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Lysine decarboxylase mutant library construction method and high-throughput screening method of high-activity or low-activity mutant strains

A technology of lysine decarboxylase and construction method, which can be used in microorganism-based methods, chemical libraries, biochemical equipment and methods, etc., and can solve problems such as difficult amplification

Pending Publication Date: 2018-11-13
CATHAY R&D CENT CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the conventional error-prone PCR based on taq enzyme also has its shortcomings, mainly because it is suitable for gene fragments with a length of about 1000bp, and it is difficult to amplify genes with a length of more than 2000bp.

Method used

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  • Lysine decarboxylase mutant library construction method and high-throughput screening method of high-activity or low-activity mutant strains
  • Lysine decarboxylase mutant library construction method and high-throughput screening method of high-activity or low-activity mutant strains
  • Lysine decarboxylase mutant library construction method and high-throughput screening method of high-activity or low-activity mutant strains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1. Construction of Escherichia coli Lysine Decarboxylase CadA Expression Vector

[0057] (1) Amplification of CadA gene

[0058] Escherichia coli KG1665 (Beijing Tianenze Biotechnology Co., Ltd.) was cultured overnight in LB medium (37°C, 220rpm), and 4ml of bacterial solution was taken the next day, and the genome extraction kit (Axygen, AP-MN-P-250G) ) Extract its genome. The primers CadAF 5'-GGAATTCCATATGATGAACGTTATTGCA ATAT-3' (SEQ ID NO:1) and CadAR 5'-CCCAAGCTTCCTTGTACGAGCT AATTAT-3' (SEQ ID NO: 2) designed according to the gene sequence of E. coli lysine decarboxylase CadA were used to Genome (GenBank: U00096.3) was used as a template to amplify the CadA gene fragment. Design NdeI and HindIII restriction sites on the amplification primers. The amplification method is a conventional PCR method, and the DNA polymerase used is from Takara Bioengineering (Dalian) Co., Ltd. (TAKARA) Max DNA polymerase.

[0059] The amplification system is as follows:

[0060] ...

Embodiment 2

[0071] Example 2. Induced expression of CadA

[0072] (1) Transform the plasmid pET30CA into E. coli BL21 (DE3) to obtain the strain BLpET30CA. The empty plasmid pET30 was transformed into BL21(DE3) to obtain the control strain BLpET30.

[0073] (2) Inoculate control BLpET30 and BLpET30CA into 5ml test tubes of LB liquid medium (containing kanamycin 50mg / ml), inoculate two tubes each, and cultivate overnight in a shaker at 37°C and 220rpm.

[0074] (3) Transfer the bacterial solution from step (2) overnight culture to a new 5ml liquid LB medium (containing kanamycin 50mg / ml) at a transfer volume of 2% (v / v), and continue at 37 Cultivate in a shaker at 220 rpm.

[0075] (4) Cultivate to OD 600 When it reached 0.6, IPTG was added to induce expression, and the final concentration of IPTG was 0.1 mM. Continue to incubate in a shaker at 37°C and 220 rpm.

[0076] (5) Collect 1ml of bacterial solution and centrifuge after 6 hours of inducing expression. Collect the supernatant and bacteri...

Embodiment 3

[0080] Example 3. Construction of mutant library

[0081] To build a library of CadA segmented mutations, according to the length, the 800-1200bp after the start codon of CadA ATG is divided into the "upper half", and the 800-1200bp before the stop codon TAA of CadA is divided into the "lower half" Segment"; According to function, the plp binding region (552-1241bp) in CadA is divided into "plp segment". Design mutant primers UF 5'-TGTTTAACTTTAAGAAGGAGA-3' (SEQ ID NO: 3), UR 5'-TCTTCGTTTACGTCACCT-3' (SEQ ID NO: 4) according to the gene sequence information of the upper, lower and plp segments; DF 5'- CCAACTTCTCACCGATTTA-3' (SEQ ID NO: 5), DR 5'- TCAAGACCCGTTTAGA GGC-3' (SEQ ID NO: 6); plpF 5'-GTAGCCTGTTCTATGATTTCTT-3' (SEQ ID NO: 7), plpR 5'-TTACCT GCATTGCCTTTC-3' (SEQ ID NO: 8). Add the following reagents to the PCR tube: 5ul of 10×rTaq Buffer (Takara), 8ul of dNTPx (2.5mM dATP, 1.25mM dTTP, 1.25mM dCTP, 2.5mM dGTP), 1.5ul UF or DF or plpF, 1.5ul UR or DR or plpR (UR with UF,...

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Abstract

The invention relates to a lysine decarboxylase mutant library construction method and a high-throughput screening method of high-activity or low-activity mutant strains, in particular to an error-prone PCR orthogenesis mutant library construction method applicable to genes with the length being higher than 2000bp such as the lysine decarboxylase and the high-throughput screening method of the high-activity or low-activity mutant strains. The lysine decarboxylase mutant library construction method and the high-throughput screening method have the advantages that the method using error-prone PCR subsection mutation, PCR integration and DpnI enzymatic digestion and using series of pH indicators to indicate pH change is adopted, the difficulties that a conventional error-prone PCR method cannot effectively build a long-fragment-gene mutant library and is low in connecting efficiency, and the pH change of the method exceeds the indicating range of indicators are solved, and the fast and simple method is provided to screen enzyme-activity-increased or enzyme-activity-lowered mutant strains from a lysine decarboxylase mutant library.

Description

Technical field [0001] The present invention relates to methods for constructing biological enzyme mutant libraries and high-throughput screening of high or low activity mutant strains, and specifically relates to error-prone PCR directed evolution mutant libraries suitable for genes longer than 2000bp, such as lysine decarboxylase genes Construction methods and methods for high-throughput screening of high or low vigor mutant strains. Background technique [0002] Pentylenediamine (1,5-pentanediamine), also known as cadaverine, is named after it was first isolated from a corrupted body. Because of its wide application in agriculture, medicine and industry, it has attracted more and more attention. In industry, it can be polymerized with dibasic acid to produce a new type of nylon-a high-quality polymer material, which is used to replace nylon 6.6, which is highly dependent on petroleum. [0003] Pentylenediamine can be prepared by biological methods. There are two main methods. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12Q1/04C12R1/19
CPCC12N15/1065C12Q1/04C40B50/06G01N2333/245
Inventor 陆文强周豪宏刘修才
Owner CATHAY R&D CENT CO LTD
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