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Chemical transferring method for transferring hpalXoo gene into brevibacillus brevis HAB-5

A technology of Bacillus brevis and HAB-5, applied in the biological field, can solve problems such as low transformation efficiency, and achieve the effects of high transformation efficiency, efficient and stable expression, and easy operation.

Inactive Publication Date: 2015-07-08
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Peng Qingzhong also used this method to try to transform 5 strains of wild Brevibacillus strains, only No. 50 and No. 735 were successful, and the transformation efficiency was very low (the highest transformation rate was 102 transformants / ugDNA).

Method used

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  • Chemical transferring method for transferring hpalXoo gene into brevibacillus brevis HAB-5
  • Chemical transferring method for transferring hpalXoo gene into brevibacillus brevis HAB-5
  • Chemical transferring method for transferring hpalXoo gene into brevibacillus brevis HAB-5

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] (1) Activate Bacillus brevis HAB-5 on the LB plate, pick a single colony and inoculate it in 2mL LB liquid medium, and culture at 28°C for 12-16 hours with shaking at 160rpm;

[0038] (2) Transfer 2ml of HAB-5 bacterial solution to 50ml of MD medium, shake at 160rpm at 28°C and incubate for 4h;

[0039] (3) Centrifuge at 8000rpm for 10min to collect the cells, pour off part of the supernatant, and leave 5ml of suspended cells to obtain the HAB-5 suspended cells solution;

[0040] (4) Add HAB-5 suspended bacteria solution hpa1 Xoo get the mixed bacteria solution, hpa1 Xoo The amount of added is 2.5 ug; 28 ° C vigorous shaking 200rpm culture 2h;

[0041] (5) Add 10 times the volume of the mixed bacterial solution in LB, and shake vigorously at 200 rpm at 28°C to recover for 2 hours;

[0042] (6) After centrifuging at 8000rpm for 10min, leave a small amount of supernatant to suspend the bacteria, which contains hpa1 Xoo Gene HAB-5;

Embodiment 2

[0044] (1) Activate Bacillus brevis HAB-5 on the LB plate, pick a single colony and inoculate it in 2mL LB liquid medium, and culture at 27°C for 12-16 hours with shaking at 180rpm;

[0045] (2) Transfer 2ml of HAB-5 bacterial solution to 50ml of MD medium, shake at 180rpm at 27°C and incubate for 3h;

[0046] (3) Centrifuge at 10,000rpm for 8 minutes to collect the cells, pour off part of the supernatant, and leave 5ml of suspended cells to obtain the HAB-5 suspended cell solution;

[0047] (4) Add HAB-5 suspended bacteria solution hpa1 Xoo get the mixed bacteria solution, hpa1 Xoo 3 ug was added; cultured at 27°C with vigorous shaking at 250rpm for 3 h;

[0048] (5) Add LB which is 10 times the volume of the mixed bacterial solution, and shake vigorously at 250 rpm at 27°C to recover for 3 h;

[0049] (6) After centrifuging at 10000rpm for 10 min, leave a small amount of supernatant to suspend the bacteria, which contains hpa1 Xoo Gene HAB-5;

Embodiment 3

[0051] (1) Activate Bacillus brevis HAB-5 on the LB plate, pick a single colony and inoculate it in 2mL LB liquid medium, and culture at 29°C for 12-16 hours with shaking at 180rpm;

[0052] (2) Transfer 2ml of HAB-5 bacterial solution to 50ml of MD medium, shake at 180rpm at 29°C and incubate for 5 hours;

[0053] (3) Centrifuge at 10,000rpm for 12 minutes to collect the cells, pour off part of the supernatant, and leave 5ml of suspended cells to obtain the HAB-5 suspended cell solution;

[0054] (4) Add HAB-5 suspended bacteria solution hpa1 Xoo get the mixed bacteria solution, hpa1 Xoo The amount of added is 4 ug; 29 ° C vigorous shaking 200rpm culture 2h;

[0055] (5) Add LB which is 10 times the volume of the mixed bacterial solution, and shake vigorously at 200 rpm at 29°C to resume cultivation for 2.5 h;

[0056] (6) After centrifuging at 8000rpm for 12min, leave a small amount of supernatant to suspend the bacteria, which contains hpa1 Xoo Gene HAB-5.

[005...

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Abstract

The present invention relates to biological technical field, and provides a chemical transferring method for transferring hpalXoo gene into brevibacillus brevis HAB-5, and the transferred hpalXoo gene can stably and efficiently express in the brevibacillus brevis. The chemical transferring method for transferring hpalXoo gene into brevibacillus brevis HAB-5 has the advantages of simple operation, low nutritional requirements, no special equipment requirements, short time consumption, good repeatability, and high conversion efficiency.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a hpa1 Xoo A chemical transformation method for gene transfer into Bacillus brevis HAB-5. Background technique [0002] It is undoubtedly of great theoretical and practical significance to transform wild-type strains in a targeted manner to make them suitable for biological control of different environments and targets. Therefore, in recent years, people have been keen to use the means of molecular biology and molecular genetics to genetically improve biocontrol bacteria, improve their adaptability to environmental conditions, and expand the scope of application. Bacillus subtilis is a more mature strain in the Bacillus expression system, but because of its strong extracellular protease activity, it will degrade a large amount of foreign proteins, so it has great limitations in the construction of engineering biocontrol strains; Bacillus thuringiensis Although the activity of extr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/87C12N15/31C12R1/01
Inventor 缪卫国汪惠刘文波政服丛
Owner HAINAN UNIVERSITY
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