Method for reducing viscosity of bacillus fermentation liquid

A technology of Bacillus and Bacillus subtilis, which is applied in the direction of fermentation, introduction of foreign genetic material by using vectors, recombinant DNA technology, etc. It can solve problems such as low repeatability and versatility, no knockout resistance markers, and inability to transform strains , achieve high repeatability and versatility, reduce viscosity and increase concentration

Active Publication Date: 2018-03-09
CHENGDU MYTECH BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the method in this patent will have the problem of not knocking out the resistance marker, and cannot continue to transform the strain or transfer the plasmid technology, and the repeatability and versatility are not high.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1

[0030] 1. The pBAV1K-T5-GFP (http: / / www.addgene.org / vector-database / ) plasmid was cleaved by EcoR I and Apa I endonucleases, and cloned by homologous recombination with the synthesized MCS fragment to obtain the plasmid pBAV1K.

[0031] In this experiment and subsequent experiments:

[0032] The endonuclease used Thermo's rapid endonuclease, and the fragment was recovered using the gel recovery kit (DE-02011) of Chengdu Fuji Biotechnology Co., Ltd. The MCS fragment was synthesized by Jinweizhi Biotechnology Co., Ltd. EsayGeno Rapid Recombination Cloning Kit (VI201-02), E. coli strain is top10, KCM method was used to prepare competent cells, and plasmid extraction was performed using Fuji Bio's Universal Plasmid Mini-Extraction Kit (DE-01001).

[0033] 2. Design primers, amplify the pBAV1K fragment with synonymous mutation deletion of Nde I restriction site, and connect the fragments by homologous recombination cloning to obtain a plasmid named pBTS. The pr...

Embodiment 2

[0035] 1. For the strain transformation method, see the hyperosmolarity transformation method (High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis, 1999). Take the newly streaked single colony of the strain to be transformed, inoculate it into about 3ml of GM (LB+0.5M sorbitol) medium, and cultivate overnight at 37°C and 180rpm. The next morning, inoculate it into 50ml of GM medium at 1:100, and cultivate at 37°C at 180rpm. When the bacterial solution grows to an OD of 0.85-0.95, put the bacterial solution on ice to pre-cool for 10min; 4°C, 5000g, 5min, centrifuge to remove the supernatant, resuspend the cells with an equal volume of pre-cooled EM (0.5M sorbitol + 0.5M mannitol + 10% glycerol aqueous solution), and centrifuge again at 4°C, 5000g, 5min Washing was repeated 4 times in total; about 1 / 40 volume of EM was added to resuspend the bacterial cells to ensure that the bacterial concentr...

Embodiment 3

[0037] 1. Design primers to amplify the upstream 558bp fragment pgs-F of the pas gene of the Z12 strain, and insert it between the KpnI and BamHI sites of pBTS to obtain the plasmid pBTS-pgs-F; amplify the downstream 693bp fragment aprE-R and insert it into the plasmid pBTS -Between BamHI and XhoI of pgs-F, a plasmid pBTS-pgs knocking out the pgs gene was obtained.

[0038] Toyobo's KOD-Plus-Neo enzyme was used for amplification, and the amplification conditions were as follows: 94°C, 2min; (94°C, 30s; 60°C, 30s; 68°C, 1min) x25; 72°C, 2min.

[0039] Primer sequence:

[0040] pgs -F -F gaggtaccAGCTGTTCCGATTTTATACTGGTC;

[0041] pgs-F-R gaggatccGTTTGTTGCCGTAGCCATCGTG;

[0042] pgs-R-F gaggatccCTAATCGACTAAGCCAAACTTCTC;

[0043] pgs-R-R gactcgagTAGCCAAAACAGCTCCTCTTG.

[0044] 2. Using the hyperosmotic transformation method in Example 2, pBTS-pgs was transformed into the Z12 strain (preserved in the General Microbiology Center CGMCC of China Microbial Culture Collection Manageme...

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Abstract

The invention belongs to the fermentation engineering field and particularly relates to a method for reducing the viscosity of bacillus fermentation liquid. According to the method, a gamma-PGA gene (pgs) is synthesized by directly knocking out bacillus subtilis. The method comprises the steps of inserting pgs-F into a space between KpnI and BamHI of a pBTS plasmid, inserting pgs-R into a space between BamHI and XhoI so as to construct a new plasmid pBTS-pgs, introducing the converted plasmid into to-be-knocked strain, and carrying out primary recombination and secondary recombination and thelike, so as to obtain a strain which is not provided with a resistance maker and is capable of knocking out the pgs gene. According to the strain, the synthesis of gamma-PGA through bacillus is impeded, furthermore, the viscosity of the fermentation liquid is reduced, the growth of thalli is promoted, and the concentration of a fermentation product is increased.

Description

technical field [0001] The invention belongs to the field of fermentation engineering, in particular to a method for reducing the viscosity of Bacillus fermentation broth. Background technique [0002] During the growth of some Bacillus, poly-γ-glutamic acid (γ-PGA) is secreted from the cell wall to form the capsular structure of the bacteria, and can also be dissolved in the fermentation broth. When the gas, solid content and gas and solid particles are dispersed to a certain critical value in the fermentation broth, the continuous mobile phase and the abrupt change of the gas and solid dispersed phases will occur, which is manifested in the non-Newtonian change of the fermentation broth and the increase in viscosity. [0003] γ-PGA is a high molecular polymer with a degree of polymerization of about 1,000-15,000. Due to the presence of hydrophilic groups on the glutamic acid residues in γ-PGA, a large amount of water can be bound by hydrogen bonds, increasing the molecula...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12P13/02
CPCC12N15/75C12P13/02
Inventor 任钧张韬唐旭
Owner CHENGDU MYTECH BIOTECH
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