3. 70% ethanol (Thermo Fisher Scientific).
4. 100% ethanol.
5. Primers (100 μM, PAGE purified, Sigma Aldrich, Note 1).
6. dNTPs (10 mM, Thermo Fisher Scientific).
7. QIA Quick PCR Purification Kit.
8. Bsa I-HF (20 U μL
À1 , NEB).
9. Dpn I (20 U μL
À1
, NEB).
10. T4 DNA Ligase (5 U μL
À1 , Thermo Fisher Scientific).
11. 10Â T4-Ligase-Buffer (Thermo Fisher Scientific).
12. Hind III (10 U μL
À1 , Thermo Fisher Scientific).
13. Electroporation Cuvette (2 mm, sterile, VWR).
14. Recovery Medium (Invitrogen).
15. Thermocycler (SensoQuest).
16. Centrifuge 5424R (Eppendorf).
17. Vortex Genie 2 (Scientific Industries).
18. Thermomixer comfort (Eppendorf).
19. NanoDrop (ND-1000, Peqlab).
20. Electroporation System GenePulser Xcell (BioRad).
2.3 Selection
of Lysine Deacetylases
1. M9 Medium: 42.5 g/l Na 2 HPO 4 ·2H 2 O, 15.0 g/l KH 2 PO 4 ,
2.5 g/l NaCl, 5.0 g/l NH 4 Cl, 1 mM MgSO 4 , 0.4% glucose,
0.1 mM CaCl 2 in sterile ddH 2 O.
2. LB medium: 1% tryptone, 0.5% yeast extract, 1% NaCl.
3. LB agar plates: LB medium, 2% agar, 15 μg/mL tetracycline
(tet), and 50 μg/mL kanamycin (kan).
4. Positive selection agar plates: M9 medium, 2% agar, 0.2% arabinose, 1% glycerol, 4.1 mg/mL tryptophan, 16 mg/mL valine,
16 mg/mL leucine, 16 mg/mL isoleucine, 50 μg/mL kan,
15 μg/mL tet, 10 mM N(ε)-acetyl-L-lysine (or 1 mM of any
other modified lysine).
5. Negative selection agar plates: M9 minimal medium, 2% agar,
0.2% arabinose, 1% glycerol, 4.1 mg/L tryptophan, 16 mg/L
valine, 16 mg/L leucine, 16 mg/L isoleucine, 50 μg/mL kan,
15 μg/mL tet, 10 mM N(ε)-acetyl-L-lysine (or 1 mM of any
other modified lysine), 0.1% 5-fluoroorotic acid, 0.1 mM
uracil.
6. Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl,
10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 .
7. Petri dishes (150 Â 20 mm, Greiner Bio-One).
8. 1 M in H 2 O. H-Lys(Ac)-OH (BACHEM).
9. 50 mg/mL kanamycin (Applichem).
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Martin Spinck et al.
4. 100% ethanol.
5. Primers (100 μM, PAGE purified, Sigma Aldrich, Note 1).
6. dNTPs (10 mM, Thermo Fisher Scientific).
7. QIA Quick PCR Purification Kit.
8. Bsa I-HF (20 U μL
À1 , NEB).
9. Dpn I (20 U μL
À1
, NEB).
10. T4 DNA Ligase (5 U μL
À1 , Thermo Fisher Scientific).
11. 10Â T4-Ligase-Buffer (Thermo Fisher Scientific).
12. Hind III (10 U μL
À1 , Thermo Fisher Scientific).
13. Electroporation Cuvette (2 mm, sterile, VWR).
14. Recovery Medium (Invitrogen).
15. Thermocycler (SensoQuest).
16. Centrifuge 5424R (Eppendorf).
17. Vortex Genie 2 (Scientific Industries).
18. Thermomixer comfort (Eppendorf).
19. NanoDrop (ND-1000, Peqlab).
20. Electroporation System GenePulser Xcell (BioRad).
2.3 Selection
of Lysine Deacetylases
1. M9 Medium: 42.5 g/l Na 2 HPO 4 ·2H 2 O, 15.0 g/l KH 2 PO 4 ,
2.5 g/l NaCl, 5.0 g/l NH 4 Cl, 1 mM MgSO 4 , 0.4% glucose,
0.1 mM CaCl 2 in sterile ddH 2 O.
2. LB medium: 1% tryptone, 0.5% yeast extract, 1% NaCl.
3. LB agar plates: LB medium, 2% agar, 15 μg/mL tetracycline
(tet), and 50 μg/mL kanamycin (kan).
4. Positive selection agar plates: M9 medium, 2% agar, 0.2% arabinose, 1% glycerol, 4.1 mg/mL tryptophan, 16 mg/mL valine,
16 mg/mL leucine, 16 mg/mL isoleucine, 50 μg/mL kan,
15 μg/mL tet, 10 mM N(ε)-acetyl-L-lysine (or 1 mM of any
other modified lysine).
5. Negative selection agar plates: M9 minimal medium, 2% agar,
0.2% arabinose, 1% glycerol, 4.1 mg/L tryptophan, 16 mg/L
valine, 16 mg/L leucine, 16 mg/L isoleucine, 50 μg/mL kan,
15 μg/mL tet, 10 mM N(ε)-acetyl-L-lysine (or 1 mM of any
other modified lysine), 0.1% 5-fluoroorotic acid, 0.1 mM
uracil.
6. Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl,
10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 .
7. Petri dishes (150 Â 20 mm, Greiner Bio-One).
8. 1 M in H 2 O. H-Lys(Ac)-OH (BACHEM).
9. 50 mg/mL kanamycin (Applichem).
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Martin Spinck et al.