3. Liquid EMJH medium (with or without spectinomycin,
depending on the step).
4. Solid EMJH plates (with or without spectinomycin, depending
on the step).
5. Sterile water (autoclaved or 0.22 μm filtered).
6. 30
C shaker and incubator.
7. Spectrophotometer (420 nm).
8. Sterile 50 mL polypropylene centrifuge tubes.
9. 0.2 cm electroporation cuvettes.
10. Electroporator.
2.4 SDSPolyacrylamide Gel
Electrophoresis
(SDS-PAGE)
and Immunoblotting
1. Thirty percent acrylamide/bis-acrylamide solution (29:1):
Wearing gloves and mask, weigh out 29 g of acrylamide and
1 g of bis-acrylamide, and mix the two reagents in water at a
final volume of 100 mL. Shake the mixture until dissolved and
filter the solution using a 0.22 μm filter. Store at 4
C, in a
Fig. 1 Expressed dCas9 and sgRNA complex blocks RNA elongation. The plasmid pMaOri.dCas9 contains the
R6k γ origin of replication, requiring the Pi protein for replication. dCas9 transcription is driven by the native
S. pyogenes promoter (pNative). Unique XmaI, SmaI, and SalI restriction sites can be used for sgRNA cassette
ligation. Plasmids with pMaOri backbone can be used to transform Leptospira spp. by electroporation or
conjugation, due to oriT. After sgRNA cassette ligation (here represented in XmaI site), recombinant pMaOri.
dCas9sgRNA plasmid is able to code for both dCas9 and sgRNA (directed by lipL32, p32, promoter), conferring
a one-step gene silencing tool. dCas9 is positioned in genomic targets by Watson-Crick base pairing between
spacer sequence and DNA coding strand, provoking a blockage of RNA polymerase elongation, abolishing
transcription, and consequently, affecting protein levels
112
Luis Guilherme Virgı ´lio Fernandes and Ana Lucia Tabet Oller Nascimento
depending on the step).
4. Solid EMJH plates (with or without spectinomycin, depending
on the step).
5. Sterile water (autoclaved or 0.22 μm filtered).
6. 30
C shaker and incubator.
7. Spectrophotometer (420 nm).
8. Sterile 50 mL polypropylene centrifuge tubes.
9. 0.2 cm electroporation cuvettes.
10. Electroporator.
2.4 SDSPolyacrylamide Gel
Electrophoresis
(SDS-PAGE)
and Immunoblotting
1. Thirty percent acrylamide/bis-acrylamide solution (29:1):
Wearing gloves and mask, weigh out 29 g of acrylamide and
1 g of bis-acrylamide, and mix the two reagents in water at a
final volume of 100 mL. Shake the mixture until dissolved and
filter the solution using a 0.22 μm filter. Store at 4
C, in a
Fig. 1 Expressed dCas9 and sgRNA complex blocks RNA elongation. The plasmid pMaOri.dCas9 contains the
R6k γ origin of replication, requiring the Pi protein for replication. dCas9 transcription is driven by the native
S. pyogenes promoter (pNative). Unique XmaI, SmaI, and SalI restriction sites can be used for sgRNA cassette
ligation. Plasmids with pMaOri backbone can be used to transform Leptospira spp. by electroporation or
conjugation, due to oriT. After sgRNA cassette ligation (here represented in XmaI site), recombinant pMaOri.
dCas9sgRNA plasmid is able to code for both dCas9 and sgRNA (directed by lipL32, p32, promoter), conferring
a one-step gene silencing tool. dCas9 is positioned in genomic targets by Watson-Crick base pairing between
spacer sequence and DNA coding strand, provoking a blockage of RNA polymerase elongation, abolishing
transcription, and consequently, affecting protein levels
112
Luis Guilherme Virgı ´lio Fernandes and Ana Lucia Tabet Oller Nascimento