12 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 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 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 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 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 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 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.