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
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[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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