3. Side product formation severely decreases the efficiency of
ligation, requiring more PCRs for sufficient DNA amounts
after gel purification.
4. High concentrations of DNA will lead to the formation
of concatemers, scale volume to adjust DNA concentration
<10 ng/μl.
5. Longer centrifugation times improve DNA recovery.
6. Do not overly dry the DNA.
7. More than 50% of the DNA should be circular for efficient
electroporation.
8. If the cells continue to stick to the wall of the tube and do not
get resuspended easily, add one more washing step.
9. The cells tend to grow very slowly at this step. An OD600 of
1 would be ideal, but values as low as 0.3 are also sufficient if
the growth takes too much time.
10. The default procedure for screening is one round of positive
selection in the beginning, then one round of negative selection and finally one more round of positive selection with the
same conditions as in the first round. If there are only very few
colonies growing on the selection plates, the screening can be
stopped after one or two rounds. In this case, the single colonies can be picked directly for plasmid DNA isolation, and step
7 can be skipped.
11. The 96-well block can be stored at À20
C, and the isolation of
plasmid DNA can be continued on another day.
12. Titrate dilution series to find the optimal working concentration for your deacetylase.
13. Due to low transformation efficiency, it is recommended to
successively transform the plasmids. A stock of E. coli DH10B
ΔcobB ΔpyrF competent cells with pPylT-Ura3 plasmid can be
stored at À80
C. The pPylT-Ura3 plasmid is required to
provide sufficient AraC protein for the induction of CobB
expression. Since CobB isolates are initially encoded on minimal pBK plasmids, this method avoids an intermediate
recloning.
Acknowledgments
The authors are grateful for the financial support by the
Max-Planck Institute of Molecular Physiology and the German
Research Foundation (DFG) [NE1589/5-1 to H.N.].
A Directed Evolution System for Lysine Deacetylases
335
ligation, requiring more PCRs for sufficient DNA amounts
after gel purification.
4. High concentrations of DNA will lead to the formation
of concatemers, scale volume to adjust DNA concentration
<10 ng/μl.
5. Longer centrifugation times improve DNA recovery.
6. Do not overly dry the DNA.
7. More than 50% of the DNA should be circular for efficient
electroporation.
8. If the cells continue to stick to the wall of the tube and do not
get resuspended easily, add one more washing step.
9. The cells tend to grow very slowly at this step. An OD600 of
1 would be ideal, but values as low as 0.3 are also sufficient if
the growth takes too much time.
10. The default procedure for screening is one round of positive
selection in the beginning, then one round of negative selection and finally one more round of positive selection with the
same conditions as in the first round. If there are only very few
colonies growing on the selection plates, the screening can be
stopped after one or two rounds. In this case, the single colonies can be picked directly for plasmid DNA isolation, and step
7 can be skipped.
11. The 96-well block can be stored at À20
C, and the isolation of
plasmid DNA can be continued on another day.
12. Titrate dilution series to find the optimal working concentration for your deacetylase.
13. Due to low transformation efficiency, it is recommended to
successively transform the plasmids. A stock of E. coli DH10B
ΔcobB ΔpyrF competent cells with pPylT-Ura3 plasmid can be
stored at À80
C. The pPylT-Ura3 plasmid is required to
provide sufficient AraC protein for the induction of CobB
expression. Since CobB isolates are initially encoded on minimal pBK plasmids, this method avoids an intermediate
recloning.
Acknowledgments
The authors are grateful for the financial support by the
Max-Planck Institute of Molecular Physiology and the German
Research Foundation (DFG) [NE1589/5-1 to H.N.].
A Directed Evolution System for Lysine Deacetylases
335