6. Add 24 mL of 10% glycerol to bring up to the final volume to
25 mL.
7. Repeat steps 4–6 twice for a total of three washes.
8. Resuspend the washed cells in 1 mL 10% glycerol. This is
sufficient for at least 10 transformations. Competent cells
should be used the same day.
3.6 Electroporation
of CRISPRi Constructs
into Electrocompetent
Mycobacteria
1. Aliquot ~1 μL (at least 100 ng) of the CRISPRi construct (see
Subheading 3.4, step 4) to the bottom of a labeled 2 mL screw
cap microfuge tubes.
2. Add 100 μL of electrocompetent M. tuberculosis to the CRISPRi construct in the 2 mL screwcap tube.
3. Pipette up and down to mix and transfer to a 2 mm electroporation cuvette.
4. Place the cuvette in the electroporation pod and electroporate
the bacilli with the following settings: 2500 V, 700 Ω, 25 μF.
5. Recover the cells by adding 1 mL of 7H9-OADC-Tween80 to
the cuvette and transferring the cells to the 2 mL screw cap
microfuge tube for recovery and outgrowth.
6. Incubate the transformation recovery standing in a 37
C incubator overnight.
7. Pellet the recovered cells by centrifugation in a microcentrifuge
at 4000 Â g for 5 min.
8. Discard the supernatant and resuspend the cell pellet in 100 μL
of 7H9-OADC-Tween80.
9. Plate cells on 7H10-OADC-Tween80 with 20 μg/mL kanamycin to select for transformants. If knockdown of your gene
of interest is expected to produce a phenotype on agar plates
(e.g., lack of growth), additional plating of half of the transformation on a separate kanamycin plate in the presence of
100 ng/mL anhydrotetracycline (ATc) can confirm the knockdown phenotype (see Notes 6 and 7).
3.7 Quantification of
Transcriptional
Knockdown by qRTPCR
1. Grow a 5 mL starter culture of the desired M. tuberculosis
CRISPRi strains, prepared as in Subheading 3.6. As a control,
also include a nontargeting control strain containing the parent
CRISPRi plasmid, plJR965.
2. Grow in a T-25 flask in stationary conditions at 37
C with 5%
CO 2 in 7H9-OADC-Tween80 with 20 μg/mL kanamycin to
saturation (OD 600 ~ 1.0).
3. Dilute the culture in a new T-25 flask to an OD 600 of 0.05 in
5 mL of the same media.
4. Grow for 3 days to log phase (OD 600 0.4–0.8) in stationary
conditions at 37
C with 5% CO 2 .
354
Andrew I. Wong and Jeremy M. Rock
25 mL.
7. Repeat steps 4–6 twice for a total of three washes.
8. Resuspend the washed cells in 1 mL 10% glycerol. This is
sufficient for at least 10 transformations. Competent cells
should be used the same day.
3.6 Electroporation
of CRISPRi Constructs
into Electrocompetent
Mycobacteria
1. Aliquot ~1 μL (at least 100 ng) of the CRISPRi construct (see
Subheading 3.4, step 4) to the bottom of a labeled 2 mL screw
cap microfuge tubes.
2. Add 100 μL of electrocompetent M. tuberculosis to the CRISPRi construct in the 2 mL screwcap tube.
3. Pipette up and down to mix and transfer to a 2 mm electroporation cuvette.
4. Place the cuvette in the electroporation pod and electroporate
the bacilli with the following settings: 2500 V, 700 Ω, 25 μF.
5. Recover the cells by adding 1 mL of 7H9-OADC-Tween80 to
the cuvette and transferring the cells to the 2 mL screw cap
microfuge tube for recovery and outgrowth.
6. Incubate the transformation recovery standing in a 37
C incubator overnight.
7. Pellet the recovered cells by centrifugation in a microcentrifuge
at 4000 Â g for 5 min.
8. Discard the supernatant and resuspend the cell pellet in 100 μL
of 7H9-OADC-Tween80.
9. Plate cells on 7H10-OADC-Tween80 with 20 μg/mL kanamycin to select for transformants. If knockdown of your gene
of interest is expected to produce a phenotype on agar plates
(e.g., lack of growth), additional plating of half of the transformation on a separate kanamycin plate in the presence of
100 ng/mL anhydrotetracycline (ATc) can confirm the knockdown phenotype (see Notes 6 and 7).
3.7 Quantification of
Transcriptional
Knockdown by qRTPCR
1. Grow a 5 mL starter culture of the desired M. tuberculosis
CRISPRi strains, prepared as in Subheading 3.6. As a control,
also include a nontargeting control strain containing the parent
CRISPRi plasmid, plJR965.
2. Grow in a T-25 flask in stationary conditions at 37
C with 5%
CO 2 in 7H9-OADC-Tween80 with 20 μg/mL kanamycin to
saturation (OD 600 ~ 1.0).
3. Dilute the culture in a new T-25 flask to an OD 600 of 0.05 in
5 mL of the same media.
4. Grow for 3 days to log phase (OD 600 0.4–0.8) in stationary
conditions at 37
C with 5% CO 2 .
354
Andrew I. Wong and Jeremy M. Rock