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Method for scale selection of microorganism alkaliproof related genes

A microbial and alkali-resistant technology applied in the field of microbial engineering

Inactive Publication Date: 2007-10-31
RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Abstract
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Problems solved by technology

But so far, no one has used this strain to clone alkali resistance-related genes from microorganisms on a large scale

Method used

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Examples

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Embodiment Construction

[0039] The following examples will further illustrate the present invention.

[0040] The genes related to microbial alkali resistance were cloned from the soil samples collected from the alkali lake in Xinjiang. The detailed steps are:

[0041] 1. Use a sample collector to collect sedimentary soil samples about 20cm deep along the perimeter of the alkali lake, and then distribute them in sterilized 50ml centrifuge tubes. After the samples were brought back to the laboratory, they were stored at -80°C for future use.

[0042] 2. Extraction of soil microbial metagenomics. The specific method is:

[0043] (1) Weigh 1g of soil sample into a 15mL centrifuge tube, add about 50mg of PVPP and 2.7mL of preheated DNA extraction solution, then add 50μL of lysozyme, shake at 37°C and 250r / min for 30min;

[0044] (2) Add 0.3mL of 20% SDS, and turn it upside down several times to make it evenly mixed;

[0045] (3) 65 ℃ water bath for 2 hours (every 20 minutes or so, gently invert the ...

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Abstract

The invention discloses a method to sieve scale microbe alkali proof relative gene in microbe engineering domain, which is characterized by the following: collecting typical basic environment soil sample; rough-extracting; purifying; getting macro-genom DNA of soil sample; electrization-inverting bacillus coli defect strain E with connecting product between macro-genom DNA part enzyme cutting fragment and carrier; sieving; getting positive colony; proceeding sub-clone and functional appraise for alkali proof related gene of positive clone. This invention can be used to breeding of alkali proof and molecule of microbe.

Description

Technical field: [0001] The invention belongs to the field of microbial engineering and relates to a method for large-scale screening of genes related to microbial alkali resistance. Background technique: [0002] Internationally, Escherichia coli defective strain E is used to clone salt-tolerance-related genes from microorganisms. The method is: extract the DNA of the purely cultured microorganism, partially digest it with a restriction endonuclease, connect the digested fragment to an appropriate vector, and use the ligated product to transform E. coli defective strain E, and then, in a certain concentration The transformants were screened on the solid medium of NaCl or LiCl, so as to obtain the recombinants carrying the genes related to salt tolerance. In fact, it is also possible to use E. coli defective strain E to clone genes related to alkali resistance. Alkali resistance-related genes refer to genes related to intracellular pH homeostasis. But so far, no one has u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12P19/34
Inventor 杨礼富王真辉
Owner RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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