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Method for improving efficiency of screening of 1,3-propylene glycol fusion bacteria

A technology for screening efficiency and propylene glycol, applied in the field of industrial biology, can solve problems such as lack and interference, and achieve the effect of improving the chance of success, reducing interference, and improving economic and economic benefits.

Inactive Publication Date: 2012-01-11
CHINA PETROLEUM & CHEM CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fusion screening has always been faced with the interference of normal bacteria, and there is still a lack of effective solutions. Therefore, improving the efficiency of screening suitable strains during the genome rearrangement process is an urgent problem in this field.

Method used

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  • Method for improving efficiency of screening of 1,3-propylene glycol fusion bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Genome rearrangement breeding, using Klebsiella pneumoniae as the initial parent bacterial strain

[0037] Bacterial species: Klebsiella pneumoniae was screened by Fushun Petrochemical Research Institute and preserved in China Microorganism Culture Collection Center (CGMCC), No. 0798.

[0038] Regeneration medium (solid): A, sucrose 171.5g / L, tryptone 10g / L, glucose: 10g / L, yeast extract 5g / L, MgCl 2 4.273g / L, NaCl 5g / L, agar 15g / L.

[0039] LB medium (solid): tryptone 10g / L, yeast extract 5g / L, NaCl 5g / L, agar 15g / L.

[0040] Lysozyme: Dissolve lysozyme with 10mM / L Tris-Cl (pH8.0), make it into a 20mg / mL stock solution, and divide it for use.

[0041] SMM buffer: 0.5mol / L sucrose, 0.02mol / L MgCl, 0.02mol / L maleic acid.

[0042] Tris-Cl buffer: also contains 0.01mol / L EDTA, 0.5mol / L sucrose.

[0043] Feed fermentation medium: yeast extract 5g / l, K 2 HPO 4 ·3H 2 O 10g / l, KH 2 PO 4 2g / l, NH 4 Cl0.8-1g / l, NaCl 0.5g / l, MgSO 4 ·7H 2 O 0.1g / l, FeCl 3 ...

Embodiment 2

[0056] Example 2 Using Klebsiella pneumoniae and Escherichia coli as parent strains, genome rearrangement technology breeding

[0057] Klebsiella pneumoniae and Escherichia coli were selected as parental strains, pre-activated with LB liquid medium for 14 hours, and the parental strains (Klebsiella pneumoniae and Escherichia coli) were subjected to UV mutagenesis and NTG mutagenesis respectively. The enzymatically detached protoplasts were filtered through a filter membrane with a pore diameter of 0.75 μm to remove bacteria, and then filtered through a filter membrane of 0.25 μm to remove bacterial fragments, followed by subsequent operations such as protoplast inactivation and fusion. The remaining steps and methods are the same as in Example 1. After 1 to 3 rounds of genome rearrangement screening, strains with excellent performance can be obtained.

Embodiment 3

[0058] Example 3 Using Klebsiella pneumoniae and Klebsiella oxytoca as parent strains, genome rearrangement technology breeding

[0059] Klebsiella pneumoniae and Klebsiella oxytoca were selected as parental strains, pre-activated with LB liquid medium for 14 hours, and the parental strains (Klebsiella pneumoniae and Escherichia coli) were subjected to UV mutagenesis and NTG induction respectively. Change. The inactivated protoplasts were filtered through a filter membrane with a pore diameter of 0.45 μm to remove bacteria, and then filtered through a filter membrane of 0.25 μm to remove bacterial fragments, followed by subsequent operations such as protoplast fusion. The remaining steps and methods are the same as in Example 1. After 1 to 3 rounds of genome rearrangement screening, strains with excellent performance can be obtained.

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Abstract

The invention discloses a method for improving the efficiency of the screening of 1,3-propylene glycol fusion bacteria. The main process of the method comprises: selecting a parent strain; subjecting the parent strain to enzymolysis wall removal to obtain a protoplast; inactivating a protoplast; subjecting the inactivated protoplast to protoplast fusion; selecting a fusant, and selecting target fusion bacteria with high performance; and subjecting the high-quality fusion strain serving as a parent to a next genomic rearrangement process till a strain with high comprehensive performance is obtained, wherein the protoplast obtained by the enzymolysis wall removal of the parent strain or inactivated protoplast are subjected to membrane filtration treatment first and then to subsequent treatment operation. Compared with the convention gene engineering fusion bacterium screening method, the method has the advantages of high target strain screening efficiency, convenience for operation, no need of expensive experimental instrument, suitability for industrial strain breeding and the like.

Description

technical field [0001] The invention relates to a screening method for preparing 1,3-propanediol-producing genetic engineering fusion bacteria by applying genome rearrangement technology, in particular to a method for improving the screening efficiency of genetic engineering fusion bacteria, and belongs to the field of industrial biotechnology. Background technique [0002] 1,3-Propanediol (PDO) is an important chemical and pharmaceutical intermediate raw material, which can be used as an organic solvent in ink, printing and dyeing, emulsifier, antifreeze, etc. The main purpose of PDO is to synthesize the monomer of new polyester polymer material polytrimethylene terephthalate (PTT). PTT is currently one of the six new petrochemical products recognized internationally. Compared with polyethylene terephthalate (PET), polybutylene terephthalate (PBT) and nylon, PTT has good printing and dyeing properties, high elasticity, anti-ultraviolet, good static performance, biodegradab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/03
Inventor 金平王领民师文静李晓姝王崇辉
Owner CHINA PETROLEUM & CHEM CORP
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