3.2.1 Preparation
of Electrocompetent Cells
(E. coli ΔpyrF ΔcobB
pURA3K93TAG-PylS/
AcKRS3)
1. Grow an overnight culture of DH10B ΔcobB ΔpyrF pPylTURA3K93TAG-PylS or DH10B ΔcobB ΔpyrF pPylTURA3K93TAG-AcKRS3 (5 mL LB medium containing
15 μg/mL tet).
2. Inoculate 100 mL LB with 1 mL overnight culture and incubate at 37
C for 2.5–3 h until the OD 600 reaches 0.3.
3. Harvest the cells by centrifugation at 2500 Â g for 5 min at
4
C and remove the medium.
4. Suspend the cells with 30–40 mL ice-cold, autoclaved, particle
free ddH 2 O.
5. Centrifuge at 2000 Â g for 5 min at 4
C and discard the
supernatant.
6. Repeat steps 4 and 5 once again (see Note 8).
7. Centrifuge at 2000 Â g, remove remaining water.
8. Suspend cells in 200 μL ddH 2 O.
9. Keep the cells on ice until further use.
3.2.2 Selection of KDACs
1. Electroporate freshly prepared electrocompetent DH10B
ΔcobB ΔpyrF pPylT-URA3K93TAG-PylS or DH10B ΔcobB
ΔpyrF pPylT-URA3K93TAG-AcKRS3 with 1 μg of the CobB
mutant library.
2. Follow steps 3–7 of Subheading 3.1.3.
3. After the recovery time, add all transformed cells to 100 mL LB
medium containing 50 μg/mL tet and 50 μg/mL kan. Prepare
a small-scale serial dilution down to a factor of 10
À7 and plate
them on appropriate LB agar plates. Incubate media and plates
at 37
C overnight.
4. Estimate the number of transformants from the colony count
of plates. It should exceed three times the number of all possible mutants in the library.
5. Inoculate 50 mL LB containing 50 μg/mL tet and 50 μg/mL
kan with 1 mL of overnight culture and incubate at 37
C until
the OD 600 exceeds 0.3 (see Note 9).
6. Harvest the cells by centrifugation at 500 Â g for 5 min at 4
C
and remove the medium.
7. Resuspend the cells in sterile PBS. For first positive selection,
plate out aliquots of 8 Â 10
8 cells on each positive selection
agar plate and incubate at 37
C for 2–3 days. For negative
selection and all successive selections, plate aliquots of
8 Â 10
5 cells on each negative selection plate and incubate at
37
C for 16 h (see Note 10).
8. Add ~3 mL sterile PBS onto the plate and scrape the colonies
from the agar with the help of a sterile cell spreader. Distribute
A Directed Evolution System for Lysine Deacetylases
331
of Electrocompetent Cells
(E. coli ΔpyrF ΔcobB
pURA3K93TAG-PylS/
AcKRS3)
1. Grow an overnight culture of DH10B ΔcobB ΔpyrF pPylTURA3K93TAG-PylS or DH10B ΔcobB ΔpyrF pPylTURA3K93TAG-AcKRS3 (5 mL LB medium containing
15 μg/mL tet).
2. Inoculate 100 mL LB with 1 mL overnight culture and incubate at 37
C for 2.5–3 h until the OD 600 reaches 0.3.
3. Harvest the cells by centrifugation at 2500 Â g for 5 min at
4
C and remove the medium.
4. Suspend the cells with 30–40 mL ice-cold, autoclaved, particle
free ddH 2 O.
5. Centrifuge at 2000 Â g for 5 min at 4
C and discard the
supernatant.
6. Repeat steps 4 and 5 once again (see Note 8).
7. Centrifuge at 2000 Â g, remove remaining water.
8. Suspend cells in 200 μL ddH 2 O.
9. Keep the cells on ice until further use.
3.2.2 Selection of KDACs
1. Electroporate freshly prepared electrocompetent DH10B
ΔcobB ΔpyrF pPylT-URA3K93TAG-PylS or DH10B ΔcobB
ΔpyrF pPylT-URA3K93TAG-AcKRS3 with 1 μg of the CobB
mutant library.
2. Follow steps 3–7 of Subheading 3.1.3.
3. After the recovery time, add all transformed cells to 100 mL LB
medium containing 50 μg/mL tet and 50 μg/mL kan. Prepare
a small-scale serial dilution down to a factor of 10
À7 and plate
them on appropriate LB agar plates. Incubate media and plates
at 37
C overnight.
4. Estimate the number of transformants from the colony count
of plates. It should exceed three times the number of all possible mutants in the library.
5. Inoculate 50 mL LB containing 50 μg/mL tet and 50 μg/mL
kan with 1 mL of overnight culture and incubate at 37
C until
the OD 600 exceeds 0.3 (see Note 9).
6. Harvest the cells by centrifugation at 500 Â g for 5 min at 4
C
and remove the medium.
7. Resuspend the cells in sterile PBS. For first positive selection,
plate out aliquots of 8 Â 10
8 cells on each positive selection
agar plate and incubate at 37
C for 2–3 days. For negative
selection and all successive selections, plate aliquots of
8 Â 10
5 cells on each negative selection plate and incubate at
37
C for 16 h (see Note 10).
8. Add ~3 mL sterile PBS onto the plate and scrape the colonies
from the agar with the help of a sterile cell spreader. Distribute
A Directed Evolution System for Lysine Deacetylases
331