Plasmid pcdsp for increasing the content of squalene in Escherichia coli and its preparation and use
A technology of Escherichia coli and squalene, applied in biochemical equipment and methods, chemical instruments and methods, microorganism-based methods, etc., can solve the problems of low content, low fermentation density, limiting the final output of squalene, etc. The effect of increasing accumulation, increasing storage space, and increasing expression
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Embodiment 1
[0033] A plasmid pCDSP for increasing the squalene content in Escherichia coli comprises a vector pCDFDuet-1, and the vector pCDFDuet-1 is inserted with a protein expression hindering gene lacI, a replication origin of Escherichia coli CDF ori, a streptomycin resistance gene and Scp-2 protein gene scp2, the upstream of Scp-2 protein gene scp2 has a PT7 promoter to start the expression of Scp-2 protein gene scp2, the nucleotide sequence of plasmid pCDSP is shown in sequence 1, the nucleus of Scp-2 protein gene scp2 The nucleotide sequence is shown in SEQ ID NO:2.
Embodiment 2
[0035] A preparation method of the above-mentioned plasmid pCDSP is specifically implemented according to the following steps:
[0036] Step 1, according to the amino acid sequence of the rat Scp-2 protein, and according to the codon preference of E. coli to design the Scp-2 protein gene scp2 suitable for E. coli expression, then insert the Scp-2 protein gene scp2 into the vector pUC57 to obtain a plasmid pUCSP, the nucleotide sequence of Scp-2 protein gene scp2 is shown in sequence 2;
[0037] Step 2, the Scp-2 protein gene scp2 in the plasmid pUCSP is inserted into the vector pCDFDuet-1 through enzyme digestion and ligation reaction to form the plasmid pCDSP, specifically:
[0038] First, the plasmid pUCSP and the vector pCDFDuet-1 were double digested with NcoI and EcoRI restriction enzymes respectively, and then the scp-2 protein gene scp2 fragment excised from the plasmid pUCSP and the linearized scp2 fragment were recovered by gel electrophoresis. vector pCDFDuet-1, and...
Embodiment 3
[0041] A method of utilizing the above-mentioned plasmid pCDSP to improve the content of squalene in Escherichia coli is implemented according to the following steps:
[0042] In step 1, the wild strain E.coli C43 (DE3) was first prepared into calcium chloride competent cells, and then the plasmid pTsqs was transformed by the heat shock method. Finally, the recombinant strain E.coli CSQ1 was obtained by ampicillin resistance screening and colony PCR verification. , the nucleotide sequence of plasmid pTsqs is shown in sequence 3;
[0043] Step 2, transform plasmid pCDSP by heat shock method, and obtain recombinant strain E.coli CSSP1 by streptomycin resistance screening and colony PCR verification;
[0044] Step 3, analyze the content of squalene in the recombinant bacteria E.coli CSSP1, specifically:
[0045] First, E.coli CSSP1 was cultured in LB liquid medium, and then IPTG was added to induce the expression of Scp-2 protein gene scp2; then, the synthesized squalene was sep...
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