37 results about "Yeast plasmid" patented technology

The invention provides a SOD gene from animal and a method for cloning and expressing the SOD gene in colibacillus and yeast cells, wherein a nucleotide sequence of superoxide dismutase is shown as SEQ NO.1; an amino acid sequence of the superoxide dismutase is shown as SEQ NO.2; carriers of nucleotide molecules are colibacillus plasmids or yeast plasmids; cells of the nucleotide molecules are formed by carrier conversion; and cells of the nucleotide molecules of the superoxide dismutase contain colibacillus containing the nucleotide molecules or converted by the carriers or pichia yeast containing the nucleotide molecules or converted by the carriers. The invention can prepare recombinant production strains which can efficiently express and secrete Cu / Zn-SOD, realizes the production industrialization of the Cu / Zn-SOD, and achieves good pH stability, favorable thermal stability and Cu / Zn-SOD products with anti-protease hydrolyzation capacity.

The invention provides agarase and a preparation method thereof and a method for cloning and expression of agarase in escherichia coli and yeast cells. The nucleotide sequence of agarase is as shown in SEQ NO.1, and the amino acid sequence of agarase is as shown in SEQ NO.2. Vectors of nucleotide molecules are escherichia coli plasmids or yeast plasmids. Cells of the nucleotide molecules are obtained by transforming the vector. Cells of the nucleotide molecules of agarase are escherichia coli containing the nucleotide molecules or transformed by the vectors or Pichia pastoris containing nucleic acid molecules or transformed by the vectors. A recombination production strain which can be expressed efficiently and secrete agarase is prepared, the production industrialization of agarase is achieved, and the agarase has good capability of hydrolysis of agar and gracilaria lemaneiformis.

The invention provides a method for improving the lipase activity persistency, comprising the steps of: connecting a lipase gene (Genbank No.: AF229435) (with a conventional method) to the N end of a saccharomyces cerevisiae cell wall component FLO sequence (Genbank No.: S73336), then inserting the lipase gene (in a conventional approach) to the C end of a downstream MF alpha1 signal peptide sequence (Genbank No.: M17301) of the GAL1 promoter (Genbank No.: AY428072) of a saccharomyces cerevisiae expression vector so as to construct an expression cassette, which is, from the N end to the C end, as follows: GAL1 promoter + MF alpha1 signal peptide sequence + lipase gene + FLO sequence; shifting a yeast plasmid containing the expression cassette into the saccharomyces cerevisiae cell. According to the invention, the lipase is connected to the saccharomyces cerevisiae cell wall component FLO, so that as long as the saccharomyces cerevisiae cell maintains alive, the lipase can keep active.

The invention provides a chitinase derived from a marine environment and a gene thereof and a method for cloning and expressing the chitinase in escherichia coli and yeast cells. The nucleotide sequence of the chitinase gene is shown as SEQ ID NO. 1; the amino acid sequence of chitinase is shown as SEQ ID NO. 2; a carrier containing the gene is an escherichia coli plasmid or a yeast plasmid; a cell containing the gene is transformed from the carrier; the cell of the gene of the chitinase is escherichia coli which comprises the nucleotide molecule or is transformed by the carrier or pichia pastoris which comprises nucleic acid molecule or is transformed by the carrier.

Methods and compositions for making stable recombinant yeast 2 μm plasmids are provided. Homologous recombination is performed to clone a nucleic acid of interest into the yeast 2 μm plasmid. Heterologous nucleic acid subsequences are recombined between an FLP and a REP2 gene of the plasmid.

The invention relates to a method for improving continuity of alpha galactosidase activity. The method comprises the following steps that: alpha galactosidase gene is linked to a N-terminal of FLO sequence of saccharomycescerevisiae cell wall composition; the resulting sequence is inserted into a C-terminal of MF alpha 1 signal peptide sequence positioned on the downstream of a GAL1 promoter of a galactosidase expression vector to construct a expression cassette, wherein the expression cassette sequentially comprises the GAL1 promoter, the MF alpha 1 signal peptide sequence, the alpha galactosidase gene and the FLO sequence from the N-terminal to the C-terminal; yeast plasmids containing the expression cassette are transformed into the saccharomycescerevisiae cells. According to the present invention, the alpha galactosidase is linked to the FLO sequence of the saccharomycescerevisiae cell wall composition, such that if the saccharomycescerevisiae cells are in survival states, the alpha galactosidase can remain the activity.

The invention provides chitinase for preparing chitin oligose, a gene of the chitinase and a method for cloning and expressing the gene in escherichia coli and yeast cells. The nucleotide sequence of the gene of the chitinase is shown in SEQ ID NO.1, and the amino acid sequence of the chitinase is shown in SEQ ID NO.2. A recombinant vector containing the gene is formed by connecting the gene of the chitinase to an escherichia coli plasmid or a yeast plasmid. Cells of the gene are formed through transformation of the recombinant vector. Cells containing the gene of the chitinase are escherichia coli which contains nucleotide molecules or is transformed through the recombinant vector, or are Pichia pastoris which contains nucleotide molecules or is transformed through the recombinant vector.

The invention provides superoxide dismutase from pig blood cells and a preparation method thereof. The nucleotide sequence of the superoxide dismutase is shown in a figure 1; the amino acid sequence of the superoxide dismutase is shown in a figure 2 in the specification; the vector of the nucleotide molecules is yeast plasmid; the cells of the nucleotide molecules are converted from the vector; and the nucleotide molecular cells of the superoxide dismutase contain nucleic acid molecules or pichia pastoris converted from the vector. The method can be used for preparing a recombinant production strain which can effectively express and secret Cu, Zn-SOD to realize production industrialization of Cu, Zn-SOD (superoxide dismutase) to obtain a Cu, Zn-SOD product with good pH stability, excellent thermal stability and protease hydrolysis capability.

The invention discloses a standardized, high-accuracy and general functional module construction method, wherein the construction method comprises the following steps: (1) adding a standardized site to a source gene to form a genetic element, linking the genetic element into a plasmid pLD-Blunt to form a plasmid with the genetic element, and carrying out sequence verification; (2) taking the genetic element from the plasmid with the genetic element, and linking to a following notch in a module tool plasmid: terminator 1-promoter-AATG-notch-TAAA-terminator 2; and constructing plasmids with different functional modules; and (3) taking different functional modules from the plasmids with the different functional modules, and co-transforming with a yeast plasmid vector subjected to restriction enzyme digestion into saccharomyces cerevisiae, constructing the standardized, high-accuracy and general functional modules, and storing in the plasmids. The construction method has the advantages of universality, standardization, high accuracy, and free selection.

The invention provides alginate lyase and a gene and application thereof and further provides a method of cloning and expression of the alginate lyase in escherichia coli and yeast cells. A nucleotide sequence of the alginate lyase is shown as SEQ ID NO.1, and an amino acid sequence of the alginate lyase is shown as SEQ ID NO.3. A recombinant vector comprising the gene is formed by connection of the alginate lyase gene to escherichia coli plasmids or yeast plasmids. A cell comprising the gene is obtained by conversion of the recombinant vector. The cell of the alginate lyase gene comprises the nucleotide molecule or escherichia coli converted from the recombinant vector or comprises the nucleic acid molecule or Pichia pastoris converted from the recombinant vector.

The invention relates to a construction method of a shuttle plasmid taking yeast G418 as a selective marker, which solves the problem that the present yeast plasmids typically take auxotroph as the selective mark and are difficult to be used in the gene engineering reconstruction of full nutritious yeasts. The construction method comprises the following steps: cloning kanR genes, recycling and purifying gene segments, and connecting with pYES2 carriers. A carrier constructed by using the method enables Saccharomyces cerevisiae which is originally sensitive to G418 to become resistant to G418,so that the gene operation of full nutritious yeasts can be conducted without auxotroph mutagenesis, thereby reducing the gene reconstruction time.

The invention discloses a standardized, high-precision, and general-purpose functional module construction method, which includes the following steps: (1) Adding standardized sites to the source gene to form a genetic element, and connecting the genetic element to the plasmid pLD-Blunt to form a genetic (2) Get the gene element from the plasmid with the gene element and connect it to the following gap in the module tool plasmid: terminator 1-promoter-AATG-gap-TAAA-terminator 2 ; Construct plasmids with different functional modules; (3) Transfer different functional modules from plasmids with different functional modules, and co-transform S. The functional modules are stored in the plasmid; the present invention has versatility, standardization, high precision and free selectivity.

The invention provides a method for improving activity of lipase. The method comprises the following steps: connecting a lipase gene (Genebank number: AF229435) to the N terminal of a Saccharomyces cerevisiae cell wall component alpha lectin sequence (Genebank number: M28164); then inserting into the C terminal of a downstream MFalpha 1 signal peptide sequence of a Saccharomyces cerevisiae expression vector GAL1 promoter (Genebank number: AY428072) so as to construct an expression frame, wherein from the N end to the C end, the expression frame successively comprises: a GAL1 promoter, a MFalpha signal peptide sequence, a lipase gene and an alpha lectin sequence; and transforming yeast plasmid containing the expression frame into a Saccharomyces cerevisiae cell. The method has the advantages that linoleate isomerase is revealed and expressed on the outer surface of a Saccharomyces cerevisiae cell wall, and the complete contact of the linoleate isomerase and an extracellular substrate is achieved, thereby obviously improving the activity of the linoleate isomerase.

The invention discloses a method for rapidly constructing a filamentous fungus expression vector. The method comprises the following main processes: respectively cloning a yeast plasmid replication module, a bacterial plasmid replication module, a filamentous fungal replication module, and the corresponding promoter sequences, wherein each fragment has a homologous arm of about 20 to 50 bp connected end to end, and employing an electrotransformation technology to obtain a plasmid containing three modules, which can be used for rapid identification of the strength of a non-mode filamentous fungi self-promoter, key enzyme gene expression, secondary metabolite biosynthesis, and interaction between endophytes and plants. The method provided by the invention has the advantages of rapidity and high precision, and no need of restriction endonuclease, and can be used for the construction or replacement of multi-fragment and long-fragment vectors, which makes up the defects of difficult selection of restriction endonuclease during the construction of the existing filamentous fungal vector and non adaption in multiple segments or long segments.

The invention relates to universal carriers constructed based on methylotrophic hansenula polymorpha and pichia pastoris, in partituclar to a construction method of a universal carrier for performing heterologous protein expression on pichia pastoris and hansenula polymorpha. The construction method comprises the following steps that (1) a plasmid pHanMOX-GFP is digested through restriction enzymes Bgl II and BamH I, so that a pHan carrier is obtained; (2) a carrier plasmid pPinkAOX1-HPV16L1 is digested through restriction enzymes Bgl II and BamH I, a fragment PAOX1-HPV16L1-CYC1TT is recovered and then connected with the pHan carrier obtained in the step (1) through a T4 ligase at 16 DEG C overnight, an LB flat plate containing 0.1 mg / mL kanamycin is converted and coated, and plasmid enzyme digestion is extracted for identification, so that a restructured hansenula polymorpha plasmid pHanAOX1-HPV16L1 is obtained. The universal carrier is used for guiding expression of human papillomavirus type 16 major capsid protein L1 in pichia pastoris and hansenula polymorpha.

The invention belongs to the technical field of biological gene engineering, and provides a method for preparing a food grade Saccharomyces cerevisiae recombinant plasmid. The method comprises: firstly, constructing shuttle plasmid yeast carrier part which having a yeast 2mu plasmid complete sequence, a yeast nutritional deficiency screening gene, a yeast promoter and a yeast terminator; constructing an exogenous gene expression box which has a yeast promoter, a yeast terminator and a target exogenous gene; using the shuttle plasmid yeast carrier part and exogenous gene expression box in combination to convert a corresponding auxotrophic Saccharomyces cerevisiae strain, and screening a culture convertor in an auxotrophic selective culture medium; and finally, separating and purifying to obtain target Saccharomyces cerevisiae recombinant plasmid. The expression plasmid can survive only in the cells of the Saccharomyces cerevisiae and does not transfer, and can be widely used for expressing various exogenous genes safely and efficiently.

The invention discloses a selecting method of gene participating in transfection of exogenous DNA from sperm, relates to a method for screening a gene participating in transfection of exogenous DNA from sperm, and aims at providing the method for screening the gene participating in transfection of the exogenous DNA from the sperm. The method comprises the following steps: 1 building a recombinant plasmid pGB-mCD4; 2 screening from a cDNA library to obtain a to-be-tested gene; 3 positively cloning the to-be-tested gene, extracting a yeast plasmid and amplifying; 4 transferring the plasmid pGB-mCD4 and the positively cloned yeast plasmid of the to-be-tested gene into a double-hybrid strain Y190 of the yeast; and 5 carrying out beta-galactosidase chromogenic reaction, if the chromogenic result becomes blue, judging to be positive, and analyzing, thereby obtaining the gene participating in the transfection of the exogenous DNA from the sperm. Through the method disclosed by the invention, the gene for transferring the exogenous DNA together with CD4 can be greatly and rapidly obtained; a new idea is developed for research on the sperm-mediated gene transfer mechanism; and a foundation is laid for building of simple, stable and efficient sperm-mediated genetically modified methodology.

The invention relates to the technical field of fermentation yeast screening genes, and discloses an experimental method of a yeast two-hybrid screening polypeptide library, which comprises the following steps: preparation of materials: bait clone pGBKT7-gA9-nosc-221, a bait carrier pGBKT7, library yeast plasmid pGADT7-polypeptide cDNA, a Clontech yeast two-hybrid system and a culture medium are prepared; the culture medium is prepared from an escherichia coli culture medium, a yeast complete culture medium, a yeast defect type screening culture medium and other stock solutions. The method specifically comprises the following steps: transforming bait plasmids into recipient bacteria AH109, carrying out self-activation detection on the bait plasmids, carrying out library screening, carrying out yeast positive clone identification and sequencing comparison, carrying out yeast positive clone rotation verification, screening a yeast two-hybrid polypeptide library by taking a gA9-nosc-221 gene constructed on a pGBKT7 vector as bait, and carrying out multiple report gene detection, DNA sequencing and BLAST comparative analysis on the positive clone, so as to obtain the yeast two-hybrid polypeptide library. According to the method, the polypeptide interacting with the pGBKT7-gA9-nosc-221 is determined, the method for determining the polypeptide protein is more efficient, and the research efficiency is greatly improved.

The invention discloses a toxic protein gene leakage expression cloning method. The method comprises the following steps: S1, adding an SsrA polypeptide sequence to a C terminal of CcdB protein; S2, obtaining a gene segment through a PCR amplification method, and recombining the gene segment to a target vector PUC57 by utilizing Gibson to construct a complete sequence; and S3, connecting and cloning a target gene sequence into an expression vector, transforming escherichia coli, culturing for 12-16 h, observing a spot growing condition, carrying out PCR bacterial colony screening positive cloning, extracting plasmids, sequencing, and verifying the sequence. The gene can be cloned in yeast, the yeast belongs to eukaryotes, most toxic proteins can be cloned, the gene is cloned in the yeast firstly, then yeast plasmids are extracted, the plasmid concentration is improved, escherichia coli is transformed for plasmid amplification, and downstream experiments are carried out.

The invention discloses a non-homology, multi-long-fragment, high-efficiency and zero-background assembly method. The method comprises the following steps: firstly, adding a B fragment Yeast ori at the unique position of the Not I site of restriction enzyme of whole cyclization plasmid, and simultaneously ensuring that the left and right of the B fragment have just two Not I enzyme digestion sites; designing two Primer 1 / 2 primers between an A fragment and a B fragment which are not homologous for example, designing two Primer 3 / 4 primers between the B fragment and a C fragment which are not homologous, and using the primers as bridges for connecting the two corresponding segments; transferring all 7 fragments ABCDEFG and 7 pairs of primer1-14 at the joints of the corresponding fragments into competent yeast cells for shaking culture for two days; extracting yeast plasmid cultured for two days; and finally, performing amplification and detection. The invention provides the non-homology, multi-long-fragment, high-efficiency and zero-background assembly method, the zero-background cloning is completed through yeast assembly and Escherichia coli screening, no empty vector is produced, and the method saves a large amount of time compared with a traditional cloning mode.

A method for raising linoleate isomerase activity of the present invention comprises steps of: connecting a linoleate isomerase gene (Genbank No: DQ22732) to an N end of an alpha agglutinin sequence (Genbank No: M28164) of a saccharomyces cerevisiae cell wall component; inserting into a C end of a downstream MFalpha1 signal peptide sequence (Genbank No: M17301) of a saccharomyces cerevisiae expression vector GAL1 promoter (Genbank No: AY428072) to construct an expression frame of, from an N end to a C end, GAL1 promoter + MFalpha1 signal peptide sequence + linoleate isomerase gene + alpha agglutinin sequence; and conversing a yeast plasmid containing the expression frame to a saccharomyces cerevisiae cell. The invention has the advantage that the linoleate isomerase is expressed in exposure on an external surface of the saccharomyces cerevisiae cell wall to realize complete contact between the enzyme and an extracellular substrate, so as to substantially raise linoleate isomerase activity.

The invention relates to the technical field of membrane protein experiments, and discloses a method for discovering membrane protein based on membrane system-yeast two-hybrid. Comprising the following material bait cloning: pBT3-ST-A, a bait carrier: pBT3-STE, library yeast plasmids: a grass membrane library, a Clontech yeast two-hybrid system, a culture medium and a stock solution. The invention discloses a method for discovering membrane protein based on membrane system-yeast two-hybrid. A bait plasmid is transformed into recipient bacteria NMY51, and the steps of self-activation detection, library screening, library screening results, yeast positive clone identification and sequencing comparison and sequencing comparison results, yeast positive clone identification and sequencing comparison and sequencing comparison results, yeast positive clone rotation verification and the like are performed. 34 yeast positive clones obtained through screening can grow on SD-TL, SD-TLH + 30mM 3AT and SD-TLHA + 30mM 3AT defective plates through yeast positive clone rotation verification, so that the final membrane protein is obtained, and the problem that the protein acting with pBT3-ST-A is not easy to find is solved.

The invention relates to a method for improving beta glucanase activity. The method comprises the following steps that: beta glucanase gene is linked to a N-terminal of alpha agglutinin sequence of saccharomycescerevisiae cell wall composition; the resulting sequence is inserted into a C-terminal of MF alpha 1 signal peptide sequence positioned on the downstream of a GAL1 promoter of a galactosidase expression vector to construct a expression cassette, wherein the expression cassette sequentially comprises the GAL1 promoter, the MF alpha 1 signal peptide sequence, the beta glucanase gene and the alpha agglutinin sequence from the N-terminal to the C-terminal; yeast plasmids containing the expression cassette are transformed into the saccharomycescerevisiae cells. According to the present invention, the beta glucanase is exposed and expressed on the outer surface of the saccharomycescerevisiae cell wall, such that complete contacts of the beta glucanase and the extracellular sunstrates are realized so as to substantially improve the beta glucanase activity.

A method for raising glutamate decarboxylase activity of the present invention comprises steps of: connecting a glutamate decarboxylase gene (Genbank No: NM_168056) to an N end of an alpha agglutinin sequence (Genbank No: M28164) of a saccharomyces cerevisiae cell wall component; inserting into a C end of a downstream MFalpha1 signal peptide sequence (Genbank No: M17301) of a saccharomyces cerevisiae expression vector GAL1 promoter (Genbank No: AY 428072) to construct an expression frame of, from an N end to a C end, GAL1 promoter + MFalpha1 signal peptide sequence + glutamate decarboxylase gene + alpha agglutinin sequence; and conversing a yeast plasmid containing the expression frame to a saccharomyces cerevisiae cell. The invention has the advantage that the glutamate decarboxylase is expressed in exposure on an external surface of the saccharomyces cerevisiae cell wall to realize complete contact between the enzyme and an extracellular substrate, so as to substantially raise glutamate decarboxylase activity.

The invention provides a method for improving the persistency of glutamic acid decarboxylase activity. The method comprises the steps of: connecting a glutamic acid decarboxylase gene (Genbank No.: NM_168056) (with a conventional method) to the N end of a saccharomyces cerevisiae cell wall component FLO sequence (Genbank No.: S73336), then inserting the glutamic acid decarboxylase gene (in a conventional approach) to the C end of a downstream MF alpha1 signal peptide sequence (Genbank No.: M17301) of the GAL1 promoter (Genbank No.: AY428072) of a saccharomyces cerevisiae expression vector so as to construct an expression cassette, which is, from the N end to the C end, as follows: GAL1 promoter + MF alpha1 signal peptide sequence + glutamic acid decarboxylase gene + FLO sequence; shifting a yeast plasmid containing the expression cassette into the saccharomyces cerevisiae cell. According to the invention, the glutamic acid decarboxylase is connected to the saccharomyces cerevisiae cell wall component FLO, so that as long as the saccharomyces cerevisiae cell maintains alive, the glutamic acid decarboxylase can keep active.

The invention relates to a construction method of a shuttle plasmid taking yeast G418 as a selective marker, which solves the problem that the present yeast plasmids typically take auxotroph as the selective mark and are difficult to be used in the gene engineering reconstruction of full nutritious yeasts. The construction method comprises the following steps: cloning kanR genes, recycling and purifying gene segments, and connecting with pYES2 carriers. A carrier constructed by using the method enables Saccharomyces cerevisiae which is originally sensitive to G418 to become resistant to G418,so that the gene operation of full nutritious yeasts can be conducted without auxotroph mutagenesis, thereby reducing the gene reconstruction time.