3.2 Construction
of Dual Fluorescent
Reporter
M. tuberculosis Strains
To use the reporter M. tuberculosis strains in the context of infection, the characterized GFP reporter construct must first be moved
into a plasmid containing a constitutively expressed mCherry (e.g.,
pCherry3). This enables visualization of the bacteria within host
cells, independent of GFP reporter induction status/presence (see
Note 9).
1. PCR amplify the GFP reporter construct from step 3 of Subheading 3.1 using primers that contain appropriate restriction
sites.
2. TOPO clone the purified PCR product and sequence verify the
plasmid obtained (see Note 1).
3. Digest and ligate the GFP reporter into pCherry3, or other
appropriate mCherry-expressing plasmid.
4. Transform the ligation into E. coli TOP10.
5. Conduct diagnostic digests on isolated plasmids to verify successful generation of the reporter construct (see Note 9).
6. Transform the newly generated reporter plasmid into
M. tuberculosis to obtain the dual fluorescent reporter
M. tuberculosis strain. Colonies should appear pink/red on
agar plates, except in cases where the GFP reporter is induced
during growth on agar.
7. Test the dual fluorescent reporter M. tuberculosis strains in
defined broth conditions as described in Subheading 3.3,
prior to use in infection studies.
3.3 Characterization
of Fluorescent
Reporter
M. tuberculosis Strains
Environmental reporter M. tuberculosis strains generated should be
tested in defined broth conditions to verify that GFP reporter signal
is induced in the expected conditions. Tests of response to other
environmental signals known to be experienced by M. tuberculosis
during infection are also important, for analysis of the specificity of
the reporter. Here we describe as an example tests for the rv2390c’::
GFP reporter, where GFP expression is induced specifically in acidic
pH and high [Cl
À
] conditions, but not by hypoxia or nitric oxide
(NO) stress [6].
1. Maintain all M. tuberculosis cultures at 37
C with 5% CO 2 .
2. For tests of reporter response to acidic pH and high [Cl
À ]
signals, grow the reporter M. tuberculosis strain in standing
T-25 flasks to log-phase (OD 600 ~ 0.6) in 10 mL 7H9,
pH 7.0 medium containing 50 μg/mL hygromycin for maintenance of the reporter plasmid.
3. Subculture the log phase M. tuberculosis culture to
OD 600 ¼ 0.05 in standing T-25 flasks containing 10 mL of
the various test media: (1) 7H9, pH 7.0, (2) 7H9, pH 5.7,
(3) 7H9, pH 7.0, 250 mM NaCl, and (4) 7H9, pH 5.7,
250 mM NaCl, all containing 50 μg/mL hygromycin.
Exploiting Fluorescent Reporter M. tuberculosis Strains
371
of Dual Fluorescent
Reporter
M. tuberculosis Strains
To use the reporter M. tuberculosis strains in the context of infection, the characterized GFP reporter construct must first be moved
into a plasmid containing a constitutively expressed mCherry (e.g.,
pCherry3). This enables visualization of the bacteria within host
cells, independent of GFP reporter induction status/presence (see
Note 9).
1. PCR amplify the GFP reporter construct from step 3 of Subheading 3.1 using primers that contain appropriate restriction
sites.
2. TOPO clone the purified PCR product and sequence verify the
plasmid obtained (see Note 1).
3. Digest and ligate the GFP reporter into pCherry3, or other
appropriate mCherry-expressing plasmid.
4. Transform the ligation into E. coli TOP10.
5. Conduct diagnostic digests on isolated plasmids to verify successful generation of the reporter construct (see Note 9).
6. Transform the newly generated reporter plasmid into
M. tuberculosis to obtain the dual fluorescent reporter
M. tuberculosis strain. Colonies should appear pink/red on
agar plates, except in cases where the GFP reporter is induced
during growth on agar.
7. Test the dual fluorescent reporter M. tuberculosis strains in
defined broth conditions as described in Subheading 3.3,
prior to use in infection studies.
3.3 Characterization
of Fluorescent
Reporter
M. tuberculosis Strains
Environmental reporter M. tuberculosis strains generated should be
tested in defined broth conditions to verify that GFP reporter signal
is induced in the expected conditions. Tests of response to other
environmental signals known to be experienced by M. tuberculosis
during infection are also important, for analysis of the specificity of
the reporter. Here we describe as an example tests for the rv2390c’::
GFP reporter, where GFP expression is induced specifically in acidic
pH and high [Cl
À
] conditions, but not by hypoxia or nitric oxide
(NO) stress [6].
1. Maintain all M. tuberculosis cultures at 37
C with 5% CO 2 .
2. For tests of reporter response to acidic pH and high [Cl
À ]
signals, grow the reporter M. tuberculosis strain in standing
T-25 flasks to log-phase (OD 600 ~ 0.6) in 10 mL 7H9,
pH 7.0 medium containing 50 μg/mL hygromycin for maintenance of the reporter plasmid.
3. Subculture the log phase M. tuberculosis culture to
OD 600 ¼ 0.05 in standing T-25 flasks containing 10 mL of
the various test media: (1) 7H9, pH 7.0, (2) 7H9, pH 5.7,
(3) 7H9, pH 7.0, 250 mM NaCl, and (4) 7H9, pH 5.7,
250 mM NaCl, all containing 50 μg/mL hygromycin.
Exploiting Fluorescent Reporter M. tuberculosis Strains
371