5. Repeat centrifugation, discard supernatant, and resuspend the
resulting pellet in 1:100 volumes of water with respect to
original culture volume. Aliquots of bacterial suspensions
(100 μL) are stored in sterile 1.5 mL microtubes.
6. Centrifuge the microtubes (4000 Â g, room temperature for
15 min), and completely remove the supernatant with pipet (see
Note 7).
7. Resuspend the bacterial pellets with 100 μL of DNA solution in
water (DNA volume varies according to the original concentration and final amount used). Incubate for 15 min at room
temperature. We have performed transformation with DNA
amounts ranging from 100 ng to 1 μg. However, even when
the same amount of DNA is used, variations in transformation
rates are observed between experiments.
8. Keep 0.2 cm electroporation cuvettes at À20
C until right
before the electroporation step. Transfer ~100 μL of cell suspensions (leptospires plus DNA) to the cuvettes; the electroporator should be set at 1.8 kV, 25 μF, and 200 Ω.
9. Electroporate the bacterial cells. The resulting pulse should
render a time constant of 5–6 ms. After electroporation, add
1 mL of fresh liquid EMJH to the cells.
10. Transfer the cells to sterile 50 mL tubes and incubate with
shaking overnight at 30
C. This step allows the successful
expression of the antibiotic resistance genes.
11. Spread 100–250 μL of solution into EMJH plates containing
spectinomycin, and incubate the plates at 30
C until colonies
are visible, normally 7–14 days. Plates must be sealed with
Parafilm or plastic film to avoid desiccation.
12. Validate the transformants by picking a few colonies from the
plates. Since leptospires grow as subsurface colonies, make sure
to “dig” below the medium surface. The collected colonies
should have a bit of solid medium.
13. Resuspend the selected colony in 50 μL of fresh EMJH, and
then place 5 μL of the suspension on glass slides for visualization of leptospires.
14. Transfer the remaining volume to 3–5 mL of liquid EMJH plus
spectinomycin, and incubate with moderate shaking at 30
C.
15. When growth is observed with the naked eye (“cloudy”
appearance), confirm transformation by PCR using pMaOri2
primers. In our experience, it is not necessary to purify genomic DNA from the cultures (see Note 6).
16. Leptospira cells can be stored at À80
C in EMJH medium plus
2.5% dimethyl sulfoxide (DMSO). Add 25 μL of DMSO to
1.5 mL microtubes and then 975 μL of leptospiral culture, mix
gently, and then freeze immediately.
Gene Silencing by Single-Guide RNA and dCas9
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resulting pellet in 1:100 volumes of water with respect to
original culture volume. Aliquots of bacterial suspensions
(100 μL) are stored in sterile 1.5 mL microtubes.
6. Centrifuge the microtubes (4000 Â g, room temperature for
15 min), and completely remove the supernatant with pipet (see
Note 7).
7. Resuspend the bacterial pellets with 100 μL of DNA solution in
water (DNA volume varies according to the original concentration and final amount used). Incubate for 15 min at room
temperature. We have performed transformation with DNA
amounts ranging from 100 ng to 1 μg. However, even when
the same amount of DNA is used, variations in transformation
rates are observed between experiments.
8. Keep 0.2 cm electroporation cuvettes at À20
C until right
before the electroporation step. Transfer ~100 μL of cell suspensions (leptospires plus DNA) to the cuvettes; the electroporator should be set at 1.8 kV, 25 μF, and 200 Ω.
9. Electroporate the bacterial cells. The resulting pulse should
render a time constant of 5–6 ms. After electroporation, add
1 mL of fresh liquid EMJH to the cells.
10. Transfer the cells to sterile 50 mL tubes and incubate with
shaking overnight at 30
C. This step allows the successful
expression of the antibiotic resistance genes.
11. Spread 100–250 μL of solution into EMJH plates containing
spectinomycin, and incubate the plates at 30
C until colonies
are visible, normally 7–14 days. Plates must be sealed with
Parafilm or plastic film to avoid desiccation.
12. Validate the transformants by picking a few colonies from the
plates. Since leptospires grow as subsurface colonies, make sure
to “dig” below the medium surface. The collected colonies
should have a bit of solid medium.
13. Resuspend the selected colony in 50 μL of fresh EMJH, and
then place 5 μL of the suspension on glass slides for visualization of leptospires.
14. Transfer the remaining volume to 3–5 mL of liquid EMJH plus
spectinomycin, and incubate with moderate shaking at 30
C.
15. When growth is observed with the naked eye (“cloudy”
appearance), confirm transformation by PCR using pMaOri2
primers. In our experience, it is not necessary to purify genomic DNA from the cultures (see Note 6).
16. Leptospira cells can be stored at À80
C in EMJH medium plus
2.5% dimethyl sulfoxide (DMSO). Add 25 μL of DMSO to
1.5 mL microtubes and then 975 μL of leptospiral culture, mix
gently, and then freeze immediately.
Gene Silencing by Single-Guide RNA and dCas9
117