12. Check for correct insert size using diagnostic restriction digests
and sequencing to confirm that no mutations have been introduced (see Note 10).
3.2 Obtaining
a Delinquent Strain
(Δ
0 int)
1. Inoculate 1–3 mL SCO mycobacterial strain (see Note 1) into
100 mL 7H9-Tw-KH in a roller bottle.
2. Incubate for 6 d at 37
C with rolling.
3. Add 10 mL 2 M glycine solution and incubate overnight.
4. Pellet 50 mL of cells at 3000 Â g (20
C) for 10 min.
5. Wash cells in 10 mL 10% glycerol.
6. Pellet 10 mL of cells at 3000 Â g (20
C) for 10 min.
7. Wash cells in 5 mL 10% glycerol.
8. Pellet 5 mL of cells at 3000 Â g (20
C) for 10 min.
9. Resuspend cells in 1 mL 10% glycerol.
10. Electroporate complement plasmid DNA 1μg—made either as
described in Subheading 3.1.1 or 3.1.2 and held in a volume of
no more than 5μL—into 200μL cells, using a pulse of 2.5 kV,
1000 Ω, 25μF.
11. Transfer cuvette contents to 10 mL recovery medium. Incubate at 37
C overnight.
12. Centrifuge transformations for 10 min at 3000 Â g to collect
cells. Resuspend in 1 mL liquid broth and plate out as a
dilution series (10
À1 to 10
À6
) onto 7H10-KHGmX.
13. Incubate at 37
C, colonies should be apparent after
2–5 weeks.
14. Streak a few blue transformants onto 7H10-Gm, incubate for
1 week at 37
C.
15. Scrape a loopful of cells from one of the streaks on the 7H10Gm plate and place into a 50 mL Falcon tubes containing 1 mL
volume of 1 mm sterile glass beads and 3 mL 7H9-Tw. Vortex
vigorously for 1 min, allow to stand for 10 min before opening.
16. Plate serial dilutions onto 7H10-GmXSuc plates. Use several
plates per dilution.
17. Incubate at 37
C for 4–6 weeks.
18. Pick white suc
R colonies, patch 40 individual transformants in
duplicate onto 7H10-GmX and 7H10-KHGm.
19. Incubate at 37
C for 2–3 weeks.
20. Score streaks for kanamycin, hygromycin sensitivity, growth on
7H10-GmX vs. 7H10-KHGm.
21. Scrap a loopful of cells from each white, Kan
S Hyg
S streaks
(7H10-GmX plates), using sterile loops, into individual 2 mL
screw cap tube containing 1 mL 10 mM Tris pH 8, heat at
Gene Switching and Essentiality Testing
295
and sequencing to confirm that no mutations have been introduced (see Note 10).
3.2 Obtaining
a Delinquent Strain
(Δ
0 int)
1. Inoculate 1–3 mL SCO mycobacterial strain (see Note 1) into
100 mL 7H9-Tw-KH in a roller bottle.
2. Incubate for 6 d at 37
C with rolling.
3. Add 10 mL 2 M glycine solution and incubate overnight.
4. Pellet 50 mL of cells at 3000 Â g (20
C) for 10 min.
5. Wash cells in 10 mL 10% glycerol.
6. Pellet 10 mL of cells at 3000 Â g (20
C) for 10 min.
7. Wash cells in 5 mL 10% glycerol.
8. Pellet 5 mL of cells at 3000 Â g (20
C) for 10 min.
9. Resuspend cells in 1 mL 10% glycerol.
10. Electroporate complement plasmid DNA 1μg—made either as
described in Subheading 3.1.1 or 3.1.2 and held in a volume of
no more than 5μL—into 200μL cells, using a pulse of 2.5 kV,
1000 Ω, 25μF.
11. Transfer cuvette contents to 10 mL recovery medium. Incubate at 37
C overnight.
12. Centrifuge transformations for 10 min at 3000 Â g to collect
cells. Resuspend in 1 mL liquid broth and plate out as a
dilution series (10
À1 to 10
À6
) onto 7H10-KHGmX.
13. Incubate at 37
C, colonies should be apparent after
2–5 weeks.
14. Streak a few blue transformants onto 7H10-Gm, incubate for
1 week at 37
C.
15. Scrape a loopful of cells from one of the streaks on the 7H10Gm plate and place into a 50 mL Falcon tubes containing 1 mL
volume of 1 mm sterile glass beads and 3 mL 7H9-Tw. Vortex
vigorously for 1 min, allow to stand for 10 min before opening.
16. Plate serial dilutions onto 7H10-GmXSuc plates. Use several
plates per dilution.
17. Incubate at 37
C for 4–6 weeks.
18. Pick white suc
R colonies, patch 40 individual transformants in
duplicate onto 7H10-GmX and 7H10-KHGm.
19. Incubate at 37
C for 2–3 weeks.
20. Score streaks for kanamycin, hygromycin sensitivity, growth on
7H10-GmX vs. 7H10-KHGm.
21. Scrap a loopful of cells from each white, Kan
S Hyg
S streaks
(7H10-GmX plates), using sterile loops, into individual 2 mL
screw cap tube containing 1 mL 10 mM Tris pH 8, heat at
Gene Switching and Essentiality Testing
295