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Bacillus subtilis heterologous protein expression quantity improving method

A Bacillus subtilis and exogenous protein technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve the problems of long cycle time, lower probability of obtaining high-yielding mutant strains, and heavy workload

Inactive Publication Date: 2015-08-26
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After narrowing the screening range, the mutants were fermented and cultured one by one by shaking flask primary screening, and the amylase activity was measured. This screening method has a long cycle and heavy workload, which reduces the probability of obtaining high-yielding mutants.

Method used

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  • Bacillus subtilis heterologous protein expression quantity improving method
  • Bacillus subtilis heterologous protein expression quantity improving method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Mutagenesis of B. subtilis 168 by atmospheric pressure and room temperature plasma

[0024] Streak and activate the starting strain recombinant B. subtilis 168 on a fresh LB plate, pick a single colony in fresh LB liquid medium, culture in a 250mL Erlenmeyer flask, the working concentration of kanamycin is 50μg / mL, and the rotation speed 200rpm. After the cultivation, transfer to fresh LB liquid medium. After 2-4 hours, when the bacteria are in a state of synchronous growth, absorb the bacteria liquid, wash with sterile saline after centrifugation, and dilute the cell concentration appropriately. Aspirate the bacterial solution and apply it evenly on the sterile slide. The breeding system uses He as the working gas, the radio frequency power is set to 100W, the gas flow rate is 10slm, and the processing time is 30s.

[0025] Carry out post-cultivation operation on the bacteria after ARTP mutagenesis, put the mutagenized bacteria (together with the slide) in...

Embodiment 2

[0026] Embodiment 2: the screening method of B.subtilis 168 bacterial strain with high alkaline amylase production

[0027] Starch-Trypan Blue Nutrient Plate Screening

[0028] The bacteria solution after mutagenesis treatment was serially diluted to 10 after post-cultivation -4 , and then dilute the undiluted bacterial solution to 10 -1 、10 -2 、10 -3 、10 -4 These 5 gradients were applied to 3 LB plates respectively, and the recombinant B.subtilis 168 mutant library was obtained after culture. The single colonies on the plate were planted on the starch-trypan blue nutrition plate one by one with a sterile toothpick, and selected after culture. The transparent circle is larger than the colony of the starting bacteria.

[0029] 96-well high-throughput screening

[0030] The colonies selected in the above step (1) were respectively inoculated in the seed medium (LB liquid medium), and then transferred to the medium in a 96-well plate for high-throughput screening, and each ...

Embodiment 3

[0033] Example 3: Verification of the genetic stability of the alkaline amylase high-yield mutant strain B.subtilis 168 mut-16#

[0034] Put the high-yielding mutant strain B.subtilis 168 mut-16# stored in glycerol tubes on a solid LB plate, streak and activate it, pick a single colony and inoculate it in fresh liquid LB medium for culture, then transfer to another bottle of fresh LB In the culture medium, cultured under the same conditions for 12 hours, after repeated transfer for 5 times, inoculated into the fermentation medium and fermented, the enzyme activity of alkaline amylase was measured by the DNS method. The results showed that the high-yielding mutant strain B. subtilis 168-mut16 had a 30.1% higher enzyme-producing ability than the original strain, indicating that the mutant strain obtained by this method had good genetic stability.

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Abstract

The present invention provides a bacillus subtilis heterologous protein expression quantity improving method. Recombinant plasmid-contained B.s ubtilis is directly induced for mutation by ARTP and starch-trypan blue panel prescreening, 96 orifice plate high-throughput screening and shaking flask fermentation test to obtain a high enzyme activity mutant strain, and the enzyme activity is increased by 31.4%. The ARTP technology used in the method is a biological mutagenesis technology very wide in application range; during induced mutation, the recombinant plasmid-contained B.s ubtilis strain is directly induced for mutation, the recombinant plasmid conversion step can be eliminated after the induced mutation, efficiency is high; at the same time, and by combination with a 96 orifice plate for high-throughput screening, the efficiency for screening of direct mutation strains can be greatly improved, so that the method can rapidly screen a heterologous protein high yield B.s ubtilis strain, and has important economic value.

Description

technical field [0001] The invention relates to the fields of microbial mutation breeding, fermentation engineering and enzymology. In particular, it relates to a method for increasing the expression level of exogenous protein of Bacillus subtilis in combination with ARTP mutagenesis breeding. Background technique [0002] B. subtilis is a spore-forming Gram-positive bacterium with a clear genetic background, good protein secretion ability and a series of high-efficiency signal peptides. Compared with the commonly used prokaryotic expression host E.coli, B.subtilis does not contain endotoxin and is non-pathogenic. It is a food-grade microorganism that can be used for food fermentation and the production of important enzyme preparations. In addition, B. subtilis has long been used to produce protease and amylase, and the culture conditions required for it are very simple. Therefore, B. subtilis is an ideal host for secreting and expressing foreign proteins in the current pr...

Claims

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Application Information

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IPC IPC(8): C12N13/00C12N15/01C12N9/26C12R1/125
Inventor 杨海泉沈微马樱芳陈献忠马道程陈坚堵国成樊游刘龙
Owner JIANGNAN UNIV
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