is performed among the drug-resistant transformants, increasing
the frequency of SNP identification to up to 10% of the cell population [17]. With the use of an oligo that repairs an amber codoncontaining hygromycin-resistant marker in an integrated plasmid,
frequencies of SNP transfer can reach close to 20% of the cells
following electroporation, depending on the position of the target
in the chromosome and the timing of platings following outgrowth
of the electroporated cells (unpublished observations).
The second consideration is to recognize that once an oligo
containing a SNP anneals to the lagging strand template of the
replication fork, a base pair mismatch will be formed, which can be
recognized and repaired by the mismatch repair (MMR) system of
the host. Active MMR systems are known to inhibit oligo-mediated
mutagenesis in vivo [21–23]. It has recently been reported that
deletion of the MSMEG_4923 gene in Mycobacterium smegmatis
generates a strain with a mutagenic phenotype, as observed by
increased frequencies of spontaneous resistance to rifampicin
[24]. The protein expressed by MSMEG_4923 is homologous to
the EndoMS/NucS protein of actinobacterium Corynebacterium
glutamicum, which has been shown to recognize and cut dsDNA
species containing mismatched bases in vitro [25, 26]. However,
not all base pair mismatches are cut, as only substrates containing
G-G, T-T, and G-T mismatches are efficiently recognized and cut
in vitro. These same mismatches are the only ones recognized by
the NucS protein of M. smegmatis in vivo (Flores and Murphy,
unpublished observations). Thus, when designing oligos to transfer
SNPs to mycobacterial chromosomes by recombineering, it is
important to avoid creating these types of mismatches. Otherwise,
the SNP-encoded base in the oligo is specifically replaced at the
replication fork by MMR, resulting in the inability to transfer the
SNP to the chromosome. There are, however, steps that can be
taken in designing oligos to transfer SNPs containing these types
of mismatches, so as to avoid repair by the mycobacterial MMR
system, as described below in the SNP-transfer protocol.
The third consideration to improve the frequency of oligomediated recombineering is to design the oligo to carry a sitespecific recombination site, such as the attP site from the mycobacterial Bxb1 phage [27–29]. The presence of a large insertion in the
oligo (such as the 48 bp attP site) lowers the frequency of ssDNA
recombineering, but makes finding the recombinant a selectable
event. This selectivity comes about by virtue of a site-specific
recombination event between the nascent chromosomal attP site
and a coelectroporated nonreplicating plasmid that contains an
attB site and an antibiotic selection marker. In order to carry this
out, the cells need to express both RecT and Bxb1 Integrase from a
replicating plasmid. This process is known as Oligo-mediated
Recombineering followed by BxB1 Integrase Targeting (ORBIT)
[14]. Oligos, along with one of a set of attB-containing plasmids
304
Kenan C. Murphy
the frequency of SNP identification to up to 10% of the cell population [17]. With the use of an oligo that repairs an amber codoncontaining hygromycin-resistant marker in an integrated plasmid,
frequencies of SNP transfer can reach close to 20% of the cells
following electroporation, depending on the position of the target
in the chromosome and the timing of platings following outgrowth
of the electroporated cells (unpublished observations).
The second consideration is to recognize that once an oligo
containing a SNP anneals to the lagging strand template of the
replication fork, a base pair mismatch will be formed, which can be
recognized and repaired by the mismatch repair (MMR) system of
the host. Active MMR systems are known to inhibit oligo-mediated
mutagenesis in vivo [21–23]. It has recently been reported that
deletion of the MSMEG_4923 gene in Mycobacterium smegmatis
generates a strain with a mutagenic phenotype, as observed by
increased frequencies of spontaneous resistance to rifampicin
[24]. The protein expressed by MSMEG_4923 is homologous to
the EndoMS/NucS protein of actinobacterium Corynebacterium
glutamicum, which has been shown to recognize and cut dsDNA
species containing mismatched bases in vitro [25, 26]. However,
not all base pair mismatches are cut, as only substrates containing
G-G, T-T, and G-T mismatches are efficiently recognized and cut
in vitro. These same mismatches are the only ones recognized by
the NucS protein of M. smegmatis in vivo (Flores and Murphy,
unpublished observations). Thus, when designing oligos to transfer
SNPs to mycobacterial chromosomes by recombineering, it is
important to avoid creating these types of mismatches. Otherwise,
the SNP-encoded base in the oligo is specifically replaced at the
replication fork by MMR, resulting in the inability to transfer the
SNP to the chromosome. There are, however, steps that can be
taken in designing oligos to transfer SNPs containing these types
of mismatches, so as to avoid repair by the mycobacterial MMR
system, as described below in the SNP-transfer protocol.
The third consideration to improve the frequency of oligomediated recombineering is to design the oligo to carry a sitespecific recombination site, such as the attP site from the mycobacterial Bxb1 phage [27–29]. The presence of a large insertion in the
oligo (such as the 48 bp attP site) lowers the frequency of ssDNA
recombineering, but makes finding the recombinant a selectable
event. This selectivity comes about by virtue of a site-specific
recombination event between the nascent chromosomal attP site
and a coelectroporated nonreplicating plasmid that contains an
attB site and an antibiotic selection marker. In order to carry this
out, the cells need to express both RecT and Bxb1 Integrase from a
replicating plasmid. This process is known as Oligo-mediated
Recombineering followed by BxB1 Integrase Targeting (ORBIT)
[14]. Oligos, along with one of a set of attB-containing plasmids
304
Kenan C. Murphy