187
1. Transform competent E. coli BL21 (DE3) cells separately with
the pET29a_TEM1-β-lactamaseE104D and pET29a_BLIP
plasmids (see Note 3).
2. For each construct, inoculate 20 mL of LB medium supplemented with 30 mg/L kanamycin with a single colony of
transformed bacteria and incubate overnight at 37 °C in a
shaking incubator at 250 rpm.
3. Transfer the seed culture to 2 L of LB medium supplemented
with 30 mg/L kanamycin.
4. Grow the bacteria in a shaking incubator at 200 rpm at
37 °C. At an optical density at λ = 600 nm of 0.6, add 2 mL of
IPTG stock yielding a final concentration of 100 μM IPTG
and incubate at 16 °C for 20 h.
5. Harvest the cells by centrifugation at 10,000 × g for 10 min
at 4 °C.
6. Resuspend the pelleted cells in 300 mL osmotic shock solution
and incubate for 10 min at room temperature.
7. Pellet the cells by centrifugation at 12,000 × g for 20 min.
8. To extract the periplasmic fraction, resuspend the pelleted
cells in 300 mL ice-cold low salt solution and incubate for
10 min on ice prior to centrifugation at 40,000 × g for 40 min
at 4 °C.
9. Add 10× Tris–HCl buffer to the supernatant (i.e., the periplasmic
fraction) to yield a final concentration of 20 mM Tris–HCl.
10. Charge a 2 mL His-bind column (gravity flow) with Ni
2+
ions
by washing the column with 10 column volumes of charge
buffer and subsequently equilibrate with 10 column volumes
bind buffer.
11. Load the periplasmic fraction on the His-bind column and
allow it to enter the column bed completely. Wash the column
with 10 column volumes of wash buffer and subsequently elute
the protein of interest with 10 column volumes of elution
buffer.
12. Concentrate the eluted proteins to a final volume of ~2.5 mL
using the centrifugal filter unit.
13. Exchange the buffer to storage buffer by gel filtration using a
PD-10 desalting column and determine the concentration by
measuring the optical absorption at λ = 280 nm with the
Nanodrop UV-VIS spectrophotometer and calculated extinction
coefficients (see Note 4).
14. Flash-freeze the purified proteins in liquid nitrogen and store
at −80 °C.
3.1 Expression
and Purification
of Recombinant
Proteins
DNA Sensors Based on β-Lactamase
1. Transform competent E. coli BL21 (DE3) cells separately with
the pET29a_TEM1-β-lactamaseE104D and pET29a_BLIP
plasmids (see Note 3).
2. For each construct, inoculate 20 mL of LB medium supplemented with 30 mg/L kanamycin with a single colony of
transformed bacteria and incubate overnight at 37 °C in a
shaking incubator at 250 rpm.
3. Transfer the seed culture to 2 L of LB medium supplemented
with 30 mg/L kanamycin.
4. Grow the bacteria in a shaking incubator at 200 rpm at
37 °C. At an optical density at λ = 600 nm of 0.6, add 2 mL of
IPTG stock yielding a final concentration of 100 μM IPTG
and incubate at 16 °C for 20 h.
5. Harvest the cells by centrifugation at 10,000 × g for 10 min
at 4 °C.
6. Resuspend the pelleted cells in 300 mL osmotic shock solution
and incubate for 10 min at room temperature.
7. Pellet the cells by centrifugation at 12,000 × g for 20 min.
8. To extract the periplasmic fraction, resuspend the pelleted
cells in 300 mL ice-cold low salt solution and incubate for
10 min on ice prior to centrifugation at 40,000 × g for 40 min
at 4 °C.
9. Add 10× Tris–HCl buffer to the supernatant (i.e., the periplasmic
fraction) to yield a final concentration of 20 mM Tris–HCl.
10. Charge a 2 mL His-bind column (gravity flow) with Ni
2+
ions
by washing the column with 10 column volumes of charge
buffer and subsequently equilibrate with 10 column volumes
bind buffer.
11. Load the periplasmic fraction on the His-bind column and
allow it to enter the column bed completely. Wash the column
with 10 column volumes of wash buffer and subsequently elute
the protein of interest with 10 column volumes of elution
buffer.
12. Concentrate the eluted proteins to a final volume of ~2.5 mL
using the centrifugal filter unit.
13. Exchange the buffer to storage buffer by gel filtration using a
PD-10 desalting column and determine the concentration by
measuring the optical absorption at λ = 280 nm with the
Nanodrop UV-VIS spectrophotometer and calculated extinction
coefficients (see Note 4).
14. Flash-freeze the purified proteins in liquid nitrogen and store
at −80 °C.
3.1 Expression
and Purification
of Recombinant
Proteins
DNA Sensors Based on β-Lactamase