3 Methods
3.1 Preparation
of Recombinogenic/
Electrocompetent
M. tuberculosis Cells
for SNP Transfer
1. Add 0.5 mL of a glycerol stock of H37Rv carrying both the
pKM402 and pKM427 plasmids to 10 mL 7H9-OADCTween 80, 20 μg/mL kanamycin, and 25 μg/mL zeocin in a
30 mL inkwell bottle (see Note 3). All steps in this and in the
following protocols using M. tuberculosis should be carried out
in a Biosafety Level 3 (BSL3) laboratory, using institutional
guidelines.
2. Grow the culture with shaking at 37
C until it reaches saturation (OD 600 ~ 1.0).
3. Transfer an aliquot of the starting culture to 30 mL of
7H9-OADC-Tween 80 medium containing 20 μg/mL kanamycin and 25 μg/mL zeocin in a 125 mL inkwell bottle so that
the starting OD 600 is between 0.05 and 0.1.
4. Grow the culture with shaking at 37
C. The 30 mL culture
contains enough cells for ~7 electroporations (see Note 4).
5. When the culture reaches an OD 600 of 0.8–1.0 (3–4 days),
induce the expression of RecT by adding 30 μL of anhydrotetracycline (ATc) stock solution. Final concentration of ATc
should be 500 ng/mL.
6. Incubate with shaking at 37
C for ~8 h.
7. Add 3 mL of 2 M glycine to the culture and continue shaking at
37
C for an additional 16–20 h.
8. Collect the cells by centrifugation at 3500 Â g for 10 min at
20
C in a 50 mL conical tube.
9. At RT, add 2 mL of 10% glycerol to the pellet and gently
resuspend the cells by pipetting back and forth with a 1 mL
pipet tip.
10. Bring the volume of the culture up to 30 mL with 10%
glycerol.
11. Cap the tube securely and invert it a few times to promote
mixing.
12. Repeat the centrifugation and washing procedures (steps 8–
11).
13. Collect the cells by centrifugation and resuspend the pellet in
3 mL of 10% glycerol. The cells are now electrocompetent and
recombinogenic (see Note 5).
3.2 Electroporation
of Oligonucleotide
Substrates for SNP
Transfer
in M. tuberculosis
1. Design the oligonucleotide for SNP transfer. The oligo containing the SNP of interest should by 70 bases long, with
the 1–4 base changes in the middle of the oligo sequence.
The SNP-containing oligo sequence should correspond to
the lagging strand of the replication fork (see Note 6). Avoid
selection of base pair changes in the oligo that would create
Oligo-Mediated Recombineering in Mycobacteria
309
3.1 Preparation
of Recombinogenic/
Electrocompetent
M. tuberculosis Cells
for SNP Transfer
1. Add 0.5 mL of a glycerol stock of H37Rv carrying both the
pKM402 and pKM427 plasmids to 10 mL 7H9-OADCTween 80, 20 μg/mL kanamycin, and 25 μg/mL zeocin in a
30 mL inkwell bottle (see Note 3). All steps in this and in the
following protocols using M. tuberculosis should be carried out
in a Biosafety Level 3 (BSL3) laboratory, using institutional
guidelines.
2. Grow the culture with shaking at 37
C until it reaches saturation (OD 600 ~ 1.0).
3. Transfer an aliquot of the starting culture to 30 mL of
7H9-OADC-Tween 80 medium containing 20 μg/mL kanamycin and 25 μg/mL zeocin in a 125 mL inkwell bottle so that
the starting OD 600 is between 0.05 and 0.1.
4. Grow the culture with shaking at 37
C. The 30 mL culture
contains enough cells for ~7 electroporations (see Note 4).
5. When the culture reaches an OD 600 of 0.8–1.0 (3–4 days),
induce the expression of RecT by adding 30 μL of anhydrotetracycline (ATc) stock solution. Final concentration of ATc
should be 500 ng/mL.
6. Incubate with shaking at 37
C for ~8 h.
7. Add 3 mL of 2 M glycine to the culture and continue shaking at
37
C for an additional 16–20 h.
8. Collect the cells by centrifugation at 3500 Â g for 10 min at
20
C in a 50 mL conical tube.
9. At RT, add 2 mL of 10% glycerol to the pellet and gently
resuspend the cells by pipetting back and forth with a 1 mL
pipet tip.
10. Bring the volume of the culture up to 30 mL with 10%
glycerol.
11. Cap the tube securely and invert it a few times to promote
mixing.
12. Repeat the centrifugation and washing procedures (steps 8–
11).
13. Collect the cells by centrifugation and resuspend the pellet in
3 mL of 10% glycerol. The cells are now electrocompetent and
recombinogenic (see Note 5).
3.2 Electroporation
of Oligonucleotide
Substrates for SNP
Transfer
in M. tuberculosis
1. Design the oligonucleotide for SNP transfer. The oligo containing the SNP of interest should by 70 bases long, with
the 1–4 base changes in the middle of the oligo sequence.
The SNP-containing oligo sequence should correspond to
the lagging strand of the replication fork (see Note 6). Avoid
selection of base pair changes in the oligo that would create
Oligo-Mediated Recombineering in Mycobacteria
309