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Cytoskeleton polymerase as well as preparation method and application of cytoskeleton polymerase

A cell scaffold and polymerase technology, applied in the field of molecular biology, can solve the problems of inability to meet the demand of L-lysine, low acid production level and conversion rate of L-lysine, and achieve the improvement of sugar-acid conversion rate, Increase concentration, improve the effect of metabolic processes

Pending Publication Date: 2020-02-04
SHANDONG SHOUGUANG JUNENG GOLDEN CORN CO LTD +3
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Problems solved by technology

[0005] Aiming at the technical problem that the acid production level and conversion rate of L-lysine obtained in the fermentation process are relatively low in the prior art and cannot meet the needs of the existing industry for L-lysine, the technical solution provided by the present invention is:

Method used

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  • Cytoskeleton polymerase as well as preparation method and application of cytoskeleton polymerase

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preparation example Construction

[0029] The embodiment of the present invention provides a method for preparing a cell scaffold polymerase, which is characterized in that it comprises the following steps:

[0030] Using the Escherichia coli gene as a template, perform PCR amplification in a pre-set amplification system to obtain the capsid protein gene, two complementary helical peptide genes and the aspartokinase gene;

[0031] amplifying the capsid protein gene and two complementary helical peptide genes by an overlapping PCR method to obtain a cell scaffold gene;

[0032] The cell scaffold gene and the plasmid vector are digested and ligated to obtain the cell scaffold gene recombination vector;

[0033] The cell scaffold gene recombination vector and the aspartokinase gene are digested and ligated to obtain the cell scaffold polymerase gene recombination vector;

[0034] The cell scaffold polymerase gene recombination vector is transformed into Escherichia coli competent cells by a heat shock method, and...

Embodiment 1

[0047] Amplification primer design: Design the capsid protein PduA gene, two complementary helical peptide CC-Di-A, CC-Di-B genes and aspartokinase LysC in the Escherichia coli genome through primer design principles and various computer programs Gene amplification primers, and through a large number of experiments to screen out the primer sequence with high specificity, wherein the capsid protein PduA gene amplification primers are primer 1PduA-up, SEQ ID No.1 and primer 2PduA-down, SEQ ID No.2; Helical peptide CC-Di-A gene amplification primers are primer 3CC-Di-A-up, SEQ ID No.3 and primer 4CC-Di-A-down, SEQ ID No.4; helical peptide CC-Di-B gene Amplification primers are primer 5CC-Di-B-up, SEQ ID No.5 and primer 6CC-Di-B-down, SEQ ID No.6; aspartokinase LysC gene amplification primer is primer 7LysC-up, SEQ ID No.7 and primer 8LysC-down, SEQ ID No.8, see Table 1 for details.

[0048] Table 1 Amplification Primer Nucleotide Sequence

[0049] Primer name Primer...

Embodiment 2

[0055] The cell scaffold gene CC-Di-A-PduA-CC-Di-B obtained in Example 1 and the Escherichia coli expression plasmid PET-24a were double-digested with BamHI and ScaI restriction endonucleases respectively, and the enzyme digestion system was: 2.5 μl of 10x digestion buffer, 1 μl of each restriction enzyme, 16 μl of plasmid / scaffold gene, ddH 2 O4.5 μl, heated in a water bath at 37°C for 30 minutes, recovered by enzymatic digestion, and stored at -20°C.

[0056] The cell scaffold gene CC-Di-A-PduA-CC-Di-B and the expression plasmid PET-24a recovered from the above enzyme-digested gel were connected to the expression vector and the target fragment using T4 DNA ligase. The connection system was: double enzyme digestion Rear Fragment C-Di-A-PduA-CC-Di-B 5 μl, double digested plasmid PET-24a 3 μl, T4 DNA Ligase (DNA ligase) 1 μL, 10xT4 DNA Ligase Buffer (DNA ligation buffer) 1 μL, at room temperature The ligation reaction was carried out for 10 minutes under the condition, and the...

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Abstract

The invention relates to the field of molecular biology, in particular to cytoskeleton polymerase as well as a preparation method and application of the cytoskeleton polymerase. The preparation methodof the cytoskeleton polymerase comprises the steps as follows: performing PCR amplification with an Escherichia coli gene as a template to obtain a capsid protein gene, helical peptide genes and an aspartokinase gene; obtaining a cytoskeleton gene by an overlap PCR method; carrying out two times of digestion and linking reactions to obtain a cytoskeleton polymerase gene recombinant vector; transforming the recombinant vector into Escherichia coli competent cells to obtain Escherichia coli engineering bacteria; and culturing the engineering bacteria to extract the cytoskeleton polymerase. A target gene is transformed into Escherichia coli by gene modification, an amplification technology, a heat shock method and other methods, and the obtained cytoskeleton polymerase can fill whole cytoplasm, improve the metabolic process of the Escherichia coli, significantly increase the concentration of lysine in a product and raise saccharic acid conversion rate.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a cell scaffold aggregation enzyme and its preparation method and application. Background technique [0002] Lysine is one of the essential amino acids for the human body. It can promote human development, enhance immune function, and improve the function of central nervous tissue. It is widely used in feed additives, food fortifiers and pharmaceuticals. [0003] In the prior art, there are four main production methods of L-lysine, which are extraction method, chemical synthesis method, enzymatic method and microbial fermentation method. The first three methods are due to the disadvantages of high precursor cost and complicated process. It is difficult to achieve the purpose of industrial production, and the biological fermentation method is the current main production method for preparing L-lysine. At present, the common strains used to produce L-lysine include Escherichia coli...

Claims

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Application Information

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IPC IPC(8): C12N9/12C07K19/00C12N15/70C12P13/08C12R1/19
CPCC07K14/245C07K2319/00C12N9/1217C12N15/70C12P13/08C12Y207/02004
Inventor 王志强刘强吕鑫杨秋霞李刚伦学宁吴泽华孙敬善
Owner SHANDONG SHOUGUANG JUNENG GOLDEN CORN CO LTD
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