7. Use the total reaction mixture to transform 50 μL of chemically
competent E. coli π1 cells. Transformation steps include 30 min
on ice (cells plus DNA), 90 s at 42
C, and then 5 min on ice;
300 μL LB medium are added and cells are incubated at 37
C
for 1 h. Streak the bacteria on a LB agar plate containing
0.3 mM dT and 40 μg/mL spectinomycin. Incubate overnight
at 37
C.
8. Evaluate the recovered colonies by PCR with flanking primers
pMaOri2F and R (see Note 6). Because only one restriction site
is used, ligation efficiency may be lower than usual (when no
CIP is used). We have observed around 20–50% ligation rate
after checking the colonies by PCR. It is worth mentioning
that, as the cloning is not directional, the insert can be ligated
in either orientation.
9. Grow recombinant E. coli cells containing pMaOri.
dCas9sgRNA in LB medium for plasmid purification.
10. Confirm sgRNA cassette ligation by sequencing and digestion
of the recombinant plasmid with XmaI restriction enzyme.
3.2 Transformation
Due to the pMaOri backbone, the recombinant plasmid containing
dCas9 and sgRNA cassette can be used to transform Leptospira spp.
by conjugation and electroporation. Here, we describe briefly the
electroporation steps. The detailed protocol for conjugation has
been described by Picardeau [17]. We recommend, for each desired
gene, the use of empty pMaOri.dCas9 (control) and this plasmid
containing sgRNA for both the coding (full gene silencing) and
template (partial) strand of DNA.
1. Each 10 mL of exponential-phase L. biflexa cells generates one
aliquot of competent bacteria. Therefore, the final volume of
culture will depend on the number of aliquots needed. Inoculate 1 volume of L. biflexa culture in 100 volumes of fresh
liquid EMJH, and incubate with moderate shaking (100 rpm)
at 30
C until the culture reaches an OD 420 between 0.3 and
0.5 (around 3 days). All procedures should be performed in a
laminar flow cabinet, previously UV-irradiated to avoid contamination. Make sure all glass used for culture growth is
properly washed.
2. Monitor the culture for possible contamination.
3. When the desired OD 420 is reached, collect the bacteria by
centrifugation (4000 Â g, room temperature for 15 min) in
50 mL centrifuge tubes.
4. Discard the supernatant and resuspend the bacterial pellet with
the same volume of sterile water by gently shaking the cells
(avoid vortexing).
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Luis Guilherme Virgı ´lio Fernandes and Ana Lucia Tabet Oller Nascimento
competent E. coli π1 cells. Transformation steps include 30 min
on ice (cells plus DNA), 90 s at 42
C, and then 5 min on ice;
300 μL LB medium are added and cells are incubated at 37
C
for 1 h. Streak the bacteria on a LB agar plate containing
0.3 mM dT and 40 μg/mL spectinomycin. Incubate overnight
at 37
C.
8. Evaluate the recovered colonies by PCR with flanking primers
pMaOri2F and R (see Note 6). Because only one restriction site
is used, ligation efficiency may be lower than usual (when no
CIP is used). We have observed around 20–50% ligation rate
after checking the colonies by PCR. It is worth mentioning
that, as the cloning is not directional, the insert can be ligated
in either orientation.
9. Grow recombinant E. coli cells containing pMaOri.
dCas9sgRNA in LB medium for plasmid purification.
10. Confirm sgRNA cassette ligation by sequencing and digestion
of the recombinant plasmid with XmaI restriction enzyme.
3.2 Transformation
Due to the pMaOri backbone, the recombinant plasmid containing
dCas9 and sgRNA cassette can be used to transform Leptospira spp.
by conjugation and electroporation. Here, we describe briefly the
electroporation steps. The detailed protocol for conjugation has
been described by Picardeau [17]. We recommend, for each desired
gene, the use of empty pMaOri.dCas9 (control) and this plasmid
containing sgRNA for both the coding (full gene silencing) and
template (partial) strand of DNA.
1. Each 10 mL of exponential-phase L. biflexa cells generates one
aliquot of competent bacteria. Therefore, the final volume of
culture will depend on the number of aliquots needed. Inoculate 1 volume of L. biflexa culture in 100 volumes of fresh
liquid EMJH, and incubate with moderate shaking (100 rpm)
at 30
C until the culture reaches an OD 420 between 0.3 and
0.5 (around 3 days). All procedures should be performed in a
laminar flow cabinet, previously UV-irradiated to avoid contamination. Make sure all glass used for culture growth is
properly washed.
2. Monitor the culture for possible contamination.
3. When the desired OD 420 is reached, collect the bacteria by
centrifugation (4000 Â g, room temperature for 15 min) in
50 mL centrifuge tubes.
4. Discard the supernatant and resuspend the bacterial pellet with
the same volume of sterile water by gently shaking the cells
(avoid vortexing).
116
Luis Guilherme Virgı ´lio Fernandes and Ana Lucia Tabet Oller Nascimento