immediately stabbed with the loop on a fresh 7H10-OADCtween plate that is incubated at 37
C for subsequent recovery
of the candidates. The cell suspensions are then heat inactivated
and used in the PCR analysis (steps 6 and 7). The advantage
here is that the PCR analysis can be done right away (i.e., no
need to grow up cultures from all the picked colonies), though
positive SNP-containing candidates, one identified, will have
to be regrown from the stabs.
16. In lieu of using diagnostic PCRs to identify the
SNP-containing colonies, one can simply sequence PCRs
from the SNP-containing region of all 24 candidates. The
decision to use diagnostic PCRs or sequence 24 PCR products
(or more) containing the targeted region will depend on both
costs and the expected frequency of SNP transfer. What frequency should one expect the SNP be present among the Hyg
R
recombinants? This is a difficult question to answer, because
that frequency will depend on the recombinogenic potential of
the cells, unanticipated effects of the SNP on the growth of the
recombinant, and perhaps the position of the target gene in the
chromosome (the reasons of which are not quite clear). Generally, using pKM427 as a coselection target, the best estimate
is between 5% and 20% of the Hyg
R cells will also contain the
SNP of interest. However, lower rates of oligo cointegration
do occur, ranging from 2% to 5% [17] (and unpublished
observations). In these latter cases, if sequencing directly has
failed to isolate a SNP recombinant in the first round, it might
be best to use SNP-specific-PCR with pools of candidates to
find the SNP-containing recombinant.
17. For an ORBIT oligo designed to create a deletion, the oligo
should retain ~10 codons of the N-terminal and C-terminal
encoding regions of the target gene. This is important where
critical regulatory signals reside in these regions for expression
of neighboring genes. Also, by keeping the “residual” gene
sequences a multiple of 3, excision of the Orbit integration
plasmid (following construction of the knockout) will allow
formation of an in-frame deletion of the target gene (see Subheading 3.6).
18. For verification of a gene knockout, it is important to not only
verify the presence of both junctions of the cassette with the
chromosome, but also to verify the absence of the target gene.
Duplications can occur (in a minority of cases). If so, one could
find that a replacement takes place in one copy of the gene,
with the second copy of the gene remaining intact.
19. The pKM512 plasmid expresses both RecT and gp47, a directionality factor that promotes excision of the ORBIT integrating plasmid. It also contains sacB, allowing for curing the
recombinant of pKM512 on sucrose plates following excision
of the integrating plasmid.
Oligo-Mediated Recombineering in Mycobacteria
319
C for subsequent recovery
of the candidates. The cell suspensions are then heat inactivated
and used in the PCR analysis (steps 6 and 7). The advantage
here is that the PCR analysis can be done right away (i.e., no
need to grow up cultures from all the picked colonies), though
positive SNP-containing candidates, one identified, will have
to be regrown from the stabs.
16. In lieu of using diagnostic PCRs to identify the
SNP-containing colonies, one can simply sequence PCRs
from the SNP-containing region of all 24 candidates. The
decision to use diagnostic PCRs or sequence 24 PCR products
(or more) containing the targeted region will depend on both
costs and the expected frequency of SNP transfer. What frequency should one expect the SNP be present among the Hyg
R
recombinants? This is a difficult question to answer, because
that frequency will depend on the recombinogenic potential of
the cells, unanticipated effects of the SNP on the growth of the
recombinant, and perhaps the position of the target gene in the
chromosome (the reasons of which are not quite clear). Generally, using pKM427 as a coselection target, the best estimate
is between 5% and 20% of the Hyg
R cells will also contain the
SNP of interest. However, lower rates of oligo cointegration
do occur, ranging from 2% to 5% [17] (and unpublished
observations). In these latter cases, if sequencing directly has
failed to isolate a SNP recombinant in the first round, it might
be best to use SNP-specific-PCR with pools of candidates to
find the SNP-containing recombinant.
17. For an ORBIT oligo designed to create a deletion, the oligo
should retain ~10 codons of the N-terminal and C-terminal
encoding regions of the target gene. This is important where
critical regulatory signals reside in these regions for expression
of neighboring genes. Also, by keeping the “residual” gene
sequences a multiple of 3, excision of the Orbit integration
plasmid (following construction of the knockout) will allow
formation of an in-frame deletion of the target gene (see Subheading 3.6).
18. For verification of a gene knockout, it is important to not only
verify the presence of both junctions of the cassette with the
chromosome, but also to verify the absence of the target gene.
Duplications can occur (in a minority of cases). If so, one could
find that a replacement takes place in one copy of the gene,
with the second copy of the gene remaining intact.
19. The pKM512 plasmid expresses both RecT and gp47, a directionality factor that promotes excision of the ORBIT integrating plasmid. It also contains sacB, allowing for curing the
recombinant of pKM512 on sucrose plates following excision
of the integrating plasmid.
Oligo-Mediated Recombineering in Mycobacteria
319