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Yeast for large-scale gene rearrangement and construction method thereof

A technology of gene rearrangement and construction method, applied in the field of genetic engineering, can solve the problems of complex method and low efficiency of yeast, and achieve the effect of rapid production

Active Publication Date: 2021-06-29
TIANJIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, the efficiency of gene rearrangement in yeast is low, and the method is relatively complicated

Method used

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  • Yeast for large-scale gene rearrangement and construction method thereof
  • Yeast for large-scale gene rearrangement and construction method thereof
  • Yeast for large-scale gene rearrangement and construction method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Using the Vika / vox system to obtain yeast strains that can produce large-scale rearrangements, including the following steps:

[0050] 1. Fragment construction

[0051] a) Design upstream and downstream primers to PCR amplify the KanMX (or hphNT1) resistance gene, the gene is synthesized between the two vox sites and the left end has 20bp homology with the KanMX (or hphNT1) resistance gene;

[0052] b) Extract the genome of the BY4742 strain, design the left and right ends of the upstream primers to carry 20bp pUC19 plasmid homology and 20bpKanMX (hphNT1) resistance gene respectively, and the left and right ends of the downstream primers respectively carry 20bp pUC19 plasmid homology and 20bp vox sequence to PCR amplify six 1000bp homologous sequences at both ends of the centromere of the wild type chromosome;

[0053] c) Using Gibson assembly to assemble the digested linearized pUC19 plasmid and double-stranded DNA, and construct six plasmids in total;

[0054] d) Tr...

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Abstract

The invention relates to the technical field of gene engineering, and particularly relates to yeast for large-scale gene rearrangement and a construction method thereof. According to the yeast for large-scale gene rearrangement and the construction method thereof, a complete yeast chromosome is cut off by using a Vika / vox technology, so that homologous recombination is generated by chromosome centromeres, the whole chromosome is knocked out, six synthetic chromosomes are integrated, and a large-scale rearrangement event is generated. Compared with existing achievements, the yeast for large-scale gene rearrangement and the construction method thereof have the following beneficial effects that cutting of the yeast chromosome is achieved simply, efficiently and quickly; cross exchange of sister dyeing monomers is avoided, and haploid yeast integrated by the six synthetic chromosomes is obtained; and very large-scale genome rearrangement is generated simply, efficiently and quickly.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to large-scale gene rearrangement yeast and its construction method. 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 Synthetic Yeast Genome Project (Sc2.0) aims to construct a fully designed and synthetic eukaryotic genome as a platform for systematic study of eukaryotic chromosomes. Synthetic genomes increase genetic flexibility while reducing genome instability, and maintain traits consistent with wild-type cells. In March 2017, the Sc2.0 project achieve...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/90C12N15/65C12N15/54C12N15/31C12N15/04C12R1/865
CPCC12N9/1241C07K14/28C12N15/81C12N15/905C12N15/65C12N15/04C12N2800/30Y02E50/10
Inventor 元英进吴毅周嗣杰
Owner TIANJIN UNIV
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