19 results about "Cotransformation" patented technology

This application discloses a recombinant bacterial strain that highly expresses nonspecific peroxygenase, its construction method, and its applications, relating to the field of microbial engineering technology. The strain of this invention uses *Escherichia coli* BL21(DE3) as the host. First, the key gene *hemA* for heme synthesis is integrated into the genome using the CRISPR-transposon system. Then, a chassis engineered strain A2 with 2 copies of optimal integration is screened and co-transformed with the rDcaUPO-A161C expression plasmid and the molecular chaperone plasmid pG-KJE8. The intracellular heme level of this strain is increased by 2.84 times compared to the original strain, and the total enzyme activity of rDcaUPO-A161C is cumulatively increased by 8.9 times and the specific enzyme activity is increased by 6.21 times compared to the wild type. This fundamentally solves the problems of insufficient cofactors, low folding efficiency, and poor activity in heterologous expression of nonspecific peroxygenases, and can be used for the efficient production of nonspecific peroxygenases.

The invention discloses a yarrowia lipolytica gene editing system based on CRISPR / AsCas12f1 and application of the yarrowia lipolytica gene editing system, and belongs to the field of molecular biology. A gene editing helper plasmid containing an AsCas12f1 expression cassette, a corresponding AssgRNA expression cassette, a related selection marker and a replication starting element is constructed through a gene cloning technology, and the helper plasmid and a proper donor DNA fragment are co-transformed into yarrowia lipolytica, so that efficient traceless knockout of a target gene can be realized. The gene editing kit of the yarrowia lipolytica is enriched, and the gene editing kit has very important application value in the field of targeted genetic modification of the yarrowia lipolytica.

The application provides a MhPHR2-MhSCL29 double-gene co-transformation synergistic regulation module and application, and belongs to the field of microbial genetic engineering.In the application, the phosphate-starvation response factor MhPHR2 is used as an upstream regulation hub, directly binds to a P1BS cis-acting element on a promoter of a downstream GRAS family transcription factor MhSCL29, positively activates the transcription of the MhSCL29, and forms a complete phosphate signal-mycorrhizal symbiosis regulation cascade.The nucleotide sequence of MhPHR2 in the synergistic regulation module is as shown in SEQ ID NO.1, and the nucleotide sequence of MhSCL29 is as shown in SEQ ID NO.2.The application improves the mycorrhizal symbiosis potential and the phosphorus utilization efficiency of a stock from the root.

This invention belongs to the field of plant genetic engineering technology and discloses a method for constructing transgenic hairy roots with multi-gene co-expression of the tanshinone biosynthesis pathway. The method first constructs a multi-gene expression vector containing the SmCPS1, SmKSL1, and SmCYP76AH1 genes, and then introduces the multi-gene expression vector into Agrobacterium rhizogenes. Subsequently, recombinant Agrobacterium rhizogenes is used to infect tanshinone explants, and transgenic hairy roots are induced after co-culture, sterilization culture, and resistance screening. Positive hairy roots are then identified and their expression analyzed by PCR and qRT-PCR, achieving stable co-expression of key genes in the tanshinone biosynthesis pathway. This invention achieves a single co-transformation of multiple key genes, simplifies the multi-gene transformation process, reduces the risk of gene segregation and expression instability caused by multiple transformations, and can provide stable transgenic materials for research on the tanshinone biosynthesis pathway and the metabolic regulation of active components in tanshinone.

The invention discloses a transgenic method of donor DNA, and belongs to the field of plant biotechnology and genetic breeding. The transgenic method of the donor DNA comprises the following steps: (1) taking a transgenic plant containing a resistance gene as a donor plant, extracting genome total DNA of the donor plant as donor DNA, and directly transferring the donor DNA into callus of a receptor plant; (2) carrying out recovery culture on the transformed calluses, and then transferring the calluses into a screening culture medium for screening; transferring the screened resistant calluses to a differential medium, and simultaneously carrying out subculture in the differential medium; and when 4-5cm resistant buds grow out, transferring the resistant buds into a rooting culture medium to obtain positive seedlings. Compared with the existing transgenic technology, the method has the advantages that the dilution ratio of the resistance gene to the DNA to be co-transformed is greatly improved, so that the co-transformation frequency is remarkably improved, and the co-transformation frequency can reach 100% by adopting the transgenic method.

The invention provides a method for improving plant genome editing efficiency based on a transcription factor AtSOG1 and application of the method, and belongs to the technical field of gene editing. The invention finds that the AtSOG1 transcription factor has multiple influences on gene editing, a target plant is co-transformed by using an expression vector constructed by the AtSOG1 transcription factor and a gene editing vector, or an expression element of the AtSOG1 transcription factor is constructed into the gene editing vector, and then the target plant is transformed, so that the plant genome editing efficiency can be improved. The method provided by the invention not only can promote targeted gene knock-in based on a PE editor and HDR mediation, but also can improve the knockout efficiency of long fragments. Meanwhile, in the embodiment of the invention, an experiment is further designed, it is found that the AtSOG1 can promote a T-DNA unconformity knock-in event, and a T-DNA free knock-in plant is directly obtained in a T0 generation.

The application provides a kind of biotinylated ferritin complex and its preparation method and application, belong to virus immune detection technical field.The biotinylated ferritin complex of the application inserts biotin receptor peptide (BAP) into Loop region, is obtained by co-transforming escherichia coli with recombinant plasmid pET28a-BAP-HFn and pETDuet-BirA (Loop-Biotin-HFn).It is assembled with HRP labeled streptavidin (SA-HRP) according to 1:26 molar ratio, and Loop-BNC complex is obtained.The Loop-BNC can be used as signal amplification probe, and is used for the detection of SARS-CoV-2 N protein by using sandwich ELISA method, and the detection sensitivity is lower than 0.0001 ng / mL, the detection effect is optimal in 0.0001-0.1 ng / mL, with the advantages of high signal-to-noise ratio and strong specificity.The application retains the high loading capacity and structural stability of ferritin, realizes the early and accurate detection of SARS-CoV-2 N protein, and provides efficient and reliable technical support and probe material for novel coronavirus detection.

This invention relates to the field of biotechnology and provides a method for gene editing in soybean using Cas12i, comprising the following steps: Step 1, constructing a plant binary vector expressing the Cas12i nuclease, wherein amino acids at positions 369 and 433 of the Cas12i nuclease are mutated to arginine, respectively; Step 2, designing gRNAs targeting the soybean GmFAD2-1A and GmFAD2-1B genes; Step 3, co-transforming the gRNA expression cassette and the Cas12i nuclease expression cassette into soybean recipient material using Agrobacterium-mediated transformation; Step 4, screening to obtain T0 generation transgenic plants and identifying the target gene editing type by molecular detection. Simultaneously mutating amino acids at positions 369 and 433 of the Cas12i nuclease to arginine significantly enhances the nuclease's cleavage activity and targeting specificity in plant cells, doubling the efficiency of dual gene knockout. Furthermore, the Cas12i nuclease used recognizes the TTC-PAM sequence, allowing it to target more AT-rich regions, significantly expanding the editing window of the soybean genome while reducing off-target risks.

The invention relates to the technical field of biology, in particular to an anti-root-knot nematode method and system for regulating and expressing hpRNA by targeting a plant root white body and application of the anti-root-knot nematode method and system. According to the invention, it is found for the first time that root-knot nematode can uptake a plastid endogenous nucleic acid RNA molecule rbcL and dsRNA of an expressed exogenous non-nematode target, so that a vector for starting hpPAS4 expression by a T7 promoter is constructed, and plant plastid transformation is mediated by a gene gun, so that a plastid-transformed transgenic plant is obtained. Besides, T-DNA insertion is mediated by agrobacterium tumefaciens, a root-specific promoter TobRB7 and a transport peptide RecA1 plastid are subjected to localization expression of T7RNA polymerase, and the T7RNA polymerase is integrated into a plant nuclear genome, so that nucleoplasm co-transformation is realized, and the plastid of the plant can efficiently express hpPAS4 by means of a T7 system. By utilizing the system, the expression of RNAi interference molecules is realized in the plastid (especially the white body of the root), the insufficient cell nucleus transformation is made up, a new way is opened up for the prevention and control of the root-knot nematode, and the system has great prevention and control value and application potential.

The invention discloses a traceless gene editing system and method in cephalosporium acremonium based on a CRISPR-Cas9 system, and belongs to the technical field of gene engineering. The method comprises the following steps: constructing a CRISPR-Cas9 gene editing DNA fragment containing Cas9 nuclease and an sgRNA expression cassette, designing a homologous recombination donor plasmid of a corresponding gene, co-transforming the editing fragment and the donor plasmid into a cephalosporium acremonium protoplast through a PEG-mediated protoplast transformation method, and sequentially realizing ku70 and sorB gene knockout and cefR gene promoter in-situ replacement, so as to obtain the recombinant cephalosporium acremonium protoplast. Meanwhile, by constructing a traceless knockout donor plasmid, a resistance selection marker and a Cas9 gene are removed, and traceless editing is achieved. The traceless editing system constructed by the method can realize multiple genetic modification, provides a new way for improving the production efficiency of cephalosporin C, and is suitable for genetic engineering modification of cephalosporium acremonium industrial strains.

The invention provides engineering escherichia coli pMki-nfss as well as a construction method and application thereof. The engineering escherichia coli comprises an mki gene and an nfss sequence. Wherein the nucleotide sequence of the mki gene is as shown in SEQ ID NO: 1, and the nucleotide sequence of the nfss sequence is as shown in SEQ ID NO: 2. According to the invention, a P-M recombinant plasmid containing an mki gene and a P-N recombinant plasmid containing an nfss sequence are co-transformed into E.coli BL21, such that engineering escherichia coli pMki-nfss is obtained. The engineering Escherichia coli can be used for producing sesterterpene fish oil alcohol through a whole-cell catalytic reaction and can be used for producing triterpenoid squalene through an enzyme catalytic reaction. The method disclosed by the invention is convenient to operate, high in efficiency and simple in equipment, has a better industrial application prospect in the field of biosynthesis of terpenoids, and is of great significance to research on biocatalysis of synthesis of terpenoids in the future.

The invention discloses a method for constructing a vitamin E production strain by synchronously editing and recombining three sites of a yeast genome and application, and relates to the technical field of synthetic biology and metabolic engineering. According to the invention, a helper plasmid for expressing CRISPR-Cas9 protein and three targeted specific site guide RNA (sgRNA) is constructed, and the helper plasmid and three vitamin E pathway gene donor fragments which are assembled and cloned in escherichia coli in advance are co-transformed into saccharomyces cerevisiae, so that efficient editing and large-scale gene fragment integration can be simultaneously carried out on three chromosome sites of HO, GAL80 and ARO10. The three-site synchronous editing and recombination efficiency of the method exceeds 80%. By utilizing the method, an engineering strain of a complete vitamin E synthesis route can be rapidly constructed at one time, and fermentation verifies that the yield of the vitamin E can reach 5.9 g / L. The invention provides an efficient and universal tool for quickly constructing complex metabolic pathways in yeast.

The invention discloses a rapid traceless insertion method for a yarrowia lipolytica exogenous gene based on CRISPR / Cas9. According to the method, gRNA can be reset and a new knockout site can be targeted only through one-time reverse ring-opening amplification and re-cyclization of plasmids used in the method; only one selection marker corresponding to the gRNA is needed to realize the integration of the exogenous gene for any times; the whole process of screening marker recovery can be completed by only one plasmid; the used plasmids can be autonomously lost only through simple passage, and the subsequent experiment is not influenced; a plasmid and gene co-transformation method is adopted, exogenous genes can be expressed and a marker recovery system can be selected only through one-time co-transformation operation, the workload is greatly reduced, and the working time is saved; compared with a common Cre / loxP screening marker recovery system, the screening marker does not need to be specially modified, the screening marker does not interfere with the Cre / loxP screening marker recovery system after being used for many times, and universality and generalization performance are higher.

The invention discloses a porphyra pythium gene editing method and application thereof, and belongs to the technical field of algal disease science and gene engineering. In order to solve the technical problem of lack of porphyra pythium gene function research tools, the method provided by the invention comprises the following steps: a) screening to obtain a nuclear localization signal and an SgRNA promoter suitable for porphyra pythium; b) designing sgRNA of a targeted target gene by using a bioinformatics tool; c) constructing a CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR associated D) co-transforming the plasmid into a pythium aphanidermatum protoplast by a PEG (Polyethylene Glycol) mediated method; and e) screening positive transformants by using antibiotics and a PCR (Polymerase Chain Reaction) technology. The CRISPR-Cas9 gene editing system aiming at the pythium aphanidermatum is established for the first time, has the characteristics of accuracy and high efficiency, and provides powerful technical support for deep analysis of gene functions and pathogenic mechanisms of the pythium aphanidermatum and development of new strategies for prevention and control of red rot.

The invention belongs to the technical field of gene engineering, and particularly relates to a preparation method of a mouse interleukin 23 heterodimer, which comprises the following steps: S1, constructing a double-vector expression system which is composed of a first expression vector and a second expression vector, the first expression vector comprises a first purification tag, a first flexible connecting peptide and a p19 gene, and the second expression vector comprises a second purification tag; the second expression vector comprises a second purification tag, a second flexible connecting peptide and a p40 gene, and the first purification tag and the second purification tag are different; and S2, co-transforming the dual-vector expression system into eukaryotic host cells, culturing, collecting a cell culture solution, and carrying out affinity purification to obtain the mouse interleukin 23 heterodimer. A p19 subunit and a p40 subunit are expressed through a double-vector expression system respectively, different purification tags are arranged at two N ends, the subunits and the purification tags are connected through flexible connecting peptides, and high-purity and high-activity mouse IL-23 can be obtained through two-step affinity purification.

The invention discloses a saccharomyces cerevisiae engineering bacterium constructed based on a CRISPR-Cas9 system and used for producing radix pseudostellariae cyclic peptide HB and a construction method. The construction method comprises the following steps: (1) constructing a pESC-URA-prePhHB-PhPOP2 plasmid; (2) constructing a gRNA-1 plasmid; (3) constructing Donor DNA (deoxyribonucleic acid); (4) co-transforming cas9 plasmid, gRNA-1 plasmid and Donor DNA into saccharomyces cerevisiae, and selecting transformants to carry out bacterial colony PCR (Polymerase Chain Reaction) verification, so as to obtain saccharomyces cerevisiae engineering bacteria for constructing and producing the radix pseudostellariae cyclic peptide HB on the basis of a CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) system; based on a saccharomyces cerevisiae recombinant expression system, the radix pseudostellariae cyclic peptide HB is generated in saccharomyces cerevisiae engineering bacteria, the saccharomyces cerevisiae subjected to gene editing can be used for constructing industrial strains which are stably produced for a long time and are free of antibiotic fermentation, the method is suitable for large-scale production and environment-friendly, and the saccharomyces cerevisiae is harmless to human beings and the environment and can be used in food and biological pharmacy industries.

The invention relates to a construction method of a recombinant baculovirus shuttle vector Bacmid '. The method comprises the following steps: cloning a lacZ alpha-attTn7 sequence into a baculovirus transfer vector subjected to reverse amplification, so as to obtain a recombinant plasmid pBacPAK8-lacZ alpha-attTn7; the recombinant virus AcNPV-lacZalpha-attTn7 is obtained by co-transfecting Sf9 cells with a defective virus Bacmid, and a recombinant virus AcNPV-lacZalpha-attTn7 is generated; carrying out amplification and ultra-high-speed centrifugation on the obtained recombinant virus, and extracting through phenol chloroform to obtain virus DNA (Deoxyribonucleic Acid); the recombinant baculovirus shuttle vector Bacmid'is obtained by co-transforming competent cells of recombinant virus DNA (deoxyribonucleic acid) and pBvBAC (obtained by cloning Vcat, miniF and Chitinase to pUC19), and carrying out recombinant cloning by utilizing blue-white spot screening. According to the present invention, the miniF replicon co-localized with lacZalpha-attTn7 is separated, such that the recombinant baculovirus shuttle vector Bacmid'with the high gene cloning stability is successfully constructed; according to the technology, the expression stability of the exogenous gene is remarkably improved, and a powerful research tool is provided for vaccine development and virology application based on the baculovirus vector.

The invention discloses a marine microalgae efficient gene editing system as well as a construction method and application thereof. An efficient'all-in-one 'CRISPR / CAS9 expression vector pKSdiaCas9-sgRNAble is constructed by inserting an expression cassette of a bleomycin resistance gene ble into a pKSdiaCas9sgRNA plasmid, meanwhile, four gRNAs targeting different sites of the same gene are respectively designed to obtain four CRISPR / CAS9'all-in-one' plasmids, and the four CRISPR / CAS9'all-in-one 'plasmids are co-transformed into algae cells, so that the total editing probability and the probability of homozygote mutation are greatly improved. Through optimization of a gene editing tool, improvement of a gene gun transformation method and an identification strategy of a genome level, efficient and stable marine microalgae gene editing is realized, and an important technical support is provided for microalgae gene function research and application.

The invention relates to the technical field of biology, and particularly discloses a method for rapidly screening a high-affinity shark source nano antibody based on a BACTH system. The method comprises the following steps: fusing an antigen and an adenylate cyclase T25 fragment on a pKT25c plasmid, fusing a shark source nano antibody variable region library and an adenylate cyclase T18 fragment on a pUT18tc plasmid, and co-transforming into adenylate cyclase defective escherichia coli BTH101. When the antigen interacts with the nano antibody, the reconstructed adenylate cyclase activates downstream lacZ and chloramphenicol resistance double reporter genes. A blue monoclonal clone with chloramphenicol resistance is screened on a flat plate containing X-Gal, IPTG and chloramphenicol at one time, so that a high-affinity positive clone can be quickly obtained. The method overcomes the defects that a phage display technology is tedious in process, long in period and high in false positive, the whole screening process only needs one week, and the method has the remarkable advantages of being efficient, simple, convenient and low in cost.