100
C for 20 min on a heat block. Allow tubes to cool to room
temperature before briefly spinning in a microfuge. Filter sterilizing the supernatant, using a 0.2μM 15 mm syringe filter and
3 mL LuerLok syringe, into a clean tube.
22. Re-incubate 7H10-GmX plates to allow regrowth.
23. Test boilates by PCR, using primers which overlap the deletion, to identify potential delinquents, i.e., transformants
which have the chromosomal gene copy deleted, thus will
have a smaller product size than those retaining the gene
copy; typically 4μL of the boiled, syringe-filtered extract in a
20μL PCR gives good results.
24. Prepare genomic DNA from potential delinquents, identified
by PCR, from 7H10-GmX plates and confirm gene deletion by
Southern blotting (see Notes 11 and 12).
3.3 Confirming
Essentiality by Gene
Switching
1. Inoculate 1–3 mL Δ-int mycobacterial strain (from Subheading 3.2) into 100 mL 7H9-Tw-Gm 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 plasmid DNA 1μg (held in a volume of no more
than 5μL) (see Note 13) into 200μL cells, using a pulse of
2.5 kV, 1000 Ω, 25μF.
11. Transfer cuvette contents to 10 mL recovery media. 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-Gm, 7H10-H,
7H10-GmH.
13. Incubate at 37
C colonies should be apparent after 2–5 weeks.
14. Inspect plates for colonies—if switching has been successful
(e.g., the gene of interest is either nonessential, or the gene/
operon being switched in is able to compensate for the loss of
the gene), there should be colonies present on the 7H10-H
plates, at either the same number or greater than on the 7H10Gm plates; if switching is not successful (e.g., the gene of
interest is essential, and the gene/operon being switched in is
unable to compensate for the loss of the gene), there will be no
296
Amanda Claire Brown
C for 20 min on a heat block. Allow tubes to cool to room
temperature before briefly spinning in a microfuge. Filter sterilizing the supernatant, using a 0.2μM 15 mm syringe filter and
3 mL LuerLok syringe, into a clean tube.
22. Re-incubate 7H10-GmX plates to allow regrowth.
23. Test boilates by PCR, using primers which overlap the deletion, to identify potential delinquents, i.e., transformants
which have the chromosomal gene copy deleted, thus will
have a smaller product size than those retaining the gene
copy; typically 4μL of the boiled, syringe-filtered extract in a
20μL PCR gives good results.
24. Prepare genomic DNA from potential delinquents, identified
by PCR, from 7H10-GmX plates and confirm gene deletion by
Southern blotting (see Notes 11 and 12).
3.3 Confirming
Essentiality by Gene
Switching
1. Inoculate 1–3 mL Δ-int mycobacterial strain (from Subheading 3.2) into 100 mL 7H9-Tw-Gm 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 plasmid DNA 1μg (held in a volume of no more
than 5μL) (see Note 13) into 200μL cells, using a pulse of
2.5 kV, 1000 Ω, 25μF.
11. Transfer cuvette contents to 10 mL recovery media. 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-Gm, 7H10-H,
7H10-GmH.
13. Incubate at 37
C colonies should be apparent after 2–5 weeks.
14. Inspect plates for colonies—if switching has been successful
(e.g., the gene of interest is either nonessential, or the gene/
operon being switched in is able to compensate for the loss of
the gene), there should be colonies present on the 7H10-H
plates, at either the same number or greater than on the 7H10Gm plates; if switching is not successful (e.g., the gene of
interest is essential, and the gene/operon being switched in is
unable to compensate for the loss of the gene), there will be no
296
Amanda Claire Brown