Gene traceless editing carrier and application of gene traceless editing carrier to organism gene editing

A gene and vector technology, applied in the field of invisible gene editing vectors, can solve the problem of lack of invisible gene knockout method, and achieve the effect of safe industrial production

Active Publication Date: 2018-10-19
河北福赛农业科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is a lack of an efficient and traceless gene knockout method

Method used

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  • Gene traceless editing carrier and application of gene traceless editing carrier to organism gene editing
  • Gene traceless editing carrier and application of gene traceless editing carrier to organism gene editing
  • Gene traceless editing carrier and application of gene traceless editing carrier to organism gene editing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] A gene-free editing vector of the present invention is characterized in that: the gene-free editing vector is composed of a genome homology sequence A, a promoter sequence, a cell wall protein CWP1 gene sequence, a resistance screening reporter gene sequence and a genome homology sequence Sequence B consists of;

[0037] Described genome homology sequence A has the nucleotide sequence of SEQ ID No.1;

[0038] Described cell wall protein CWP1 gene sequence has the nucleotide sequence of SEQ ID No.2;

[0039] The genome homologous sequence B has the nucleotide sequence of SEQ ID No.3.

[0040] The cell wall protein CWP1 gene is composed of CWP1 gene or its homologous gene.

[0041] The genome homologous sequence A consists of the upstream sequence I of the target gene and the downstream sequence III of the target gene, and the lengths of the sequence I and the sequence III are 10-100 bp;

[0042] The target gene upstream sequence I has the nucleotide sequence of SEQ ID...

Embodiment 2

[0055] 1. The preparation and transformation of E. coli competent cells, plasmid extraction and restriction endonuclease digestion, DNA fragment recovery, DNA fragment ligation, screening and identification of recombinant plasmids, etc. refer to the relevant chapters of the "Molecular Cloning Experiment Guide" conduct.

[0056] 2. Restriction enzymes, kit reagents and other related materials are all commercially available products.

[0057] like figure 1 As shown, the vector uses the pRS306-Gal-CWP1-13myc plasmid as the starting plasmid and the Saccharomyces cerevisiae ADE8 gene as the knockout gene.

[0058] 1) Design primers to amplify Gal-CWP1 fragment with homology arm sequence and amplify

[0059] F: ACTTGCAGCAAGCGCAGGTGAGAGCCAACACACATCAATAATCTTTCCAAAAGCTCTCGCGTCGTAAATCATGATCATGGATTGTGACAAAACGATCTTAAAGGTTTCGAACCTTCTCTTTGGAACTTTC

[0060] R: ATGTTTCGCGCCTCACTTTGAAGAATGCCAAATATAAAAGTATAAATATGGGAACTATTCAGATTGTACTGAGAGTGCAC

[0061]

[0062] (1) Mix the sample evenly i...

Embodiment 3

[0088] like figure 2 As shown, the vector uses the pFA6A-Gal-CWP1-KanMX-3HA plasmid as the starting plasmid and the Saccharomyces cerevisiae ADE8 gene as the knockout gene.

[0089] 1) Design primers to amplify Gal-CWP1 fragment with homology arm sequence and amplify

[0090] F':

[0091] ACTTGCAGCAAGCGCAGGTGAGAGCCAACACACATCAATAATCTTTCCAAAAGCTGAATAGTTCCCATATTTATACTTTTATATTTGGCATTCTTCAAAGTGAGGCGCGAgacatggaggcccagaatac

[0092] R':

[0093] TTATTTGTGAAGCTGCTGTAAAACCTTATATGTAGCTTCTACAATCGCGATGTGCTCAGCagatCCGCGGTTAACAACAA

[0094] plasmid

[0095]

[0096] The samples were mixed evenly in the plate, and the amplification reaction was carried out. PCR amplification program: 95°C for 5 min; 94°C for 30s, 55°C for 30s, 72°C for 3min, 35 cycles; 72°C for 5min, 12°C∞.

[0097] 2) Lithium acetate chemical transformation method is introduced into the starting strain of Saccharomyces cerevisiae

[0098] The yeast-derived strain was cultured on YPD solid medium, and single clone...

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Abstract

The invention discloses a gene traceless editing carrier and application of the gene traceless editing carrier to organism gene editing. The gene traceless editing carrier consists of a genome homology sequence A, a promoter sequence, a cell wall protein CWP1 gene sequence, a resistance screening report gene sequence and a genome homology sequence B. By aiming at the problems that during the saccharomyces cerevisiae traditional gene knockout, screening markers are remained, the multigene knockout research is inconvenient, and the exogenous gene is remained, the invention provides a saccharomyces cerevisiae efficient gene traceless editing method. The method can be used for saccharomyces cerevisiae gene editing and can also be applicable to other microorganisms of the gene editing principle. The gene traceless editing carrier can be used for studying the function and metabolic mechanism of the yeast gene; no any exogenous gene is remained at an obtained mutant strain; the gene tracelessediting carrier can be safely used for industrial production.

Description

technical field [0001] The invention relates to the field of microbial genetic engineering, in particular to a vector for traceless gene editing and its application. Background technique [0002] As a model fungus, Saccharomyces cerevisiae not only has the characteristics of rapid growth and simple operation of prokaryotes, but also has a gene expression regulation mechanism similar to that of higher eukaryotes. Currently commonly used screening marker genes, such as Cre / Loxp system and 5-FOA, are all non-self exogenous genes, and their production and application are limited. Therefore, it is necessary to develop a gene-free knockout technology suitable for yeast to ensure that no exogenous DNA is introduced, which is convenient for the practical application of engineered bacteria. There are two main methods to remove the selectable marker: one is to use a recombinase-mediated knockout system, such as the Cre / Loxp system, by transforming the knockout element with recombinas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12R1/865
CPCC07K14/395C12N15/81
Inventor 郝志敏曾凡力胡玉笑贾艳荣赵相东董金皋申珅
Owner 河北福赛农业科技有限公司
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