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Myxobacteria specific plasmid and its use

A technology of plasmid and Myxococcus aurantiacus, which is applied in the direction of bacteria, application, and the use of vectors to introduce foreign genetic material, etc., and can solve the problems of inability to effectively identify and utilize heterologous hosts

Inactive Publication Date: 2007-04-11
SHANDONG UNIV +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Moreover, the promoter sequences in myxobacteria also have the characteristics of high GC%, which are often not effectively recognized and utilized by heterologous hosts.

Method used

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  • Myxobacteria specific plasmid and its use
  • Myxobacteria specific plasmid and its use

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1. Isolation and cultivation of Myxococcus fulvus 124B02 CCTCC M 206081

[0026] Prepare myxobacteria enrichment medium WCX [CaCl 2 0.15%, agar 1.5%, cycloheximide 0.5% (added after sterilization), KOH adjusted to pH 7.0, prepared with distilled water]. Spread live E. coli cells on solidified WCX plates as a substrate for myxobacteria. Soil samples collected from the Changchun field in Jilin, China were evenly sprinkled on the fungus. The fruiting body of myxococcus was picked by micromanipulation and purified to obtain pure strain 124B02.

Embodiment 2

[0027] Example 2. Extraction of circular plasmids from myxobacteria

[0028]Inoculate the myxobacteria to be detected in 3ml of CTT [Casitone 1%, MgSO 4 8mM, Tris HCl (pH7.6) 10mM, potassium phosphate (pH7.6) 1mM, pH7.6] or VY-2 (Baker yeast 0.5%, VB 12 0.5mg / L, CaCl 2 0.1%, MgSO 4 ·7H 2 O 0.05%, pH7.2) liquid medium, and cultured at 30°C with shaking at 200rpm, and grown for 60-72h. Collect the cells by centrifugation, add 500ul TE25S [sucrose 10.3%, Tris HCl (pH8.0) 25mM, EDTA (pH8.0) 25mM] and lysozyme (final concentration 2mg / ml), suspend the cells, and incubate at 37°C for 1h , mix by inverting from time to time, add 250ul alkali lysate (NaOH 0.3M, SDS 2%), invert and mix 4-6 times, incubate at 55°C for 30min, add 250ul water-saturated phenol / chloroform / isoamyl alcohol (25:24: 1) Invert and mix well, centrifuge to take the supernatant, add an equal volume of Tris-saturated phenol / chloroform / isoamyl alcohol (25:24:1) to extract protein, centrifuge to take the supern...

Embodiment 3

[0029] Example 3. Extraction of linear plasmids from myxobacteria

[0030] The myxobacteria to be detected were inoculated in 3ml of CTT or VY-2 liquid medium, cultured at 30°C with shaking at 200rpm, and grown for 60-72h. Collect the cells by centrifugation, add 500ul TE25S and lysozyme (final concentration 2mg / ml), suspend the cells, incubate at 37°C for 1h, mix by inversion from time to time, add SDS (final concentration 1%) and protein K (final concentration 50ug / ml ), incubate at 37°C for 1 h, add an equal volume of Tris saturated phenol / chloroform / isoamyl alcohol (25:24:1) to extract protein, centrifuge to get the supernatant, and reuse an equal volume of Tris saturated phenol / chloroform / isoamyl alcohol (25:24:1) extract the protein until there is no obvious protein layer at the interface. The supernatant was collected by centrifugation, 1 / 10 volume of 3M NaAc and an equal volume of isopropanol were added to precipitate DNA for 0.5-1 h, the supernatant was discarded by ...

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Abstract

The present invention discloses one Myxococcus fulvus 124B02 strain in the preservation number of CCTCC M 206081, and one plasmid, pMX1, originated from Myxococcus fulvus 124B02 and with the nucleotide sequence as shown in SEQ ID No. 1. The recombinant carrier or recombinant plasmid via reconstituting pMX1 plasmid may convert or conjugating myxobacteria, especially myxococcus, and is used in the genetic analysis and genetic engineering research of myxobacteria resource.

Description

technical field [0001] The present invention relates to a strain of Myxococcus aurantiacus, a proprietary plasmid derived from said Myxobacterium, a recombinant vector or recombinant plasmid containing the above-mentioned plasmid fragment, and a microorganism transformed or conjugated by the above-mentioned plasmid and its derivatives or the recombinant vector or recombinant plasmid And the application of these microorganisms, as well as the application of the plasmid and its derivatives, recombinant vector and recombinant plasmid. Background technique [0002] Myxobacteria (myxobacteria) is a class of Gram-negative single-celled rod-shaped bacteria, belonging to the delta branch of Proteobacteria, mainly found in soil, herbivore manure, bark and decaying trees everywhere, with complex multicellular Behavioral and morphogenetic processes are a class of higher prokaryotes. According to its morphological characteristics, it can be divided into two suborders, four families, an...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/31C12N15/63C12N1/21
Inventor 李越中覃重军赵静宜沈美娟夏志洁钟莉
Owner SHANDONG UNIV
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