166results about How to "Clear genetic background" patented technology

The invention relates to a rapid construction method of a conditional gene knockout animal model, which is implemented by using a CRISPR / Cas9 technology. The specific method can be implemented as follows: designing a gRNA target spot sequence, for a to-be-knocked-out gene of an animal, of a CRISPR / Cas9 system, designing a corresponding promoter, constructing a plasmid, thereby implementing conditional gene knockout. According to the invention, the rapid, efficient, simple, feasible, economic, efficient, and wide-applicable-scope construction of a conditional gene knockout animal model can be realized, and the defects existing in traditional methods (specific recombination enzyme system Cre-LoxP) can be overcome.

The invention relates the construct method for transgenosis rhinestone algae bioreactor. The method comprises the following steps: using the rhinestone algae as transgenosis receptor biology, highly effective expressing the carrier construct by constructing the exogenesis gene containing screening mark expression frame, leading the destination genes in rhinestone algae with bead milling method, electric punching method and other methods, integrating them into nucleus genome or chloroplast genome, and adopting a series of gene expression regulate and control technology to construct the transgenosis rhinestone algae bioreactor. The transgenosis rhinestone algae bioreactor possesses the characters of fast growth rate, short breeding cycle, easy segregation, photoautotroph and large scale culture, fast and cheap producing medicinal protein, industrial raw materials, and feeding vaccination, and possessing the bioactivity secondary metabolite.

The invention discloses a genetically engineered bacterium co-generating geraniol and nerol and a construction method and an application thereof, and belongs to the technical field of generic field. According to the genetically engineered bacterium disclosed by the invention, acetyl coenzyme A acyltransferase / hydroxymethyl glutaryl coenzyme A reductase, 3-hydroxyl-3-methyl glutaryl coenzyme A synthase, mevalonic acid kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-diphosphonic acid decarboxylase, isopentenylpyrophosphate isomerase, geranyl diphosphonic acid synthetase and geraniol synthetase or phosphatase are expressed. The metabolic pathways of geraniol and nerol synthesized in escherichia coli are successfully constructed by genetic engineering means, and glucose is biologically converted into geraniol and nerol.

The present invention provides a high-yield L-histidine genetically engineered bacterium strain and a constructing method thereof. The bacterium integrates a nucleotide sequence of a corynebacterium glutamicum ATP phosphoribosyl transferase HisG mutant encoding gene hisG* represented by SEQ ID NO:1 based on genome of escherichia coli to enhance activity of histidine-synthesizing key enzyme HisG, also increases copy number of histidine operon genes in the genome to enhance a terminal synthesis pathway of histidine, and also integrates an encoding gene lysE of arginine / lysine transportprotein derived from the corynebacterium glutamicum to promote extracellular secretion of the intracellular histidine. The genetically engineered bacterium is used for producing the L-histidine by a fermentation method, can stably produce 40-55 g / L of the histidine by fermentation in a 5L fermentation tank for 40-50 h, has a production intensity reaching 1.0-1.5 g / (Lxh), and has a conversion rate of 0.18-0.22 g histidine / g glucose.

The invention provides a genetic engineering bacterium for L-theanine production and a fermentation method thereof. A construction method of the genetic engineering bacterium comprises the steps of carrying out single copying on an ribonucleic acid (RNA) polymerase gene T7RNAP derived from T7 phage on an Escherichia coli W3110 genome, wherein the gene is controlled by a xylose promoter; carrying out dual copying on a gamma-glutamyl methylamine synthetase gene gmas derived from methylovorusmays, wherein the gene is controlled by a T7 promoter; knocking out a xylose operon repressor protein genexylR; and knocking out a succinyl CoA synthetase gene sucCD. The genetic engineering bacterium has the beneficial effects that the yield of L-theanine fermented by using the genetic engineering bacterium can be up to 40g / L and the sugar acid conversion rate can be up to 25%; and the L-theanine production method provided by the invention has the advantages of being low in raw material price, shortin cycle, simple and convenient to operate, green and friendly to environment and high in yield and has good industrial application value.

The invention provides an escherichia coli genetically engineered bacterium for the synchronous high yield of L-tryptophan and L-valine, and application thereof. The genetically engineered bacterium is obtained through the steps that on a genome of escherichia coli, trpE (S40F) mutation is introduced while a promoter of tryptophan operon is replaced with a Ptrc promoter; an aroG (S180F) gene controlled by the Ptrc promoter is integrated to a tyrR locus; a serA (H344A, N364A) gene controlled by a Plac promoter is integrated to a yjiV pseudo gene locus; a glnA gene controlled by the Plac promoter is integrated to a ycjV pseudo gene locus; and then a bacillus subtilis alsS gene controlled by the Ptrc promoter is integrated to a yghx pseudo gene locus. Shaking flask fermentation is conducted through a strain to be able to accumulate the L-tryptophan within 22-28 h to 10-14 g / L, meanwhile the accumulation amount of valine reaches 5-7 g / L, and the total acid-producing ability is improved by50% or so compared with that of a tryptophan producing strain; and meanwhile, strain OD600 is different slightly, growth problems are avoided, but the acid-producing ability per strain is obviously improved by 120%, and effective utilization of carbon sources and cells is improved greatly.

The invention provides a preparation method of small molecular proteins or polypeptides. The method comprises the following steps: (a) establishing a gene engineering strain, wherein the gene engineering strain comprises a recombinant expression vector with a fusion protein coding nucleotide sequence, the fusion protein is an F-L-polypeptide, the F is fusion chaperonin and selected from aldosterone isomerase protein, aldosterone isomerase protein mutants, human glucagon-like peptide-1 and human glucagon-like peptide-1 mutants; L is a connecting peptide; the polypeptides are insulin or analogues thereof, GLP-1 or analogues thereof, GLP-2 or analogues thereof, antibacterial peptides or thymosin; (b) culturing the gene engineering strain so as to express the fusion protein; (c) purifying thefusion protein, and performing protease cleavage, thereby obtaining the small molecular proteins or polypeptides with biological activities. The method disclosed by the invention overcomes the defectthat the small molecular proteins or polypeptides are difficultly expressed, and provides a new thought for expressions of the proteins.

The invention relates to a method for breeding high-temperature-resistant Paralichthys olivaceus new varieties and belongs to the technical field of agricultural mariculture. In the method, Paralichthys olivaceus half sibs family and sibs family with a clear pedigree are established; multi-generation breeding is performed on the Paralichthys olivaceus family at half lethal temperature; inbreeding possibly occurring in the breeding process and tolerance of the Paralichthys olivaceus at different growth stages of juvenile fish and adult fish to the lethal temperature are taken into full consideration; and screening is repeated for two or more times, so that the high-temperature-resistant Paralichthys olivaceus new varieties can be bred effectively, and the problem that the Paralichthys olivaceus in some provinces in south China are difficult to aestivate.

The invention relates to an unmarked gene-deleted and attenuated mutant strain of a vibrio alginolyticus wild strain, which is the attenuated active vaccine of the vibrio alginolyticus wild strain and in which the sRNA molecular chaperone gene hfq of the vibrio alginolyticus wild strain is deleted. The vibrio alginolyticus wild strain is EPGS020401, and the collection number of the vibrio alginolyticus wild strain is CCTCC NO.AB 209306. The invention also provides the preparation of the unmarked gene-deleted and attenuated mutant strain of the vibrio alginolyticus wild strain and the use thereof in prevention and control of vibrio alginolyticus disease in cultured fish. The unmarked gene-deleted and attenuated mutant strain of the vibrio alginolyticus wild strain or the preparation thereof, which are disclosed by the invention, eliminates universal potential environment and product safety risks of the conventional attenuated vaccine and is a safe, effective and economic vaccine for preventing and controlling vibrio alginolyticus diseases in cultured fish.

The invention relates to a marker-free gene deletion attenuated mutant of a vibrio alginolyticus wild strain. The mutant is an attenuated live vaccine of the vibrio alginolyticus wild strain, wherein sRNA molecular chaperone protein gene hfq of the vibrio alginolyticus wild strain is deleted; and the vibrio alginolyticus wild strain is the vibrio alginolyticus wild strain EPGS020401 with a collection number of CCTCC No. AB209306. The invention also provides a preparation of the marker-free gene deletion attenuated mutant of the vibrio alginolyticus wild strain and related application to prevention and treatment of vibrio alginolyticus diseases of cultured fishes. The marker-free gene deletion attenuated mutant of the vibrio alginolyticus wild strain or the related preparation eliminates common potential security risk of environment and product existing in the conventional attenuated live vaccine, and is a safe, high-efficiency and economic vaccine aiming at the vibrio alginolyticus diseases of the cultured fishes.

The invention discloses a method of target-integrating a foreign DNA (Deoxyribonucleic Acid) sequence to Rosa26 sites of rats and mice as well as application thereof. According to the invention, a stable cell line can be obtained for expressing various foreign genes through constructed TALEN plasmid, rat homologous recombination plasmid and mouse homologous recombination plasmid by means of respectively transferring corresponding plasmids to cells of rats and mice. The stable cell line obtained can be stably handed from generation to generation with clear genetic background, so that the biological function of the foreign gene is accurately represented.