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Shewanella EPA synthetic gene cluster and gene engineering bacterium containing same

A technology of genetic engineering bacteria and synthetic genes, applied in the field of Shewanella EPA synthesis gene cluster, can solve the problems of reducing the fluidity of cell membranes and hindering the normal physiological functions of cell membranes

Inactive Publication Date: 2011-01-12
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recently, it has been found that high pressure can have a similar effect on the cell membrane, which can also reduce the fluidity of the cell membrane and hinder the normal physiological function of the cell membrane.

Method used

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  • Shewanella EPA synthetic gene cluster and gene engineering bacterium containing same
  • Shewanella EPA synthetic gene cluster and gene engineering bacterium containing same
  • Shewanella EPA synthetic gene cluster and gene engineering bacterium containing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Screening of Escherichia coli comprising the WP3EPA gene cluster

[0035] Bacterial strains and culture conditions: WP3 is generally cultured at 15°C under normal pressure, and the medium used is 2216E medium. The clones of the cosmid library are generally cultured at 37°C under normal pressure, and the medium used is LB medium plus ampicillin at a final concentration of 100ug / ml. When the fatty acid content of the cosmid library clones was detected by GC-MS, the culture temperature of the clones was 20°C.

[0036] Construction of the WP3 genome cosmid library: the kit used was the pWEBTM cosmid cloning kit from EPICENTER. The entire cosmid library was constructed in accordance with the instructions provided by the kit. That is, the genomic DNA of large molecular weight is extracted and prepared according to the standard method of extracting total DNA, and dissolved in a certain amount of TE buffer. Next, the total DNA was mechanically fragmented to 30-45 ...

Embodiment 2

[0039] Example 2 Determination of the function of WP3 EPA synthetic gene cluster

[0040] Gene knockout method:

[0041] First, the DNA fragment of the target gene deletion was obtained by two rounds of PCR amplification, which was cloned into the suicide vector pRE112 to obtain the target gene deletion mutation vector pRE112-EPA. The pRE112-EPA was introduced into the diaminopimelic acid (DAP) auxotrophic Escherichia coli WM3064 by chemical transformation method, and then the plasmid pRE112-EPA was introduced from the donor strain WM3064 into WP3 by the method of combined transfer, and then passed two rounds DNA Homologous Recombination Screening to Obtain Genetic Engineering Strain WP3 with Target Deletion ΔEPA .

[0042] 1. Materials:

[0043] 1. PRE112 plasmid, the structure is shown in the appendix image 3 , in the figure:

[0044] Cm: chloramphenicol resistance gene;

[0045] oriT: mediates conjugation and plasmid transfer;

[0046] oriV: origin of replication (c...

Embodiment 3

[0071] The mensuration of embodiment 3 bacterial strain cultivation and bacterial fatty acid content

[0072] Bacterial strains and culture conditions: WP3 is generally cultured at 15°C under normal pressure, and the medium used is 2216E medium. The clones of the cosmid library are generally cultured at 37°C under normal pressure, and the medium used is LB medium plus ampicillin at a final concentration of 100ug / ml. When the fatty acid content of the cosmid library clones was detected by GC-MS, the culture temperature of the clones was 20°C.

[0073] 2216E Medium (1000ml)

[0074]Yeast extract 1g

[0075] Tryptone 2g

[0076] NaCl 34g

[0077] Fe 3 PO 4 0.1g

[0078] (1) Cultivation of WP3 under different temperature growth conditions: after overnight culture in normal 2216E medium (salinity 3.4%), the bacterial liquid was diluted 50 times into fresh medium, and placed in low temperature (4°C) and Cultivate at room temperature (20°C).

[0079] (2) Cultivat...

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Abstract

The invention relates to a shewanella EPA synthetic gene cluster and a gene engineering bacterium containing the same. The invention obtains colibacillus gene engineering bacterium of EPA synthetic gene cluster of shewanella piezotolerans WP3, and determines that the gene cluster is in charge of synthesis of EPA in WP3. The EPA synthetic gene cluster contains a phosphopantetheine transferase (PPTase) gene and pfaA, B, C and D genes, and the genetic transcription amount is controlled by the forward Fis transcription control factor of pfaA. Low temperature and high pressure can enhance the synthesis of EPA.

Description

technical field [0001] The invention relates to a shewanella EPA synthesis gene cluster and belongs to the field of biotechnology. The present invention particularly relates to obtaining the escherichia coli genetically engineered bacterium comprising the Shewanella piezotolerans WP3 EPA synthetic gene cluster and the EPA synthetic gene cluster in WP3, the gene cluster comprising a phosphopantetheinyl transferase (PPTase) gene and pfaA, B, C, D genes, and synthesize EPA through the polyketide enzyme pathway (PKS). Background technique [0002] Eicosapentaenoic acid (EPA) is an important component of most animal membrane lipids. It is very beneficial to human health. It can prevent many diseases and has high nutritional value. As early as the 1960s, it was proved that EPA had the effect of resisting experimental thrombosis, and it was applied clinically in the 1970s. At present, it is believed that EPA is not only related to cardiovascular diseases, but also closely related...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/54C12N15/52C12N15/31C12N1/21C12N15/70C12P7/64C12R1/19C12R1/01
Inventor 王风平肖湘姜丽晶王峰彭华煜
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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