the cellular metabolome as arguably the most physiologic chemical
library of potential substrates that can be tested, in a label and
synthesis-free manner. Moreover, candidate activities assigned by
this method can be confirmed using independent biochemical
(including reconstitution with purified components) and genetic
(using wild-type and genetic knockout, knockdown, or overexpression strains) techniques.
1.7 Carbon Tracing
and Flux Analysis
An additional application of metabolomics in M. tuberculosis is
carbon tracing, and the resultant tool of flux analysis, which allow
for the measurement of changes in individual metabolite levels over
time using nonradioactive heavy isotopes such as
13
C or
15
N. This
allows for a more complete picture of the constantly changing
metabolic status of M. tuberculosis in response to an external stimulus. While measurements using the LC/MS analysis with the filter
system described herein cannot generate continuous measurements
from a single sample, measurements at rapid time intervals can
provide insight into the constantly evolving metabolic status of
M. tuberculosis in response to different conditions.
2 Materials
2.1 Culture System
1. 7H9-ADN medium: Dissolve 4.7 g Middlebrook 7H9 broth in
900 mL deionized water and add 4 mL of 50% (v/v) glycerol.
Mix and autoclave to sterilize. Add 0.04% (v/v) sterile Tyloxapol and 100 mL of a sterile solution of 5% (w/v) bovine serum
albumin, 2% (w/v) D-glucose, and 0.85% (w/v) NaCl (ADN)
(see Note 1). Filter sterilize immediately and store at 4
C.
2. Durapore membrane filters (25 mm diameter)—Millipore 0.2μm PVDF filters (see Note 2).
3. Swinnex filter holder, 25 mm (Millipore).
4. Vacuum flask.
5. M. tuberculosis culture at a density of 5 Â 10
8 cells/mL
(OD 600 ¼ 1).
6. 7H10 agar plates: Dissolve 19 g Middlebrook 7H10 agar in
900 mL deionized water and add 10 mL of 50% (v/v) glycerol.
Mix and autoclave to sterilize. Add 100 mL sterile ADN solution. Pour 24 mL into each round, plastic Petri dish
(100 mm  15 mm) and allow to dry (yields approximately
40 plates).
7. Sterile tweezers.
8. Sterile 6-well plates.
9. 15-mL centrifuge tube caps (Celltreat), autoclaved.
10. Cyanoacrylate-based glue.
582
Kyle A. Planck and Kyu Rhee
library of potential substrates that can be tested, in a label and
synthesis-free manner. Moreover, candidate activities assigned by
this method can be confirmed using independent biochemical
(including reconstitution with purified components) and genetic
(using wild-type and genetic knockout, knockdown, or overexpression strains) techniques.
1.7 Carbon Tracing
and Flux Analysis
An additional application of metabolomics in M. tuberculosis is
carbon tracing, and the resultant tool of flux analysis, which allow
for the measurement of changes in individual metabolite levels over
time using nonradioactive heavy isotopes such as
13
C or
15
N. This
allows for a more complete picture of the constantly changing
metabolic status of M. tuberculosis in response to an external stimulus. While measurements using the LC/MS analysis with the filter
system described herein cannot generate continuous measurements
from a single sample, measurements at rapid time intervals can
provide insight into the constantly evolving metabolic status of
M. tuberculosis in response to different conditions.
2 Materials
2.1 Culture System
1. 7H9-ADN medium: Dissolve 4.7 g Middlebrook 7H9 broth in
900 mL deionized water and add 4 mL of 50% (v/v) glycerol.
Mix and autoclave to sterilize. Add 0.04% (v/v) sterile Tyloxapol and 100 mL of a sterile solution of 5% (w/v) bovine serum
albumin, 2% (w/v) D-glucose, and 0.85% (w/v) NaCl (ADN)
(see Note 1). Filter sterilize immediately and store at 4
C.
2. Durapore membrane filters (25 mm diameter)—Millipore 0.2μm PVDF filters (see Note 2).
3. Swinnex filter holder, 25 mm (Millipore).
4. Vacuum flask.
5. M. tuberculosis culture at a density of 5 Â 10
8 cells/mL
(OD 600 ¼ 1).
6. 7H10 agar plates: Dissolve 19 g Middlebrook 7H10 agar in
900 mL deionized water and add 10 mL of 50% (v/v) glycerol.
Mix and autoclave to sterilize. Add 100 mL sterile ADN solution. Pour 24 mL into each round, plastic Petri dish
(100 mm  15 mm) and allow to dry (yields approximately
40 plates).
7. Sterile tweezers.
8. Sterile 6-well plates.
9. 15-mL centrifuge tube caps (Celltreat), autoclaved.
10. Cyanoacrylate-based glue.
582
Kyle A. Planck and Kyu Rhee