Red/ET recombination method for luminous light shaped bacillus

A bacillus and recombinant plasmid technology, applied in the biological field, can solve the problems of unapplied luminous photobacteria

CN100558891CInactive Publication Date: 2009-11-11INST OF SUBTROPICAL AGRI CHINESE ACAD OF SCI
1 Cites 0 Cited by

Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Publication Date
2009-11-11
Estimated Expiration
Not applicable · inactive patent

Smart Images

  • Figure 1
    Figure 1
  • Figure 2
    Figure 2
  • Figure 3
    Figure 3
Patent Text Reader

Abstract

The invention discloses a red / ET recombination method for Photobacterium luminescent. The recombinant plasmid pASK-αβγA is constructed as follows: a plasmid containing αβγA gene is used as a template, and ASK homologous arms are used as primers to amplify by PCR The PCR product αβγA with homology arms was obtained, pASK-IBA was used as the vector, and the plasmid pASK-αβγA was constructed by Red / ET recombination, and the plasmid was identified. The red / ET recombination is as follows: firstly, primers with homology arms of the gene to be modified are synthesized; secondly, the target gene with the homology arm of the gene to be modified is obtained by PCR amplification; thirdly, the red / ET recombinant plasmid pASK-αβγA is introduced into photorhabdus luminescens; The fourth is to inoculate the overnight bacteria of photorhabdus luminescens containing pASK-αβγA in LB, and culture it; the fifth is to induce with anhydrotetracycline; the sixth is to transform the target gene PCR product with the homology arm of the modified gene into pASK-αβγA; finally Spread it on selective LB plates, culture, and remove plasmid pASK-αβγA. The method has short homologous sequences, high recombination efficiency, quickness and convenience.
Need to check novelty before this filing date? Find Prior Art

Description

technical field

[0001] The invention relates to the field of biotechnology, and more specifically relates to a red / ET recombination method for Photobacterium luminescent, which can be used for genetic modification of Photobacter luminescent. Background technique

[0002] The genus photorhabdus and Heterorhaditis nematodes live in symbiosis, the symbiotic bacteria parasitize in the intestinal tract of the nematode, the nematode carries the symbiotic bacteria into the body of the host insect, and releases the symbiotic bacteria into the hemocoel of the insect, and the symbiotic bacteria reproduce in the hemocoel of the insect , Produce insecticidal toxins, kill host insects within 48 hours, and produce antibacterial active substances to inhibit the pollution of other bacteria. Symbiotic bacteria have a broad antibacterial spectrum, and it has been reported that symbiotic bacteria can inhibit various bacteria, fungi, yeasts, etc. (including plant pathogenic bacteria and human p...

Examples

Embodiment Construction

[0054] A specific implementation of the red / ET recombination method used in Photobacterium luminescent is as follows:

[0055] 1 material

[0056] 1.1 Strain and carrier

[0057] photorhabdus luminescens DSM15138: purchased from the German Resource Center for Biological Material (ie the German Resource Center for Biological Material, DSMZ www.dsmz.de). E.coliYZ2005, pSC101-705-flp-cm r , pSC101-BAD-αβγA was purchased from the German gene brigdes company (www.genebrigdes.com). pASK-IBA was purchased from IBA GmbH (http: / / www.iba-go.de). E.coliYZ2005, pSC101-BAD-αβγA and Red / ET kits were purchased from the German gene brigdes company (www.genebrigdes.com), and pASK-IBA was purchased from IBA GmbH. pASK-αβγA is obtained by the present invention, its characteristics are shown in Table 2, and used for recombination of Photobacterium luminescent red / ET.

[0058] 1.2 Tool enzyme

[0059] RNaseA, Proteinase K, and restriction endonucleases were purchased from New England Biolabs...