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DNA (deoxyribonucleic acid) molecule, recombinant plasmid and escherichia coli

A DNA molecule, Escherichia coli technology, applied in the biological field, can solve the problems of inability to tolerate L-threonine, low protein content, and low acid production capacity of Escherichia coli

Active Publication Date: 2014-12-10
新疆梅花氨基酸有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although wild-type E. coli has a gene expressing L-threonine tolerance protein, the amount of protein expressed by this gene is low, which is only required for the normal growth of E. coli, and cannot tolerate L-threonine at a concentration greater than 0.1M. Threonine, making wild-type E. coli less acid-producing

Method used

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  • DNA (deoxyribonucleic acid) molecule, recombinant plasmid and escherichia coli
  • DNA (deoxyribonucleic acid) molecule, recombinant plasmid and escherichia coli
  • DNA (deoxyribonucleic acid) molecule, recombinant plasmid and escherichia coli

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1: Optimization and synthesis of DNA molecule of the present invention

[0032] The present invention obtains a protein sequence from Yersinia enterocolitica subsp.enterocolitica8081 from the NCBI database, its amino acid sequence is shown in SEQ ID NO: 4, and the NCBI protein number is Gi: 123440599. The present invention optimizes codons according to the codon preference of Escherichia coli W3110, synthesizes a DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 1 through a nucleic acid synthesizer, verifies the correctness of the sequence by sequencing, and synthesizes a gene enzyme cut site Points are SmaI / HindIII. Using SmaI / HindIII as the insertion site, it was cloned into the high-copy plasmid vector pUC57, and Escherichia coli DH5α was used as the recipient strain for amplification.

[0033] The optimization and synthesis work were completed by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

Embodiment 2

[0034] Example 2: Construction of recombinant plasmid pKR-E

[0035] Extract the pUC57 plasmid from the above-mentioned Escherichia coli DH5α, digest with SmaI / HindIII, 50 μl system, T buffer+BSA, perform 1% agarose gel electrophoresis on the digested product, take off the gel at a suitable time, and cut the gel to recover the target gene fragment Take 3 μl and mix it with 1 μl of pKK223-3 plasmid that has been cut with SmaI / HindIII, and add 4 μl of high-efficiency ligase to make 8 μl of ligation system. Ligate overnight at 16°C, transform the prepared Escherichia coli Trans T1 competent cells the next day, screen with ampicillin at a final concentration of 100 μg / ml on an LB plate, pick a single colony after 20 hours of cultivation, and verify the plasmid by double digestion with HindIII and BamHI. See image 3 . The results show that the DNA molecule has been constructed to pKK223-3 to form a new recombinant plasmid pKR-E, see the plasmid map figure 1 .

[0036] Depend o...

Embodiment 3

[0037] Example 3: Construction of recombinant plasmid pKTR-E

[0038] Extract the plasmid pKR-E in Escherichia coli in Example 2 as a template, and use the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3 in the sequence listing to amplify the target gene fragment, which has a section on pKTR-E Tac promoter sequence, rrnB T1 Terminator, rrnB Terminator terminator sequence derived from basic plasmid pKK223-3. BamHI / SphI digested amplified product, took 3 μl and mixed it with 1 μl of pKK223-3-thrA’BC plasmid that had also been double-digested with BamHI / SphI, and added 4 μl of high-efficiency ligase to make 8 μl of ligation system. Ligate overnight at 16°C, transform the prepared Escherichia coli Trans T1 competent cells the next day, screen with ampicillin at a final concentration of 100 μg / ml on an LB plate, pick a single colony after 20 hours of cultivation, and extract the plasmid for HindIII digestion verification, see image 3 . The results show that the fragment has been ...

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Abstract

The invention relates to the technical field of organisms, and discloses a DNA (deoxyribonucleic acid) molecule of a nucleotide sequence shown in SEQ ID NO:1, a recombinant plasmid pKR-E obtained by plasmid pKK223-3 and the DNA molecule inserted between the SmaI and HindI II enzyme cutting sites, and a recombinant plasmid pKTR-E obtained by plasmid pKK223-3-ThrABC and a segment composed of a Tac promoter, the DNA molecule, rrnB T1 Terminator and rrnB Terminator inserted between the BamHI and SphI enzyme cutting sites. The recombinant plasmid disclosed by the invention is converted into W3110 escherichia coli to obtain two strains with high L-threonine tolerance activity, and can be applied to fermentation of L-threonine.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a DNA molecule, a recombinant plasmid and Escherichia coli. Background technique [0002] L-threonine is one of the 8 kinds of amino acids necessary for the human body. It is widely used in medicine, food, feed, etc. At present, L-threonine is mainly produced by microbial fermentation. A variety of bacteria can be used for L-threonine Production, such as Corynebacterium glutamicum, Escherichia coli, etc. Escherichia coli has gradually become a commonly used strain for L-threonine production due to its advantages of rapid reproduction, high fermentation temperature, and in-depth physiological and biochemical research. [0003] With the continuous increase of the world's demand for L-threonine, more and more attention has been paid to the research on strains with high L-threonine production. Among them, the most critical factor affecting the high yield of Escherichia coli is its abil...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12N15/70C12N15/66C12N1/21C12P13/08C12R1/19C12R1/01
Inventor 李岩宫卫波王秋岩胡炎华胡征宇
Owner 新疆梅花氨基酸有限责任公司
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