5. Dry the cuvette with a paper towel and place it in the electroporation system.
6. Electroporate using E. coli electroporation settings: 2500 V,
25 μF, 200 Ω.
7. Immediately add 1 mL recovery medium (RT).
8. Transfer the reaction into a 1.5 mL reaction tube.
9. Incubate at 37
C for 1 h.
10. Pool the transformations in a volume of 250 mL LB medium
containing 50 μg/mL kan and plate a dilution series (10
À4 to
10
À7
) on LB-kan plates. Incubate both overnight at 37
C, the
flask shaking at 200 rpm.
11. Estimate transformation efficiency from the colony count of
the dilution series.
12. Isolate plasmid DNA from the pool and from at least 12 individual clones and sequence the KDAC ORF.
13. If transformation efficiency exceeds three times the diversity of
the library and the majority of individual sequences are unique
and without frameshifts continue to the next step.
14. Use the plasmid preparation isolated from the pool as template
in the next round of mutagenesis.
3.2 Selection
of Lysine Deacetylases
The mutant libraries generated above contain a large number of
different mutants due to the numerous possible combinations of
randomized positions (20
5 different proteins coded for by 32
5
codon combinations if five positions are mutated). Most of the
mutations will be detrimental to protein function or folding, rendering the mutants inactive. Hence, an efficient selection system is
required to identify the mutants of interest in a large background of
inactive ones. Therefore, we designed a bacterial selection system
that allows parallel screening of each mutant in a single step [24]. We
created an E. coli strain without deacetylase activity and impaired
uracil biosynthesis by inactivating the genes for CobB and PyrF
using CRISPR/Cas9 and Lambda Red Recombineering [30]. We
complemented the uracil auxotrophic strain with Ura3 (orotidine
5
0 -phosphate decarboxylase), which contains an amber codon in
Table 4
Restriction digest of PCR product (scale to match volume of PCR product)
PCR product
30 μL
ddH 2 O
1 1 μL
Cut smart buffer (10Â)
5 μL
Bsa I-HF (20 U μL
À1 )
4 μL
Total volume
50 μL
A Directed Evolution System for Lysine Deacetylases
329
6. Electroporate using E. coli electroporation settings: 2500 V,
25 μF, 200 Ω.
7. Immediately add 1 mL recovery medium (RT).
8. Transfer the reaction into a 1.5 mL reaction tube.
9. Incubate at 37
C for 1 h.
10. Pool the transformations in a volume of 250 mL LB medium
containing 50 μg/mL kan and plate a dilution series (10
À4 to
10
À7
) on LB-kan plates. Incubate both overnight at 37
C, the
flask shaking at 200 rpm.
11. Estimate transformation efficiency from the colony count of
the dilution series.
12. Isolate plasmid DNA from the pool and from at least 12 individual clones and sequence the KDAC ORF.
13. If transformation efficiency exceeds three times the diversity of
the library and the majority of individual sequences are unique
and without frameshifts continue to the next step.
14. Use the plasmid preparation isolated from the pool as template
in the next round of mutagenesis.
3.2 Selection
of Lysine Deacetylases
The mutant libraries generated above contain a large number of
different mutants due to the numerous possible combinations of
randomized positions (20
5 different proteins coded for by 32
5
codon combinations if five positions are mutated). Most of the
mutations will be detrimental to protein function or folding, rendering the mutants inactive. Hence, an efficient selection system is
required to identify the mutants of interest in a large background of
inactive ones. Therefore, we designed a bacterial selection system
that allows parallel screening of each mutant in a single step [24]. We
created an E. coli strain without deacetylase activity and impaired
uracil biosynthesis by inactivating the genes for CobB and PyrF
using CRISPR/Cas9 and Lambda Red Recombineering [30]. We
complemented the uracil auxotrophic strain with Ura3 (orotidine
5
0 -phosphate decarboxylase), which contains an amber codon in
Table 4
Restriction digest of PCR product (scale to match volume of PCR product)
PCR product
30 μL
ddH 2 O
1 1 μL
Cut smart buffer (10Â)
5 μL
Bsa I-HF (20 U μL
À1 )
4 μL
Total volume
50 μL
A Directed Evolution System for Lysine Deacetylases
329