8. At this stage, cells may be frozen and stored in aliquots at
À70
C for future use. Cells frozen in this way should be
thawed on ice and used as required (see Note 29).
9. Add 0.5 to 5 μg salt-free DNA in no more than 5 μL volume to
0.2 mL mycobacterial suspension and leave on ice for 10 min
(see Notes 6 and 18).
10. Mix the cells and transfer to a 0.2-cm electrode gap electroporation cuvette. The cuvette should be chilled on ice before use
(see Notes 19 and 30).
11. Dry the cuvette and place into the electroporation chamber
and subject to one single pulse of 2.5 kV, 25 μF, with the pulsecontroller resistance set a 1000 Ω resistance (see Note 20).
12. Put cuvette back on ice for 10 min, transfer cell suspension to a
sterile universal bottle containing 5 mL Lemco broth, then
incubate at 37
C for 2–3 h (see Note 23).
13. Harvest the cells by centrifugation at 3000 Â g for 10 min and
plate out suitable dilutions (to give 30–300 colonies per plate)
on Lemco agar and appropriate antibiotic (see Note 24).
14. Incubate plates at 37
C until colonies become visible; this will
take 3–7 days.
15. Count transformants to calculate transformation efficiency.
16. Streak out transformants onto solid medium (Lemco) plus
selection or inoculate 5 mL Lemco broth plus Tween and
selection antibiotic with transformant colonies. Incubate at
37
C with shaking (100 rpm) for 2–3 days (see Note 25).
17. Analyze transformants as required.
4 Notes
1. Pathogenic mycobacteria represent an important biohazard;
therefore, all culture and genetic manipulation must be carried
out in appropriate containment facilities inside a biosafety cabinet. In most countries, genetic manipulation involving pathogenic mycobacteria or their DNA requires approval by the
relevant authorities. In any case, risk assessment must form
the first part of any experiment with pathogenic mycobacteria.
A list of mycobacterial species and the type of containment
required should be consulted prior to use.
2. Mycobacteria, particularly M. tuberculosis, have a tendency to
form clumps in culture due to the waxy nature of the mycobacterial coat. The addition of Tween-80, a non-ionic detergent,
to media reduces clumping to generate a homogenous suspension of cells. Tween-80 can be metabolized by mycobacteria.
Other detergents can be used, for example, the
non-metabolizable detergent Tyloxapol.
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Tanya Parish
À70
C for future use. Cells frozen in this way should be
thawed on ice and used as required (see Note 29).
9. Add 0.5 to 5 μg salt-free DNA in no more than 5 μL volume to
0.2 mL mycobacterial suspension and leave on ice for 10 min
(see Notes 6 and 18).
10. Mix the cells and transfer to a 0.2-cm electrode gap electroporation cuvette. The cuvette should be chilled on ice before use
(see Notes 19 and 30).
11. Dry the cuvette and place into the electroporation chamber
and subject to one single pulse of 2.5 kV, 25 μF, with the pulsecontroller resistance set a 1000 Ω resistance (see Note 20).
12. Put cuvette back on ice for 10 min, transfer cell suspension to a
sterile universal bottle containing 5 mL Lemco broth, then
incubate at 37
C for 2–3 h (see Note 23).
13. Harvest the cells by centrifugation at 3000 Â g for 10 min and
plate out suitable dilutions (to give 30–300 colonies per plate)
on Lemco agar and appropriate antibiotic (see Note 24).
14. Incubate plates at 37
C until colonies become visible; this will
take 3–7 days.
15. Count transformants to calculate transformation efficiency.
16. Streak out transformants onto solid medium (Lemco) plus
selection or inoculate 5 mL Lemco broth plus Tween and
selection antibiotic with transformant colonies. Incubate at
37
C with shaking (100 rpm) for 2–3 days (see Note 25).
17. Analyze transformants as required.
4 Notes
1. Pathogenic mycobacteria represent an important biohazard;
therefore, all culture and genetic manipulation must be carried
out in appropriate containment facilities inside a biosafety cabinet. In most countries, genetic manipulation involving pathogenic mycobacteria or their DNA requires approval by the
relevant authorities. In any case, risk assessment must form
the first part of any experiment with pathogenic mycobacteria.
A list of mycobacterial species and the type of containment
required should be consulted prior to use.
2. Mycobacteria, particularly M. tuberculosis, have a tendency to
form clumps in culture due to the waxy nature of the mycobacterial coat. The addition of Tween-80, a non-ionic detergent,
to media reduces clumping to generate a homogenous suspension of cells. Tween-80 can be metabolized by mycobacteria.
Other detergents can be used, for example, the
non-metabolizable detergent Tyloxapol.
278
Tanya Parish