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30 results about "PBR322" patented technology

PBR322 is a plasmid and was one of the first widely used E. coli cloning vectors. Created in 1977 in the laboratory of Herbert Boyer at the University of California, San Francisco, it was named after Francisco Bolivar Zapata, the postdoctoral researcher who constructed it. The p stands for "plasmid," and BR for "Bolivar" and "Rodriguez."

Newcastle disease virus heat resistant live vaccine vector system and application thereof

The invention belongs to the field of virus genetic operation, and in particular to a Newcastle disease virus (NDV) heat resistant live vaccine vector system and application thereof. The Newcastle disease virus (NDV) heat resistant live vaccine vector system comprises a) a transcription plasmid, b) three auxiliary plasmids and c) host cells. The transcription plasmid is obtained by cloning genomic full-length cDNA of a NDV heat resistant vaccine strain to pBR322 vector; and the three auxiliary plasmids are obtained by cloning nucleoprotein, phosphoprotein and large polymerase protein gene of the NDV heat resistant vaccine strain to pcDNA3.1 vector. The artificial recombinant Newcastle disease virus has the characteristic of heat resistance, and the Newcastle disease virus (NDV) heat resistant live vaccine vector system is established for the first time. The artificial recombinant Newcastle disease virus has great application prospect in the aspects of research and development of multiple (multivalent) heat resistant genetic engineering live vaccines of the NDV, avian influenza and other major diseases of poultry, research on virus heat-resistant mechanism, and the like.
Owner:INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI

Brewing yeast integrated expression vector with recyclable selective marker and construction method thereof

The invention provides a brewing yeast integrated expression vector with a recyclable selective marker as well as a construction method and application thereof. The Brewing yeast integrated expression vector with the recyclable selective marker comprises a loxp-KanMX-URA-PBR322 replicon-Loxp (SEQ ID NO:1), a yeast primary expression cassette and a homologous arm insertable section, wherein the yeast primary expression cassette is any one of a yeast promoter, a multiple cloning site and a transcription terminator. According to the brewing yeast integrated plasmid, replacement of an expression cassette element, cloning of a foreign gene in escherichia coli, integration and screening of the gene in yeast, rapid, simple and convenient removal of the selective marker and recycling of the tool can be realized. According to the invention, removal of the selective marker is represented and verified; the brewing yeast integrated expression vector can be widely applied to long-way multi-gene integration of brewing yeast.
Owner:ZHEJIANG UNIV

Biological mothballing system suitable for synechococcus elongatus, and construction method and application of biological mothballing system

The invention discloses a biological mothballing system suitable for synechococcus elongatus, and a construction method and application of the biological mothballing system. The construction method comprises the steps that a virulent gene sepT2, an antitoxic gene sepA2 and an iron-deficiency inducible promoter PisiAB are obtained from the synechococcus elongatus 7942, and a promoter PpsbA2 is obtained from synechocystis 6803; a terminator Trbcl is obtained from integrative plasmid pBA3031 through amplification; pBR322 is taken as a carrier, the virulent gene sepT2 is expressed with the iron-deficiency inducible promoter PisiAB, and the terminator Trbcl is used for terminating transcription; and the antitoxic gene sepA2 is expressed with the promoter PpsbA2, and plasmid is constructed and transferred into the synechococcus elongatus. According to the system, the synechococcus elongatus grows normally under the non-inducing situation and rapidly dies after being induced. The important theoretical and practical significance for development and optimization of the biological mothballing system of the synechococcus elongatus is achieved, and reference is also provided for solving the bio-safety problem of other cyanobacteria.
Owner:TIANJIN UNIV

Engineering strain for efficiently expressing MccJ25 and fermentation process of engineering strain

ActiveCN113774006AEfficient expressionThe post-processing purification process is simpleBacteriaMicroorganism based processesBiotechnologyEscherichia coli
The invention provides an engineering strain for efficiently expressing MccJ25. The engineering strain is used for solving the problem of low expression level of Microcin J25 in the prior art. The engineering strain is constructed by the following method: genetic operation is separately carried out on mcjABCD to become a direction, a promoter T7 is shared in Escherichia coli and is cloned on a vector pBR322, high-efficiency expression in the Escherichia coli is realized, and the expression level is 2.4 g / L. According to the scheme, new expression vectors are constructed, an Escherichia coli expression vector and a lactobacillus plantarum expression vector are separately constructed to efficiently express the MccJ25, meanwhile, a directed evolution technology is adopted, molecular evolution is performed on an MccJ25 gene cluster, and efficient expression of the MccJ25 is realized. Extracellular expression is carried out on the Escherichia coli, intracellular expression is carried out on lactobacillus plantarum, the expression levels reach 4.1g / L and 2.3g / L separately, an industrialization level is achieved, and a foundation is laid for further application of biological veterinary drugs and feed additives.
Owner:安杰利(重庆)生物科技有限公司

Method for preparing rebaudioside M through fermentation catalysis of bacillus subtilis

The invention relates to a method for preparing rebaudioside M by fermentation catalysis of bacillus subtilis, which is characterized by comprising the following steps: (1) connecting glycosyl transferase UGT76G1 and UGT11 genes to a PBR322 plasmid vector, guiding into the bacillus subtilis, inoculating into an LB culture medium, and culturing for 20-24 hours at the temperature of 30-37 DEG C and the speed of 100-250 rpm; (2) inoculating into a seed tank according to the inoculum size of 1%-10%, and culturing for 20-24 hours at the temperature of 30-37 DEG C at the speed of 200-400 rpm and the ventilation ratio of 0.1-1 V / V.min; (3) inoculating into a fermentation tank according to the ratio of 1%-10%, and culturing for 48-72 hours at the temperature of 30-37 DEG C and the pH value of 6-8 at the speed of 200-1000 rpm and the ventilation ratio of 0.1-2 V / V.min; (4) carrying out ceramic membrane ultrafiltration on the fermentation liquor, carrying out resuspension and high-pressure homogenization on the thallus, and carrying out ceramic membrane ultrafiltration on the thallus crushing liquor to obtain crude enzyme liquor; and (5) mixing stevioside, uridine diphosphate glucose, a phosphate buffer and the crude enzyme according to a mass ratio of (5-10): (1-5): (40-60): (5-20), and reacting at 25-40 DEG C for 12-48 hours. The method has the advantages that two crude enzyme solutions are obtained through one-time fermentation, the operation is simple, the cost is low, and the conversion rate is 88.4% or above.
Owner:ANHUI JINGHE IND

Method for preparing rebaudioside A by fermentation and catalysis of bacillus subtilis

PendingCN111593062AIncrease productionSolve the problems of low enzyme activity, complicated operation and high production costTransferasesMicroorganism based processesBiotechnologyPhosphate
The invention relates to a method for preparing rebaudioside A by fermentation and catalysis of bacillus subtilis. The method is characterized by comprising the following steps of: 1, connecting a glycosyltransferase UGT76G1 gene onto a PBR322 plasmid vector, transferring the plasmid vector to a competent state of bacillus subtilis, inoculating the bacillus subtilis into an LB culture medium, andculturing at 30-37 DEG C for 20-24 h; 2, inoculating into a seed tank according to an inoculation amount of 1-10% volume ratio, culturing, introducing air, and culturing at 30-37 DEG C for 20-24 h; 3,inoculating into a fermentation tank according to an inoculation amount of 1-10% volume ratio, culturing, introducing air, controlling the pH to be 6-8, and culturing at 30-37 DEG C for 48-72 h; 4, carrying out filter pressing to obtain bacillus subtilis thalli, re-suspending, carrying out high-pressure homogeneous crushing, and carrying out filter pressing to obtain crude enzyme solution; and 5,mixing stevioside, uridine diphosphate glucose, phosphate buffer solution and the crude enzyme solution according to a mass ratio of (5-10): (1-5): (40-60): (5-20), and carrying out a reaction at 25-40 DEG C for 12-48 h. The method in the invention has the advantages that: the yield of the rebaudioside A is increased, and can reach 85.3 g/L; and the method is few in operation step, low in production cost and easy for industrial production.
Owner:ANHUI JINGHE IND

Method for preparing rebaudioside D through fermentation catalysis of bacillus subtilis

PendingCN114150031ASolve the problems of low enzyme activity, complicated operation and high production costReduce stepsBacteriaMicroorganism based processesBiotechnologyUltrafiltration
The invention relates to a method for preparing rebaudioside D by fermentation catalysis of bacillus subtilis, which is characterized by comprising the following steps: (1) inoculating glycosyl transferase UGT76G1 and AtSUSY genes to a PBR322 plasmid vector, guiding into the bacillus subtilis, inoculating into an LB culture medium, and culturing for 20-24 hours at the temperature of 30-37 DEG C and the speed of 100-250 rpm; (2) inoculating into a seed tank according to the inoculation amount of 1%-10%, and culturing for 20-24 hours at the temperature of 30-37 DEG C at the speed of 200-400 rpm and the ventilation ratio of 0.1-1 V/V.min; (3) inoculating into a fermentation tank according to the volume ratio of 1%-10%, and culturing for 48-72 hours under the conditions that the speed is 200-1000 rpm, the ventilation ratio is 0.1-2 V/V.min, the temperature is 30-37 DEG C and the pH value is 6-8; (4) carrying out ultrafiltration on the fermentation liquor by adopting a ceramic membrane, resuspending bacteria, carrying out high-pressure homogeneous crushing, and carrying out ultrafiltration on the crushed liquor by adopting the ceramic membrane to obtain crude enzyme liquor; and (5) mixing stevioside, uridine diphosphate glucose, a Tris-HCl buffer solution and the crude enzyme solution according to a mass ratio of (5-10): (1-5): (40-60): (5-20), and reacting at 25-40 DEG C for 12-48 hours. The method has the advantages that two crude enzymes are obtained through one-time fermentation, the cost is low, and the conversion rate of rebaudioside D reaches 88-90 g/L.
Owner:ANHUI JINGHE IND
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