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Gene directed molecular evolution system in vitro based on half-rational evolutionary design

An out-directed, gene-based technology, applied in organic chemistry, sugar derivatives, etc., can solve the problems of unclear genes, difficulties in gene expression and analysis, and lack of clarity of key sites.

Inactive Publication Date: 2008-11-26
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it's unclear whether it's better to mutate the entire gene, or just the key sites
Moreover, the protein structure of most gene expression products has not yet been elucidated, and the key sites have not yet been clarified
In addition, most gene expression and analysis are still difficult

Method used

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  • Gene directed molecular evolution system in vitro based on half-rational evolutionary design
  • Gene directed molecular evolution system in vitro based on half-rational evolutionary design
  • Gene directed molecular evolution system in vitro based on half-rational evolutionary design

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: PCR amplification β-galactosidase gene (HLacZ)

[0053] (1) Test method:

[0054] 1. Synthesis of oligonucleotide primers

[0055] 1) According to the nucleotide sequence ( figure 1 ), design two oligonucleotide primers, respectively numbered as Hlacz-sh-S1 and Hlacz-sh-S2 primers. In order to facilitate cloning and construction, BamHI and SacI restriction site sequences were added to the 5' ends of the primers, respectively. For the designed primer sequences, see Figure 5 :

[0056] 2) Primer synthesis was completed by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0057] 3) Purify by PAGE referring to the molecular cloning literature method.

[0058] 2. PCR amplification reaction:

[0059] HLacZ gene template: 1μl; Hlacz-sh-S1: 0.2ng; Hlacz-sh-S2: 0.2ng; 10×PCRbuffer: 5μl; dNTP (2.5mmol / L): 4μl; Taq polymerase: 2U; add ddH 2 O to a final volume of 50 μl.

[0060] The reaction program was 94°C, 10min pre-denaturation, 94°C denatur...

Embodiment 2

[0066] Embodiment 2: Construction of β-galactosidase gene (HLacZ) mutant library

[0067] (1) Test method:

[0068] 1. DNase I degradation and recovery of β-galactosidase gene (HLacZ) DNA fragment

[0069] 1) DNase I degradation:

[0070] Dissolve the β-galactosidase gene (HLacZ) DNA fragment recovered in Example 1 with 100 μl DNase I buffer, add 0.1 U DNase I, treat at 25° C. for 15 minutes, and then treat at 70° C. for 10 minutes (inactivation of DNase). The degradation products were identified by 10% acrylamide gel electrophoresis. In this step, the composition of DNase I buffer is: 50mmol / L Tris-C1pH7.4+1mmol / LMgCl 2 .

[0071] 2) Recovery of degradation products:

[0072] A small fragment of 50-100bp was recovered by the dialysis bag method. The precipitate was dissolved with 10 μl of 10× primer-free PCR buffer (50 mmol / L KCl+10 mmol / LTris-Cl pH9.0+1% Triton).

[0073] 2. Primer-free PCR amplification of degradation products:

[0074] reaction system:

[0075] De...

Embodiment 3

[0085] Embodiment 3: Construction and screening of the Escherichia coli transformant of the mutant library containing β-galactosidase gene (HLacZ) DNA fragment

[0086] 1. Construction of mutant library E. coli plasmid

[0087] According to the basic operation of Sambrook molecular biology (Sambrook J, Frets E F, Mannsdes Tet al. In: Molecular Cloning. 2nd ed. Cold Spring Harbor Laboratory Press, 1989.), the expression vector pYPX251 was first digested with BamHI and SacI, and connected with T4DNA The enzyme ligation connects the β-galactosidase gene (HLacZ) DNA fragment, and the obtained connection product is the Escherichia coli expression plasmid containing the mutant library of the β-galactosidase gene (HLacZ) DNA fragment.

[0088] 2. Construction of mutant library transformants

[0089] The constructed Escherichia coli expression plasmid containing the mutant library of the β-galactosidase gene (HLacZ) DNA fragment was transformed into Escherichia coli strain DH5α by el...

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Abstract

The invention relates to a simple and efficient gene in vitro directed evolution system based on half-reasoning design; the system can achieve larger mutation potential and more definite mutation region compared with the conventional reshuffle mutation by combining DNA reshuffle mutation and the half-reasoning design and can provide more diversified mutant population for the modification of gene and protein, wherein, the mutant sites of the diversified mutant population are positioned in critical area.. With the combination of a screening system, reliable improved gene or protein can be obtained more easily; moreover, the system of the invention has effectiveness as well as simple and convenient operation.

Description

technical field [0001] The invention relates to a gene in vitro directed evolution system, in particular to gene synthesis modification, semi-reasonable design and gene in vitro directed evolution. Background technique [0002] Directed evolution in vitro is an important method for discovering and modifying biologically active molecules. In vitro directed molecular evolution provides an efficient method to obtain diversity. DNA shuffling (DNA shuffling) is an important in vitro molecular evolution technology, combined with high-throughput screening, it can transform many important commercial enzymes such as medicine, industry, and environmental protection. DNA shuffling is an in vitro directed molecular evolution technique that combines with PCR technology to cause gene changes through homologous recombination and mutation of random fragments. The mature conventional technical route of DNA shuffling is that the gene is treated with DNase to become a random short fragment, ...

Claims

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Application Information

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IPC IPC(8): C07H21/00
Inventor 姚泉洪熊爱生彭日荷
Owner SHANGHAI ACAD OF AGRI SCI
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