Method for extracting microorganism macrogenome from oil/gas pool environment

A gas reservoir environment and metagenome technology, which is applied in the field of direct extraction of microbial metagenomes to achieve the effects of high repeatability, low cost and a wide range of target microorganisms

Active Publication Date: 2012-10-17
DALIAN BITEOMICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0022] At present, there is no relevant research and applicati...

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  • Method for extracting microorganism macrogenome from oil/gas pool environment
  • Method for extracting microorganism macrogenome from oil/gas pool environment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Simple method for extracting microbial metagenomics from crude oil

[0041] 1. Take about 100mL of crude oil and put it in a glass beaker, and put it in a water bath at 70°C for 30 minutes.

[0042] 2. Add an equal volume of 30% (V / V) ethanol and half volume of crude oil petroleum ether: n-hexane (V / V=1:1) mixture, shake well.

[0043] 3. Stand still to separate the oil and water phases, and use a rubber hose to extract the lower water phase. Use filter paper to filter out a small amount of crude oil and other impurities in the aqueous phase.

[0044] 4. After filtration, the liquid is vacuum-filtered with a 0.22 μm microporous filter membrane, and the bacteria are enriched on the filter membrane.

[0045] 5. Cut the filter membrane into pieces and put it into a 10mL centrifuge tube, add 360μL lysozyme (10mg / mL dissolved in 10mM Tris-HCl, pH8.0), and bathe in 37℃ water for 1h.

[0046] 6. Add 115 μL of 10% SDS and 25 μL of proteinase K (20 mg / mL), and bathe...

Embodiment 2

[0052] Example 2 Simple method for extracting microbial metagenomics from oil-water mixture

[0053] 1. Take about 100mL of oil-water mixture and put it in a glass beaker, and put it in a water bath at 70°C for 30min.

[0054] 2. Filter the oil-water mixture with filter paper and collect the water phase.

[0055] 3. Mix the crude oil left on the filter paper with twice the volume of sterile PBS, stir well, and collect the aqueous phase by filtering with filter paper, repeat three times.

[0056] 4. Combine and filter the aqueous phase, vacuum filter with a 0.22 μm microporous membrane, and enrich the bacteria on the membrane.

[0057] 5. Cut the filter membrane into pieces and put it into a 10mL centrifuge tube, add 360μL lysozyme (10mg / mL dissolved in 10mM Tris-HCl, pH8.0), and bathe in 37℃ water for 1h.

[0058] 6. Add 115 μL of 10% SDS and 25 μL of proteinase K (20 mg / mL), and bathe in water at 37°C for 2 hours.

[0059] 7. Add 400 μL of 5M NaCl and mix by inversion, add...

Embodiment 3

[0065] Example 3 Simple method for extracting microbial metagenomics from oily soil (sludge)

[0066] 1. Take about 10g of oily soil and put it in a glass beaker, add 100mL of PBS, stir repeatedly, and bathe in 70℃ water for 30min.

[0067] 2. Filter the mixture with filter paper and collect the aqueous phase.

[0068] 3. Dissolve the crude oil and soil left on the filter paper with 100mL sterile PBS, stir well, collect the water phase by filtering with filter paper, and repeat three times.

[0069] 4. Combine and filter the aqueous phase, vacuum filter with a 0.22 μm microporous membrane, and enrich the bacteria on the membrane.

[0070] 5. Cut the filter membrane into pieces and put it into a 10mL centrifuge tube, add 360μL lysozyme (10mg / mL dissolved in 10mM Tris-HCl, pH8.0), and bathe in 37℃ water for 1h.

[0071] 6. Add 115 μL of 10% SDS and 25 μL of proteinase K (20 mg / mL), and bathe in water at 37°C for 2 hours.

[0072] 7. Add 400 μL of 5M NaCl and mix by inversion,...

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Abstract

The invention provides a simple method for directly extracting microorganism macrogenome from an oil/gas pool environment sample, which comprises the following steps: an organic phase/water phase is extracted to obtain an microbe component in an oil sample/oil-water sample, vacuum filtering is carried out on the water phase enrichment microbes, cells are cracked by lysozyme, protease, sodium dodecyl sulfate (SDS) and the like, passes through the cetyl trimethyl ammonium bromide (CTAB) under high salt condition to remove organic macro-molecule components like protein and polysaccharide, extraction is carried out through nucleic acid and deposition is carried out, thereby the unbiased oil/gas pool microorganism macrogenome component can be obtained. The method of the invention has the advantages that the process is fast and simple, the cost is low, the whole extraction process can be completed in 10 hours; the obtained genome enables high purity, the ratio of OD260nm to OD280nm is between 1.7 and 2.0, the electrophoresis result detects that the integral genome possesses no degradation condition. The method of the invention aims at the oil/gas pool complex environments, and is suitable for extraction of other microbe genome in the soil and water body polluted by petroleum.

Description

technical field [0001] The invention relates to the technical field of molecular ecology, in particular to a simple method for directly extracting microbial metagenomes from the complex environment of oil / gas reservoirs. Background technique [0002] One of the key points of microbial oil / gas recovery technology and oil / gas reservoir microbial exploration technology is to understand the overall information of microorganisms in the complex environment of oil / gas reservoirs. Due to the limitations of current technical means, only about 1% of microorganisms can be artificially cultivated and recognized. For this reason, at the end of the last century, researchers developed metagenomics technology. 1. A method of constructing a genomic library and screening the library to find and discover new functional genes and active metabolites. [0003] In 1998, Handelsman et al. proposed the concept of metagenomics for the first time, and defined it as: a kind of microbial diversity, pop...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 梅晓丹刘铭楠赵静王平项迎霞王金良马雯雯吴迪杭晓明
Owner DALIAN BITEOMICS INC
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