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A kind of culture medium combination and its transformation method for promoting the natural growth transformation of Bacillus subtilis

A technology of natural growth of Bacillus subtilis, applied in the field of microorganisms, can solve the problems of low transformation efficiency, difficulty in preparing protoplasts of Bacillus subtilis, complicated operation, etc., achieve high transformation efficiency, improve complex methods, and simple and easy methods

Active Publication Date: 2021-06-15
SOUTH CHINA AGRI UNIV
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  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art: (1) traditional electric shock transformation requires special instruments, and not all plasmids can successfully transform Bacillus subtilis by electric shock; (2) common chemical transformation needs to transform the recipient bacteria The gene of comK gene is up-regulated, the plasmid is transformed, and the operation is complicated; (3) Bacillus subtilis is difficult to prepare protoplasts, and the transformation efficiency is extremely low. A kind of medium and transformation method thereof for promoting the natural growth transformation of Bacillus subtilis are provided, and the Special medium to obtain competent cells of Bacillus subtilis that grow naturally, and then use natural growth transformation technology, so that Bacillus subtilis can be transformed into ordinary plasmids more simply and easily

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 A kind of culture medium combination of Bacillus subtilis natural growth transformation

[0038] Medium SP I: (NH 4 ) SO 4 0.1g; K 2 HPO 4 0.2g; KH 2 PO 4 0.4g; Sodium citrate dihydrate (Na-Citrate-2H 2 O) 0.2g; MgSO 4 ·7H 2 O 0.01g; 20% (mass fraction)*glucose (Glucose) 2.4mL; 1% (mass fraction)*yeast extract (Yeast extract) 9mL; 10mg / mL*L-leucine (L-Leucine) 450μL; 10mg / mL*L-Methionine (L-Methionine) 450μL; 0.2% (mass fraction)*casein hydrolyzate (Casamino acid) 9mL (the above with * should be sterilized separately), with dH 2 O was dissolved and adjusted to 100mL for sterilization.

[0039] Medium SP II: (NH 4 ) SO 4 0.1g; K 2 HPO 4 1.3g; KH 2 PO 4 0.5g; Sodium citrate dihydrate (Na-Citrate-2H 2 O) 0.06g; MgSO 4 ·7H 2 O 0.01g; 20% (mass fraction)*glucose (Glucose) 2.4mL; 1% (mass fraction)*yeast extract (Yeast extract) 1.8mL; 15mg / mL*L-leucine (L-Leucine) 48μL ; 10mg / mL*L-Methionine (L-Methionine) 48μL; 500mmol / L*MgCl 2 0.8mL (...

Embodiment 2

[0040] Embodiment 2 A kind of culture medium combination of Bacillus subtilis natural growth transformation

[0041] Medium SP I: (NH 4 ) SO 4 0.3g; K 2 HPO 4 0.3g; KH 2 PO 4 0.45g; Sodium citrate dihydrate (Na-Citrate-2H 2 O) 0.25g; MgSO 4 ·7H 2 O 0.014g; 20% (mass fraction)*glucose (Glucose) 2.45mL; 1% (mass fraction)*yeast extract (Yeast extract) 10.5mL; 10mg / mL*L-leucine (L-Leucine) 470μL ; 10mg / mL* L-methionine (L-Methionine) 470μL; 0.2% (mass fraction)* casein hydrolyzate (Casaminoacid) 9.6mL (the above with * should be sterilized separately), use dH 2 O was dissolved and adjusted to 100mL for sterilization.

[0042] Medium SP II: (NH 4 ) SO 4 0.21g; K 2 HPO 4 1.36g; KH 2 PO 4 0.55g; Sodium citrate dihydrate (Na-Citrate-2H 2 O) 0.07g; MgSO 4 ·7H 2 O 0.016g; 20% (mass fraction) * glucose (Glucose) 2.35mL; 1% (mass fraction) * yeast extract (Yeast extract) 1.9mL; 15mg / mL * L-leucine (L-Leucine) 49.2 μL; 10mg / mL*L-Methionine (L-Methionine) 49.6μL; 5...

Embodiment 3

[0043] Embodiment 3 A kind of culture medium combination of Bacillus subtilis natural growth transformation

[0044] Medium SP I: (NH 4 ) SO 4 0.3g; K 2 HPO 4 0.5g; KH 2 PO 4 0.5g; Sodium citrate dihydrate (Na-Citrate-2H 2 O) 0.26g; MgSO 4 ·7H 2 O 0.021g; 20% (mass fraction)*glucose (Glucose) 2.56mL; 1% (mass fraction)*yeast extract (Yeast extract) 10.2mL; 10mg / mL*L-leucine (L-Leucine) 510μL ; 10mg / mL* L-methionine (L-Methionine) 510μL; 0.2% (mass fraction)* casein hydrolyzate (Casaminoacid) 10mL (the above with * should be sterilized separately), with dH 2 O was dissolved and adjusted to 100mL for sterilization.

[0045] Medium SP II: (NH 4 ) SO 4 0.26g; K 2 HPO 4 1.42g; KH 2 PO 4 0.62g; Sodium citrate dihydrate (Na-Citrate-2H2O) 0.11g; MgSO 4 ·7H 2 O 0.02g; 20% (mass fraction) * Glucose (Glucose) 2.53mL; 1% (mass fraction) * Yeast extract (Yeast extract) 2.2mL; 15mg / mL * L-leucine (L-Leucine) 50μL ; 10mg / mL*L-Methionine (L-Methionine) 50μL; 500mmol / L*...

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Abstract

The invention discloses a culture medium combination for promoting natural growth and transformation of Bacillus subtilis and a transformation method thereof. The medium combination includes Medium 1 and Medium 2. The invention utilizes the culture medium containing special nutrient formula to efficiently obtain competent cells of Bacillus subtilis, and cooperates with the natural transformation method to obtain beneficial recombinants more efficiently and quickly. Furthermore, the complex method of Bacillus subtilis that can only rely on electrotransformation is improved, the cost of transformation is greatly reduced, and the foundation is laid for the directed evolution and metabolic engineering of Bacillus subtilis-based proteins. The technology of the invention overcomes the disadvantages of the traditional conversion technology, and the method is simple and easy, the conversion efficiency is high, and it is easy to popularize.

Description

technical field [0001] The invention relates to the technical field of microorganisms, more specifically, relates to a technology for preparing a culture medium combination of competent cells of Bacillus subtilis and promoting their natural growth and transformation. Background technique [0002] Bacillus subtilis is a widely distributed mesophilic, aerobic and spore-forming Gram-positive rod-shaped bacterium, which has been identified as GRAS (Generally recognized assafe) strain by the US Food and Drug Administration (FDA). Bacillus subtilis has strong stress resistance, and can secrete a large number of enzymes and produce antibacterial substances. Therefore, on the one hand, it is widely used as a live bacterial agent in the fields of feed, biocontrol, and environmental protection; on the other hand, it plays an important role in the production of exogenous proteins and metabolites as an expression system. However, as an expression system, Bacillus subtilis has a transfo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12R1/125
CPCC12N1/20
Inventor 张玲华贾军皓楚品品曹丁马苗鹏邓锦波明飞平
Owner SOUTH CHINA AGRI UNIV