Gene element for accurately controlling gene rearrangement and recombinant plasmid and application thereof

A gene rearrangement and gene element technology, applied in the biological field, can solve the problem of unfavorable control of gene rearrangement, and achieve the effect of avoiding leakage expression

Active Publication Date: 2018-07-31
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In summary, it is not difficult to find that the pSCW11-Cre-EBD plasmid has the problem of leaky expression, which is very unfavorable for controlling gene rearrangement

Method used

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  • Gene element for accurately controlling gene rearrangement and recombinant plasmid and application thereof
  • Gene element for accurately controlling gene rearrangement and recombinant plasmid and application thereof
  • Gene element for accurately controlling gene rearrangement and recombinant plasmid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: Effects of Gene Elements of the Present Invention and pSCW11-Cre-EBD on Synthetic Yeast Chromosome Growth

[0061] According to the gene sequences of the pGAL1 promoter, Cre-EBD fusion protein gene and tCYC1 terminator, the genetic elements of the present invention are artificially synthesized by sequential splicing (see the schematic diagram image 3), and connected to the pRS413 plasmid to obtain the recombinant plasmid pCRE4 (pGal1-Cre-EBD), see the plasmid map Figure 4 At the same time, according to the gene sequence of the pSCW11 promoter, the Cre-EBD fusion protein gene and the tCYC1 terminator, the artificially synthesized control gene elements were sequentially spliced ​​(see the schematic diagram in image 3 ), and connected to the pRS413 plasmid to obtain the recombinant plasmid pCRE1 (pSCW11-Cre-EBD), see the plasmid map Figure 5 ;

[0062] Transform pCRE1 and pCER4 into synthetic synIII yeast and synthetic synV yeast (both synthetic chromosome...

Embodiment 2

[0064] Example 2: Effects of Different Gene Elements on Chromosomal Gene Rearrangement in Synthetic Yeast

[0065] In order to compare the control of Cre activity by different promoters and different control methods, pCRE1 (pSCW11-Cre-EBD) and pCRE2 (pZEO1-Cre-EBD) were constructed in this example. For the plasmid map, see Figure 7 ), pCRE3 (pGAL1-Cre, see the plasmid map Figure 8 ) and pCRE4 (pGal1-Cre-EBD) four "switch" plasmids ( Figure 9 ), where pZEO1 is a constitutively expressed weak promoter, which is a comparative setting considering whether pSCW11 is too strong to cause leaky expression; the EBD junction domain of pCRE3 is removed. The above four "switch" plasmids and the control plasmid pRS413 were respectively transformed into the synthetic synV Saccharomyces cerevisiae, and the recombinant strains pRS413-synV, pCRE1-synV, pCRE2-synV, pCRE3-synV and pCRE4-synV were cultured in glucose medium After culturing for 32 hours, samples were taken at intervals of 2 ho...

Embodiment 3

[0067] Example 3: Effects of Gene Elements According to the Present Invention on Gene Rearrangement of Synthetic Yeast Chromosome in Different Induction Modes

[0068] In order to evaluate the performance of the gene elements of the present invention, pRS413-synV (control) and pCRE4-synV were cultivated under 4 different conditions in Example 2: (1) SC-His glucose medium; (2) with 1 μ M estradiol (3) SGal-His galactose medium; (4) SGal-His galactose medium supplemented with 1 μM estradiol. The growth curve was recorded by a microplate reader, and the doubling time of the yeast was calculated according to the formula.

[0069] Such as Figure 11 As shown, when the cells were cultured with SC-His glucose medium, there was no significant difference in passage time between pCRE4-synV and the control group. When 1 μM estradiol was added to the glucose medium, the generation time of pCRE4-synV was prolonged compared with the control group, which indicated that the pGAL1 promoter a...

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Abstract

The invention relates to the field of biotechnology, and particularly discloses a genetic element for accurately controlling gene rearrangement and recombinant plasmid and application thereof. The gene element is formed by sequentially splicing a pGAL1 promoter, a Cre-EBD fusion protein gene and a terminator. According to the the gene element, the pGAL1 promoter, the Cre-EBD fusion protein gene and the terminator are combined to form a 'switch' of the SCRaMbLE-based gene rearrangement, and the gene element can control the expression of Cre, accurately control the opening and closing of the gene rearrangement, and avoid a leakage expression problem. The gene element can be applied to the subsequent construction of the recombinant plasmid and recombinant yeast strains and screening of dominant phenotypic yeast strains.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a gene element for precisely controlling gene rearrangement, its recombinant plasmid and its application. Background technique [0002] In recent years, with the continuous improvement of genome sequencing capabilities, it has been possible to resolve ubiquitous genome rearrangements that were difficult to find in the past. The main forms of genomic variation discovered in the 20th century are single nucleotide polymorphism (SNP) and heterochromatin polymorphism (HP). However, in recent years, studies have found that genome rearrangements are commonly found in microorganisms (such as Yersinia, yeast), plants (such as grass, rice), animals (such as lampreys, mice), and human genomes , in particular, a large number of structural variations (SVs) in the genome (including fragment deletions, duplications, horizontal migration, reciprocal translocations, inversions, etc.)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12Q1/04C12R1/865
CPCC12N15/81C12N2800/30C12N2830/34C12N2830/36C12Q1/04
Inventor 元英进贾斌潘硕吴毅李炳志
Owner TIANJIN UNIV
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