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Plasmid pTsqs for synthesizing squalene through Escherichia coli as well as preparation method and using method thereof

A technology of Escherichia coli and squalene

Inactive Publication Date: 2018-12-11
XIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Escherichia coli cannot naturally synthesize squalene due to the lack of squalene synthase

Method used

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  • Plasmid pTsqs for synthesizing squalene through Escherichia coli as well as preparation method and using method thereof
  • Plasmid pTsqs for synthesizing squalene through Escherichia coli as well as preparation method and using method thereof
  • Plasmid pTsqs for synthesizing squalene through Escherichia coli as well as preparation method and using method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] A plasmid pTsqs for Escherichia coli to synthesize squalene, including vector pTrc99A, inserted into the vector pTrc99A is the hindered protein expression gene lacIq, the replication initiation site pBR_322ori, the ampicillin resistance gene and the squalene synthase gene ERG9, squalene The upstream of the ene synthase gene ERG9 has a promoter Ptrc, and the downstream of the squalene synthase gene ERG9 contains a multiple cloning site (Multiple cloning site, MCS). The nucleotide sequence of the plasmid pTsqs is shown in sequence 1, The nucleotide sequence of the squalene synthase gene ERG9 is shown in SEQ ID NO:3.

Embodiment 2

[0035] A method for preparing the above-mentioned plasmid pTsqs, specifically implemented according to the following steps:

[0036] Step 1, first select the wild-type Saccharomyces cerevisiae S288C gene ERG9, and optimize the nucleotide sequence of the wild-type Saccharomyces cerevisiae S288C gene ERG9 according to the codon preference of Escherichia coli to obtain the squalene synthase gene ERG9, and then the squalene The synthase gene ERG9 was inserted into the vector pUC57 to obtain the plasmid pUCsqs. The upstream and downstream ends of the squalene synthase gene ERG9 were respectively added with NcoI (CCATGG) restriction site and KpnI (GGGTACC) restriction site. Wild-type Saccharomyces cerevisiae S288C The nucleotide sequence of gene ERG9 is shown in sequence 2, and the nucleotide sequence of squalene synthase gene ERG9 is shown in sequence 3;

[0037] Step 2, the squalene synthase gene ERG9 in pUCsqs is inserted into the vector pTrc99A by enzyme digestion and ligation r...

Embodiment 3

[0041] A kind of method utilizing above-mentioned plasmid pTsqs to make escherichia coli synthesize squalene, specifically implement according to the following steps:

[0042] Step 1, first prepare the wild strain E.coli C43 (DE3) to be competent for calcium chloride, then transform the plasmid pTsqs by heat shock method, and finally use ampicillin resistance screening and colony PCR verification to obtain the recombinant strain E.coli CSQ1;

[0043] Step 2, verify the squalene synthesized in the recombinant bacteria E.coli CSQ1, specifically:

[0044]First, the recombinant E.coli CSQ1 was cultured in LB liquid medium, and the inducer IPTG was added to induce the expression of squalene synthase gene ERG9; then the synthesized squalene was separated and extracted by saponification; finally, it was detected by high performance liquid chromatography The squalene.

[0045] Among them, the DNA sequence of the wild strain E.coli C43(DE3) is the sequence of GenBank number CP011938.1...

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Abstract

The invention discloses a plasmid pTsqs for synthesizing squalene through E.coli (Escherichia Coli). The plasmid pTsqs comprises a carrier pTrc99A, wherein a protein expression inhibition gene lacIq,a replication origin site pBR_322ori, an amicillin resistance gene and a squalene synthase gene ERG9 optimized according to E.coli codon preference are inserted in the carrier pTrc99A; a promoter Ptrcis located in an upstream position of the squalene synthase gene ERG9, and a downstream position of the squalene synthase gene ERG9 comprises one MCS (Multiple Cloning Site). The invention disclosesa preparation method and a using method of the plasmid pTsqs. The plasmid disclosed by the invention is capable of expressing that the E.coli is facilitated to synthesize the squalene by the squalenesynthase, the plasmid comprises the squalene synthase gene ERG9 controlled by the strong promoter Ptrc, and the squalene synthase gene ERG9 is optimized according to the E.coli codon preference. A carrier tool is provided for producing the squalene through fermentation of the E.coli.

Description

technical field [0001] The invention belongs to the field of biogenetic engineering, and in particular relates to a plasmid pTsqs for Escherichia coli to synthesize squalene, a method for preparing the plasmid pTsqs, and a method for using the plasmid pTsqs. Background technique [0002] Squalene is a triterpene compound naturally present in a variety of microorganisms and animal and plant cells. It has many functions such as anti-oxidation, protecting the heart, improving immunity, reducing blood lipids and inhibiting the development of cancer. It has been widely used In the field of medicine and health care products. Currently, the squalene on the market is mainly derived from the extraction of plant seeds and animal livers, so the price is relatively expensive. In addition, the extraction method also has the disadvantages of long cycle, high cost, and environmental damage. Therefore, new replacements are urgently needed. Environmentally friendly and cheaper production me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/54C12P5/02
CPCC12N9/1085C12N15/70C12N2800/22C12P5/026C12Y205/01021
Inventor 徐文姚佳马茜李薇孙晓敬汪洋
Owner XIAN MEDICAL UNIV
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