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Method and kit for in-situ construction of gene mutation library

A library, in situ technology, applied in the fields of molecular biology, protein engineering, and genetic engineering, can solve the problems of lack of versatility, limitation of mutant gene size, and complicated steps, so as to facilitate high-throughput screening and plate screening. , the effect of convenient operation

Inactive Publication Date: 2009-09-16
SHINE E BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The large primer amplification method has improved the efficiency of constructing the gene mutation library, but there are still many deficiencies, for example, the large primers used are the mutant populations of specific genes, which are not universal; only when the large primer size Only 500-1000bp can obtain good amplification effect, and the size of the mutant gene is limited; the amplified by large primers is linear DNA, and it is difficult to form a circular plasmid with two gaps during annealing, and the transformation efficiency is low; large primers induce The modified method also needs to digest the PCR product and digest the plasmid template, the steps are cumbersome

Method used

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  • Method and kit for in-situ construction of gene mutation library

Examples

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Effect test

Embodiment 1

[0040] Embodiment 1: Construction and screening of a mutant library of xylanase gene of Thermomyces lanuginosus:

[0041] (1) Synthesize the complete gene encoding the mature peptide according to the thermomyces lanuginosus xylanase gene sequence (GenBank: U35436), and insert it into the expression vector pHsh-amp with the ampicillin resistance gene to construct recombinant expression Plasmid pHsh-xynA, for specific methods, refer to Yin Erkang et al. (World J Microbiol Biotechnol, 2008, 24: 275-280).

[0042] (2) Using pHsh-xynA as a template and using a linear carrier fragment with a kanamycin resistance gene as a primer, prepare the following PCR reaction system (20 μl):

[0043] Linear vector fragment 1μl

[0044] Thermostable DNA Ligase 1 μl

[0045] Plasmid template (pHsh-xynA) 1μl (30ng / μl)

[0046] 10mM MnCl 2 0.8μl

[0047] 10×PCR buffer 2μl

[0048] 10mM NAD + (Sigma company) 1μl

[0049] wxya 2 O 12.8 μl

[0050] Taq DNA polymerase ...

Embodiment 2

[0053] Example 2: Construction and screening of thermotoga maritima cellulase gene celB mutant library

[0054] The kit consists of 8 reagents: thermostable ligase, 2 linear vector fragments, expression vector pHsh, Taq DNA polymerase, 10mM MnCl 2 , 10mM NAD + , 10×PCR buffer (containing MgCl 2 and dNTPs).

[0055] The method for constructing the Thermotoga maritima cellulase CelB gene mutation library using the above kit is as follows:

[0056] (1) Design primers (5'-GGGCT CGAGT TATTT TACAA CTTCG ACAG-3' and 5'-CTAGC GTTGG TGCAACGGAC-3') according to the gene sequence of Thermotoga maritima cellulase CelB (GenBank Accession No.NC_000853), Using the Thermotoga maritima genomic DNA as a template, all the coding genes except the signal peptide were amplified, and the gene was inserted into the expression vector pHsh-kan with kanamycin resistance gene according to the standard molecular cloning method to construct a recombinant Expression plasmid pHsh-celB.

[0057] (2) Usin...

Embodiment 3

[0069] Example 3: Level 2 Random Mutation and Screening of Thermotoga maritima Cellulase Gene celB

[0070] Using the plasmid pHsh-celB-m1 containing the first-grade positive mutation gene obtained in Example 2 as a template, repeat steps (2) to (5) in Example 2, except that the step (2) is replaced with The linear plasmid fragment with the kanamycin resistance gene is used as a primer; the culture medium plate in step (4) is replaced with an LB plate containing kanamycin instead of ampicillin. After the positive mutation was confirmed, it was named pHsh-celB-m2.

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Abstract

The invention provides a novel method for quick and universal in-situ construction of a gene mutation library for directed gene evolution, and a kit for constructing the gene mutation library by the method. The method is characterized in that: (1) thermostable DNA ligase mediates the amplification of an annular plasmid during PCR; (2) the in-situ error-prone PCR is performed on target genes or partial target genes in a small expression vector; (3) random mutant genes and an original template are separated by different screening markers; and (4) a set of reagents are suitable for various target genes, and are convenient for screened positive mutant genes to re-construct the mutation library, and the positive mutation with multi-stage accumulation is acquired by screening. The EvoGen Kit provides various reagents needed by constructing the gene mutation library by the method.

Description

technical field [0001] The invention relates to the fields of protein engineering, molecular biology technology and genetic engineering, in particular to a method for constructing a gene mutation library in situ, and a kit for constructing one to multi-level mutation libraries using the method. Background technique [0002] Directed evolution in vitro not only provides a powerful method for studying the substrate specificity of enzymes, determining the catalytic active sites of enzymes, and improving the thermal stability of enzymes, but also helps to understand the relationship between protein structure and function. The directed evolution strategy is mainly to simulate the natural evolution process in vitro and quickly and efficiently establish a random mutation gene library, which has become an important measure for protein engineering research. [0003] Error-prone PCR is a basic method for constructing a gene mutation library, the principle of which is to adjust the Mg ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12N15/70C12R1/19
CPCC40B40/08C40B50/06C12N15/102C12N15/10C12N15/1093C12N15/64
Inventor 邵蔚蓝乐易林裴建军
Owner SHINE E BIOTECH CO LTD
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