4. 7H9-OADC-Tween 80 media and 7H10 plates (see items 4
and 5 in Subheading 2.1).
5. Items 5–11 in Subheading 2.2.
2.5 Verification
of ORBIT-Mediated
Recombinants
in M. tuberculosis
1. 7H9-OADC-Tween 80 media and 7H10 plates (see items 4
and 5 in Subheading 2.1).
2. 50 μg/mL Hygromycin B: Supplied as a solution in PBS
(Roche); store at 4
C in the dark.
3. Nalgene Inkwell bottles (30 mL capacity).
4. Items 5–9 in Subheading 2.3.
2.6 Curing
of pKM461
and Creation of a
Markerless Deletion
1. pKM512: For use in constructing markerless deletions using
the ORBIT protocol (addgene.com). The plasmid expresses
Bxb1 Integrase, gp47 (a directionality factor) from the Ptet
promoter, and SacB.
2. 7H9-OADC-Tween 80 media and 7H10 plates (see items 4
and 5 in Subheading 2.1).
3. 25 mg/mL Zeocin (1000Â): Dilute in sterile water. Toxic:
handle with gloves.
4. 20 mg/mL Kanamycin (1000Â): Dissolve in deionized water
and filter sterilize. Store at 4
C for 6 months.
5. 50 μg/mL Hygromycin B: Supplied as a solution in PBS
(Roche); store at 4
C in the dark.
6. 500 μg/mL Anhydrotetracycline (ATc) (1000Â): Dissolve ATc
in methanol. Store at À20
C in the dark.
7. 7H10 plates containing 3% sucrose. Prepare plates as described
in Subheading 2.1 (see step 5); dissolve 30 g of sucrose to
mixture prior to autoclaving.
8. Items 6–11 in Subheading 2.2.
2.7 SNP Transfer
and ORBIT
in M. smegmatis
1. AD enrichment: For 10Â stock, add 25 g of BSA, 10 g of
glucose, and 4.25 g NaCl to 500 mL of deionized water. When
dissolved, filter sterilize through 0.2 μm membrane filter.
2. 7H9-AD-Tween 80: To 900 mL of 7H9 broth, add 100 mL of
10Â AD enrichment and 2.5 mL of 20% (v/v) Tween 80, filter
sterilize through 0.2 μm membrane filter. Store at 4
C; stable
for 4 months.
3. LB plates: Add 40 g of Difco LB-Agar powder to 1 L of
distilled water, mix and autoclave the suspension, and pour
30 mL per plate after cooling the solution to 50
C.
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Kenan C. Murphy
and 5 in Subheading 2.1).
5. Items 5–11 in Subheading 2.2.
2.5 Verification
of ORBIT-Mediated
Recombinants
in M. tuberculosis
1. 7H9-OADC-Tween 80 media and 7H10 plates (see items 4
and 5 in Subheading 2.1).
2. 50 μg/mL Hygromycin B: Supplied as a solution in PBS
(Roche); store at 4
C in the dark.
3. Nalgene Inkwell bottles (30 mL capacity).
4. Items 5–9 in Subheading 2.3.
2.6 Curing
of pKM461
and Creation of a
Markerless Deletion
1. pKM512: For use in constructing markerless deletions using
the ORBIT protocol (addgene.com). The plasmid expresses
Bxb1 Integrase, gp47 (a directionality factor) from the Ptet
promoter, and SacB.
2. 7H9-OADC-Tween 80 media and 7H10 plates (see items 4
and 5 in Subheading 2.1).
3. 25 mg/mL Zeocin (1000Â): Dilute in sterile water. Toxic:
handle with gloves.
4. 20 mg/mL Kanamycin (1000Â): Dissolve in deionized water
and filter sterilize. Store at 4
C for 6 months.
5. 50 μg/mL Hygromycin B: Supplied as a solution in PBS
(Roche); store at 4
C in the dark.
6. 500 μg/mL Anhydrotetracycline (ATc) (1000Â): Dissolve ATc
in methanol. Store at À20
C in the dark.
7. 7H10 plates containing 3% sucrose. Prepare plates as described
in Subheading 2.1 (see step 5); dissolve 30 g of sucrose to
mixture prior to autoclaving.
8. Items 6–11 in Subheading 2.2.
2.7 SNP Transfer
and ORBIT
in M. smegmatis
1. AD enrichment: For 10Â stock, add 25 g of BSA, 10 g of
glucose, and 4.25 g NaCl to 500 mL of deionized water. When
dissolved, filter sterilize through 0.2 μm membrane filter.
2. 7H9-AD-Tween 80: To 900 mL of 7H9 broth, add 100 mL of
10Â AD enrichment and 2.5 mL of 20% (v/v) Tween 80, filter
sterilize through 0.2 μm membrane filter. Store at 4
C; stable
for 4 months.
3. LB plates: Add 40 g of Difco LB-Agar powder to 1 L of
distilled water, mix and autoclave the suspension, and pour
30 mL per plate after cooling the solution to 50
C.
308
Kenan C. Murphy