and Mycobacterium smegmatis [5]. A number of plasmids carrying
different selectable markers have been used, including resistance to
kanamycin, hygromycin, gentamicin, apramycin, although transformation efficiencies in different species can vary (Table 1) [1, 3–6, 9–
16]. Selection markers should be chosen carefully due to the natural
resistance of mycobacteria to many antibiotics and the requirement
to use stable drugs with a low frequency of spontaneous resistance.
Two commonly used selection markers are for kanamycin or
hygromycin resistance. Most slow-growers possess only one rrn
operon encoding ribosomal RNAs; this unusual situation means
that the resistance to agents such as kanamycin can easily arise
through modification of the target by spontaneous mutation in
the rrn operon itself [17]b. This is not likely to occur in fast
growers which contain two rrn operons. This can be an important
consideration for approaches in which a low transformation efficiency is expected, for example where suicide vectors are used as
substrates for homologous recombination [18].
This chapter presents methods for introducing DNA into a
representative slow-growing species, M. tuberculosis, and a representative fast-growing species, M. smegmatis. Other mycobacteria
can be transformed using variations of these methods, although the
efficiency of transformation will vary.
2 Materials
2.1 Electroporation
of M. tuberculosis
1. Mycobacterium tuberculosis strain (see Note 1).
2. Tween-80: Prepare as a 10% w/v stock, filter-sterilize through
a 0.2-μm membrane and store at 4
C (see Note 2).
3. 7H9-OADC-Tw: Dissolve 4.7 g of Middlebrook 7H9 base
(Difco) in 900 mL deionized water. Add 100 mL Middlebrook
OADC enrichment (Becton Dickinson), containing oleic acid,
bovine albumin fraction V, dextrose, catalase, and NaCl. Add
5 mL of 10% w/v Tween-80 (see item 2). Sterilize by autoclaving
or filtering and store at 4
C for up to 1 week (see Notes 3 and 4).
4. 450-cm
2 roller bottles (Corning).
5. 2 M glycine, autoclave.
6. 10% w/v glycerol; sterilize by autoclaving or filtration.
7. Electroporation apparatus with pulse controller (see Note 5).
8. Electroporation cuvettes; 0.2-cm gap electrodes.
9. DNA solution: 0.2–1 mg/mL. This should be free from salts,
enzymes and other substances (see Note 6).
10. 7H10-OADC plates: Dissolve 9.5 g Middlebrook 7H10 agar
base in 450 mL and sterilize by autoclaving. Add 50 mL 10%
v/v OADC supplement. Add antibiotics if required. Pour
plates and use within 1 week (see Notes 3, 4 and 7).
274
Tanya Parish
different selectable markers have been used, including resistance to
kanamycin, hygromycin, gentamicin, apramycin, although transformation efficiencies in different species can vary (Table 1) [1, 3–6, 9–
16]. Selection markers should be chosen carefully due to the natural
resistance of mycobacteria to many antibiotics and the requirement
to use stable drugs with a low frequency of spontaneous resistance.
Two commonly used selection markers are for kanamycin or
hygromycin resistance. Most slow-growers possess only one rrn
operon encoding ribosomal RNAs; this unusual situation means
that the resistance to agents such as kanamycin can easily arise
through modification of the target by spontaneous mutation in
the rrn operon itself [17]b. This is not likely to occur in fast
growers which contain two rrn operons. This can be an important
consideration for approaches in which a low transformation efficiency is expected, for example where suicide vectors are used as
substrates for homologous recombination [18].
This chapter presents methods for introducing DNA into a
representative slow-growing species, M. tuberculosis, and a representative fast-growing species, M. smegmatis. Other mycobacteria
can be transformed using variations of these methods, although the
efficiency of transformation will vary.
2 Materials
2.1 Electroporation
of M. tuberculosis
1. Mycobacterium tuberculosis strain (see Note 1).
2. Tween-80: Prepare as a 10% w/v stock, filter-sterilize through
a 0.2-μm membrane and store at 4
C (see Note 2).
3. 7H9-OADC-Tw: Dissolve 4.7 g of Middlebrook 7H9 base
(Difco) in 900 mL deionized water. Add 100 mL Middlebrook
OADC enrichment (Becton Dickinson), containing oleic acid,
bovine albumin fraction V, dextrose, catalase, and NaCl. Add
5 mL of 10% w/v Tween-80 (see item 2). Sterilize by autoclaving
or filtering and store at 4
C for up to 1 week (see Notes 3 and 4).
4. 450-cm
2 roller bottles (Corning).
5. 2 M glycine, autoclave.
6. 10% w/v glycerol; sterilize by autoclaving or filtration.
7. Electroporation apparatus with pulse controller (see Note 5).
8. Electroporation cuvettes; 0.2-cm gap electrodes.
9. DNA solution: 0.2–1 mg/mL. This should be free from salts,
enzymes and other substances (see Note 6).
10. 7H10-OADC plates: Dissolve 9.5 g Middlebrook 7H10 agar
base in 450 mL and sterilize by autoclaving. Add 50 mL 10%
v/v OADC supplement. Add antibiotics if required. Pour
plates and use within 1 week (see Notes 3, 4 and 7).
274
Tanya Parish