or very few colonies on 7H10-H, compared with 7H10-Gm.
Occasionally both the Gm and Hyg complementing vectors
will both be taken up—these mutants will grow on both the
7H10-H and 7H10-Gm plates, and also the 7H10-HGm.
Therefore, to confirm if switching has occurred colonies
should be taken from the 7H10-H plate and patched in duplicate onto 7H10-H and 7H10-Gm, to identify if the double
plasmid is present. Only hygromycin-resistant, gentamicinsensitive colonies represent successful switching (see Notes
14 and 15).
4 Notes
1. The method described here is based on use of a single crossover strain which has been generated by homologous recombination using a suicide vector carrying an unmarked deletion of
the gene with approximately 1 kb of flanking DNA (as described by Parish & Stoker, 2000) [3]. Using this method the
suicide vector contains hygromycin and kanamycin resistance
genes, plus lacZ and sacB, (which confers sucrose sensitivity).
The method can be adapted for use with SCOs made using
different vectors/methods, however since antibiotic resistance
and sucrose sensitivity are dependent on the vectors used to
construct the SCO, the selection/screening plates will need to
be changed as appropriate for the system employed.
2. If not using a vector with a promoter, e.g., pAPA3 or pHAPY1,
the natural prompter sequence of the gene will need to be
included. The addition of PacI sites (5
0
. . .TTAATTAA. . .3
0 )
to the 5
0 end of primers facilitates cloning into pAPA3/
pHAPY1, this restriction site is absent from the M. tuberculosis
genome.
3. Typical PCR reaction mixes contain: 5μL 10Â Pfu Buffer, 5μL
DMSO, 2.5μL forward primer, 2.5μL reverse primer (both at
10 pmol/μL), 1μL dNTP mix (10 mM of each dNTP), 0.5μL
Pfu, 10–100 ng DNA, and SDW to 50μL total volume. Typical
PCR cycling parameters are 94
C for 2 min, followed by
35 cycles of 94
C for 30 s, annealing of typically 2–4
C
lower than lowest primer T m for 1 min, and extension of
72
C for 1 min per kb of amplified product. We have found
that Pfu works well with GC-rich mycobacterial DNA, however
there are many other proof-reading polymerases available
which may also be suitable.
4. The products from proof-reading polymerase PCR reactions
typically are blunt ended and therefore require A-tailing prior
to introduction into T-A cloning vectors.
Gene Switching and Essentiality Testing
297
Occasionally both the Gm and Hyg complementing vectors
will both be taken up—these mutants will grow on both the
7H10-H and 7H10-Gm plates, and also the 7H10-HGm.
Therefore, to confirm if switching has occurred colonies
should be taken from the 7H10-H plate and patched in duplicate onto 7H10-H and 7H10-Gm, to identify if the double
plasmid is present. Only hygromycin-resistant, gentamicinsensitive colonies represent successful switching (see Notes
14 and 15).
4 Notes
1. The method described here is based on use of a single crossover strain which has been generated by homologous recombination using a suicide vector carrying an unmarked deletion of
the gene with approximately 1 kb of flanking DNA (as described by Parish & Stoker, 2000) [3]. Using this method the
suicide vector contains hygromycin and kanamycin resistance
genes, plus lacZ and sacB, (which confers sucrose sensitivity).
The method can be adapted for use with SCOs made using
different vectors/methods, however since antibiotic resistance
and sucrose sensitivity are dependent on the vectors used to
construct the SCO, the selection/screening plates will need to
be changed as appropriate for the system employed.
2. If not using a vector with a promoter, e.g., pAPA3 or pHAPY1,
the natural prompter sequence of the gene will need to be
included. The addition of PacI sites (5
0
. . .TTAATTAA. . .3
0 )
to the 5
0 end of primers facilitates cloning into pAPA3/
pHAPY1, this restriction site is absent from the M. tuberculosis
genome.
3. Typical PCR reaction mixes contain: 5μL 10Â Pfu Buffer, 5μL
DMSO, 2.5μL forward primer, 2.5μL reverse primer (both at
10 pmol/μL), 1μL dNTP mix (10 mM of each dNTP), 0.5μL
Pfu, 10–100 ng DNA, and SDW to 50μL total volume. Typical
PCR cycling parameters are 94
C for 2 min, followed by
35 cycles of 94
C for 30 s, annealing of typically 2–4
C
lower than lowest primer T m for 1 min, and extension of
72
C for 1 min per kb of amplified product. We have found
that Pfu works well with GC-rich mycobacterial DNA, however
there are many other proof-reading polymerases available
which may also be suitable.
4. The products from proof-reading polymerase PCR reactions
typically are blunt ended and therefore require A-tailing prior
to introduction into T-A cloning vectors.
Gene Switching and Essentiality Testing
297