84 results about "PUC19" patented technology

The invention discloses a preparation method of a zebrafish notch1b gene mutant. The preparation method includes: determining positions of knock-out target spots of the notch1b gene; utilizing pUC19-gRNA scaffold plasmid as a template, and performing PCR (polymerase chain reaction) amplification with primers T7-notch1b-sfd and tracr rev; subjecting a PCR product to purification and in-vitro transcription to obtain gRNA (guide ribose nucleic acid); introducing the gRNA and Cas9mRNA into a cell-stage embryo of zebrafish, and culturing to obtain the notch1b gene mutant stable in inheritance. Thepreparation method has the advantages that by the aid of the CRISPR/Cas9 (clustered regularly interspersed short palindromic repeats, CRISPR/CRISPR-associated genes, cas gene) technology, and by meansof selecting a specific section of targeting domain, the notch1b gene in the zebrafish is knocked out, other genes are protected from being 'injured accidentally', the zebrafish without the Notch1b gene is formed, and great significance is achieved on study of Notch signal channels.

The invention discloses a DNA extraction method of Duck Enteritis Virus genome and the genome sequence. Firstly, the pyrolysis of chicken embryo fibroblast which is infected with DEV is processed by hypertonic buffer; then the nuclease is added for the digestion of cell DNA; and the virus genome DNA is obtained through phenol chloroform extracting method. The obtained sample is broken randomly into 2kb to 2.5kb fragments; the pUC19 vector is linked to transform host E. coli and construct genomic library. The positive clones in the library are randomly sequenced; the total sequence length has 6 times coverage rate for the virus genome. The sequence result is assembled and clustered to obtain the whole DEV genome sequence with the length of156512bp. The analysis shows that the genome structure is the same with that of the members of Varicella virus in Alpha- herpesus subfamily, which provides theoretical basis for the classification of DEV.

The invention discloses a building method of a silkworm BmNPV Polh+Bac-to-Bac rhabdovirus expression system, comprising: taking silkworm wild type BmNPV genome DNA as the template; respectively taking P10-upF/P10-upB and P10-downF/P10-down B as a primer PCR for amplification to obtain p10-up and p10-down; after processed, inserting into BamHI-PstI-HindIII locus in pUC 19 to obtain recombinant plasmid pUC19-p10-up-down; using pUC-19-p10-up-polh-down, liposome lipofectin and MilliQ H2O to prepare DNA-Lipofectin mixed liquor; adding the mixed liquor into BmN cells for cultivating; collecting transfection cell supernatant; inoculating BmN cells, and recovering a polyhedral body; extracting virus DNA from the polyhedral body and electrically converting DH10 beta competent cells; screening locus ceruleus and cultivating; after cultivating PCR positive bacterial plaque, extracting macro-molecular DNA to transfect to the BmN cells; separating to obtain helper plasmids from DH10Bac culture bacteria of AcMNPV Bac-to-Bac; converting the helper plasmids into E.coli DH10 beta containg Ploh+BmBacmid; and screening the DH10 beta bacterial strain of the helper plasmid containg Ploh+BmBacmid. The invention can produce recombinant virus capable of infecting by eating with mouth, and recombinant virus does not need to infect by intracutaneous inoculation, thus improving the production efficiency of the silkworm rhabdovirus expression system.

The invention relates to a double-tagging microbial cell biosensor for detecting N-acyl homoserine lactone (AHL), and the biosensor is suitable for detection of AHL and characterization of host cells in the environment. The cell biosensor comprises: Escherichia coli used as host cells for carrying recombinant plasmids; recombinant plasmid containing an ahlI promoter responding to AHL, a cherry red fluorescent protein reporter gene mCherry and an AHL regulatory protein gene ahlR; and a neomycin phosphotransferase promoter gene nptII for characterization of host bacteria, a green fluorescent protein reporter gene gfp and a T1-4 pUC19 plasmid of four terminator rrnb tandem sequence. According to the biosensor, constitutive type of expression green fluorescent protein is used to characterize the host bacteria; the response time to AHL is 6 h; and the minimum detectable concentration of AHL is 50 nmol / L. The biosensor has the advantages of high sensitivity, fast response, low cost and simple operation, and can be widely applied to detection of AHL and host characterization in the environment.

The invention discloses an aptazyme modified sgRNA carrier with theophylline regulated expression. Aptazyme P1-F5 is inserted to tetraloop and stem loop 2 positions of an expressed sgRNA-AZ 2.0 skeleton; by means of Kpn I and EcoR I sites, and EcoR I and Spe I sites, a U6 promoter and the sgRNA-AZ 2.0 skeleton are connected into the carrier pUC19/EKSHL in order to obtain the carrier pU6-sgRNA-AZ 2.0; using Bsa I for enzyme digestion of the carrier pU6-sgRNA-AZ 2.0, and using cohesive end design and connecting an sgRNA sequence directing at the target gene so as to obtain the pU6-sgRNA-AZ 2.0-target site. The carrier can effectively mediate the genome editing function of Cas9 protein, and bring the editing activity under regulation of theophylline, also can mediate the transcription inhibition of dCas9-KRAB protein to the target gene, and enables regulation of inhibitory activity by theophylline.

The invention relates to preparation of escherichia coli competent cells and a transformation method of the escherichia coli competent cells. The preparation comprises following the steps of culturing the escherichia coli in a SOB liquid medium, adding a buffer solution I when OD 600 is 0.4 to 0.6, centrifugally collecting cells after ice bath, after a buffer solution II resuspends the cells, respectively putting into pre-cooling sterile centrifuge tubes to obtain the scherichia coli competent cells. In a transformation process, uniformly mixing DNA (deoxyribonucleic acid) with the competent cells, and directly coating the culture medium with the mixture after heat-shock, wherein ice bath and recovery steps are not needed. The escherichia coli competent cells are prepared by adopting buffer solutions of a special formula, the transformation efficiency can reach 1.23X109 cfu/mug pUC19 DNA. The method is simple in operation, high in transformation efficiency and good in repeatability, the preparation of the escherichia coli competent cells and DNA transforming operation are simplified, and a technological base is provided for high-efficiency and rapid transformation of the DNA.

The invention discloses a fused antibacterial peptide and a preparation method thereof. According to the amino acid sequence of the antibacterial peptide YFGAP, the gene sequence of the amino acid sequence is optimally designed and connected to pUC19 to obtain pUC19-YFGAP; the pUC19-YFGAP is connected to pET-22b-S-ELPs to obtain pET-22b-YSELP; the pET-22b-YSELP is transformed to E.coli BL21 (DE3) competent cells to obtain E.coli BL21 (DE3)/pET-22b-YSELP; inoculating and culturing are performed and IPTG is added to induce the expression of the YSELP. The fused antibacterial peptide YSELP has an inhibition effect on a plurality of testing strains without being cut off from the tag protein, and does not kill host bacteria; as a result, the problem of large-scale application of the existing antibacterial peptides is expected to be solved.

The present invention relates to a noninfectious virus recombinant gene SARS-CoV-EGFP for screening medicine for resisting SARS virus and making basic research of virology. In 5' end of total genome of SARS coronate virus the promotor sequence T7 Pro. is introduced, the coding sequence of external structural domain related to viral pathogenicity and toxicity in S protein can be sustituted with EGFD gene of code enhanced green fluorescin, and in the 3' end of total genome HDV antisense genome ribozyme sequence HDV R2 and T7 transcription terminator sequence T7 Ter are successively introduced to create recombinant gene SARS-CoV-EGFP, then it is inserted into carrier to construct recombinant plasmid pUC19/SARS-CoV-EGFP, after cell transfection, through replication, transcription, traslation, processing and packaging to obtain recombinant virus capable of making transfected cell give out green fluorescence under the fluorescent microscope.

The invention discloses a T carrier and a preparation method thereof. A known carrier is a carrier after an improvement of a starting carrier which is pBlueScript II SK(-), pUC18, pUC19, pUC118, pUC119, pUC19, or pGEM serial carriers the polyclone sites of which are replaced with XcmI cases, wherein the XcmI cases comprise ccdB gene sequences and an XcmI restriction enzyme cutting site sequence which is connected to each of two sides of the ccdB gene sequences, and resistance genes in the starting carrier are lengthened by inserting an antisense gene with the length of 1000-2000bp in front and/or back of the antisense gene. The T carrier provided by the invention has higher TA clone efficiency, the TA clone efficiency can reach 100% especially for fragments which are difficult to distinguish for cloning conventional business clone carriers and have the lengths between 2500bp and 3500bp, and the T carrier plays an important role in the filed of genetic engineering.

The present invention discloses a method which utilizes the in-vitro expression system of the posterior silkgland of Bombyx mori to synthesize a protein. pUC19-FL-GFP-PolyA recombinant plasmid or pUC19-FL-GFP-PolyA recombinant plasmid with an exogenous gene or a pUC19-FL-PolyA recombinant plasmid or a pUC19-FL-PolyA recombinant plasmid with an exogenous gene and protease inhibitor are added into the tissue extract of the posterior silkgland of Bombyx mori, and after water bathing under the temperature between 25 DEG C and 29 DEG C, the expression of the targeted protein is obtained. The method has the following advantages: since the protein is expressed under a temperature between 25 DEG C and 29 DEG C, the low-temperature condition (normally, 37 DEG C) helps to generate the expressed protein in a dissolved state, and the formation of inclusion bodies is reduced, which is favorable for the separation and purification of the protein. Meanwhile, the method is not limited by cells and can synthesize the protein in a short period.

The invention discloses construction of a bacillus subtilis integrated vector capable of being cloned by using homologous recombination and application thereof. The construction of the bacillus subtilis integrated vector capable of being cloned by using homologous recombination and the application thereof belong to the technical field of biology. The vector pMLK-BN can transfer an exogenous gene or a promoter cloned on a common cloning vector puc18, puc19 or puc57 to self by a homologous recombination method so as to integrally express exogenous protein in bacillus subtilis or test the promoting effect of the promoter. Compared with a common connecting cloning method, the cloning method of homologous recombination is simple and efficient; and the pMLK-BN can be effectively used as a choice for cloning the exogenous gene or the promoter to the bacillus subtilis to perform integrated expression.

A method of constructing a zebrafish notch1a mutant using CRISPR/Cas9 technique. The method includes: determining a target for knocking out notch1a; using primers T7-notch1a-sfd and tracr rev for PCR amplification with a pUC19-gRNA scaffold plasmid as a template; transcribing PCR product in vitro followed by purification to obtain gRNA; and microinjecting the gRNA and a Cas9 mRNA into a zebrafish embryo followed by culture to obtain an notch1a mutant of stable inheritance. The invention selects a specific target and utilizes CRISPR/Cas9 technique to knock out the notch1a in the zebrafish without destroying other genes, generating the zebrafish notch1a mutant. Moreover, the invention also discloses the phenotype of the zebrafish notch1a mutant, which plays a significant role in studying the effect of the Notch1a receptor in the Notch signaling pathway.

The invention discloses a construction method and an application of an annular RNA overexpression system of protoplast of moso bamboo and belongs to the technical field of genetic engineering. The system constructs an annular RNA overexpression vector and converts the vector to the protoplast of moso bamboo. The construction method comprises the following steps: amplifying an annular RNA host genewith a high fidelity Taq enzyme and constructing an annular RNA overexpressed recombinant plasmid by a Gateway method by taking pUC19-35s-sGPF as a final vector; and extracting the recombinant plasmids massively and converting the plasmids to the protoplast of moso bamboo mediated by PEG to research the influence of annular RNA overexpression to transcription and post-transcription levels of thehost gene.

The invention discloses a constructing method of a zein gene RNAi (Ribonucleic Acid Interference) carrier. The method comprises the following steps of: constructing a recombinant carrier which is inserted into a forward target fragment and an endospem-specific expression promoter P-zp22/6 as well as a recombinant carrier which is inserted into a reverse target fragment and a terminator poly(A) by utilizing pUC19 as a frame carrier, and respectively carrying out digestion and connection on the two recombinant carriers to obtain a recombinant carrier pUC19-p22/6-22KD1-22KD2-poly(A); and inserting the GUS gene intron into the recombinant carrier to obtain the zein gene RNAi carrier of which the target fragment is partial cDNA (complementary Deoxyribonucleic Acid) fragment of a 22-KD alcohol-soluble protein gene. Another zein gene RNAi carrier is constructed by an identical method, and the target fragment of the zein gene RNAi carrier is partial cDNA fragment of a 19-KD alcohol-soluble protein gene. By adopting the method, the carrier constructing time is shortened and a transgenic plant with high lysine content can be successfully obtained.

The invention discloses a construction method of a drug-resistant Escherichia coli intergrase integration vector pUC19-attI-400-attC. The recombinant containing attI and attC recombinant sites is constructed, a 440bp DNA sequence exists between the attI and attC recombinant sites, the vector is small and only contains 3270bp, the attI and attC sites can be simultaneously cut, the attC site also can be individually cut, and the vector also contains more than 10 multiple cloning sites, so a user can conveniently select suitable restrictive endonuclease to research the action site of intergrase; and the vector has aminobenzyl resistance, is convenient to screen, can be used to carry out in vitro detection of the vector model of the integration activity of the drug-resistant bacterial intergrase, and also can be used to explore the integration mechanism of other bacterial integrons.

The invention discloses a universal transfer vector for a pseudorabies virus capable of independently expressing dual genes and a construction method of the universal transfer vector. The universal transfer vector is obtained by inserting a dual-gene expression cassette containing two CMV promoters, multiple cloning sites respectively at downstream positions of the two CMV promoters and a BGH pA signal sequence between gEL and gER contained in pUC19/HEK-gE recombinant plasmids containing gEL and gER. The universal transfer vector is capable of independently expressing two exogenous genes by utilizing two promoters and can be used for constructing a recombinant pseudorabies virus capable of independently expressing two exogenous genes.

The invention provides a novel bacterial artificial chromosome of a turkey herpes virus, a construction method and application and relates to the field of animal medicine. The bacterial artificial chromosome is obtained by knocking out gC gene in HVT genome and replacing with a mini-F plasmid sequence. The construction method includes: amplifying upstream and downstream homologous arms of the gC gene in the HVT genome, and inserting into a pUC19 vector to obtain a recombinant vector A; inserting a mini-F plasmid segment into the recombinant vector A to obtain a recombinant transfer vector B, performing cotransfection (CEF) with DNA of HTV, and screening to obtain mini-F recombinant HVT; extracting DNA of the mini-F recombinant HVT to convert DH10B cells, adopting chloromycetin for screening to obtain positive recombinant bacteria, culturing, and then extracting plasmid DNA. The bacterial artificial chromosome has high stability, and the construction method is simple, efficiency and applicable to construction of recombinant vector live vaccines.

This invention relates to a YENB culture medium for preparing high-efficiency electroporation-competent cells. The YENB culture medium comprises (per liter): yeast extract for cell culture 6-9 g, nutrient broth for cell culture 6-9 g, and water. The YENB culture medium can prepare high-efficiency electroporation-competent cells by controlling cell primary and secondary growth time, centrifugal speed and time. The conversion rate is 2.19X1010 cfu/mu.g pUC19 DNA, which is higher than imported culture medium, while the cost is only 5% that of imported culture medium. The culture medium lays a basis for high-efficiency conversion of large fragment DNA genomic library.

A recombinant mononuclear capsid nucleopolyhedrovirus of bollw orm able to express polyhedron and chitinase fusion protein is prepared through PCR amplification of polyhedron gene HaSNPV and chitinase gene, cloning to carrier pUC19 to configure fusion gene, inserted it in eukaryotic carrier pHaWHL5 to obtain eukaryotic transfer carrier pHaWD6, transfecting insection cell by it along with wild virus DNA, and homologous recombination.