285 results about "Microbial Genes" patented technology

A probe and a primer capable of collectively detecting microorganisms of the same species while differentiating microorganisms of other species with an object of classification by species of fungus. An oligonucleotide probe for detecting an infectious etiologic microorganism gene includes at least one base sequence selected from the base sequences belonging to one group of the following first to ninth groups. The base sequence groups of first to ninth groups are: first group (SEQ ID NOS:1 to 5); second group (SEQ ID NOS:6 to 10); third group (SEQ ID NOS:11 to 15); fourth group (SEQ ID NOS:16 to 21); fifth group (SEQ ID NOS:22 to 26); sixth group (SEQ ID NOS:27 to 31); seventh group (SEQ ID NOS:32 to 36); eighth group (SEQ ID NOS:37 to 41); and ninth group (SEQ ID NOS:42 to 46).

The invention discloses a nucleic acid composition for detecting novel coronavirus COVID-19 and application. The nucleic acid composition is prepared by the following steps: firstly, carrying out nucleic acid sample amplification based on a transcription-mediated amplification technology (TMA); then, specifically detecting amplified virus target nucleic acid in combination with activity of 'associated cleavage' of CRISPR-Cas13a protease; adding sgRNA, Cas13a protein, an ssRNA signal report probe and a reaction buffer solution into a reaction system containing target nucleic acid to be detected; and reading and detecting signals when the reaction is carried out so as to detect a target gene. By using the method disclosed by the invention, whether a sample contains a target nucleic acid sequence or not can be rapidly detected; and the nucleic acid composition is combined with the transcription-mediated nucleic acid amplification technology, so that the sensitivity of the detection methodcan reach a nanomole level, and the nucleic acid composition for detecting the novel coronavirus COVID-19 is suitable for rapidly detecting pathogenic microorganisms, gene mutation, specific target RNA and the like.

The invention discloses a corynebacteria expression system without depending on antibiotic being selection pressure, and belongs to the microbe gene engineering field. Alr knock out carrier and expression carrier used for plasmid anaplerosis are constructed to bring conveniences for lab research and utilization, and a fermentation process no longer needs antibiotic. Through test results, the expression system realizes thalline inner plasmid stability in fermentation without adding the antibiotic; when the expression system is used to construct overexpression vector of branched chain amino acid-valine synthesis path main gene ilvBNC, valine output can be obviously improved; the corynebacteria expression system has wide application value.

An Escherichia coli recombinant strain producing shikimic acid, and a construction method and application thereof belong to the technical field of microbial gene engineering. The invention firstly utilizes a molecular biology technique to delete shikimic acid kinase I gene (aroK) and shikimic acid kinase II gene (aroL) of Escherichia coli CICIMB0013, and a gene (ptsG) of a key protein EIIBC<Glc> and a quinin acid/shikimic acid dehydrogenase gene (ydiB) of a glucose phosphotransferase system to obtain an Escherichia coli mutant strain CICIMB0013.SA4 (delta aroK, delta aroL, delta ptsG, delta ydiB). The invention also constructs a recombinant expression plasmid pTHGAA containing key genes comprising aroG*,ppsA and tktA in a metabolic pathway of shikimic acid; and the recombinant expression plasmid pTHGAA is transferred into the recombinant strain CICIMB0013.SA4 to obtain a recombinant Escherichia coli B0013 (SA4/pTHGAA) capable of producing shikimic acid efficiently. The Escherichia coli recombinant strain provided by the invention can realize efficient accumulation of shikimic acid in a fermentation process.

Disclosed are bacillus licheniformis engineering bacteria for nattokinase production and a method for producing nattokinase by using the bacillus licheniformis engineering bacteria, which belong to the technical fields of microorganism genetic engineering and enzyme engineering. According to the invention, a nattokinase gene of bacillus subtilis MBS04-6 is obtained through PCR technology augmentation. Bacillus licheniformis BL10 is utilized as an expression host; promoter P43 of bacillus subtilis 168 is taken as a promoter; a signal peptide of extracellular serine protease Vpr of bacillus licheniformis WX-02 is taken a signal peptide; and a terminator sequence of alpha-amylase of bacillus licheniformis WX-02 is taken as a terminator. The expression elements are connected to shuttle vector pHY300PLK to form an expression vector, and the expression vector is transformed into the bacillus licheniformis BL10 to obtain the bacillus licheniformis engineering bacteria BL10 (pP43SNT) for nattokinase production. The bacterial strain has been deposited with the China Center for Type Culture Collection (CCTCC) at September 10th, 2013, and the accession number is CCTCC NO:M2013401. The invention also provides a method of the bacterial strain for producing nattokinase, and the maximum enzyme activity can reach 11.37 FU/mL in a liquid fermentation medium.

Disclosed is a beta-mannase gene and preparation, which belongs to the field of biological genetic engineering. The invention provides the beta-mannase gene man originated from Bacillus licheniformis ATCC14580, the nucleic acid sequence of the gene man and the amino acid sequence of the corresponding protein, cloned carrier containing the gene man and the bacillus expression carrier pBL-man, and the highly performance expression in bacillus coli or bacillus. the invention can be applied to the construction of genetic engineering bacterium for the industrial production of the beta-mannase, the molecule transformation for the present beta-mannase genes, and the increase of level and quality for beta-mannase by means of biofermentation.

The invention discloses genetically engineered bacteria capable of increasing the yield of lactoyl-N-trisaccharide II and a production method, and belongs to the field of microbial genetic engineering. According to the invention, the expression of glmM, glmU, glmS and lgtA in a lactoyl-N-trisaccharide II synthetic pathway is regulated and controlled in a combined manner, so the carbon flux of a metabolic pathway is accurately regulated and controlled, and the metabolic pressure is relieved; the expression of wecB, nagB and lacZ in an escherichia coli host, namely the lactoyl-N-trisaccharide IIsynthetic pathway is knocked out, so the yield of the lactoyl-N-trisaccharide II is further increased; in a flask shaking experiment, the capacity of escherichia coli for producing the lactoyl-N-trisaccharide II is increased to 4.82 g/L from 0.53 g/L; in a fermentation tank with a volume of 3 L, the yield of the lactoyl-N-trisaccharide II reaches 46.2 g/L; and thus, thegenetically engineered bacteria have industrial application prospect.

The invention belongs to the technical field of microorganism genetic engineering and discloses separation and cloning of nematicidal crystal protein gene originated from thuringiensis and biological activity of the nematicidal crystal protein gene on nematode. In the invention, a new insecticidal crystal protein gene cry1518-35 is obtained by being separated from bacterial strain of activated thuringiensis YBT-1518 of root-knot nematodes in north of China; an encoded product of the gene is a new insecticidal crystal protein Cry1518-35; the protein Cry1518-35 features high insecticidal activity on root-knot nematodes in north of China. The invention also includes the preparation of colon bacillus recombinant bacteria EMB0225 which expresses the insecticidal crystal protein Cry1518-35, the recombinant colon bacillus is preserved in CCTCC, the preservation number is CCTCC NO: M209009.

The invention discloses a kit and an extraction method for quickly extracting microbial genome DNA from animal fecal microorganisms. The extraction method for extracting microbial genome DNA from animal fecal microorganisms comprises the following five steps: pre-treatment of feces; splitting decomposition of microbial cells in feces; dissociation of DNA and abstraction of impure proteins; DNA adsorption by a DNA adsorption column and removal of impurities; and elution of DNA. The kit disclosed by the invention is capable of absorbing the genome DNA by a silicone mold adsorption column and removing impurities such as proteins by using special deproteinization liquid and wash liquid. Compared with conventional extraction methods for extracting microbial genome DNA from animal fecal microorganisms, the microbial genome DNA from animal fecal microorganisms can be extracted within 4 hours, so that not only is a lot of extraction time shortened, but also the experimental steps are simplified. Therefore, the microbial genome DNA from animal fecal microorganisms can be effectively, quickly and economically extracted.

The invention provides a method for analyzing the diversity and the differences of intestinal and oral florae based on a new generation of high-throughput sequencing technology. The method comprises the following steps of (1) collecting a to-be-detected sample and carrying out microbial genome DNA extraction to obtain a genome DNA extracting solution; (2) detecting the genome DNA extracting solution to determine the concentration and the purity of a microbial genome DNA in the sample; (3) carrying out PCR amplification on the genome DNA extracting solution by using 16SrDNA-specific V3-V4 region primers and carrying out library construction; (4) sequencing by using an IlluminaMiSeq sequencing system; and (5) carrying out bioinformatics analysis by using FLASH, QIIME and usearch61. Compared with a traditional flora detection method, by using the new generation of high-throughput technology, compositions and functions of the florae can be analyzed more deeply in more detail.

The invention belongs to the technical field of microbial genetic engineering, specifically relates to a recombinant bovine tuberculosis specific antigen protein with three fused genes and a preparation method thereof. The gene of the bovine tuberculosis specific fusion antigen protein has a nucleotide sequence and an amino acid sequence shown as in a sequence list SEQ ID NO:1. The invention discloses a recombinant Escherichia coli BL21/pET28a-RCE capable of expressing the bovine tuberculosis specific fusion antigen protein. The Escherichia coli is conserved in China Center for Type Culture Collection (CCTCC) with a conservation number CCTCC NO:M208244. The invention further discloses a preparation method of the three gene fused antigen protein.

A Strain producing dynamic controlling recombinant strain and a method for preparing D-lactic acid with the recombinant strain belong to the technical field of genetically engineered agricultural microorganisms. The recombinant strain is named (Escherichia coli)B0013-070B, and is preserved in the China center for type culture collection, and the preservation number is CCTCCNO:M2012071. A lactate dehydrogenase gene promoter ldhAp in the genome of the strain is replaced with a culture environment/nutritional factors control-type promoter. Through the utilization of the recombinant strain, fermentation is conducted for 28 to 40 hours in stages at 25 to 50 DEG C, and the level of producing D-lactic acid reaches 12.5%; the optical purity lives up to 99.9%; and the chemical purity reaches to 98.4%. The dynamic controlling to the D-lactic acid dehydrogenase encoding gene expression on D-lactic acid high-producing strain B0013-070 chromosomes is conducted, so that the gene expression is controlled only through changing the fermentation temperature during the D-lactic acid production process to achieve the purpose of efficiently synthesizing D-lactic acid through taking glucose as a raw material. After simple modification, the invention can be used for producing important microbial metabolites in other industries.

The invention discloses a long-life nano bioluminescent probe used for detecting pathogenic microorganisms, a preparation method and a detecting method thereof. Long-life luminous nano particles used for the nano probe are prepared; a capture probe DNA1 on a synthetic glass chip, a discriminating probe DNA2 connected on the long-life luminous nano particle and a target probe on the gene fragment of the pathogenic microorganism are designed to construct a detecting model which is similar to a sandwich structure. The invention has the advantage that the interference of source fluorescence and background signals can be effectively eliminated by utilizing the long-life luminous nano particles, and has the advantages of high sensitivity, stability, function of signal transformation and the like of nano technology. The detecting method has the advantages of simple and convenient operation, low cost, high sensitivity and specificity and good stability.

The invention provides an analytical method of correlation of xylanase heat resistance and an N-terminal disulfide bond, and belongs to the technical field of microbial genetic engineering. A result that the N-terminal disulfide bond is one of factors which affect the xylanase heat resistance is proved by adopting a scheme of combining three kinds of bioinformatics methods including the homologous comparison, the homologous modeling and the molecular dynamics simulation of a primary structure of the zymoprotein, and the result is also proved by combining an experimental means of site-specific mutagenesis and by analyzing a heat resistance mechanism of the EvXyn11TS. The research result establishes a solid foundation for the heat resistance transformation of the 11 family normal temperature high specific activity xylanase which has a similar primary structure with the EvXyn11TS.

The invention relates to a microbial genome DNA (deoxyribonucleic acid) indirect extraction method for evaluating diversity of animal intestinal microflora. Before cell lysis, the method performs pretreatment on a sample and separates microbial cells from the excrement sample to avoid the problems that pollutants are difficult to remove and the DNA recovery rate is low in a purification step. According to the invention, phenol and chloroform are not used in the extraction process, thus harm to the physical health of experimenters is reduced. The OD260/OD230 and OD260/OD280 of the extracted intestinal microorganism DNA are approximate to standard values, and the intestinal microorganism DNA can be directly applied to molecular operation to evaluate the diversity of animal intestinal microflora.

The invention discloses a pseudomonas putida gene knockout and genome simplification system, and belongs to the field of microbial gene engineering. According to the pseudomonas putida gene knockout and genome simplification system disclosed by the invention, a suicide plasmid knockout system and a site specific recombination system are skillfully combined, so that the correction rate of two turns of screening is greatly improved to be higher than 90%, and the correction rate is still higher than 90% when a gene cluster, which is 69 genes long, is knocked out. The system disclosed by the invention achieves the continuous knockout of the gene cluster, simple and convenient to operate, efficient, high in correction rate and low in cost. The system disclosed by the invention lays a molecular basis for the simplification and the optimization of a pseudomonas putida genome; therefore, the system is conducive to the implementation of efficient construction of chassis cells of synthetic biology.

The invention belongs to the fields of soil microorganisms, biochemistry and molecular biology and relates to a method for separation and purification of a large-fragment DNA from soil. The method is used for indirect separation and purification of a large-fragment DNA having molecular weight more than 30kb from various types of soil, wherein the large-fragment DNA is used for construction of a metagenomic library, or is used for separation of soil microbial gene clusters. The method solves the problem that separation of microbial cells from soil and preparation of large-fragment soil DNA having high purity and satisfying various biological study demands are realized difficultly by the existing indirect method, and is an efficient method for extraction of soil microorganism DNA having a large fragment and high purity.

The invention relates toOmega3- fatty acid dehydrogenase promoter sequence separated from Pichia Pastoris and its application. The invention comprises SEQ ID NO:1. The invention includes the microbiological gene engineering method of changing microbiological cells protein components with the promoter sequence, and comprises: connecting heterogenic or homologous gene to the promoter downstream, transferring into microbiological expression vector, transforming microorganism host for the expression of target protein in its cells to realize quality improvement or medical use. Omega3- fatty acid dehydrogenase promoter sequence separated from Pichia Pastoris can be used in all the transgenic microorganisms.

The invention relates to the technical field of microbial enhanced oil recovery, and in particular relates to an extraction method of oil reservoir microbial genome DNA. The method comprises the following steps: removing interference of crude oil in samples with extraction by means of organic solvent extraction; releasing microbes on an oil-water interface; filtering water phase by using a hollow fiber membrane to enrich microbes; removing organic macromolecular components such as proteins by phenol and chloroform in cooperation with lysis cells such as lysozyme, helicase, protease and lauryl sodium sulfate according to a physical wall-breaking method under a high-salinity condition; subsequently, performing nucleic acid extraction and precipitation to obtain the oil reservoir microbial genome DNA. The method is quick and simple, and the whole extraction process can be completed in 10 hours; the extraction efficiency is high, and the purity of the obtained genome is high.

The invention relates to a non-citrinin monascus gene engineering fungus construct method, belonging to animalcule gene engineering field. The invention offers (1) citrinin coding gene pksCT sequence origin from CICC5006;(2) construct CICC5006 citrinin gene clone carrier pBpksCTHyg;(3)REMI induces CICC5006 citrinin gene remove method. Using this invention, it can interdict the metabolizing approach of citrinin in monascus, solve associated citrinin and pollution during pigment producing and make sure of the security of red bend, fulfilling the purpose of ameliorating the equality and depth development of red bend product.

The invention provides a bacillus licheniformis industrially producing bacterial strain genetic transformation method, which belongs to microbial gene engineering and fermentation technology field. The invention provides a bacillus licheniformis industrially producing bacterial strain genetic transformation method; firstly DNA molecule which needs to be genetically transformed is methylated by the bacillus licheniformis specificity methyltransferase medium; specifically methylated DNA molecule adopts optimized electroporation method to realize the genetic transformation of bacillus licheniformis industrially producing bacterial strain. By the method, the genetic transformation rate can reach 25-1250 strains/microgram DNA. In particular, the invention can be used in the genetic transformation of bacillus licheniformis bacterial strain that the prior genetic transformation cannot be realized.

The invention discloses an LAMP-based visual fluorogenic and chromogenic genetic testing method for microorganisms, which relates to a genetic testing method for microorganisms. The testing method includes the following steps: (1) buffered peptone water (BPW) is used for culturing a sample to be tested according to a national standard method for 4 hours; (2) a water boiling method is used for extracting DNA (deoxyribonucleic acid) from the sample to be tested; (3) the DNA is added into an LAMP reaction system, and the temperature of 65 DEG C is kept for 1 hour; (4) by means of comparison with a control group, the result of the sample to be tested is observed with naked eyes, and qualitative analysis is carried out on the sample. Compared with the conventional culture method, the genetic testing method has the advantages that: testing is rapid, only taking 5 hours, and plus the extraction of the sample DNA, testing takes less than 6 hours in total; the specificity is good, and the sensitivity is high; the steps are simple, and the repeatability is high; and a plurality of samples can be tested at the same time. The genetic testing method can qualitatively test microorganisms, and can take the place of the conventional culture method and the serological testing method which are used up to now.

The invention relates to a marker for intestinal microecology and application thereof, in particular to a biomarker used for early diagnoses of liver cancer. The biomarker used for early diagnoses ofliver cancer is composed of 34 kinds of genes shown as SEQ ID NO:1-34, and the genes are enriched in intestinal tracts. The invention also provides a detection reagent, and the reagent includes a primer used for detecting the 34 kinds of genes shown as SEQ ID NO:1-34. Feces of patients in groups are collected through a noninvasive method, and 16S rRNA Miseq sequencing of intestinal florae is conducted. During a discovery period of the biomarker, through a random forest law, microorganism gene markers with specificity of early liver cancer are identified among patients suffering from early cirrhosis and liver cancer, patients suffering from cirrhosis and healthy comparisons, an index of a probability of disease (POD) of liver cancer is created, and verification of the markers during the discovery period is achieved. During the verification period of the markers, the POD index of the patients during the verification period is calculated, and verification of the diagnosis value of the patients suffering from liver cancer during the verification period according to the POD index is achieved.

The invention discloses a high efficient expression method for an actinomyces-based nitrile hydratase gene in escherichia coli, and belongs to the field of microbial genetic engineering technology. The method processes high efficiency and safety. It is benefit for large scaled nitrile hydratase extraction and purification, and further theoretical research about nitrile hydratase that a large amount of soluble nitrile hydratase can be obtained in a short expression period by applications of the method.