Method for forming biomembrane by using burkholderia pyrrocinia

A technology of Burkholderia pyrrole and biofilm, applied in the field of forming biofilm of Burkholderia pyrrole, can solve the problem of difficult vitality and biofilm formation, complex medium composition, and difficulty in producing biofilm. and other problems, to achieve the effect of easy biofilm formation, strong stress resistance and short time consumption

Active Publication Date: 2020-01-07
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, it is difficult to produce biofilm, the composition of the medium is complicated, and it takes a long time. It is difficult to ensure that JK-SH007 has strong vitality

Method used

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  • Method for forming biomembrane by using burkholderia pyrrocinia
  • Method for forming biomembrane by using burkholderia pyrrocinia

Examples

Experimental program
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Effect test

Embodiment 1

[0026] A method for forming a biofilm by Burkholderia pyrrole, including the following steps:

[0027] 1) Take out the JK-SH007 glycerol bacteria stored in the refrigerator at -80℃, dip a small amount of glycerol bacteria with the inoculation loop, transfer it to the solid plate LB for activation, and place it in a dark incubator at 28℃ for 2 days to produce a single colony. It turns yellow, then use a picking needle to pick a single colony into the LB liquid medium, and purely culture it at 180r / min at 28°C for 24h to obtain a turbid bacterial suspension;

[0028] 2) Configure LB medium (10g / L tryptone, 5g / L yeast extract, 10g / L NaCl), TSB medium (15g / L tryptone, 5g / L soya peptone, 5g / L NaCl) , Test medium TMG (TSB medium + 1% glycerol + 25mM MgSO 4 );

[0029] The configuration method of TMG medium is as follows: add 15g tryptone, 5g soyapeptone, 5g NaCL in 500mL distilled water, stir well and dilute to 1L, then add 100uL glycerol, 6.16g MgSO 4 ·7H 2 O, stir well, sterilize at 121...

Embodiment 2

[0042] A method for the formation of biofilm by Burkholderia pyrrolei includes the following steps:

[0043] 1) Use LB medium to activate the JK-SH007 strain on a solid plate to obtain a single colony, pick a single colony into the LB liquid medium, and purely culture at 180r / min at 28°C for 24h to obtain a bacterial suspension;

[0044] 2) Configure TSB medium (15g / L tryptone, 5g / L soya peptone, 5g / L NaCl), test medium TMG (TSB medium + 1% glycerol + 25mM MgSO 4 ). Glucose medium (TSB medium + 1% glucose + 25mMMgSO 4 ).

[0045] 3) Inoculate the bacterial suspension 1% v / v into TMG liquid medium and glucose medium, add 3 mL per well to a 12-well cell culture plate, 180r / min, 28°C for 24 hours, take it out and let it stand 4- 6 days. Results: No biofilm formed in glucose medium; JK-SH007 formed visible biofilm in TMG liquid medium ( Figure 5 ).

[0046] Using crystal violet to fix the biofilm, semi-quantitatively determine the amount of biofilm.

[0047] Crystal violet staining meth...

Embodiment 3

[0049] A method for the formation of biofilm by Burkholderia pyrrolei includes the following steps:

[0050] 1) Use LB medium to activate the JK-SH007 strain on a solid plate to obtain a single colony, pick a single colony into the LB liquid medium, and purely culture at 180r / min at 28°C for 24h to obtain a bacterial suspension;

[0051] 2) Configure TSB medium (15g / L tryptone, 5g / L soya peptone, 5g / L NaCl), test medium TMG (TSB medium + 1% glycerol + 25mM MgSO 4 ). Iron ion medium (TSB medium + 1% glycerol + 25mMFeCl 3 ·6H 2 O).

[0052] 3) Inoculate the bacterial suspension 1% v / v into TMG liquid medium and iron ion medium, add 3 mL per well to a 12-well cell culture plate, 180r / min, 28℃ pure culture for 24h, take it out and let it stand for 4 -6 days. A static bacterial suspension was obtained. The result: the iron ion medium did not form a biofilm; the JK-SH007 in the TMG liquid medium formed a visible biofilm ( Figure 7 ).

[0053] Using crystal violet to fix the biofilm, sem...

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Abstract

The invention discloses a method for forming a biomembrane by using burkholderia pyrrocinia. The method comprises the following steps: 1) preparing single colonies from an LB solid slab activated burkholderia pyrrocinia JK-SH007 strain, selecting the single colonies, putting the single colonies into an LB liquid culture medium, and performing pure culture for 24 hours at 180r/min at 28 DEG C so asto obtain a bacterial suspension; 2) preparing a test culture medium TMG, and adding 1%v/v glycerinum and 25mM of MgSO4 into the TSB (trypticase soy broth) culture medium, wherein the pH value is 5-7; and 3) inoculating the 1%v/v bacterial suspension into the TMG liquid culture medium, performing pure culture for 24 hours at 180r/min at 28 DEG C, taking out the solution, and allowing the solutionto stand for 4-6 days, so as to form the biomembrane. By adopting the method, the JK-SH007 can be induced to form the culture medium TMG of the biomembrane, after culture with the culture medium, theJK-SH007 can form the biomembrane which is visible by naked eyes, and thus the JK-SH007 can survive well in an environment in nutrition deficiency.

Description

Technical field [0001] The invention belongs to the technical field of microbial culture, and specifically relates to a method for forming a biofilm by Burkholderia pyrrole. Background technique [0002] Burkholderia pyrrocinia (Burkholderia pyrrocinia) JK-SH007 is a highly effective gram-negative biocontrol bacterium of poplar, belonging to the Burkholderia cepacia complex (Bcc) genotype Ⅸ. Through the use of BCESM and cblA virulence gene specific PCR, onion (Allium cepa), tobacco (Nicotiana sp.) and alfalfa (Medicago sp.) models and other Bcc commonly used safety detection techniques have been preliminarily tested for its toxicity, indicating that Burkholderia pyrrocinia JK-SH007 is a potential safe biocontrol strain. The results of previous studies have shown that this bacteria has three pathogenic bacteria for poplar canker: Phomopsis macrospora, Cytospora chrysosperm, Fusicoccum aesculi has a strong antagonistic effect, and it can also promote plant growth by secreting som...

Claims

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Application Information

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IPC IPC(8): C12N1/20A01P3/00C12R1/01
CPCC12N1/20
Inventor 叶建仁付欢欢陈飞飞刘婉慧方雪琦王朝恩
Owner NANJING FORESTRY UNIV
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