ L of ddH 2 O.
  • the reaction procedure was 94 ° C for 5 min, 94 ° C for 30 sec, 55 ° C for 30 sec, 72 ° C for 30 sec, 29 cycles, 72 ° C for 2 min, and 12 ° C.
  • 5 ⁇ L of the digested product was subjected to electrophoresis, and electrophoresis was carried out using a 1% agarose gel at 180 V for 30 min.
  • the electrophoresis results are shown in Figure 5(b), and the correct strip size is indicated by the arrow.
  • the correct bacterial extract plasmid of the bacterial solution was selected for sanger sequencing, and the plasmid with the correct assembly sequence was analyzed.
  • the correct subfragment cloning plasmid obtained in step 4 and the vector backbone were assembled in one step.
  • the 20 ⁇ L reaction system was as follows: 4 ⁇ buffer 5 ⁇ L, the subfragment cloning plasmid and the vector backbone were added in a molar ratio of 5:1, and the endonuclease was 1 ⁇ L.
  • DDH 2 O was supplemented with 20 ⁇ L and warmed for 1 h.
  • the endonuclease of the reporter construct was BswiI, and the reaction temperature was 55 ° C.
  • the endonuclease of the GIV (Gal4-crtI-VP16) plasmid construct was TspMI, and the reaction temperature was 75. °C, 10 ⁇ L was taken after the warm bath for clonal transformation.
  • the monoclonal clone was picked from the TA cloning plate in the step 5, and after the overnight culture, the plasmid was extracted using a kit, and the restriction enzyme digestion was used to identify the plasmid.
  • the enzyme digestion system 0.2 ⁇ L of XbaI (NEB), 0.2 ⁇ L of XhoI/PstI (NEB), 1 ⁇ L of buffer, 3 ⁇ L of plasmid DNA, and 10 ⁇ L of ddH 2 O.
  • the enzyme was digested at 37 ° C for 1 h. 5 ⁇ L of the digested product was subjected to electrophoresis, and electrophoresis was carried out using a 1% agarose gel at 180 V for 30 min.
  • the electrophoresis results are shown in Figure 5(c) with a schematic diagram of the simulated digestion.
  • the correct plasmid was selected for sequencing, and the plasmid with the correct assembly sequence was analyzed.
  • the two integrated plasmids with the correct sequencing were mixed and mixed with 1:1, and the concentration was measured.
  • 200 ng of the transformed lycopene yeast engineering bacteria (as a positive yeast strain) was taken out, and the corresponding auxotrophic medium was coated and picked.
  • the colonies were cultured overnight in a liquid medium, and the lycopene yeast engineering strain (as a negative yeast strain) which was not transformed with any plasmid was cultured overnight, as shown in Fig. 6, a tomato containing a biosensor was found on a fluorescent display device.
  • the erythromycin yeast strain (A) has a more pronounced green fluorescence display than the lycopene yeast strain (B) without a biosensor.
  • the above-mentioned strains as positive and negative were cultured, the OD value was detected in a time period, and the fluorescence value was read by a microplate reader (the excitation wavelength of yEGFP) It is 488 nm, the emission wavelength is 575 nm), and the data is plotted as the abscissa and the fluorescence value as the ordinate, as shown in Fig. 7. It can be seen from the figure that the positive yeast strain has a significantly higher fluorescence value than the negative yeast strain, which proves that the lycopene biosensor is successfully constructed in this embodiment, and the biosensor can be used to detect whether the strain produces lycopene. .
  • Resveratrol is formed by the synthesis of p-coumaroyl-CoA by resveratrol under the action of p-diphenylene synthase (STS) (Fig. 3). Therefore, the STS can be used as a recognition group of the biosensor, and the oligonucleotide sequence used for construction is as shown in Table 2.
  • the other construction elements are the same as those in Embodiment 1, and the experimental construction method is the same as that in Embodiment 1. For specific information, see Example 1.
  • the final assembled large fragment was the 5'-GAL1 promoter + yEGFP + CYC1-3' terminator fragment (SEQ ID NO: 133) and the Mata2-GL77F-SV fragment (SEQ ID NO: 171), which were ligated into the vector. Skeleton (same as in Example 1).
  • the assembled plasmid was subjected to restriction enzyme digestion and sequencing, and the two constructed plasmids with the correct sequencing were co-transformed into the engineering bacteria of resveratrol yeast, and the experimental results were observed.
  • Resveratrol yeast engineering bacteria from the National Gene Bank of Shenzhen Huada Gene Life Science Research Institute, which is based on the Saccharomyces cerevisiae strain SynYII (Accession No. CTCCC NO: M 2014434) as the chassis cells, synthesizing resveratrol
  • the foreign genes Z26250, CYP81B1C, TAL, 4CL3, STS and PAL
  • the foreign genes are spliced to form a recombinant plasmid, which is introduced into the chassis cells for metabolite detection, and the strain capable of expressing resveratrol is a resveratrol yeast engineering strain.
  • the recombinant strain of resveratrol transformed with two integrated plasmids was used as a positive strain, and the recombinant strain of resveratrol yeast which had not transformed any plasmid was used as a negative strain, and the monoclonal medium of each of the two strains was cultured overnight.
  • Fig. 8 it can be found on the fluorescent display that the resveratrol yeast strain (C) containing the biosensor has more obvious green fluorescence than the resveratrol yeast strain (D) without the biosensor. display.
  • the OD value of the positive and negative strains was detected by time period, and the fluorescence value was read by a microplate reader (the excitation wavelength of yEGFP was 488 nm, the emission wavelength was 575 nm), and the OD value of the bacterial solution concentration was taken as the abscissa and fluorescence value.
  • the ordinate draws the data into a graph, as shown in Fig. 9, it can be seen from the figure that the positive yeast strain has a significantly higher fluorescence value than the negative yeast strain, which proves that the resveratrol biosensor is successfully constructed in this embodiment. And the biosensor can be used to detect whether the strain produces resveratrol.

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Abstract

L'invention concerne un biocapteur permettant la détection d'un composé moléculaire cible et un système comprenant le biocapteur. Le biocapteur constitue une protéine de fusion et comprend une molécule synthase cible, un domaine de liaison à l'ADN et un domaine d'activation transcriptionnelle respectivement ligaturés aux deux extrémités de la molécule synthase cible, et un déterminant de dégradation. Lorsqu'il n'y a pas de substance précurseure de la molécule cible, le déterminant de dégradation dégrade le biocapteur ; lorsqu'il y a une substance précurseure de la molécule cible, la molécule synthase cible reconnaît et lie la substance précurseure afin de stabiliser le biocapteur, de telle sorte que le déterminant de dégradation échoue et est automatiquement détaché, que le domaine de liaison à l'ADN se lie à une région de promoteur d'un gène rapporteur, et que le domaine d'activation transcriptionnelle active l'expression du gène rapporteur. La présente invention permet de mettre en œuvre un criblage précis et à haut rendement de divers métabolites moléculaires, de réduire considérablement les opérations manuelles compliquées, de réduire les coûts de détection à l'aide d'instruments de haute précision, et peut être largement appliquée.
PCT/CN2018/110245 2017-10-18 2018-10-15 Biocapteur permettant la détection d'un composé moléculaire cible et système comprenant un biocapteur WO2019076262A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104094115A (zh) * 2012-01-31 2014-10-08 坎瓦克斯生物技术公司 基于细胞的传感器
WO2016058094A1 (fr) * 2014-10-14 2016-04-21 Université de Montréal Biocapteur faisant appel à des protéines interagissant avec la gβυ pour surveiller l'activation de la protéine g
US20160202256A1 (en) * 2015-01-12 2016-07-14 President And Fellows Of Harvard College Biosensors Engineered from Conditionally Stable Ligand-Binding Domains
WO2016161088A2 (fr) * 2015-03-31 2016-10-06 Fundamental Solutions Corporation Système de biocapteur pour la détection rapide d'analytes
WO2017048316A1 (fr) * 2015-09-18 2017-03-23 President And Fellows Of Harvard College Biocapteurs de petites molécules
WO2017117248A1 (fr) * 2015-12-28 2017-07-06 Colorado State University Research Foundation Compositions et procédés pour la détection de petites molécules
WO2017156238A1 (fr) * 2016-03-11 2017-09-14 President And Fellows Of Harvard College Biocapteurs à petites molécules basés sur la stabilité protéique et procédés associés

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104094115A (zh) * 2012-01-31 2014-10-08 坎瓦克斯生物技术公司 基于细胞的传感器
WO2016058094A1 (fr) * 2014-10-14 2016-04-21 Université de Montréal Biocapteur faisant appel à des protéines interagissant avec la gβυ pour surveiller l'activation de la protéine g
US20160202256A1 (en) * 2015-01-12 2016-07-14 President And Fellows Of Harvard College Biosensors Engineered from Conditionally Stable Ligand-Binding Domains
WO2016161088A2 (fr) * 2015-03-31 2016-10-06 Fundamental Solutions Corporation Système de biocapteur pour la détection rapide d'analytes
WO2017048316A1 (fr) * 2015-09-18 2017-03-23 President And Fellows Of Harvard College Biocapteurs de petites molécules
WO2017117248A1 (fr) * 2015-12-28 2017-07-06 Colorado State University Research Foundation Compositions et procédés pour la détection de petites molécules
WO2017156238A1 (fr) * 2016-03-11 2017-09-14 President And Fellows Of Harvard College Biocapteurs à petites molécules basés sur la stabilité protéique et procédés associés

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
FENG: "A general strategy to construct small molecule biosensors in eukaryotes", ELIFE, vol. 6, no. 14, 29 December 2015 (2015-12-29), pages 1 - 7, XP055370564 *

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