Extracting method of cellulose degradation flora metagenome

A technology of cellulose degrading bacteria and cellulose decomposing bacteria, which is applied in the field of metagenomic extraction, can solve the problems of interference, affect bacterial extraction, increase the difficulty of DNA extraction, etc., achieve easier release, ensure repeatability, and ensure integrity Effect

Active Publication Date: 2016-08-03
TSINGHUA UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the characteristics of the cellulosic flora, it is difficult to extract its genomic DNA. Therefore, a complete macrogene analysis technology for the cellulosic flora has not yet been established.
[0005] The factors affecting the extraction effect of the metagenomics of the natural cellulose flora mainly include the following three points: First, the strains in the cellulose flora are mostly tightly combined with the cellulose microfilaments through the structure of the cellulosus. The cross-linking form of "body" exists in the fermentation broth, which will significantly affect the breaking of the cell wall and the extr...

Method used

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  • Extracting method of cellulose degradation flora metagenome
  • Extracting method of cellulose degradation flora metagenome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1, Metagenome Extraction of Cellulose Degrading Bacteria

[0065] 1. Screening of natural cellulose degrading bacteria

[0066] 1. Selection of cellulose degrading bacteria

[0067] With the lignocellulosic raw material of 10g / L (Sigma company PH-101 (Cat. No. 11365) and 5g of soil samples collected in cellulose-enriched areas were added to the flora-enrichment medium and cultured at 30-65°C. Through continuous subculture, bacteria with stable cellulose conversion ability were selected. The group is the bacterial group that stably degrades the lignocellulosic raw material.

[0068] The stability index of the cellulose-converting ability to stabilize the flora is to ferment the lignocellulosic raw material and the flora in the flora-enriched medium for 3 days, and detect the degradation rate of the lignocellulose raw material every day. If the lignocellulose raw material degrades in 3 days The rate is basically unchanged, which is to stabilize the flora for t...

Embodiment 2

[0086] Embodiment 2, adopting existing kit to extract metagenomic DNA of saliva sample (comparative example)

Embodiment 4

[0131] The metagenomic DNA that embodiment 4, embodiment 1 and embodiment 2 extract compares

[0132] 1. Concentration and purity testing

[0133] The metagenomic DNA concentration and purity (indicated by OD260 / 280) extracted in Example 1 and Example 2 were measured respectively by Nanodrop (Thermo Fisher Scientific, model Nanodrop2000c), as shown in Table 1, and detected by agarose electrophoresis DNA concentration and integrity.

[0134] Table 1 compares the concentration and purity of extracted DNA

[0135]

[0136] It can be seen from Table 1 that the metagenomic DNA obtained by the method provided by the present invention for extracting natural cellulose utilizing flora is significantly improved in terms of concentration and purity compared with the metagenomic DNA obtained by the kit method.

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Abstract

The invention discloses an extracting method of cellulose degradation flora metagenome and provides an extracting method of cellulose degradation flora metagenome DNA. The extracting method comprises the following steps that cellulose degradation florae are sequentially subjected to thallus wall breaking, DNA organic solution extraction, DNA isoamylol sediment and DNA solution so that the metagenome DNA can be obtained. Experiments prove that a high-salt solution is adopted for carrying out eluting before extraction, lysozyme and mechanical homogenate are added to reinforce cell wall breaking, proteinase K is adopted for wall breaking, cell walls of microorganisms can be broken more easily, and DNA molecules can be released more easily. Meanwhile, the process repeatability is effectively ensured, and then integrity and purity of extracted metagenome DNA fragments and flora structure integrity are ensured. The extracting method provides an effective technology support for study and application of the natural cellulose utilizing florae and the microorganism molecular ecology study.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for extracting the metagenomic group of cellulose-degrading bacteria. Background technique [0002] Lignocellulose is the most productive polysaccharide in nature, and it widely exists in forests, grasslands and farmlands. Its output is abundant, its price is low, and it mostly exists in the form of agricultural and industrial waste, so it is an ideal raw material for the production of bioenergy substances. Studies have shown that the bioenergy production process using lignocellulose as raw material can not only realize the reuse of agricultural waste, but also effectively avoid the contradiction between energy production and food resources, and has become the most potential way of industrialized bioenergy production. [0003] However, due to the complex composition and compact structure of lignocellulose, the process of hydrolyzing it into usable sugars requires the synerg...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N1/06
CPCC12N1/06C12N15/1003
Inventor 闫建斌杜然
Owner TSINGHUA UNIV
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