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Construction method of plasmid taking yeast G418 as selective marker

A construction method, selective technology, applied in the field of microbial genetic engineering

Inactive Publication Date: 2010-04-21
KUNMING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report on the plasmid construction method using G418 as a selectable marker in the prior art, and the use of this method to solve the problem that the current total nutritional yeast must undergo auxotrophic mutagenesis before it can be used for genetic engineering operations

Method used

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  • Construction method of plasmid taking yeast G418 as selective marker

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Experimental program
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Embodiment 1

[0022] (1) Cloning of kanR gene sequence:

[0023] Using the plasmid pPIC9K as a template, the knaR gene sequence was cloned by PCR amplification. Amplify the upstream primer sequence as 5′-CATG CCATGG TCTGCCAGTGTTACAACC AAT-3′, amplified downstream primer sequence is 5′-CTA GCTAGC ATGCGACTCCTGCATTAGGAAG-3'. The PCR reaction system is 50 μL, consisting of 10 μL 10×E x -Taq buffer, 0.6μL E x - Composition of Taq DNA polymerase (5U / μL), 2.6 μL 10mM dNTP, 0.5 μL upstream primer (10 pmol / μL), 0.5 μL downstream primer (10 pmol / μL), 0.2 μL pPIC9K (100 ng / μL) and 35.6 μL sterile water . PCR reaction conditions: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 1 min, annealing at 52°C for 0.5 min, extension at 72°C for 1.5 min, a total of 30 cycles, and extension at 72°C for 10 min.

[0024] The 10×E used x -Taq buffer contains Mg 2+ .

[0025] The recognition site of Nhe I introduced by the upstream primer is the underlined part, and the recognition site of Nco...

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Abstract

The invention relates to a construction method of a shuttle plasmid taking yeast G418 as a selective marker, which solves the problem that the present yeast plasmids typically take auxotroph as the selective mark and are difficult to be used in the gene engineering reconstruction of full nutritious yeasts. The construction method comprises the following steps: cloning kanR genes, recycling and purifying gene segments, and connecting with pYES2 carriers. A carrier constructed by using the method enables Saccharomyces cerevisiae which is originally sensitive to G418 to become resistant to G418,so that the gene operation of full nutritious yeasts can be conducted without auxotroph mutagenesis, thereby reducing the gene reconstruction time.

Description

technical field [0001] The invention belongs to the technical field of microbial genetic engineering, and in particular relates to a method for constructing a plasmid in which yeast G418 is a selectable marker. Background technique: [0002] In genetic engineering, the commonly used yeast screening method is to use the auxotrophy of the yeast strain as a screening marker, which requires that the host bacteria itself must be an auxotrophic strain, which limits the scope of use of the recipient bacteria in the study of yeast genetic engineering operations . Mutagenesis is currently a commonly used method for using a complete vegetative yeast for genetic engineering operations. However, mutagenesis screening is a tedious and lengthy process, and the probability of reverse mutation is high, requiring selective pressure to stabilize its traits. At the same time, for industrial strains used in actual production, adding essential nutrients will also increase production costs. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12R1/865
Inventor 魏云林谢莹莹季秀玲井申荣林连兵
Owner KUNMING UNIV OF SCI & TECH
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