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Gene-deleted Aeromonas intermedius

A technology of Aeromonas and gene deletion, which is applied in the field of microorganisms, can solve the problems of lack of model organisms, and achieve the effect of simplifying operations and saving costs

Active Publication Date: 2021-05-18
CANVEST WUHAN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Due to the lack of suitable model organisms, the research on the synthesis and biological function of melanin is still in its infancy

Method used

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  • Gene-deleted Aeromonas intermedius
  • Gene-deleted Aeromonas intermedius
  • Gene-deleted Aeromonas intermedius

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Obtaining of Aeromonas intermediate WS (Aeromonas media WS) ΔkatE

[0049] 1. Construction of knockout vector

[0050] Using the WS genome as a template, amplify the upstream homology arm of the katE gene with the katE upstream forward primer shown in SEQ ID NO.1 and the katE upstream reverse primer shown in SEQ ID NO.2, using such as SEQ ID The katE downstream forward primer shown in NO.3 and the katE downstream reverse primer shown in SEQ ID NO.4 are amplified to obtain the downstream homology arm of katE gene, and the upstream homology arm is used restriction endonuclease SalI and Cut with XbaI, cut the downstream homology arms with restriction endonucleases XbaI and SacI, then cut the vector pDM4 with restriction endonucleases SalI and SacI at both ends, then connect the upper and downstream homology arms and the vector fragment with T4 Enzymes connect. The obtained ligation product was transformed into Escherichia coli competent, and the transformants ...

Embodiment 2

[0053] Embodiment 2: the growth curve of bacterial strain and melanin synthetic curve assay

[0054] Pick single colonies of Aeromonas intermedius WS and WSΔkatE, inoculate them into 5 mL of fresh liquid LB medium respectively, cultivate the activated cells at 30°C and 200 rpm overnight, and inoculate the above bacterial liquid with an inoculation amount of 1% (v / v). Transfer to a 250mL Erlenmeyer flask filled with 100mL liquid LB medium, culture at 30°C and 200rpm for 4h, start sampling every 2h until 48h, measure and record the OD 600 value and OD 400 value, in OD 600 Reflect its growth, in OD 400 The value represents its melanin synthesis yield. Taking culture time as abscissa, OD 600 Draw the growth curve as the ordinate, and take the culture time as the abscissa, OD 400 Plot the melanin synthesis curve for the abscissa and ordinate.

[0055] Its growth curve showed that WSΔkatE had no significant difference from wild-type WS growth curve, and its melanin synthesis c...

Embodiment 3

[0056] Embodiment 3: bacterial strain cultivates 12h-24h live bacterial count change

[0057] Pick single colonies of Aeromonas intermedius WS and WSΔkatE, inoculate them into 5 mL of fresh liquid LB medium respectively, cultivate the activated cells at 30°C and 200 rpm overnight, and inoculate the above bacterial liquid with an inoculation amount of 1% (v / v). Transfer to a 250mL Erlenmeyer flask filled with 100mL liquid LB medium, and cultivate on a shaker at 30°C at 200rpm. Using the plate colony counting method, the strains were cultured until 12h, 18h, and 24h, and the bacterial liquid was taken separately for gradient dilution, and three appropriate dilution gradients were selected, and 100μL was used to coat the plate with glass beads, and cultured at 30°C for 12h. Count the effective plates with 30 to 300 colonies, calculate the average number of colonies on three plates of the same dilution, and calculate according to the following formula: Colony forming unit (CFU) pe...

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Abstract

The invention discloses a gene deletion type Aeromonas intermedia, which lacks the katE gene compared with the Aeromonas intermedia WS genome. Compared with the wild strain WS, the gene-deleted Aeromonas intermedia provided by the present invention dies earlier and can maintain a complete cell shape, and is suitable as a model organism for studying the mechanism of melanin formation and as a bioreactor for producing melanin. As a bioreactor, it has a good prospect in industrialized production, does not need subsequent treatment, can save cost and simplify operation.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a genetically modified Aeromonas intermedius. Background technique [0002] Aeromonas media WS (Aeromonas media WS) is a high-yielding melanin strain isolated from Donghu Lake, which has the characteristics of strong pigment synthesis ability and short synthesis time. Melanin is usually formed by the polymerization of phenols or indoles, which is a class of hydrophobic biomacromolecules. It widely exists in animals, plants and microorganisms. Previous studies have shown that melanin can greatly improve the survival and competitiveness of organisms. Therefore, the ubiquity of melanin is considered to be the result of long-term mutual adaptation between life and the environment during the evolution process. According to different synthetic pathways, the melanin synthesized by bacteria is usually eumelanin and pyomelanin. Aeromonas intermedius WS mainly synthesizes pyomela...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12P1/04C12R1/01
CPCC07K14/195C12N15/74C12P1/04C12N1/205C12R2001/01
Inventor 王娇徐国东袁冰
Owner CANVEST WUHAN BIOTECH
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