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Construction method of pseudomonas fluorescens capable of producing siderophore at high yield

A Pseudomonas fluorescens, construction method technology, applied in the direction of microorganism-based methods, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of lack of high-efficiency production of strains, limited increase in siderophilic production, etc. Achieve the effect of avoiding harm to the environment and reducing drug resistance

Active Publication Date: 2020-07-28
ZHEJIANG GONGSHANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the strain lacks high-efficiency production, which limits the improvement of the production of siderophilic

Method used

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  • Construction method of pseudomonas fluorescens capable of producing siderophore at high yield
  • Construction method of pseudomonas fluorescens capable of producing siderophore at high yield

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1. Screening of Pseudomonas fluorescens PF08:

[0037] Eight strains of Pseudomonas fluorescens screened from spoiled tuna preserved in the laboratory were inoculated into LB liquid medium respectively, and vibrated at 30° C. and 200 rpm for 3 days to obtain the first fermentation broth.

[0038] According to the amount of 100 μL per well, the first fermentation liquid was poured on the CAS plate, placed at 30° C., cultured for 3 days, and the color change of the strain on the CAS plate was observed.

[0039] Select the strain with a large color circle on the CAS plate as the primary screening strain, inoculate it into LB medium, and cultivate it at 30°C and 200 rpm for 24 hours to obtain a seed solution. The seed solution was inoculated into 20 mL of KMB liquid medium at a volume fraction of 1%, and cultured at 30° C. and 200 rpm for 24 hours to obtain a second fermentation solution.

[0040]Then, 5 mL of the second fermentation liquid was evenly taken and centrifuged...

Embodiment 2

[0053] The construction of Pseudomonas fluorescens with high siderophilic production, the steps are as follows:

[0054] 1. Extract the genomic DNA of Pseudomonas fluorescens PF08.

[0055] Collect 1 mL of the overnight cultured bacterial solution PF08, centrifuge at 8000 rpm for 1 min at room temperature in a 1.5 mL centrifuge tube, discard the supernatant, and collect the bacteria. Genomic DNA was extracted using the Ezup Column Bacterial Genomic DNA Extraction Kit: add 180 μL Buffer Digestion to the bacteria, then add 20 μL Proteinase K solution, shake and mix well; incubate in a water bath at 56°C for 60 minutes to fully degrade the bacteria; Add 200 μL of Buffer BD and mix well; add 200 μL of absolute ethanol; put the adsorption column into the collection tube, use a pipette to add all the solution and translucent fibrous suspension into the adsorption column, let it stand for 2 minutes, and then 12000rpm at room temperature Centrifuge for 1 min, and discard the waste li...

Embodiment 3

[0064] The PF08 strain overexpressing the second messenger c-di-GMP (i.e. the pBBR1MCS-2-GGDEF: PF08 strain constructed in Example 2) and the wild-type PF08 strain were inoculated in LB and KMB culture solutions at 1% volume fraction, respectively, Cultivate for 24 hours at 30° C. and 200 rpm. Centrifuge the culture solution at 4°C and 10,000rpm for 15min, take 3mL of the supernatant and mix it with an equal volume of CAS solution, treat the mixture in the dark at room temperature for 1h, then measure the OD value at a wavelength of 630nm, by SU(%)=((Ar -As) / Ar)×100%, to calculate the siderophilic production.

[0065] The result is as figure 2As shown, the siderophilic content in the wild-type PF08 strain was 72.69%, while the siderophilic content in the c-di-GMP overexpressed PF08 strain was 87.2%. The second messenger c-di-GMP can promote the synthesis of siderophore no matter in the LB culture medium under the condition of iron richness or the KMB culture medium under th...

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Abstract

The invention relates to a construction method of pseudomonas fluorescens capable of producing siderophore at high yield, which comprises the following steps: extracting genomic DNA of pseudomonas fluorescens PF08, and carrying out PCR amplification on a GGDEF gene by using the extracted genomic DNA as a template; after being recovered, a PCR electrophoresis product being bonded with a vector pBBR1MCS-2 subjected to double digestion of HindIII and BamHI to obtain recombinant plasmids, transforming the recombinant plasmids into E.coLi WM3064 competent cells, and obtaining positive transformantsthrough screening; and transforming the recombinant plasmid in the positive transformant into pseudomonas fluorescens PF08 by conjugational transfer, and screening and verifying to obtain the pseudomonas fluorescens capable of producing siderophore at high yield. The constructed pseudomonas fluorescens can significantly increase the yield of siderophore, siderophore has antagonistic effects on common pathogenic bacteria and putrefying bacteria, and the strain can be used for food preservation and safety control.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a Pseudomonas fluorescens with high siderophilic production. Background technique [0002] Iron is an essential micronutrient for eukaryotes and prokaryotes. Iron plays an important role in metabolic processes such as amino acid synthesis, tricarboxylic acid (TCA) cycle activity, DNA replication, cellular respiration and electron transport. Iron is one of the most abundant chemical elements on Earth, but free bioavailable iron in solution is extremely limited due to its insolubility under physiological conditions and its ability to form iron oxyhydroxide precipitates (10 -8 -10 -9 M), well below the range that supports microbial growth (~10 -6 M). In order to use iron to maintain their own growth and complete various physiological functions, bacteria have evolved a series of systems that can chelate and absorb iron. Siderophore-mediated iron uptake is t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/31C12N15/78C12R1/39
CPCC07K14/21C12N15/78
Inventor 傅玲琳王彦波王飞飞孙志华周瑾茹陈剑
Owner ZHEJIANG GONGSHANG UNIVERSITY
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