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Method for producing transformant

A manufacturing method, a technology for transformants, applied in microorganism-based methods, biochemical equipment and methods, other methods of inserting foreign genetic material, etc., to achieve the effect of efficient manufacturing

Pending Publication Date: 2022-06-07
TOYOTA JIDOSHA KK
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

On the other hand, in the case of using a linear vector to introduce a gene of interest into yeast, it is necessary to integrate the gene of interest into the genome by homologous recombination, so only about 10 -6 efficient production of transformed yeast (Non-Patent Document 2)

Method used

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  • Method for producing transformant
  • Method for producing transformant
  • Method for producing transformant

Examples

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Embodiment 1

[0089] In this example, a haploid experimental yeast S. Cerevisiae (Saccharomyces cerevisiae) BY4742 strain was used as a host.

[0090] [Preparation of a linear vector set for GRE3 disruption in which loxP and the marker are contained in the same DNA fragment]

[0091] In this embodiment, as Figure 8 As shown, three plasmids were prepared into which the following DNA fragments were inserted: DNAs containing the 5' homologous recombination region of the GRE3 gene (homologous sequence 1) and a DNA sequence for homologous recombination (homologous sequence 2) Fragment; DNA recombinase Cre gene ( Figure 8 (not shown) DNA fragments sandwiched between loxP sequences and containing DNA sequences for homologous recombination (homologous sequences 2 and 3) at both ends; DNA sequence (homologous sequence 3) and DNA fragment of the 3' homologous recombination region (homologous sequence 4) of the GRE3 gene. Homologous sequence 2 and homologous sequence 3 are about 60 bp and are des...

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Abstract

Provided is a method for producing a transformant. The present invention addresses the problem of easily determining whether or not a nucleic acid fragment containing a gene of interest is accurately integrated into a host genome. A nucleic acid fragment group containing a nucleic acid fragment having a gene of interest is introduced into a host cell, and a host cell in which the gene of interest has been cleaved from genomic DNA using a site-specific recombinase is selected.

Description

technical field [0001] The present invention relates to a method for producing a transformant obtained by deleting a predetermined region from a host genome using a site-specific recombinase and a recognition sequence of the enzyme. Background technique [0002] A site-specific recombinase is an enzyme having the activity of recognizing a specific short homologous pair of base sequences and causing homologous recombination between the pair of base sequences. When homologous recombination occurs between a pair of nucleotide sequences in a state where the homologous pair of nucleotide sequences are arranged in the same direction, the region sandwiched by the pair of nucleotide sequences is excised. In addition, when homologous recombination occurs between a pair of nucleotide sequences in a state where a pair of nucleotide sequences of homology are arranged in opposite directions, the region sandwiched by the pair of nucleotide sequences is inverted. [0003] By utilizing the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12N15/61C12N15/90C12R1/865
CPCC12N15/81C12N9/90C12Y599/01C12N15/905C12N2800/30C12N9/22
Inventor 大西彻
Owner TOYOTA JIDOSHA KK