Red/ET recombination method for luminous light shaped bacillus

A bacillus, recombinant plasmid technology, applied in the direction of recombinant DNA technology, fungi, etc., can solve the problem of not using luminous photobacteria

CN101173269AInactive Publication Date: 2008-05-07INST OF SUBTROPICAL AGRI CHINESE ACAD OF SCI
0 Cites 3 Cited by

Patent Information

Authority / Receiving Office
CN · China
Current Assignee / Owner
Publication Date
2008-05-07
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 technique used for the photobacterium. The recombinant plasmid pASK-Alpha Beta Gamma A is constructed as follows: to use the plasmid containing a Alpha Beta Gamma A gene as a template, to use a homologous arm with the ASK as a primer, to obtain the PCR product Alpha Beta Gamma A with the homologous arm through PCR amplification, to use pASK-IBA as a carrier, and to identify the plasmid using the Red / ET recombination to construct the plasmid pASK- Alpha Beta Gamma A. The red / ET recombination is as follows: firstly, to synthesize the primer with the homologous arm that needs modifying gene; secondly, to obtain a target gene with the homologous arm that needs modifying gene through PCR amplification; thirdly, to introduce the red / ET recombinant plasmid pASK- Alpha Beta Gamma A into photorhabdus luminescens; fourthly, to transfer the photorhabdus luminescens containing pASK- Alpha Beta A overnight into LB and incubate; fifthly, to add a dehydrated tetracycline to induce; sixthly, to transform the PCR product of the target gene with the homologous arm that needs modifying gene into pASK- Alpha Beta Gamma A; at last, to lay the photobacterium on the selective LB panel and incubate, and to remove the plasmid pASK- Alpha Beta Gamma A. The technique has advantages that the homologous sequence is short, recombination efficiency is high and the use is quick and convenient.
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

[0056] A specific implementation of the red / ET recombination method used in Photobacterium luminescent is as follows:

[0057] 1 material

[0058] 1.1 Strain and carrier

[0059] 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.

[0060] 1.2 Tool enzyme

[0061] RNaseA, Proteinase K, and restriction endonucleases were purchased from New England Biolabs...