7. Glycerol. Make a 10% v/v stock in water and filter sterilize.
Store at 4
C.
8. 7H9-Tw-Gm medium: Dissolve 4.7 g of Middlebrook 7H9
broth base (Difco) in 900 mL deionized water. Add 5 mL of
20% (v/v) Tween 80 (final concentration of 0.05% v/v). Mix
well and filter sterilize or autoclave. Add 100 mL of OADC and
gentamicin to give a final concentration of 10μg/mL immediately before use.
9. 7H10-HGm plates: Dissolve 19 g of Middlebrook 7H10
medium (Difco) in 900 mL deionized water. Autoclave.
When media is cooled to $50
C, add 100 mL of OADC
enrichment. Add hygromycin to give final concentration of
50μg/mL, and gentamicin to give a final concentration of
10μg/mL. Mix well and pour in standard plastic Petri dishes.
10. 7H10-Gm plates: Dissolve 19 g of Middlebrook 7H10
medium (Difco) in 900 mL deionized water. Autoclave.
When media is cooled to $50
C, add 100 mL of OADC
enrichment. Add gentamicin to give a final concentration of
10μg/mL. Mix well and pour in standard plastic Petri dishes.
11. 7H10-H plates: Dissolve 19 g of Middlebrook 7H10 medium
(Difco) in 900 mL deionized water. Autoclave. When media is
cooled to $50
C, add 100 mL of OADC enrichment. Add
hygromycin to give final concentration of 50μg/mL. Mix well
and pour in standard plastic Petri dishes.
3 Methods
3.1 Construction of a
Complementing Vector
Depending on the strategy you are adopting you should follow
either Subheading 3.1.1—for the creation of a vector where expression is driven by the Ag85a promoter; or Subheading 3.1.2—where
expression is driven by the native promoter.
3.1.1 Construction
of Ag85a Promoter-Driven
Complementing Vector
Using pAPA3
1. Design PCR primers to amplify the region of DNA required
with PacI sites included to the 5
0 end (see Note 2).
2. Amplify gene of interest using a proof reading polymerase and
standard PCR protocol (see Note 3).
3. Check PCR product, using agarose gel electrophoresis, for
band of correct size and the absence of spurious bands, etc.
Isolate PCR product with PCR extraction kit/method or gel
extract band, if required, and clean using gel extraction kit/method as per kit instructions.
4. A-tail cleaned PCR products (if required) by adding 5μL of
10Â Taq buffer, 1μL dATP, and 5μL Taq, to cleaned PCR
product to give final volume of 50μL. Incubate at 72
C for
20 min (see Note 4).
Gene Switching and Essentiality Testing
293
Store at 4
C.
8. 7H9-Tw-Gm medium: Dissolve 4.7 g of Middlebrook 7H9
broth base (Difco) in 900 mL deionized water. Add 5 mL of
20% (v/v) Tween 80 (final concentration of 0.05% v/v). Mix
well and filter sterilize or autoclave. Add 100 mL of OADC and
gentamicin to give a final concentration of 10μg/mL immediately before use.
9. 7H10-HGm plates: Dissolve 19 g of Middlebrook 7H10
medium (Difco) in 900 mL deionized water. Autoclave.
When media is cooled to $50
C, add 100 mL of OADC
enrichment. Add hygromycin to give final concentration of
50μg/mL, and gentamicin to give a final concentration of
10μg/mL. Mix well and pour in standard plastic Petri dishes.
10. 7H10-Gm plates: Dissolve 19 g of Middlebrook 7H10
medium (Difco) in 900 mL deionized water. Autoclave.
When media is cooled to $50
C, add 100 mL of OADC
enrichment. Add gentamicin to give a final concentration of
10μg/mL. Mix well and pour in standard plastic Petri dishes.
11. 7H10-H plates: Dissolve 19 g of Middlebrook 7H10 medium
(Difco) in 900 mL deionized water. Autoclave. When media is
cooled to $50
C, add 100 mL of OADC enrichment. Add
hygromycin to give final concentration of 50μg/mL. Mix well
and pour in standard plastic Petri dishes.
3 Methods
3.1 Construction of a
Complementing Vector
Depending on the strategy you are adopting you should follow
either Subheading 3.1.1—for the creation of a vector where expression is driven by the Ag85a promoter; or Subheading 3.1.2—where
expression is driven by the native promoter.
3.1.1 Construction
of Ag85a Promoter-Driven
Complementing Vector
Using pAPA3
1. Design PCR primers to amplify the region of DNA required
with PacI sites included to the 5
0 end (see Note 2).
2. Amplify gene of interest using a proof reading polymerase and
standard PCR protocol (see Note 3).
3. Check PCR product, using agarose gel electrophoresis, for
band of correct size and the absence of spurious bands, etc.
Isolate PCR product with PCR extraction kit/method or gel
extract band, if required, and clean using gel extraction kit/method as per kit instructions.
4. A-tail cleaned PCR products (if required) by adding 5μL of
10Â Taq buffer, 1μL dATP, and 5μL Taq, to cleaned PCR
product to give final volume of 50μL. Incubate at 72
C for
20 min (see Note 4).
Gene Switching and Essentiality Testing
293