Red/ET recombination method for luminous light shaped bacillus

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.

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...

AI Technical Summary

Problems solved by technology

A recombination method based on λ bacteriophage red / ET recombinase has been applied to the genetic engineering research of Escherichia coli, but it has never been applied to Luminescent bacteria

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