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High throughput screening method for salt resistant mutation bacterial strain of colloid bacillus cereus

A technology of Bacillus colloidus and mutant strains, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of heavy screening workload, low screening efficiency, and no application, and achieve improvement of salinization , Improve screening efficiency and reduce screening workload

Inactive Publication Date: 2008-12-03
ZHEJIANG SCI-TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The screening of mutant strains is generally carried out on the culture plate with screening medium. This screening method has problems such as heavy screening workload and low screening efficiency.
The 96-well culture plate has played an important role in large-scale drug screening as a relatively large-throughput screening tool, but it has not been used in the breeding of excellent bacterial strains. The 96-well culture plate is applied to the screening of bacterial mutant strains , can effectively improve screening efficiency and reduce screening workload

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Nuclear radiation was used to induce the mutation of Bacillus glialis AS1.153, and the mutant strain with salt tolerance above 0.5% was screened. Glial bacillus was activated on a nitrogen-free medium, cultured at 30°C for 36 hours, then transferred to a liquid medium that inhibited capsule growth, cultured at 30°C for 16 hours, collected by centrifugation, suspended in normal saline or phosphate buffer cells, and adjust the cell concentration to 10 4 -10 5 cfu ml -1 . The bacterial suspension was treated with Co 60 For mutagenesis, the dose of mutagenesis is 5kGy. After the cells after mutagenesis are collected by centrifugation, they are suspended in liquid screening medium, and the concentration is adjusted to 10 3 -10 4 cfu ml -1 , shake well, distribute in 96-well culture plate, 150 μl per well, culture at 30°C for 5 days, use a spectrophotometer to detect the absorbance value of the mutagenized bacteria in the 96-well culture plate, and the absorbance in the...

Embodiment 2

[0031] Bacillus glialis KNP2 mutation was induced by nuclear radiation, and salt-tolerant mutant strains with a salt tolerance of more than 0.7% were screened. Activate Bacillus coliformis on nitrogen-free medium. After culturing at 32°C for 36 hours, transfer the colony to a liquid medium that inhibits capsule growth. After culturing at 32°C for 14 hours, collect the bacteria by centrifugation and wash with normal saline or phosphate buffer. Suspend the bacteria in the liquid, and adjust the concentration of the bacteria to 10 4 -10 5 cfu ml -1 . The bacterial suspension was treated with Co 60 For mutagenesis, the dose of mutagenesis is 3kGy. After the cells after mutagenesis are collected by centrifugation, they are suspended in liquid screening medium and the concentration is adjusted to 10 3 -10 4 cfu ml -1 , after shaking well, divide into 96-well culture plates, 100 μl per well, culture at 32°C for 5 days, use a spectrophotometer to detect the absorbance value of t...

Embodiment 3

[0040] Bacillus glialis KNP13 was induced to mutate by nuclear radiation, and salt-tolerant mutant strains with a salt tolerance of more than 1.0% were screened. Activate the strain KNP13 on nitrogen-free medium, culture at 28°C for 36 hours, transfer the colony to a liquid medium that inhibits capsule growth, culture at 28°C for 16 hours, collect the bacteria by centrifugation, and suspend with normal saline or phosphate buffer cells, and adjust the cell concentration to 10 4 -10 5 cfu ml -1 . The bacterial suspension was treated with Co 60 For mutagenesis, the dose of mutagenesis is 8kGy. After the mutated bacteria are collected by centrifugation, they are suspended in liquid screening medium, and the concentration is adjusted to 10 3 -10 4 cfu ml -1 , after shaking well, distribute in 96-well culture plate, 200μl per well, culture at 28°C for 7 days, use a spectrophotometer to detect the absorbance value of the mutagenized bacteria in the 96-well culture plate, and th...

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Abstract

The invention discloses a high-throughput screening method of a colloid bacillus salt tolerant mutant strain, which comprises the following steps: firstly, after being activated in nitrogenfree medium, colloid bacillus is placed in a liquid culture medium for restraining the growth of capsula, and cultured to the logarithmic growth phase, thallus is centrifuged and collected, physiological saline or phosphate buffer is used for suspending the thallus, and the concentration of the thallus is adjusted to 10<4>-10<5>cfu ml<-1>; secondly, Co<60> is used for inducing the thallus suspending liquid, and the amount of a mutagenic agent is 1-8kGy; the thallus suspending liquid after being induced is centrifuged to obtain the induced thallus; thirdly, after being diluted to 10<3>-10<4>cfu ml <-1> by using the liquid screening culture medium, the mutate thallus is cultured after being separately packaged into a culture plate with 96 holes for the high-throughput screening, after the secondary screening is performed, the salt tolerant mutant strain is obtained. In the invention, nuclear radiation inducement and the high-throughput screening method are used for obtaining the salt tolerant mutant strain of the colloid bacillus, so as to provide the strain for the rehabilitation of greenhouse soil.

Description

technical field [0001] The invention relates to a high-throughput screening method for a salt-tolerant mutant strain of Bacillus colloidus. Background technique [0002] Facility agriculture is the most ideal planting method for human beings to obtain a large amount of green agricultural products, and has become one of the most dynamic emerging industries in the world's agricultural development. At present, the area of ​​protected agriculture in the world has reached more than 4 million hm 2 , my country's 2006 facility agriculture area was 2.5 million hm 2 , is the world's largest facility agricultural country. Facility agriculture has become the most direct and effective way to increase agricultural efficiency and farmers' income. It plays an important role in off-season and cross-regional planting, and realizes the annual production and balanced supply of economic crops such as vegetables. Economic, social and ecological The benefits are considerable. However, due to ...

Claims

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Application Information

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IPC IPC(8): C12Q1/04
Inventor 胡秀芳陈集双竺锡武方琼楼吴金光
Owner ZHEJIANG SCI-TECH UNIV
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