4. White colonies are selected, plasmids are extracted, and positive
clones are confirmed by plasmid DNA restriction; mix 3 μL of
plasmid, 1 μL of buffer 10Â, 0.25 μL of each enzyme, and
5.5 μL of water, and incubate mixture for 1 h at 37
C.
5. DNA fragments are purified and ligated into the E. coli expression vector as described in Table 3. Incubate the reaction
mixture for 50 min at 22
C.
6. Plasmids are used to transform competent E. coli BL21, which
are then plated on LB agar containing 50 μg/mL ampicillin
and 34 μg/mL chloramphenicol. Plates are incubated overnight at 37
C. Positive clones are confirmed by DNA restriction analysis, as described above.
7. Select one positive colony and prepare a pre-inoculum in LB
medium containing the antibiotics of interest. Allow it to grow
until saturation, generally, overnight at 37
C.
8. Dilute 20-fold the saturated inoculum in a larger volume of LB
medium containing antibiotics, and place the flask in a 37
C
incubator with continuous shaking until an absorbance of
0.6 at 600 nm is reached. For recombinant protein synthesis,
add 0.1–1 mM IPTG, and keep under continuous shaking at
37
C for 3 h.
9. Centrifuge the bacterial suspensions at 3,075 Â g for 15 min at
4
C. Resuspend the bacterial cell pellets in ten volumes of lysis
buffer in relation to the culture volume used, place on ice for
15 min, and lyse cells with the aid of a cell disruptor.
10. Centrifuge the suspension at 12,000 Â g for 15 m at 4
C, and
separate the soluble and insoluble fractions. If the protein is
expressed in the insoluble form, resuspend the pellet in the
resuspension buffer containing 8 M urea. Incubate overnight
with shaking. Cloning and expression steps are shown in Fig. 1.
Table 3
Recipe for a ligation reaction on pAE vector
Ligation reaction for cloning into an expression vector
Reagents
Concentration
Buffer T4 DNA ligase
1Â
pAE vector
100 ng
DNA fragment
a
T4 DNA ligase
0.5 μL
Pure water
Complete to a final volume 20 μL
a
Use 3–5 times more DNA insert in relation to vector
Cell Adhesion Assay for Leptospiral Proteins
177
clones are confirmed by plasmid DNA restriction; mix 3 μL of
plasmid, 1 μL of buffer 10Â, 0.25 μL of each enzyme, and
5.5 μL of water, and incubate mixture for 1 h at 37
C.
5. DNA fragments are purified and ligated into the E. coli expression vector as described in Table 3. Incubate the reaction
mixture for 50 min at 22
C.
6. Plasmids are used to transform competent E. coli BL21, which
are then plated on LB agar containing 50 μg/mL ampicillin
and 34 μg/mL chloramphenicol. Plates are incubated overnight at 37
C. Positive clones are confirmed by DNA restriction analysis, as described above.
7. Select one positive colony and prepare a pre-inoculum in LB
medium containing the antibiotics of interest. Allow it to grow
until saturation, generally, overnight at 37
C.
8. Dilute 20-fold the saturated inoculum in a larger volume of LB
medium containing antibiotics, and place the flask in a 37
C
incubator with continuous shaking until an absorbance of
0.6 at 600 nm is reached. For recombinant protein synthesis,
add 0.1–1 mM IPTG, and keep under continuous shaking at
37
C for 3 h.
9. Centrifuge the bacterial suspensions at 3,075 Â g for 15 min at
4
C. Resuspend the bacterial cell pellets in ten volumes of lysis
buffer in relation to the culture volume used, place on ice for
15 min, and lyse cells with the aid of a cell disruptor.
10. Centrifuge the suspension at 12,000 Â g for 15 m at 4
C, and
separate the soluble and insoluble fractions. If the protein is
expressed in the insoluble form, resuspend the pellet in the
resuspension buffer containing 8 M urea. Incubate overnight
with shaking. Cloning and expression steps are shown in Fig. 1.
Table 3
Recipe for a ligation reaction on pAE vector
Ligation reaction for cloning into an expression vector
Reagents
Concentration
Buffer T4 DNA ligase
1Â
pAE vector
100 ng
DNA fragment
a
T4 DNA ligase
0.5 μL
Pure water
Complete to a final volume 20 μL
a
Use 3–5 times more DNA insert in relation to vector
Cell Adhesion Assay for Leptospiral Proteins
177