245
be tested, as receptor and effector domain might influence each
other depending on the exact composition of the fusion site. Good
controls that mimic maximum and minimum activity for both the
receptor and the effector domain are necessary to have a good
measure for the performance range of the synthetic switch. Once
the switch shows reasonable performance, further optimizations
by specific changes or a directed evolution approach may be conducted. In the latter case, it makes sense to allow changes in both,
the signal receptor and the effector domain simultaneously, as the
best performing variant might require alterations in both parts.
Finally, a quick assay that is easily quantifiable is crucial to obtain
reliable data about the performance of the different variants. The
following part of this chapter contains a detailed description of the
engineering steps and the yeast techniques that have been used to
obtain and improve the photosensitive degron module.
2 Materials
For homologous recombination and analysis of the psd module,
no specific yeast strain is required; every wild type lab strain that
has the necessary auxotrophy markers can be used (e.g., ESM356–1
[17], S288C background).
1. High-Fidelity DNA amplification system (e.g., Phusion, KOD,
or Herculase) for amplifying large DNA fragments including
polymerase 10x reaction buffer, 10 mM dNTP stock solutions
each and oligonucleotide primers.
2. Taq DNA polymerase for error prone PCR including Taq
polymerase, 10x reaction buffer, 10 mM MnCl 2 stock solution,
separate 10 mM stock solutions for dATP, dGTP, dCTP, and
dTTP in order to optimize relative ratios for error prone PCR
conditions and oligonucleotide primers.
3. QuickChange Site Directed Mutagenesis kit (Agilent).
4. DpnI restriction enzyme (for removing residual DNA template
after PCR).
Standard media were used for yeast growth [18].
1. Yeast complete medium (YPD): 1% (w/v) yeast extract, 2%
(w/v) peptone, 2% (w/v) glucose.
2. Low-fluorescence medium [16]: 5 g/l (NH 4 ) 2 SO 4 , 1 g/l
KH 2 PO 4 , 0.5 g/l MgSO 4 , 0.1 g/l NaCl, 0.1 g/l Ca 2 Cl, 0.5
mg/l H 3 BO 4 , 0.04 mg/l CuSO 4 , 0.1 mg/l KI, 0.2 mg/l
FeCl 3 , 0.4 mg/l MnSO 4 , 0.2 mg/l Na 2 MoO 4 , 0.4 mg/l
ZnSO 4 , 2 mg/l biotin, 0.4 mg/l calcium pantothenate, 2
mg/l inositol, 0.4 mg/l niacin, 0.2 mg/l 4-aminobenzoic acid
2.1 Yeast Strains
2.2 Cloning
Procedures
2.3 Yeast Media
Controling Protein Stability with Light
be tested, as receptor and effector domain might influence each
other depending on the exact composition of the fusion site. Good
controls that mimic maximum and minimum activity for both the
receptor and the effector domain are necessary to have a good
measure for the performance range of the synthetic switch. Once
the switch shows reasonable performance, further optimizations
by specific changes or a directed evolution approach may be conducted. In the latter case, it makes sense to allow changes in both,
the signal receptor and the effector domain simultaneously, as the
best performing variant might require alterations in both parts.
Finally, a quick assay that is easily quantifiable is crucial to obtain
reliable data about the performance of the different variants. The
following part of this chapter contains a detailed description of the
engineering steps and the yeast techniques that have been used to
obtain and improve the photosensitive degron module.
2 Materials
For homologous recombination and analysis of the psd module,
no specific yeast strain is required; every wild type lab strain that
has the necessary auxotrophy markers can be used (e.g., ESM356–1
[17], S288C background).
1. High-Fidelity DNA amplification system (e.g., Phusion, KOD,
or Herculase) for amplifying large DNA fragments including
polymerase 10x reaction buffer, 10 mM dNTP stock solutions
each and oligonucleotide primers.
2. Taq DNA polymerase for error prone PCR including Taq
polymerase, 10x reaction buffer, 10 mM MnCl 2 stock solution,
separate 10 mM stock solutions for dATP, dGTP, dCTP, and
dTTP in order to optimize relative ratios for error prone PCR
conditions and oligonucleotide primers.
3. QuickChange Site Directed Mutagenesis kit (Agilent).
4. DpnI restriction enzyme (for removing residual DNA template
after PCR).
Standard media were used for yeast growth [18].
1. Yeast complete medium (YPD): 1% (w/v) yeast extract, 2%
(w/v) peptone, 2% (w/v) glucose.
2. Low-fluorescence medium [16]: 5 g/l (NH 4 ) 2 SO 4 , 1 g/l
KH 2 PO 4 , 0.5 g/l MgSO 4 , 0.1 g/l NaCl, 0.1 g/l Ca 2 Cl, 0.5
mg/l H 3 BO 4 , 0.04 mg/l CuSO 4 , 0.1 mg/l KI, 0.2 mg/l
FeCl 3 , 0.4 mg/l MnSO 4 , 0.2 mg/l Na 2 MoO 4 , 0.4 mg/l
ZnSO 4 , 2 mg/l biotin, 0.4 mg/l calcium pantothenate, 2
mg/l inositol, 0.4 mg/l niacin, 0.2 mg/l 4-aminobenzoic acid
2.1 Yeast Strains
2.2 Cloning
Procedures
2.3 Yeast Media
Controling Protein Stability with Light