10. 15 mg/mL tetracyclin (Sigma Aldrich).
11. 10% in DMSO 5-Fluoroorotic acid (Zymo Research).
12. 25 mM in H 2 O Uracil (Applichem).
13. 96 deep well block (AXYGEN).
14. 10 mm cuvettes (Sarstedt).
15. Plasmid Isolation Kit (Roti-Prep Plasmid MINI, ROTH).
16. Incubator IPP55 (Memmert).
17. Multichannel pipette (10–100 μL Research plus, Eppendorf).
18. Photometer (Biowave CO8000 Cell Density Meter, WPA).
19. Centrifuge 5810 R (Eppendorf).
2.4 KDAC Assay
Based on Acylated
Firefly Luciferase
1. 20% L(+)-Arabinose (Roth).
2. 50 mg/mL spectinomycin.
3. 1 M IPTG (Roth).
4. 2 M Nicotinamide (Sigma Aldrich).
5. Pierce™ Protease Inhibitor Mini Tablets, EDTA Free (Thermo
Fisher Scientific).
6. 200 mM Phenylmethylsulfonylfluorid (PMSF, Roth) in
isopropanol.
7. 1 M Dithiothreitol (DTT, AppliChem).
8. Lysozyme (SERVA).
9. 1 mg per purification DNase I (Roche).
10. HisPur™ Ni-NTA Resin (Thermo Fisher Scientific).
11. CobB wash buffer: 20 mM HEPES, pH 7.5, 200 mM NaCl,
20 mM DTT, 1 mM PMSF supplemented with lysozyme
(0.1 mg/mL), and DNAse (~0.1 mg).
12. Elution buffer (CobB wash buffer supplemented with 200 mM
imidazole).
13. Gel filtration buffer (20 mM HEPES, pH 7.5, 100 mM NaCl,
10 mM DTT).
14. KDAC reaction buffer: 25 mM Tris, pH 8, 137 mM NaCl,
2.7 mM KCl, 1 mM MgCl 2 , 1 mM DTT, 1 mg/mL BSA.
15. 2Â Luciferase Buffer: 40 mM Tricine, pH 7.8, 200 μM EDTA,
7.4 mM MgSO 4 , 2 mM NaHCO 3 , 34 mM DTT, 0.5 mM
ATP, 0.5 mM luciferin (Thermo Fisher Scientific).
16. 96-well PCR plates (clear, semi-skirted, Thermo Fisher
Scientific).
17. 80 mM NAD
+
, in 200 mM Tris pH 8 (Sigma Aldrich).
18. 96-well plate (Microfluor
® 2, black, flat bottom, Thermo
Fisher Scientific).
A Directed Evolution System for Lysine Deacetylases
323
11. 10% in DMSO 5-Fluoroorotic acid (Zymo Research).
12. 25 mM in H 2 O Uracil (Applichem).
13. 96 deep well block (AXYGEN).
14. 10 mm cuvettes (Sarstedt).
15. Plasmid Isolation Kit (Roti-Prep Plasmid MINI, ROTH).
16. Incubator IPP55 (Memmert).
17. Multichannel pipette (10–100 μL Research plus, Eppendorf).
18. Photometer (Biowave CO8000 Cell Density Meter, WPA).
19. Centrifuge 5810 R (Eppendorf).
2.4 KDAC Assay
Based on Acylated
Firefly Luciferase
1. 20% L(+)-Arabinose (Roth).
2. 50 mg/mL spectinomycin.
3. 1 M IPTG (Roth).
4. 2 M Nicotinamide (Sigma Aldrich).
5. Pierce™ Protease Inhibitor Mini Tablets, EDTA Free (Thermo
Fisher Scientific).
6. 200 mM Phenylmethylsulfonylfluorid (PMSF, Roth) in
isopropanol.
7. 1 M Dithiothreitol (DTT, AppliChem).
8. Lysozyme (SERVA).
9. 1 mg per purification DNase I (Roche).
10. HisPur™ Ni-NTA Resin (Thermo Fisher Scientific).
11. CobB wash buffer: 20 mM HEPES, pH 7.5, 200 mM NaCl,
20 mM DTT, 1 mM PMSF supplemented with lysozyme
(0.1 mg/mL), and DNAse (~0.1 mg).
12. Elution buffer (CobB wash buffer supplemented with 200 mM
imidazole).
13. Gel filtration buffer (20 mM HEPES, pH 7.5, 100 mM NaCl,
10 mM DTT).
14. KDAC reaction buffer: 25 mM Tris, pH 8, 137 mM NaCl,
2.7 mM KCl, 1 mM MgCl 2 , 1 mM DTT, 1 mg/mL BSA.
15. 2Â Luciferase Buffer: 40 mM Tricine, pH 7.8, 200 μM EDTA,
7.4 mM MgSO 4 , 2 mM NaHCO 3 , 34 mM DTT, 0.5 mM
ATP, 0.5 mM luciferin (Thermo Fisher Scientific).
16. 96-well PCR plates (clear, semi-skirted, Thermo Fisher
Scientific).
17. 80 mM NAD
+
, in 200 mM Tris pH 8 (Sigma Aldrich).
18. 96-well plate (Microfluor
® 2, black, flat bottom, Thermo
Fisher Scientific).
A Directed Evolution System for Lysine Deacetylases
323