2.1 Preparation
for Conjugation
1. EMJH liquid medium: Prepare 100 mL of filter-sterilized
bovine serum albumin (BSA) supplement which consists of
pre-autoclaved ultrapure (18 MΩ at 25
C) water supplemented with 10% fraction V BSA, 0.01% calcium chloride, 0.01%
magnesium sulfate, 0.004% zinc sulfate, 0.0003% copper sulfate, 0.005% ferrous sulfate, 0.00002% vitamin B12, 1.25%
Tween-80, and pH adjusted to 7.4 with 2 N NaOH. Add
100 mL of BSA supplement to 890 mL of autoclaved EMJH
base, which consists of 886.6 mL of ultrapure water supplemented with 2.3 g of Difco Leptospira Medium Base EMJH
(Becton Dickinson), 3.4 mL of 100% glycerol stock, and
10 mL of heat-inactivated rabbit serum [39, 40] (see Notes
1–3).
2. EMJH solid medium: 100 mL BSA supplement pre-warmed to
50
C is added to 890 mL of warm EMJH base (886.6 mL of
ultrapure water supplemented with 2.3 g of Difco Leptospira
Medium Base EMJH (Becton Dickinson), 3.4 mL of 100%
glycerol stock, and 1% noble agar prior to autoclaving) and
10 mL of heat-inactivated rabbit serum. Solution of 20–30 mL
is poured into 100 mm  15 mm Petri plates (see Note 4).
3. Luria-Bertani (LB) liquid medium: 10% tryptone, 5% yeast
extract, 10% sodium chloride in distilled water. Autoclave
prior to use.
4. Diaminopimelic acid (DAP) solution: 100 mM DAP in autoclaved ultrapure water, filter-sterilized.
5. Kanamycin solution: 50 mg/mL of kanamycin in autoclaved
ultrapure water, filter-sterilized.
6. Leptospira biflexa serovar Patoc strain Patoc1 (ATCC 23582/
Paris) as the recipient cell (see Notes 5 and 6).
7. E. coli strain β2163 as the donor cell.
8. Plasmid pSW29T-TKS2 or other (see Note 7).
9. Biosafety cabinet type 2A (minimum) with UV disinfection
cycle.
10. Dark-field microscope.
11. Spectrophotometer with detection available at 420 nm.
2.2 Conjugation
1. Glass filter apparatus consisting of 125 mL filter flask, filter
holder base with frit and stopper 25 mm diameter, 15 mL
glass funnel 25 mm diameter, clamp for 25 mm diameter filter
holder (see Note 8).
2. Filter forceps, blunt end.
3. Membrane filter, hydrophilic PVDF type size 0.1 μm pore size,
25 mm diameter (see Note 9).
Random Transposon Mutant Library
79
for Conjugation
1. EMJH liquid medium: Prepare 100 mL of filter-sterilized
bovine serum albumin (BSA) supplement which consists of
pre-autoclaved ultrapure (18 MΩ at 25
C) water supplemented with 10% fraction V BSA, 0.01% calcium chloride, 0.01%
magnesium sulfate, 0.004% zinc sulfate, 0.0003% copper sulfate, 0.005% ferrous sulfate, 0.00002% vitamin B12, 1.25%
Tween-80, and pH adjusted to 7.4 with 2 N NaOH. Add
100 mL of BSA supplement to 890 mL of autoclaved EMJH
base, which consists of 886.6 mL of ultrapure water supplemented with 2.3 g of Difco Leptospira Medium Base EMJH
(Becton Dickinson), 3.4 mL of 100% glycerol stock, and
10 mL of heat-inactivated rabbit serum [39, 40] (see Notes
1–3).
2. EMJH solid medium: 100 mL BSA supplement pre-warmed to
50
C is added to 890 mL of warm EMJH base (886.6 mL of
ultrapure water supplemented with 2.3 g of Difco Leptospira
Medium Base EMJH (Becton Dickinson), 3.4 mL of 100%
glycerol stock, and 1% noble agar prior to autoclaving) and
10 mL of heat-inactivated rabbit serum. Solution of 20–30 mL
is poured into 100 mm  15 mm Petri plates (see Note 4).
3. Luria-Bertani (LB) liquid medium: 10% tryptone, 5% yeast
extract, 10% sodium chloride in distilled water. Autoclave
prior to use.
4. Diaminopimelic acid (DAP) solution: 100 mM DAP in autoclaved ultrapure water, filter-sterilized.
5. Kanamycin solution: 50 mg/mL of kanamycin in autoclaved
ultrapure water, filter-sterilized.
6. Leptospira biflexa serovar Patoc strain Patoc1 (ATCC 23582/
Paris) as the recipient cell (see Notes 5 and 6).
7. E. coli strain β2163 as the donor cell.
8. Plasmid pSW29T-TKS2 or other (see Note 7).
9. Biosafety cabinet type 2A (minimum) with UV disinfection
cycle.
10. Dark-field microscope.
11. Spectrophotometer with detection available at 420 nm.
2.2 Conjugation
1. Glass filter apparatus consisting of 125 mL filter flask, filter
holder base with frit and stopper 25 mm diameter, 15 mL
glass funnel 25 mm diameter, clamp for 25 mm diameter filter
holder (see Note 8).
2. Filter forceps, blunt end.
3. Membrane filter, hydrophilic PVDF type size 0.1 μm pore size,
25 mm diameter (see Note 9).
Random Transposon Mutant Library
79