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A strain of Bacillus subtilis and its preparation method

A technology for Bacillus subtilis and strains, which is applied in the field of Bacillus subtilis and its preparation, can solve the problems of reduced biomass of Bacillus subtilis and the like, and achieves improved resistance to adverse growth conditions, improved stress resistance, and enhanced resistance to autolysis. Effect

Active Publication Date: 2021-08-13
CANVEST WUHAN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since their accumulation leads to a decrease in the biomass of Bacillus subtilis

Method used

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  • A strain of Bacillus subtilis and its preparation method
  • A strain of Bacillus subtilis and its preparation method
  • A strain of Bacillus subtilis and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Obtaining of Bacillus subtilis △WB800

[0036]The first is to construct the knockout vector. The upstream and downstream homology arms of the knockout gene were respectively amplified from the WB800 genome by PCR, and then the skfA upstream forward primer shown in Q ID NO.1 and the skfA upstream reverse primer shown in SEQ ID NO.2 Amplify the primer to obtain the upstream homology arm of the skfA gene, use the downstream forward primer of skfA shown in SEQ ID NO.3 and the downstream reverse primer of skfA shown in SEQ ID NO.4 to amplify the downstream of the skfA gene Homology arm, the upstream homology arm is cut with restriction endonuclease HindIII and PstI, the downstream homology arm is cut with restriction endonuclease PstI and EcoRI, and then the restriction endonuclease HindIII and EcoRI at both ends are cut The vector pGK12 was cut, and then the upper and lower homology arms and the vector fragment were ligated with T4 ligase. The obtained ligation...

Embodiment 2

[0039] Example 2: Detection of autolytic activity of bacterial strains

[0040] Pick a single colony of Bacillus subtilis to be tested, inoculate it in liquid LB medium (peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L), and cultivate the activated bacterium at 37°C and 200rpm overnight. The inoculum size of % (v / v) was transferred to a 250mL Erlenmeyer flask equipped with 50mL liquid LB medium, and cultivated at 37°C and 200rpm to the mid-logarithmic phase (about 3h, OD 600 value reaches about 1.0), take a sample and measure and record the OD 600 After the value, NaN3 solution with a final concentration of 0.05M was added. Continue culturing at 37°C, 200rpm, measure OD every 0.5h 600 value. Take the OD at each time point after adding NaN3 600 and starting OD 600 The ratio is used as the ordinate to plot the autolysis curve.

[0041] After adding NaN 3 After 4 hours, the starting strain WB800 had 80% cell lysis, while the cell autolysis activity of the quadruple ...

Embodiment 3

[0042] Embodiment 3: heat-resistant, acid-resistant, high-salt-resistant performance detection of bacterial strain

[0043] Pick a single colony of Bacillus subtilis to be tested, inoculate it in liquid LB medium, and cultivate the activated bacterium overnight at 37°C, 200rpm, transfer the above-mentioned bacterium solution to a 50mL container with an inoculation amount of 1% (v / v). Culture a 250mL Erlenmeyer flask with liquid LB medium at 37°C and 200rpm until the stationary phase (about 12h), take a sample and measure and record the OD 600 value. Take 0.5mL bacterial liquid, centrifuge at 8000g for 5min at room temperature, wash twice with 10mM PBS (pH 7.4).

[0044] Heat resistance: resuspended in 0.5mL PBS, treated in a water bath at 55°C for 30 minutes, sampled at different time intervals, and spread on LB solid plates after gradient dilution (peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L, agar powder 15g / L), the number of viable bacteria was recorded after ...

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Abstract

The invention relates to a strain of bacillus subtilis (Bacillus subtilis) ΔWB800 and a preparation method thereof, belonging to the technical field of bioengineering. In the present invention, by knocking out the autolysis-related genes skfA, sdpC, lytC, and xpf of the Bacillus subtilis WB800 strain, the autolysis-inhibiting strain Δ800 is obtained. The strain has excellent properties of heat resistance, acid resistance, and bile salt resistance, and shows Good prospects for industrial application.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a strain of Bacillus subtilis whose cell autolysis is inhibited after genetic modification and a preparation method thereof. Background technique [0002] Bacillus subtilis is a Gram-positive, spore-forming, non-pathogenic microbial bacterium that normally exists in the natural environment. As one of the most studied Gram-positive soil bacteria and model microorganisms, Bacillus subtilis is a recognized GRAS (Generally recognized as safe) strain, and is widely used in biological control of animal and plant diseases, plant growth promoters, and probiotics. Bacteria, production of industrial enzymes and expression of heterologous proteins and many other fields. [0003] In decades of research on Bacillus subtilis, people have made great efforts to increase the biomass and protein production of Bacillus subtilis, and achieved many excellent results. The construc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/75C12R1/125
CPCC12N1/20C12N15/75C12N1/205C12R2001/125
Inventor 赵瑞丽王轶郑从义
Owner CANVEST WUHAN BIOTECH
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