303
efficient screening or selection procedures, provides a ready
means for discrimination against the template construct (see
Subheading 3.2 step 5). In case the resultant PATCHY library
still contains an exceedingly high fraction of the initial template construct, steps (a) and (b) can be repeated.
5. The ligation reaction can alternatively be carried out at 16 °C
over night.
6. A particular strength of PATCHY is the generation of hybrid
genes of defined composition. Although this usually is the
wanted outcome, in certain scenarios the desired traits may not
manifest in any of the hybrid gene variants within the PATCHY
libraries. In these cases, the PATCHY libraries can further be
diversified via error-prone PCR [21]. Introduction of random
residue exchanges, in particular within the linker region
between protein modules, may well yield variants with the
desired functional properties.
7. The screening of PATCHY libraries can be expedited with
fluorescence- activated cell sorting. This is especially useful in
case of protein switches that are supposed to show differential
activity in response to a signal. PATCHY hybrid libraries can
alternately be screened in the absence and presence of this signal, and stringently responding variants can thus be enriched,
identified and isolated.
8. Alternatively, the activity of the PATCHY variants can be
assessed in microtiter format. Within a 96-deep-well plate,
aliquots of 600 μL LB/Kanamycin medium are inoculated
with individual variants and incubated for 16 h in darkness or
under constant blue light (470 nm, 40 μW/cm
2
). All subsequent steps are conducted as described in Subheading 3.2
steps 8 and 9.
9. Many companies offer plasmid preparation and Sanger
sequencing in microtiter format. For larger number of hybrid
variants, this approach usually is more economic in terms of
time and money.
Acknowledgments
Discussions with C. Engelhard and R. Bittl (Freie Universität
Berlin) are appreciated. Financial support through Deutsche
Forschungsgemeinschaft grant MO2192/4-1 (A.M.), through a
Sofja-Kovalevskaya Award by the Alexander- von- Humboldt
Foundation (A.M.), and through Boehringer- Ingelheim Fonds
(R.O.) is gratefully acknowledged.
Generation of Hybrid Gene Libraries
efficient screening or selection procedures, provides a ready
means for discrimination against the template construct (see
Subheading 3.2 step 5). In case the resultant PATCHY library
still contains an exceedingly high fraction of the initial template construct, steps (a) and (b) can be repeated.
5. The ligation reaction can alternatively be carried out at 16 °C
over night.
6. A particular strength of PATCHY is the generation of hybrid
genes of defined composition. Although this usually is the
wanted outcome, in certain scenarios the desired traits may not
manifest in any of the hybrid gene variants within the PATCHY
libraries. In these cases, the PATCHY libraries can further be
diversified via error-prone PCR [21]. Introduction of random
residue exchanges, in particular within the linker region
between protein modules, may well yield variants with the
desired functional properties.
7. The screening of PATCHY libraries can be expedited with
fluorescence- activated cell sorting. This is especially useful in
case of protein switches that are supposed to show differential
activity in response to a signal. PATCHY hybrid libraries can
alternately be screened in the absence and presence of this signal, and stringently responding variants can thus be enriched,
identified and isolated.
8. Alternatively, the activity of the PATCHY variants can be
assessed in microtiter format. Within a 96-deep-well plate,
aliquots of 600 μL LB/Kanamycin medium are inoculated
with individual variants and incubated for 16 h in darkness or
under constant blue light (470 nm, 40 μW/cm
2
). All subsequent steps are conducted as described in Subheading 3.2
steps 8 and 9.
9. Many companies offer plasmid preparation and Sanger
sequencing in microtiter format. For larger number of hybrid
variants, this approach usually is more economic in terms of
time and money.
Acknowledgments
Discussions with C. Engelhard and R. Bittl (Freie Universität
Berlin) are appreciated. Financial support through Deutsche
Forschungsgemeinschaft grant MO2192/4-1 (A.M.), through a
Sofja-Kovalevskaya Award by the Alexander- von- Humboldt
Foundation (A.M.), and through Boehringer- Ingelheim Fonds
(R.O.) is gratefully acknowledged.
Generation of Hybrid Gene Libraries