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Related Experiment Video

Updated: Jul 18, 2026

Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria

Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria

Published on: December 8, 2023

Recombineering in Mycobacterium tuberculosis.

Julia C van Kessel¹, Graham F Hatfull

¹Pittsburgh Bacteriophage Institute and Department of Biological Sciences, 376 Crawford Hall, 4249 Fifth Ave., University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.

|December 21, 2006

Summary

Researchers developed a new recombineering system for Mycobacterium tuberculosis, enabling easier genetic manipulation. This method uses mycobacteriophage proteins to facilitate allelic exchange, simplifying the creation of gene replacement mutants.

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Published on: June 25, 2013

Area of Science:

Microbiology
Molecular Biology
Genetics

Background:

Genetic manipulation of Mycobacterium tuberculosis is challenging due to slow growth and high rates of illegitimate recombination.
Existing methods for homologous DNA exchange are often inefficient in mycobacteria.

Purpose of the Study:

To develop a facile allelic exchange system for Mycobacterium tuberculosis and other mycobacteria.
To adapt recombineering strategies from Escherichia coli for use in mycobacteria.

Main Methods:

Identification and expression of mycobacteriophage-encoded recombination proteins (RecE and RecT homologs).
Biochemical characterization of Che9c gp60 (exonuclease) and gp61 (DNA-binding).
Application of these proteins to facilitate allelic exchange in Mycobacterium smegmatis and Mycobacterium tuberculosis.

Main Results:

Expression of Che9c gp60 and gp61 significantly enhanced recombination.
The developed system facilitated allelic exchange in both slow-growing (M. tuberculosis) and fast-growing (M. smegmatis) mycobacteria.
This approach simplifies the construction of gene replacement mutants.

Conclusions:

Mycobacterial recombineering using phage-encoded proteins is an effective and simple method for genetic manipulation.
This system overcomes limitations associated with slow growth and recombination in mycobacteria.
Facilitates the generation of gene replacement mutants for further study of mycobacterial genetics.