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Method of carrying out rapid and efficient DNA combination and evolution based on synthesis of single-chain DNA library

A single-chain, high-efficiency technology that can be used in the field of genetic engineering to solve the problem of wasting cell synthesis capacity and the burden of cell growth.

Inactive Publication Date: 2015-07-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because, if only the expression level is enhanced, it may lead to the waste of cell synthesis capacity and the burden of cell growth, but cannot fundamentally solve the problem of metabolic pathway flow balance

Method used

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  • Method of carrying out rapid and efficient DNA combination and evolution based on synthesis of single-chain DNA library
  • Method of carrying out rapid and efficient DNA combination and evolution based on synthesis of single-chain DNA library
  • Method of carrying out rapid and efficient DNA combination and evolution based on synthesis of single-chain DNA library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1 RECODE technology two-round PCR reaction system prepares combinatorial mutation library

[0050] A. Preparation of double-stranded mutant library after purification of single mutant library

[0051] Take the genes of β-galactosidase and esterase lacZ-Esterase as an example to carry out combined lethal mutations, and the nucleotide sequence is shown in SEQ ID NO.1. In this example, galactosidase and esterase genes are used for combined lethal mutation, and the diversity of the library can be indicated by plate screening. Substrates 0.1 mM IPTG, 20 μg / ml X-Gal and 1% Tris Glyceryl Butyrate. The two genes were recombined into the pMD19 vector and transformed into the JM109 host. On the screening plate, the parental gene host such as image 3 As shown, the colony is blue (lacZ degrades X-gal), and produces a hydrolysis transparent circle (Esterase hydrolyzes tributyrin).

[0052] β-galactosidase (with promoter) downstream tandem esterase gene, carry out comb...

Embodiment 2

[0066] Embodiment 2 RECODE technology one-step PCR reaction system prepares combinatorial mutation library

[0067] In this example, in order to further simplify the RECODE technique, the genes of β-galactosidase and esterase lacZ-Esterase in Example 1 were also used as an example to carry out combined lethal mutations. Editing primers JPS-MD / LacZ-P1F, JPS-MD / Est-P2F, JPS-MD / Est-P3F and downstream anchor primer JPS-LZE / DTA were mixed in equimolar ratio, and phosphorylated by T4 polynucleotide kinase For chemical reaction, the reaction system was operated according to the product instructions, reacted at 37°C for 30 minutes, and inactivated the enzyme at 70°C for 10 minutes. One-step reaction to prepare a double-stranded mutant library is a simultaneous reaction of PCR cycles for DNA extension, ligation, and amplification of mutant strands.

[0068] In a 50 μl reaction system: 5 μl 10x reaction buffer, upstream anchor primer JPS-LZE / UTA, phosphorylated downstream anchor primer...

Embodiment 3

[0071] Example 3 In vitro transformation of the constitutive promoter of the rpoS gene of Escherichia coli

[0072] Taking the constitutive promoter of the rpoS gene of Escherichia coli as an example, the nucleotide sequence is shown in SEQ ID NO.2, and the four internal spacer sequences in the -10 and -35 intervals were combined and mutated simultaneously to construct a mutant library. Four editing primers were designed according to the sequence of the promoter segment, editing primers (Jps / rpoSp-F, Jps / rpoSp4-F, Jps / rpoSp3-F, Jps / rpoSp21-F), anchor primers (Jps / rpoSp-HM -F, Jps / rpoSp-HM-R) outer primers (rpoSp-F, rpoSp-R) and vector preparation primers are as follows:

[0073] Jps / rpoSp-F:TTCCACCGTTGCTGTTGCGTNNNNNNNNNNNNNNNNTATTCTGAGTCTTCGGGTGAAC

[0074] Jps / rpoSp4-F:CATAACGACACAATGCTGGTNNNNNNNNNNNNNAAGTTAAGGCGGGGCAAAAAATAGC

[0075] Jps / rpoSp3-F:TAGCACCGGAACCAGTTCAANNNNNNNNNNNNNNNNAATTCGTTACAAGGGGAAATCCG

[0076] Jps / rpoSp21-F:GCAGCGATAAATCGGCGGAACNNNNNNNNNNNNNNNNNTGNTC...

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Abstract

The invention discloses a method of carrying out rapid and efficient DNA combination and evolution based on synthesis of single-chain DNA library and belongs to the technical field of genetic engineering. The method comprises the following steps: constructing DNA single-chain mutant library and double-chain mutant library sequentially or simultaneously; and after an expression vector of the mutation library is recombined, transforming a host, and high throughput screening evolved mutant strains. According to the method disclosed by the invention, with the adoption of the technology, transformation in vitro is successfully carried out on a promoter, enzyme and metabolic pathway, and the mutant strain with very excellent property is obtained; rapid evolution in vitro of the gene is realized, and the method is simple to operate and is particularly suitable for carrying out rapid oriented evolution for introducing rich mutation diversity in the gene.

Description

technical field [0001] The invention relates to a method for rapidly and efficiently combining and evolving DNA based on a synthetic single-stranded DNA library, especially a method for rapidly and efficiently combining and evolving gene expression regulatory elements, enzymes and metabolic pathways based on a synthetic single-stranded DNA library, belonging to the technical field of genetic engineering . Background technique [0002] With the development of molecular biology technology and genetic engineering technology, thousands of natural enzyme genes have been discovered. Because enzyme-mediated biocatalysis has the advantages of environmental friendliness, high efficiency and specificity, and mild reaction conditions, it is used as a biocatalyst. The leader of catalysis is widely used in the synthesis of complex drugs, intermediates, compounds, and even the synthesis of biofuels. Enzymes are the core participants in synthetic biology and metabolic engineering. By cons...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1058C12N15/1031C12Q2521/101C12Q2521/501C12Q2531/113C12Q2533/101C12Q2563/179
Inventor 康振陈坚金鹏堵国成
Owner JIANGNAN UNIV
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