are coelectroporated into mycobacteria to construct insertions,
deletions, and fusions of chromosomal target genes. A diagram of
how this process takes place is shown in Fig. 1b; steps to carry out
this procedure are described in the ORBIT protocol below.
2 Materials
2.1 Preparation
of Recombinogenic/
Electrocompetent
M. tuberculosis Cells
for SNP Transfer
1. pKM402: This plasmid expresses phage Che9c RecT from the
inducible P tet promoter; it is Kan
R (addgene.com).
2. pKM427: This plasmid is an L5 integrating vector that contains a defective hygromycin phosphotransferase gene by virtue
of an early termination codon (addgene.com). During SNP
transfer, this plasmid serves as a target for a selectable oligo
(Hyg
repair
) that is coelectroporated with the SNP-containing
oligo. The plasmid confers resistance to zeocin.
3. 20% v/v Tween 80: Mix 20 mL Tween 80 with 80 mL of
distilled water, stir the solution at 50
C to dissolve the
Tween 80, cool to RT, and filter sterilize through 0.2 μm filter.
Store at 4
C for 3 months.
4. 7H9-OADC-Tween 80: Dissolve 4.7 g of Middlebrook 7H9
powder and 2 mL glycerol in 900 mL deionized water and
autoclave. Add 100 mL of Middlebrook OADC enrichment
supplement (oleic acid, albumin, dextrose, and catalase) and
2.5 mL of 20% w/v Tween 80 to 900 mL of 7H9 broth, filter
sterilize through 0.2 μm membrane filter. Store at 4
C for
2 months.
5. 7H10 plates: Add 19 g of Middlebrook 7H10 powder and
5 mL glycerol to 900 mL of deionized water. Autoclave, cool
to 50
C, and add 100 mL of OADC. Mix and pour (30 mL per
plate).
6. 20 mg/mL Kanamycin (1000Â): Dissolve in deionized water
and filter sterilize. Store at 4
C for 6 months.
7. 25 mg/mL Zeocin (1000Â): Dilute in sterile water. Toxic:
handle with gloves.
8. 500 μg/mL Anhydrotetracycline (ATc) (1000Â): Dissolve ATc
in methanol. Store at À20
C in the dark.
9. Nalgene Inkwell square bottles (30 mL and 125 mL capacity).
10. 2 M glycine: Dissolve 150.14 g in 1 L distilled water; filter
sterilize through 0.2 μm membrane filter. Store at room temperature for 6 months.
11. PBS-Tween 80: Add 2.5 mL of 20% (w/v) Tween 80 to 1 L
PBS. Filter sterilize.
12. 10% (v/v) glycerol: Mix 100 mL glycerol with 900 mL distilled
water and autoclave.
Oligo-Mediated Recombineering in Mycobacteria
305
deletions, and fusions of chromosomal target genes. A diagram of
how this process takes place is shown in Fig. 1b; steps to carry out
this procedure are described in the ORBIT protocol below.
2 Materials
2.1 Preparation
of Recombinogenic/
Electrocompetent
M. tuberculosis Cells
for SNP Transfer
1. pKM402: This plasmid expresses phage Che9c RecT from the
inducible P tet promoter; it is Kan
R (addgene.com).
2. pKM427: This plasmid is an L5 integrating vector that contains a defective hygromycin phosphotransferase gene by virtue
of an early termination codon (addgene.com). During SNP
transfer, this plasmid serves as a target for a selectable oligo
(Hyg
repair
) that is coelectroporated with the SNP-containing
oligo. The plasmid confers resistance to zeocin.
3. 20% v/v Tween 80: Mix 20 mL Tween 80 with 80 mL of
distilled water, stir the solution at 50
C to dissolve the
Tween 80, cool to RT, and filter sterilize through 0.2 μm filter.
Store at 4
C for 3 months.
4. 7H9-OADC-Tween 80: Dissolve 4.7 g of Middlebrook 7H9
powder and 2 mL glycerol in 900 mL deionized water and
autoclave. Add 100 mL of Middlebrook OADC enrichment
supplement (oleic acid, albumin, dextrose, and catalase) and
2.5 mL of 20% w/v Tween 80 to 900 mL of 7H9 broth, filter
sterilize through 0.2 μm membrane filter. Store at 4
C for
2 months.
5. 7H10 plates: Add 19 g of Middlebrook 7H10 powder and
5 mL glycerol to 900 mL of deionized water. Autoclave, cool
to 50
C, and add 100 mL of OADC. Mix and pour (30 mL per
plate).
6. 20 mg/mL Kanamycin (1000Â): Dissolve in deionized water
and filter sterilize. Store at 4
C for 6 months.
7. 25 mg/mL Zeocin (1000Â): Dilute in sterile water. Toxic:
handle with gloves.
8. 500 μg/mL Anhydrotetracycline (ATc) (1000Â): Dissolve ATc
in methanol. Store at À20
C in the dark.
9. Nalgene Inkwell square bottles (30 mL and 125 mL capacity).
10. 2 M glycine: Dissolve 150.14 g in 1 L distilled water; filter
sterilize through 0.2 μm membrane filter. Store at room temperature for 6 months.
11. PBS-Tween 80: Add 2.5 mL of 20% (w/v) Tween 80 to 1 L
PBS. Filter sterilize.
12. 10% (v/v) glycerol: Mix 100 mL glycerol with 900 mL distilled
water and autoclave.
Oligo-Mediated Recombineering in Mycobacteria
305