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Method for producing polyhydroxyalkanoate using engineering strain

A technology of polyhydroxyalkanoate and engineering strains, applied in the fields of genetic engineering and microbial fermentation

Inactive Publication Date: 2009-02-11
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the induction and production of key enzymes in the metabolic process in the current PHA fermentation process, the present invention provides a method for producing polyhydroxyalkanoate (PHA) through environment-induced fermentation using recombinant Escherichia coli

Method used

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  • Method for producing polyhydroxyalkanoate using engineering strain
  • Method for producing polyhydroxyalkanoate using engineering strain
  • Method for producing polyhydroxyalkanoate using engineering strain

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Experimental program
Comparison scheme
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Embodiment 1

[0050] Embodiment 1, construction of environment-inducible expression plasmid

[0051] 1. Extraction of Escherichia coli MG1655 genome

[0052] The genome of Escherichia coli MG1655 was extracted according to the instructions of the bacterial genome extraction kit, and detected by agarose electrophoresis. The concentration of agarose is 0.8% by mass.

[0053] 2. Cloning of Environmentally Inducible Fragment (EPI)

[0054] According to the genome sequence of Escherichia coli (2865574-2866200) published on GenBank, the primer pEPI primer 1: 5′-AAT was designed ACATGT CCATACGCGCTGAACGTTGGTCA-3′ and pEPI primer 2: 5′-ATA CCCGGG AAGGTGGCTCCTACCCGTGATCCCT-3′

[0055] Using the extracted Escherichia coli MG1655 genome as a template, PCR (polymerase chain reaction) was used to amplify the environment-induced fragment sequence in vitro.

[0056] PCR reaction system is as follows: (primer concentration is 20 μ mol / L)

[0057] 5 μl of 10× buffer;

[0058] 25mmol / L MgCl 2 4μl; ...

Embodiment 2

[0081] Embodiment 2, the construction of PHB recombinant expression plasmid

[0082] Construction of PHB recombinant Escherichia coli:

[0083] The PHB synthase gene phbCAB was inserted into the multiple cloning site of the recombinant plasmid pEPI to construct a recombinant plasmid for expressing PHB synthase, which was named pEPI-phbCAB. The constructed recombinant plasmid expressing PHB synthase was transformed into Escherichia coli, and cultured to detect the accumulation of PHB under the regulation of the environment-induced expression system.

[0084] (1) Construction of PHB recombinant plasmid pEPI-phbCAB

[0085] According to the genome sequence of Alcaligenes eutropha published by Genbank, the primers were designed as follows:

[0086] PHB primer 1: 5′-ATCCCCGGGGCGACCGGCAAAGGCGCGGCAGCTTCCA-3′

[0087] PHB primer 2: 5′-ATGGAATTCCAGCCCATATGCAGGCCGCCGTTGAGC-3′

[0088] Using the genome sequence of Alcaligenes eutropha as a template, the phbCAB gene cluster was amplif...

Embodiment 3

[0096] Embodiment 3, the construction of PHB recombinant Escherichia coli

[0097] Add the connection solution containing the recombinant environment-inducible expression plasmid pEPI to 100 μl of DH5α competent cells, mix well; put it on ice for 30 minutes; heat shock at 42°C, keep it on ice for 2 minutes, add 900 μl of LB medium, 37 °C, 100 rpm, and incubate for 1 hour. Spread the transformation solution on a solid LB medium plate containing agar with a mass volume ratio of 2.0% mass volume ratio and ampicillin with a final concentration of 100 μg / ml, and culture it statically for 16 hours at 37°C , Take a single colony to screen transformants. Pick a single colony and transfer it to LB liquid medium, and culture overnight at 37°C and 225 rpm (add ampicillin to the medium to a final concentration of 100 μg / ml). The bacteria were centrifuged, and the recombinant plasmid pEPI was extracted using a plasmid extraction kit (purchased from TIAN GEN Company), and the correctness ...

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Abstract

The invention discloses a method for producing poly-hydroxy fatty acid (PHA) by utilizing engineering strains, which can express PHB synthetase gene phbCAB by constructing environmental induction type expression plasmid; under the condition that no revulsant is added, the phbCAB gene is expressed by the external conditions such as environmental intimidation, and the like, thereby catalyzing the synthesis of the PHA. The fermentation result shows that under the condition that no revulsant is added, the content of recombined colon bacillus DH5 alpha / pEPI-phbCAB PHB reaches 85.7% of the dry weight of cells. The recombined colon bacillus DH5 alpha / pEPI-phbCAB of the invention has important application prospect and value.

Description

technical field [0001] The invention relates to a method for producing polyhydroxyalkanoate (PHA), in particular to a method for fermenting and producing polyhydroxyalkanoate (PHA) by recombinant Escherichia coli. It belongs to the fields of genetic engineering and microbial fermentation. Background technique [0002] Polyhydroxyalkanoat (PHA for short) is a functional biological polyester synthesized by microorganisms. PHA has biodegradability, biocompatibility, gas barrier property, piezoelectricity, nonlinear optical activity and other special properties caused by functional groups. The nature of PHA determines that it has many potential applications such as biodegradable plastics and scaffold materials for tissue engineering. Therefore, a large number of basic and applied development researches have been carried out at home and abroad. Polyhydroxybutyrate (Polyhydroxybutyrate, PHB) is the simplest, most studied and most thorough member of the PHA family. PHB is granul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/62C12N15/52C12N15/63C12N1/21C12R1/19
Inventor 祁庆生康振
Owner SHANDONG UNIV
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