37 results about "Yeast plasmid" patented technology

A W303a Saccharomyces cerevisiae yeast cell which contains a polynucleotide that encodes a functional Bax polypeptide under the control of a galactose- inducible promoter that is integrated at the LEU2 chromosomal locus. A kit of parts comprising the yeast cells and a yeast plasmid vector suitable for transforming a cDNA library into the yeast cells. Use of the yeast cell for screening a cDNA library for a polynucleotide that is or encodes an inhibitor of B ax-mediated apoptosis. Genes and polypeptides that inhibit Bax-mediated apoptosis and which were identified from a human hippocampus cDNA library screened in the yeast cells. A method of combating Bax-mediated apoptosis in a cell using an inhibitor of Bax-mediated apoptosis which was identified from a human hippocampus cDNA library screened in the yeast cells. A method of promoting Bax-mediated apoptosis in a cell using an inhibitor or antagonist of the anti-apoptotic polypeptides identified from a human hippocampus cDNA library screened in the yeast cells.

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.

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 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 belongs to the technical field of genetic engineering, and in particular relates to a construction method of an engineering yeast of double-channel protein expression. A genetic engineering yeast plasmid vector is constructed; two promoters, namely GAL10 and GAL1, expressing exogenous genes are carried in the plasmid vector, wherein exogenous protein is located to the surface of theyeast by virtue of the promoter GAL10; and protein of the gene, under the control of the promoter GAL1, is expressed in cytoplasm of the yeast. According to the engineering yeast provided by the invention, two different proteins can be conveyed to the surface of the yeast or located into the cytoplasm through different protein expression channels; and the protein on the surface of the yeast is applicable to researches on protein functions, and through intracellular fluorescence intensity, yeast quantity is indicated, so that purposes of being efficient, convenient and economical are achieved,and the construction method is applicable to intracellular protein researches.

The present invention provides a chitinase and its gene for the preparation of chitin oligosaccharides, and a method for cloning and expressing it in Escherichia coli and yeast cells. The nucleotide sequence of the chitinase gene is as shown in SEQ ID Shown in NO.1; The amino acid sequence of chitinase is shown in SEQ ID NO.2; The recombinant vector containing said gene is that chitinase gene is connected to Escherichia coli plasmid or yeast plasmid; The cells of said gene are composed of The recombinant vector is transformed; the cell containing the chitinase gene is Escherichia coli that contains the nucleotide molecule or is transformed with the recombinant vector or contains the nucleotide molecule or is transformed with the recombinant vector Transformed Pichia pastoris.

The present invention provides a chitinase derived from a marine environment and its gene, and a method for cloning and expressing it in Escherichia coli and yeast cells. The nucleotide sequence of the chitinase gene is as shown in SEQ ID NO.1 shown; the amino acid sequence of chitinase is shown in SEQ ID NO.2; the vector containing the gene is Escherichia coli plasmid or yeast plasmid; the cells containing the gene are transformed by the vector; The cell containing the gene of the tritinase is Escherichia coli transformed with the nucleic acid molecule or transformed with the vector, or Pichia pastoris transformed with the nucleic acid molecule or transformed with the vector.

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.

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.

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.