253
addition of manganese ions and unbalanced amounts of
nucleotides will increase the error rate. Standard protocols can
be found in the literature [16, 20, 21].
3. The FMN cofactor of the LOV2 domain is excited by blue
light (400–500 nm) with a maximum at 450 nm. Thus, the
wavelength used for illumination can be varied without affecting the protein destabilization activity of the psd module. At a
light- flux of 30 μmol m
−2
s
−1
(465 nm), strong activation of
the psd module has been observed, whereas yeast cell growth
is not affected [9, 16]. A much higher light flux is not recommended, as yeast cells are sensitive to high-intensity blue light
[30]. To characterize the behavior of the psd module at different light conditions, the light flux can be varied between 0 and
30 μmol m
−2
s
−1
. It is advisable to measure the actual light-flux
before the experiment with an optometer. The illumination
strength influences the psd module degradation rate; reduced
illumination will prolong the time that is necessary to reduce
the target protein amounts in most cases [9, 16].
4. For growth in the presence of blue light on solid medium,
yeast cells bearing a plasmid can be grown on synthetic medium
lacking the appropriate auxotrophy compound. An upright
orientation of the Petri dishes is recommended to expose the
cells directly to the light, which avoids unwanted shading. The
same LED-equipped boxes that are used for growth in liquid
culture can be used to grow yeast on solid medium.
5. Low fluorescence medium is recommended to grow cells
exposed to defined amounts of light. The carbon source can
be chosen freely. Other media can be used as well, as long as
the blue light is not completely absorbed by the medium. If
light absorption takes place, the illumination conditions have
to be adjusted accordingly. The main difference between low
fluorescence medium and synthetic complete medium is the
lack of flavin derivatives in low fluorescent medium. If a yeast
strain is used that does not produce FMN in appropriate
amounts, it can be added to the medium or synthetic complete medium can be used. In both cases, the blue light
absorption of the flavin derivative in the medium has to be
taken into account and the illumination conditions have to be
adjusted accordingly.
Depending on the experiment, different illumination regimens are applied. For cycloheximide chase experiments, cells
are kept in darkness during the initial growth phase and illumination is started after the first sample has been collected. For
protein level quantifications, cells are grown under constant
conditions, either exposed to a certain light flux or kept in
darkness.
Controling Protein Stability with Light
addition of manganese ions and unbalanced amounts of
nucleotides will increase the error rate. Standard protocols can
be found in the literature [16, 20, 21].
3. The FMN cofactor of the LOV2 domain is excited by blue
light (400–500 nm) with a maximum at 450 nm. Thus, the
wavelength used for illumination can be varied without affecting the protein destabilization activity of the psd module. At a
light- flux of 30 μmol m
−2
s
−1
(465 nm), strong activation of
the psd module has been observed, whereas yeast cell growth
is not affected [9, 16]. A much higher light flux is not recommended, as yeast cells are sensitive to high-intensity blue light
[30]. To characterize the behavior of the psd module at different light conditions, the light flux can be varied between 0 and
30 μmol m
−2
s
−1
. It is advisable to measure the actual light-flux
before the experiment with an optometer. The illumination
strength influences the psd module degradation rate; reduced
illumination will prolong the time that is necessary to reduce
the target protein amounts in most cases [9, 16].
4. For growth in the presence of blue light on solid medium,
yeast cells bearing a plasmid can be grown on synthetic medium
lacking the appropriate auxotrophy compound. An upright
orientation of the Petri dishes is recommended to expose the
cells directly to the light, which avoids unwanted shading. The
same LED-equipped boxes that are used for growth in liquid
culture can be used to grow yeast on solid medium.
5. Low fluorescence medium is recommended to grow cells
exposed to defined amounts of light. The carbon source can
be chosen freely. Other media can be used as well, as long as
the blue light is not completely absorbed by the medium. If
light absorption takes place, the illumination conditions have
to be adjusted accordingly. The main difference between low
fluorescence medium and synthetic complete medium is the
lack of flavin derivatives in low fluorescent medium. If a yeast
strain is used that does not produce FMN in appropriate
amounts, it can be added to the medium or synthetic complete medium can be used. In both cases, the blue light
absorption of the flavin derivative in the medium has to be
taken into account and the illumination conditions have to be
adjusted accordingly.
Depending on the experiment, different illumination regimens are applied. For cycloheximide chase experiments, cells
are kept in darkness during the initial growth phase and illumination is started after the first sample has been collected. For
protein level quantifications, cells are grown under constant
conditions, either exposed to a certain light flux or kept in
darkness.
Controling Protein Stability with Light