the cells into aliquots of 500 μL and isolate the plasmid DNA
using a commercial kit. Pool plasmid DNA for the next round
of selection.
9. Repeat steps 1–8 until all selection rounds are completed.
10. Transform DH10B cells with the plasmid DNA obtained by
the selection via heat shock. Plate the cells on agar plates
containing 50 μg/mL kan and incubate them overnight at
37
C.
11. On the next day, prepare a 96 deep well block by filling each
well with 1 mL LB medium containing 50 μg/mL kan. Inoculate each well by picking single colonies of the transformed
DH10B cells. Incubate the block overnight at 37
C.
12. Harvest the cells by centrifuging the 96 well block for 10 min
at 2700 Â g and remove the media.
13. Isolate the plasmid DNA of each well and sequence the KDAC
ORF (see Note 11).
3.2.3 6-AU Assay
(Optional)
This step is optional, but can help filter out the most active variants
from a pool of mutants. Therefore, repeat the last selection step of
Subheading 3.2.2 with the addition of 6-azauridine (6-AU) to the
agar plates (0.01–2 mM, at least 10 different concentrations). This
can be done by plating the pool of mutants or by replicating single
colonies from a 96-well plate onto the selection plates. 6-AU is an
inhibitor of Ura3, which requires cells to produce more active
Ura3, increasing the stringency of the positive selection assay.
3.3 KDAC Assay
Based on Acylated
Firefly Luciferase
Similar to Ura3, the firefly luciferase (FLuc) also contains an essential lysine (K529) in its active site. When modified, the luciferase is
completely inactive and therefore can be used to monitor the
activity of enzymes capable of reversing the modification [25]. We
use this to assay the activity and selectivity of the isolated mutants.
3.3.1 Preparation
of FLuc mod
1. Transform E. coli BL21 DE3 with plasmids pFluc and
pBK-AcKRS3opt or pBK-PylS depending on the desired
modification.
2. Grow the cells in LB medium (50 μg/mL spectinomycin and
50 μg/mL kan, 5 mM N(ε)-acetyl-lysine or 1 mM other AS
and 20 mM nicotinamide) at 37
C to and OD 600 of 1.0.
3. Shift cells to 30
C and induce protein expression by the
addition of 1 mM IPTG.
4. After 4 h at 30
C harvest cells by centrifugation, wash with
PBS, and suspend in Ni-wash buffer.
5. Lyse cells by passing them three times through a microfluidizer.
6. Centrifuge lysate at 20,000 Â g for 20 min and transfer the
supernatant to a falcon tube.
332
Martin Spinck et al.
using a commercial kit. Pool plasmid DNA for the next round
of selection.
9. Repeat steps 1–8 until all selection rounds are completed.
10. Transform DH10B cells with the plasmid DNA obtained by
the selection via heat shock. Plate the cells on agar plates
containing 50 μg/mL kan and incubate them overnight at
37
C.
11. On the next day, prepare a 96 deep well block by filling each
well with 1 mL LB medium containing 50 μg/mL kan. Inoculate each well by picking single colonies of the transformed
DH10B cells. Incubate the block overnight at 37
C.
12. Harvest the cells by centrifuging the 96 well block for 10 min
at 2700 Â g and remove the media.
13. Isolate the plasmid DNA of each well and sequence the KDAC
ORF (see Note 11).
3.2.3 6-AU Assay
(Optional)
This step is optional, but can help filter out the most active variants
from a pool of mutants. Therefore, repeat the last selection step of
Subheading 3.2.2 with the addition of 6-azauridine (6-AU) to the
agar plates (0.01–2 mM, at least 10 different concentrations). This
can be done by plating the pool of mutants or by replicating single
colonies from a 96-well plate onto the selection plates. 6-AU is an
inhibitor of Ura3, which requires cells to produce more active
Ura3, increasing the stringency of the positive selection assay.
3.3 KDAC Assay
Based on Acylated
Firefly Luciferase
Similar to Ura3, the firefly luciferase (FLuc) also contains an essential lysine (K529) in its active site. When modified, the luciferase is
completely inactive and therefore can be used to monitor the
activity of enzymes capable of reversing the modification [25]. We
use this to assay the activity and selectivity of the isolated mutants.
3.3.1 Preparation
of FLuc mod
1. Transform E. coli BL21 DE3 with plasmids pFluc and
pBK-AcKRS3opt or pBK-PylS depending on the desired
modification.
2. Grow the cells in LB medium (50 μg/mL spectinomycin and
50 μg/mL kan, 5 mM N(ε)-acetyl-lysine or 1 mM other AS
and 20 mM nicotinamide) at 37
C to and OD 600 of 1.0.
3. Shift cells to 30
C and induce protein expression by the
addition of 1 mM IPTG.
4. After 4 h at 30
C harvest cells by centrifugation, wash with
PBS, and suspend in Ni-wash buffer.
5. Lyse cells by passing them three times through a microfluidizer.
6. Centrifuge lysate at 20,000 Â g for 20 min and transfer the
supernatant to a falcon tube.
332
Martin Spinck et al.