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A method for knocking out Saccharomyces cerevisiae chromosomes

A technology of Saccharomyces cerevisiae and Saccharomyces cerevisiae strains, applied to other methods of inserting foreign genetic materials, stably introducing foreign DNA into chromosomes, biochemical equipment and methods, etc., can solve the problems of low efficiency, inability to ensure the integrity of a single chromosome, The workload of the complete chromosome is large, and the effect of avoiding crossover, complete knockout and high success rate is achieved.

Active Publication Date: 2020-09-04
TIANJIN UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

The existing method of using the unequal distribution of yeast chromosomes during meiosis to achieve chromosome loss, in the process of meiosis, homologous recombination will occur between homologous chromosomes of yeast, and the integrity of a single chromosome cannot be guaranteed
This method requires a lot of work and low efficiency to obtain a complete chromosome

Method used

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  • A method for knocking out Saccharomyces cerevisiae chromosomes
  • A method for knocking out Saccharomyces cerevisiae chromosomes
  • A method for knocking out Saccharomyces cerevisiae chromosomes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Knockout of chromosome X to be knocked out in commercial BY4742 Saccharomyces cerevisiae

[0033] 1. Fragment construction

[0034] Design upstream and downstream primers to PCR amplify and synthesize the KanMX resistance gene, and the gene between the two vox sites (20bp homologous KanMX resistance gene + vox + chromosome X chromosome sequence + vox), the left end of the gene between the two vox sites (upstream of vox) has 20bp homology with KanMX resistance gene;

[0035] Extract the genome of BY4742 strain, design the upstream primer with 20bp pUC19 plasmid EcoRI restriction site upstream homology and the right end with 20bp KanMX resistance gene homology sequence on the left end, and the downstream primer right end with 20bp pUC19 plasmid HindIII restriction site downstream homology and left end band 20bp vox homologous sequence, PCR amplified 1000bp homologous sequence at both ends of chromosome X chromosome of wild type BY4742 Saccharomyces cerevisiae;...

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Abstract

The invention relates to the technical field of biology and in particular discloses a method for knocking out a saccharomyces cerevisiae chromosome. According to the method, a Vika / vox technology is utilized for removing a complete yeast chromosome, and chromosome centromeres are specifically recombined into a ring by virtue of loci, so that knockout of the whole chromosome is realized. Compared with a method for realizing elimination of the whole chromosome in a reduction mitosis manner, the method disclosed by the invention has the advantages that cutting of the saccharomyces cerevisiae chromosome is more simply, efficiently and rapidly realized, crossing over of sister chromatids is avoided, and a saccharomyces cerevisiae homozygous diploid bacterial strain with the chromosome knocked out is obtained.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a method for knocking out the chromosome of Saccharomyces cerevisiae. Background technique [0002] The biological genome carries the genetic information that determines the basic traits of the organism. Artificial DNA synthesis technology and DNA large fragment manipulation technology have promoted the progress of genome artificial synthesis research. The development of synthetic biology has promoted the "writing" of genome information through artificial design and synthesis, marking the beginning of "artificial life". [0003] The length of genomic DNA is too large, and most eukaryotes have multiple chromosomes, and the length of the chromosomes is relatively large. When it comes to chromosomal diseases or functional integration of chromosomes, the entire chromosome needs to be manipulated. How to achieve the knockout of the entire chromosome is a question worth ex...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/04C12N15/81C12N15/90
CPCC12N15/04C12N15/81C12N15/905
Inventor 元英进吴毅周嗣杰马璐刘瑞刘明
Owner TIANJIN UNIV
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