Optimized culture method and application of butyribacterium methylotrophium

A technology of methylbutyric acid bacteria and methyl butyric acid bacteria, applied in the field of microbiology, can solve problems such as difficulty in accepting foreign genes

Active Publication Date: 2020-05-15
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the deficiencies of the prior art, the object of the present invention is to provide an optimized culture method and application of butyric acid bacteria methyl-eating bacteria, so as to solve the problem that the butyric acid methyl-eating bacteria are difficult to accept exogenous genes, enrich the food The research on Methylbutyricum will provide a reference for further research on genetic manipulation tools for this strain

Method used

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  • Optimized culture method and application of butyribacterium methylotrophium
  • Optimized culture method and application of butyribacterium methylotrophium
  • Optimized culture method and application of butyribacterium methylotrophium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Screening different types of medium

[0031] Six media suitable for the growth of Clostridia were selected, including

[0032] RCM (yeast extract 3g / L, beef extract 10 g / L, peptone 10 g / L, soluble starch 1 g / L, glucose 5 g / L, sodium chloride 3 g / L, sodium acetate 3 g / L),

[0033] TYA (glucose 40 g / L, beef extract 2 g / L, yeast extract 2 g / L, tryptone 6 g / L, ammonium acetate 3 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate heptahydrate 0.2 g / L, ferrous sulfate heptahydrate 0.01 g / L),

[0034] YTF (peptone 16 g / L, yeast powder 12 g / L, sodium chloride 4 g / L, glucose 5 g / L), PB-G (potassium dihydrogen phosphate 4 g / L, dipotassium hydrogen phosphate 6 g / L , ammonium chloride 1 g / L, magnesium chloride hexahydrate 0.1 g / L, calcium chloride dihydrate 0.1 g / L, yeast powder 3 g / L),

[0035] NRM (glucose 40 g / L, beef extract 2 g / L, yeast extract 2 g / L, tryptone 6 g / L, ammonium acetate 3 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate hept...

Embodiment 2

[0040] Example 2 Screening for different types of replicons

[0041] Select 5 kinds of replicons, including pIM13, pCB102, pBP1, pCD6, pIP404, corresponding to plasmid numbers 1, 2, 3, 4, 8, and select the plasmid TOP10 competent or phage containing a type 1 methylase gene to infect Bacillus subtilis The plasmid TOP10 competent for the methylase gene obtained from the bacillus, numbers 9 and 10. (The method of TOP10 competence containing the corresponding methylated gene is as follows: Transform the plasmid containing the methylase gene into commercial TOP10 competence, and spread it on solid culture. Pick a single colony from the plate Insert into a 50mL centrifuge tube containing 10mL LB culture medium, cultivate to OD at 37°C 600 After = 0.4, place the bacterial solution on ice, apply ice for 10 minutes, and centrifuge at 4000r / min for 10 minutes. Discard the supernatant, resuspend each pellet with 10ml of ice-cold 0.1mM CaCl2, place on ice for 15min, and centrifuge at 40...

Embodiment 3

[0046] Example 3 Screening of different methylases

[0047] In order to investigate the effect of different methylases on plasmid transformation, the three types of methylases including type 1 methylase, the methylase obtained by phage infection of Bacillus subtilis, and the bacteria itself were screened. The corresponding plasmids for the enzymes are pMCljS, pAN2, pACYC184-M1, pACYC184-M2, and pACYC184-M3, respectively, and TOP10 competent cells containing the above plasmids were prepared, numbered 9-13. The most stable replicons screened were selected for electroporation experiments.

[0048] Add the plasmid containing the pCB102 replicon into TOP10 competent cells containing the methylated plasmids No. Add 1mL LB culture solution to the ultra-clean workbench, place it in a shaker at 37°C for 1 hour, and then take it out and spread it on a solid plate.

[0049] Pick a single colony from the solid plate and transfer it into a 50mL centrifuge tube containing 10mL LB culture ...

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Abstract

The present invention discloses an optimized culture method and an application of butyribacterium methylotrophium. A type I methyltransferase is used to conduct methylated modification of foreign plasmids in a TOP10 strain to obtain methylated modified plasmid DNA. The used method comprises (1) screening a culture medium suitable for clostridium; (2) screening replicons that are stable in the clostridium; and (3) determining the optimal methyltransferase. Under all optimal conditions, the methylated modified plasmid DNA can be transferred into the butyribacterium methylotrophium with a restricted modification system without being sheared and degraded, and subsequent metabolic pathway transformation can be carried out.

Description

technical field [0001] The invention belongs to the field of microbes, and in particular relates to an optimized culture method and application of butyricum methylovora. Background technique [0002] In recent years, more and more studies have found that an important obstacle for Clostridium to be difficult or impossible to transform is the existing restriction modification system. The main reason is that the modification restriction system can cut and degrade most or even all of the exogenous DNA that enters the cell before it can perform its function, so transformants cannot be obtained or the number of transformants is very small. [0003] The non-type strain Bacillus methylbutyricum, referred to as Bm, is an anaerobic Clostridium spp., which belongs to the single-carbon anaerobic type, and can use a variety of C1 raw materials for fermentation at the same time, such as CO 2 , CO and methanol etc. In addition, it can also metabolize multi-carbon substances, including gl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/74C12P7/52C12R1/145
CPCC12N9/1007C12Y201/01C12N15/74C12P7/52
Inventor 王昕马琛王雪麟陈可泉马江峰王静金雨琪欧阳平凯
Owner NANJING UNIV OF TECH
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