preferred for Tn library generation as spectinomycin resistance
can occur spontaneously in Leptospira [19].
8. We recommend the Millipore filter apparatus system as it works
well with 25 mm diameter filter membranes. These items are
125 mL filter flask item no. 1002505, filter holder base with frit
and stopper 25 mm size item no. 1002502, 15 mL glass funnel
25 mm size item no. 1002514, and clamp for 25 mm filter
holder item no. 1002503.
9. We recommend Millipore Durapore membrane filter item
no. VVLP02500. Be certain to use a 0.1 μm pore size filter,
as Leptospira may pass through 0.22 μm pores.
10. We use a Welch 2546B pump (which features an inlet water
trap) which is attached to an intermediate 0.2 μm Whatman
Polydisc in-line filter between flask and vacuum pump.
11. We recommend buying silicone tubing that can be autoclaved,
since this decreases risk of contamination outside of the filtration system as well as decreases risk of equipment
contamination.
12. We have found that some filters are less effective for filtering
Leptospira. We recommend Millipore Millex-GS filters.
13. This growth can be from resurrection or from low-passage
L. biflexa. L. biflexa can be grown at 30
C or room temperature (18
C) to allow flexibility of conjugation experiment to
your work schedule. If grown at room temperature, we recommend to move L. biflexa to 30
C at least 1 day prior to
conjugation to increase growth rate.
14. Some laboratories prefer to aliquot E. coli β2163 harboring
pSW29T-TKS2 from freezer culture the morning of conjugation directly into EMJH media, bypassing the LB liquid
medium growth step. This approach also works; however, it
typically has a delayed growth (4–6 h for exponential
growth vs. 2–4 h when grown from LB liquid medium). Neither approach noticeably reduces transformation efficiency.
15. Dilution of E. coli can range from 1:10 to 1:100, with the
1:100 conferring less salts into EMJH media used for conjugation, which may be advantageous. In our lab, either dilution
has yielded no notable difference in transformation efficiency.
16. This EMJH media should not contain kanamycin, since Leptospira will still be sensitive to kanamycin until transconjugation
occurs.
17. If you are planning on completing many conjugation rounds,
scale up with several conicals of E. coli in EMJH.
18. Do not allow E. coli (or Leptospira) to reach stationary phase as
this will decrease transformation efficiency.
Random Transposon Mutant Library
89
can occur spontaneously in Leptospira [19].
8. We recommend the Millipore filter apparatus system as it works
well with 25 mm diameter filter membranes. These items are
125 mL filter flask item no. 1002505, filter holder base with frit
and stopper 25 mm size item no. 1002502, 15 mL glass funnel
25 mm size item no. 1002514, and clamp for 25 mm filter
holder item no. 1002503.
9. We recommend Millipore Durapore membrane filter item
no. VVLP02500. Be certain to use a 0.1 μm pore size filter,
as Leptospira may pass through 0.22 μm pores.
10. We use a Welch 2546B pump (which features an inlet water
trap) which is attached to an intermediate 0.2 μm Whatman
Polydisc in-line filter between flask and vacuum pump.
11. We recommend buying silicone tubing that can be autoclaved,
since this decreases risk of contamination outside of the filtration system as well as decreases risk of equipment
contamination.
12. We have found that some filters are less effective for filtering
Leptospira. We recommend Millipore Millex-GS filters.
13. This growth can be from resurrection or from low-passage
L. biflexa. L. biflexa can be grown at 30
C or room temperature (18
C) to allow flexibility of conjugation experiment to
your work schedule. If grown at room temperature, we recommend to move L. biflexa to 30
C at least 1 day prior to
conjugation to increase growth rate.
14. Some laboratories prefer to aliquot E. coli β2163 harboring
pSW29T-TKS2 from freezer culture the morning of conjugation directly into EMJH media, bypassing the LB liquid
medium growth step. This approach also works; however, it
typically has a delayed growth (4–6 h for exponential
growth vs. 2–4 h when grown from LB liquid medium). Neither approach noticeably reduces transformation efficiency.
15. Dilution of E. coli can range from 1:10 to 1:100, with the
1:100 conferring less salts into EMJH media used for conjugation, which may be advantageous. In our lab, either dilution
has yielded no notable difference in transformation efficiency.
16. This EMJH media should not contain kanamycin, since Leptospira will still be sensitive to kanamycin until transconjugation
occurs.
17. If you are planning on completing many conjugation rounds,
scale up with several conicals of E. coli in EMJH.
18. Do not allow E. coli (or Leptospira) to reach stationary phase as
this will decrease transformation efficiency.
Random Transposon Mutant Library
89