3. Heat-shock cells at 42
C for 30 s.
4. Immediately place cells back on ice.
5. Add 250 μL of SOC media.
6. Allow cells to incubate in the console incubator shaker at 37
C
for 1 h while shaking at 200 rpm.
7. Afterward, plate 50 μL of transformed cells on LB-agar plates
containing 0.1 mg mL
À1 of ampicillin or another plasmid
selective antibiotic.
8. Incubate the plates upside down for 16 h at 37
C.
9. After incubation, check to observe the presence of colonies on
the plates.
3.2 Heterologous
Expression of CCDs
in E. coli
1. For the generation of a bacterial preculture, mix 5 μL of
100 mg mL
À1 stock solution of ampicillin into 5 mL of LB
media.
2. Select a single bacteria colony from the agar plate using a sterile
pipette tip and place it into the 5 mL LB media.
3. Allow bacteria culture to grow for 16 h at 37
C in an incubator
shaker set to 200 rpm.
4. The generation of the expression culture involves adding 50 μL
of stock ampicillin into a 50 mL LB media and 100 μL of stock
iron/ascorbic acid solution.
5. Add 500 μL of fresh overnight preculture to each 50 mL LB
expression culture.
6. Incubate the expression culture at 37
C until the OD 600
equals 0.6 in an incubator shaker set to 200 rpm. The optical
density (OD) of the culture can be determined using a cell
densitometer.
7. Once an OD 600 of 0.6 is reached, expression can be induced by
adding IPTG (0.1–0.5 mM final concentration) from the stock
solution and incubated at 16
C for 24 h in an incubator shaker
set to 200 rpm.
8. Harvest cells by centrifugation at 6000 Â g for 15 min at 4
C.
9. Discard supernatant carefully and store the bacterial pellet at
À80
C until further use.
3.3 Preparation
of CCD Protein
Extracts
1. Thaw the bacterial pellet on ice.
2. Add 20 μL of 100 mM PMSF and 20 μL of 100 mM TCEP to
2 mL of the extraction buffer.
3. Add the solution to the bacterial pellet and mix homogeneously by vortexing.
4. Add 4 μL of lysozyme solution (10 mg mL
À1
), vortex again,
and allow to stand on ice for 30 min.
80
Linda Dora Thomas et al.