Chapter 13
Gene Switching and Essentiality Testing
Amanda Claire Brown
Abstract
The identification of essential genes is of major importance to mycobacterial research, and a number of
essential genes have been identified in mycobacteria, however confirming essentiality is not straightforward,
as deletion of essential genes results in a lethal phenotype. In this chapter, protocols are described which can
be used to confirm gene essentiality using gene switching, following the construction of a strain carrying its
only functional copy on an integrated plasmid (Δ
0 int). Since deletion mutants cannot be created for
essential genes, a second gene copy is introduced via an integrating vector, which allows the chromosomal
gene copy to be deleted. The integrated vector can then be replaced using the gene switching method,
where no transformants are obtained, essentiality is confirmed. This technique can also be used to confirm
functionality of gene homologs and to easily identify essential operon members.
Key words Essentiality testing, Gene switching, Gene homologs
1 Introduction
Essential genes are those which are required for growth and development, and which cannot be deleted from the chromosome.
Determining gene essentiality is a powerful tool in understanding
the biology of any organism. Much of mycobacterial research
(especially in the case of the pathogenic mycobacteria), has focused
on the identification of essential genes and establishing their function, see Ehrt et al., 2015 for a review [1]. This is of importance
because it indicates the biological significance of the enzymes transcribed from these genes and can help us further understand their
function. Also, in the case of pathogens, it can identify possible
novel drug targets.
Homologous recombination methods, employing both plasmid and phage systems, to create both marked and unmarked
mycobacterial mutants have been established and have led to a
wealth of knowledge regarding the roles of individual gene within
the bacteria. Unmarked mycobacterial deletion mutants have been
created by homologous recombination using various systems [2–
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_13, © Springer Science+Business Media, LLC, part of Springer Nature 2021
285
Gene Switching and Essentiality Testing
Amanda Claire Brown
Abstract
The identification of essential genes is of major importance to mycobacterial research, and a number of
essential genes have been identified in mycobacteria, however confirming essentiality is not straightforward,
as deletion of essential genes results in a lethal phenotype. In this chapter, protocols are described which can
be used to confirm gene essentiality using gene switching, following the construction of a strain carrying its
only functional copy on an integrated plasmid (Δ
0 int). Since deletion mutants cannot be created for
essential genes, a second gene copy is introduced via an integrating vector, which allows the chromosomal
gene copy to be deleted. The integrated vector can then be replaced using the gene switching method,
where no transformants are obtained, essentiality is confirmed. This technique can also be used to confirm
functionality of gene homologs and to easily identify essential operon members.
Key words Essentiality testing, Gene switching, Gene homologs
1 Introduction
Essential genes are those which are required for growth and development, and which cannot be deleted from the chromosome.
Determining gene essentiality is a powerful tool in understanding
the biology of any organism. Much of mycobacterial research
(especially in the case of the pathogenic mycobacteria), has focused
on the identification of essential genes and establishing their function, see Ehrt et al., 2015 for a review [1]. This is of importance
because it indicates the biological significance of the enzymes transcribed from these genes and can help us further understand their
function. Also, in the case of pathogens, it can identify possible
novel drug targets.
Homologous recombination methods, employing both plasmid and phage systems, to create both marked and unmarked
mycobacterial mutants have been established and have led to a
wealth of knowledge regarding the roles of individual gene within
the bacteria. Unmarked mycobacterial deletion mutants have been
created by homologous recombination using various systems [2–
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_13, © Springer Science+Business Media, LLC, part of Springer Nature 2021
285