Chapter 14
Oligo-Mediated Recombineering and its Use for Making
SNPs, Knockouts, Insertions, and Fusions in Mycobacterium
tuberculosis
Kenan C. Murphy
Abstract
Phage recombination systems have been instrumental in the development of gene modification technologies for bacterial pathogens. In particular, the Che9 phage RecET system has been used successfully for over
10 years for making gene knockouts and fusions in Mycobacterium tuberculosis. This “recombineering”
technology typically uses linear dsDNA substrates that contain a drug-resistance marker flanked by (up to)
500 base pairs of DNA homologous to the target site. Less often employed in mycobacterial recombineering is the use of oligonucleotides, which require only the action of the RecT annealase to align oligos to
ssDNA regions of the replication fork, for subsequent incorporation into the chromosome. Despite the
higher frequency of such events relative to dsDNA-promoted recombineering, oligo-mediated changes
generally suffer from the disadvantage of not being selectable, thus making them harder to isolate. This
chapter discusses steps and methodologies that increase the frequencies of finding oligo-mediated events,
including the transfer of single nucleotide polymorphisms (SNPs) to mycobacterial chromosomes, and the
use of oligos in conjunction with the mycobacterial phage Bxb1 site-specific recombination system for the
easy generation of knockouts, insertion, and fusions, in a protocol known as ORBIT.
Key words Recombineering, Oligonucleotide, Gene replacement, Single nucleotide polymorphism,
Mycobacteriophage, ORBIT, Mycobacterium tuberculosis, Mycobacterium smegmatis
1 Introduction
The deletion or modification of mycobacterial genes is critical
for deciphering the mechanisms of M. tuberculosis pathogenesis
and immune evasion. Many genetic modification schemes for
M. tuberculosis have been developed over the last 25 years and
include transformation with nonreplicating vectors containing target gene sequences, which integrate into the bacterial chromosome
and are subsequently resolved in a way to promote a gene replacement event [1–6]. The low frequencies of transformation using
these techniques encouraged other schemes that use phage to
deliver recombination substrates. The specialized phage
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_14, © Springer Science+Business Media, LLC, part of Springer Nature 2021
301
Oligo-Mediated Recombineering and its Use for Making
SNPs, Knockouts, Insertions, and Fusions in Mycobacterium
tuberculosis
Kenan C. Murphy
Abstract
Phage recombination systems have been instrumental in the development of gene modification technologies for bacterial pathogens. In particular, the Che9 phage RecET system has been used successfully for over
10 years for making gene knockouts and fusions in Mycobacterium tuberculosis. This “recombineering”
technology typically uses linear dsDNA substrates that contain a drug-resistance marker flanked by (up to)
500 base pairs of DNA homologous to the target site. Less often employed in mycobacterial recombineering is the use of oligonucleotides, which require only the action of the RecT annealase to align oligos to
ssDNA regions of the replication fork, for subsequent incorporation into the chromosome. Despite the
higher frequency of such events relative to dsDNA-promoted recombineering, oligo-mediated changes
generally suffer from the disadvantage of not being selectable, thus making them harder to isolate. This
chapter discusses steps and methodologies that increase the frequencies of finding oligo-mediated events,
including the transfer of single nucleotide polymorphisms (SNPs) to mycobacterial chromosomes, and the
use of oligos in conjunction with the mycobacterial phage Bxb1 site-specific recombination system for the
easy generation of knockouts, insertion, and fusions, in a protocol known as ORBIT.
Key words Recombineering, Oligonucleotide, Gene replacement, Single nucleotide polymorphism,
Mycobacteriophage, ORBIT, Mycobacterium tuberculosis, Mycobacterium smegmatis
1 Introduction
The deletion or modification of mycobacterial genes is critical
for deciphering the mechanisms of M. tuberculosis pathogenesis
and immune evasion. Many genetic modification schemes for
M. tuberculosis have been developed over the last 25 years and
include transformation with nonreplicating vectors containing target gene sequences, which integrate into the bacterial chromosome
and are subsequently resolved in a way to promote a gene replacement event [1–6]. The low frequencies of transformation using
these techniques encouraged other schemes that use phage to
deliver recombination substrates. The specialized phage
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_14, © Springer Science+Business Media, LLC, part of Springer Nature 2021
301