8. Any remaining albumin in the samples can turn them viscous.
It is very important to wash the bacterial pellets thoroughly.
9. The cell concentration of leptospiral protein extracts can vary
according to the abundance of the target protein. As a starting
point, we recommend adjusting the OD 420 of the cell suspension to 10. In some cases, as we experienced with FliG protein
(low abundance), this was not enough, and therefore, we had
to concentrate exponential-phase cells up to 300 times to
prepare the extracts. We performed this by centrifuging
30 mL of the culture and resuspending the final pellet in
100 μL.
10. It is recommended to include an antiserum against another
leptospiral protein as a loading control. For example, we have
used anti-DnaK antiserum along with the antiserum against
the target protein to show that leptospiral extracts are loaded
into the lanes.
11. Avoid using shakers with circular rotation, since the contact of
the solution with center of the membrane will be less efficient
when using small volumes. If that is the only shaker available,
we recommend using larger volumes. Using “up-and-down”
shakers ensures that the whole membrane surface will be in
contact with the solution, even with small volumes (6–10 mL
per membrane).
12. Depending on the gene silenced, there is an expected phenotype to be observed, and this could work as a complementary
result to Western blotting. For example, we have successfully
silenced the β-galactosidase gene in L. biflexa, and since chromogenic substrates for this enzyme are routinely used, gene
silencing was demonstrated by enzymatic assays. Also, when
genes involved in motility are silenced, smaller colonies in
plates can be observed.
13. Since our gene silencing tools are episomal, it is recommended
to maintain selective pressure in in vitro cultures always. In our
experience, the pMaOri backbone is quite stable, and
β-galactosidase gene silencing in L. biflexa is maintained even
after eight passages with no antibiotic addition. In the eighth
passage, we observed a remaining 50% gene silencing, which
was fully recovered after one passage in new medium with
spectinomycin (100% silencing).
Acknowledgments
This work was financially supported by FAPESP (grants 2014/
50981-0 and 2017/06731-8), CNPq (grants 301229/2017-1
and 441449/2014-0), and Fundac ¸a ˜o Butantan. Dr. A. Leyva
(USA) provided English editing of the manuscript.
Gene Silencing by Single-Guide RNA and dCas9
121
It is very important to wash the bacterial pellets thoroughly.
9. The cell concentration of leptospiral protein extracts can vary
according to the abundance of the target protein. As a starting
point, we recommend adjusting the OD 420 of the cell suspension to 10. In some cases, as we experienced with FliG protein
(low abundance), this was not enough, and therefore, we had
to concentrate exponential-phase cells up to 300 times to
prepare the extracts. We performed this by centrifuging
30 mL of the culture and resuspending the final pellet in
100 μL.
10. It is recommended to include an antiserum against another
leptospiral protein as a loading control. For example, we have
used anti-DnaK antiserum along with the antiserum against
the target protein to show that leptospiral extracts are loaded
into the lanes.
11. Avoid using shakers with circular rotation, since the contact of
the solution with center of the membrane will be less efficient
when using small volumes. If that is the only shaker available,
we recommend using larger volumes. Using “up-and-down”
shakers ensures that the whole membrane surface will be in
contact with the solution, even with small volumes (6–10 mL
per membrane).
12. Depending on the gene silenced, there is an expected phenotype to be observed, and this could work as a complementary
result to Western blotting. For example, we have successfully
silenced the β-galactosidase gene in L. biflexa, and since chromogenic substrates for this enzyme are routinely used, gene
silencing was demonstrated by enzymatic assays. Also, when
genes involved in motility are silenced, smaller colonies in
plates can be observed.
13. Since our gene silencing tools are episomal, it is recommended
to maintain selective pressure in in vitro cultures always. In our
experience, the pMaOri backbone is quite stable, and
β-galactosidase gene silencing in L. biflexa is maintained even
after eight passages with no antibiotic addition. In the eighth
passage, we observed a remaining 50% gene silencing, which
was fully recovered after one passage in new medium with
spectinomycin (100% silencing).
Acknowledgments
This work was financially supported by FAPESP (grants 2014/
50981-0 and 2017/06731-8), CNPq (grants 301229/2017-1
and 441449/2014-0), and Fundac ¸a ˜o Butantan. Dr. A. Leyva
(USA) provided English editing of the manuscript.
Gene Silencing by Single-Guide RNA and dCas9
121