Red/ET recombination method for luminous light shaped bacillus

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.

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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