Genetically engineered bacteria for producing resuscitation-promoting factor RpfE and application thereof

A technology of genetically engineered bacteria and factors, applied in the direction of genetic engineering, application, plant genetic improvement, etc., to achieve the effect of enhancing application

Active Publication Date: 2019-03-01
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, few studies have studied the recovery and growth promotion of Rpf protein from the perspective of environmental functional bacteria

Method used

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  • Genetically engineered bacteria for producing resuscitation-promoting factor RpfE and application thereof
  • Genetically engineered bacteria for producing resuscitation-promoting factor RpfE and application thereof
  • Genetically engineered bacteria for producing resuscitation-promoting factor RpfE and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Cloning of recovery-promoting factor genes and construction of genetically engineered bacteria

[0033] (1) Cultivation of Rhodococcus biphenyllivorans TG9 and extraction of its genomic DNA

[0034] Inoculate TG9 into LB medium, revive culture at 30°C.

[0035] LB medium formula: 10.0g tryptone, 5.0g yeast extract, 10.0g sodium chloride, add 1000mL of distilled water to dissolve, adjust pH=7, and sterilize under high temperature and high pressure.

[0036] Genomic DNA was extracted using a Gram-positive bacteria genomic DNA extraction kit, and the extracted DNA solution was frozen at -20°C.

[0037] (2) Cloning of recovery-promoting factor genes and construction of engineering strains

[0038] The genome of Rhodococcus biphenylphila TG9 contains the gene rpfE encoding the recovery promotion factor, and its nucleotide sequence is shown in SEQ.ID.NO.1. The rpfE gene is amplified from the genomic DNA of the above bacterial strains by PCR. According to the se...

Embodiment 2

[0045] Example 2: Construction, expression and purification of recovery-promoting factor genetically engineered bacteria

[0046]Use a plasmid extraction kit to extract the recombinant plasmid from the cloned strain in Example 1, transfer the extracted recombinant plasmid into Escherichia coli BL21(DE3), spread it on an LB plate containing 100 μg / mL ampicillin, and culture it overnight at 37°C , pick positive clones, and shake culture overnight at 37°C in LB medium containing 100 μg / mL ampicillin. The successful construction of the expression strain was verified by PCR amplification and agarose gel electrophoresis. The strain has been preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee, namely, the genetically engineered strain producing recovery-promoting factor RpfE with the preservation number CGMCC NO.16351.

[0047] Inoculate the successfully verified bacterial solution into 200mL of the same resistance medium at ...

Embodiment 3

[0050] Embodiment 3: the promoting effect of recovery promoting factor on microbial growth and organic matter degradation performance

[0051] (1) Preparation of TG9 bacterial suspension

[0052] TG9 was inoculated into LB culture medium, and cultured with shaking at 30°C for 24 hours was used as seed culture medium. The seed solution was inoculated into a certain volume of LB liquid medium according to the inoculation amount of 1% (v / v), and cultured with shaking at 30°C until the logarithmic growth phase (OD 600 =0.98), after getting the bacteria solution at this time and centrifuging, wash twice with 0.85% sterilized normal saline, add sterilized inorganic salt culture solution, adjust the bacterium biomass OD 600 = about 1.00, the TG9 bacterial suspension was obtained.

[0053] Inorganic salt medium formula: KH 2 PO 4 1g, K 2 HPO 4 ·3H 2 O 3g, MgSO 4 0.2g, FeSO 4 ·7H 2 O 0.02g, NaCl 1g, (NH 4 ) 2 SO 4 0.5g, CaCl 2 0.01g, trace salt solution (mg / L: MoO 3 ...

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Abstract

The invention discloses genetically engineered bacteria for producing resuscitation-promoting factor RpfE and application thereof. Exogenous resuscitation-promoting factor rpfE gene is introduced intoa host cell, and the resuscitation-promoting factor is expressed to realize preparation of the resuscitation-promoting factor RpfE by applying engineering bacteria. The produced resuscitation-promoting factor RpfE can be applied to the development of dormant-state microbial resources in an environmental sample and to the promotion of microbial growth so as to improve the degradation efficiency ofenvironmental pollutants by microorganisms.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to a genetically engineered bacterium producing resuscitation promoting factor (Resuscitation Promoting Factor, Rpf) RpfE and a method for rapidly preparing RpfE protein by using the genetically engineered bacterium, in particular to the application of recombinant proteins. Background technique [0002] Resuscitation promoting factor (Resuscitation Promoting Factor, Rpf) is a type of bacterial autocrine or paracrine protein that can promote the recovery and growth of dormant bacteria at picomolar concentrations. This factor was originally found in Micrococcus luteus. In addition to promoting the resurrection and growth of dormant bacteria, this factor is also effective on normal growing bacteria. The mechanism may be involved in intercellular signal transduction. The domain prediction and nuclear magnetic resonance analysis of the protein showed that it has a highly conserved domain co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/31C12N15/70C07K14/195C07K1/22A62D3/02C12R1/19A62D101/20
CPCA62D3/02A62D2101/20C07K14/195
Inventor 沈超峰贾洋洋叶哲万吉星秦智慧李鸿炫王慧苏晓梅
Owner ZHEJIANG UNIV
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