252
3. The samples are transferred in a lightproof, flat-bottom
microtiter plate and transferred to a fluorimeter.
4. The fluorescence intensity of each sample is measured according to the protocol given by the manufacturer of the
fluorimeter.
The photosensitive degron module is activated by blue light (465
nm, 30 μmol m
−2
s
−1
); cells are grown under continuous illumination (see Note 3). The illumination regimen can be easily adapted
for yeast cells growing on solid medium (see Note 4).
1. Yeast cells are grown in low fluorescence medium supplemented with 2% glucose in darkness or under continuous illumination until mid-log phase is reached (see Note 5).
2. Degradation of the psd module is initiated by exposure of the
cells to blue light (465 nm, 30 μmol m
−2
s
−1
). The time frame
until depletion of the target protein is achieved depends on the
psd module variant, but it can be expected that 2–4 h are sufficient for short-lived psd module variants (see Note 3).
Immunoblotting or fluorescence measurements can be used to
quantify RFP-psd levels. Measuring the abundance of the construct in cells kept under restrictive conditions (blue lightexposed) and permissive conditions (darkness) gives a measure
for the depletion efficiency and the overall performance of the
psd module variant. Ongoing target protein synthesis will lead
to minimal amounts of target protein even after prolonged
exposure to blue light (see Note 6). The overall performance
of a psd module variant can be assessed with mutants, in which
the degron or the photoreceptor is inactivated (see Note 7).
4 Notes
1. Homologous recombination offers high flexibility for vector
design. The illustration shows the most simplified case, in yeast
ligation of a single gene or gene fragment into an existing vector (Fig. 2). However, with the same approach, several fragments can be ligated together in one step. To do so, it is
necessary to generate DNA fragments with flanking homologous sequences. The same strategy as depicted for AtLOV2
can be used to combine several genes or gene fragments, e.g.,
one- step- fusion of a photoreceptor with a degron sequence.
Also the vector backbone can be produced by homologous
recombination of PCR fragments [19].
2. To generate photoreceptor variants, the PCR to amplify the
AtLOV2 domain can be performed with a polymerase lacking
proofreading activity like the Taq polymerase. Furthermore,
3.10 Blue Light
Illumination of Yeast
Christof Taxis
3. The samples are transferred in a lightproof, flat-bottom
microtiter plate and transferred to a fluorimeter.
4. The fluorescence intensity of each sample is measured according to the protocol given by the manufacturer of the
fluorimeter.
The photosensitive degron module is activated by blue light (465
nm, 30 μmol m
−2
s
−1
); cells are grown under continuous illumination (see Note 3). The illumination regimen can be easily adapted
for yeast cells growing on solid medium (see Note 4).
1. Yeast cells are grown in low fluorescence medium supplemented with 2% glucose in darkness or under continuous illumination until mid-log phase is reached (see Note 5).
2. Degradation of the psd module is initiated by exposure of the
cells to blue light (465 nm, 30 μmol m
−2
s
−1
). The time frame
until depletion of the target protein is achieved depends on the
psd module variant, but it can be expected that 2–4 h are sufficient for short-lived psd module variants (see Note 3).
Immunoblotting or fluorescence measurements can be used to
quantify RFP-psd levels. Measuring the abundance of the construct in cells kept under restrictive conditions (blue lightexposed) and permissive conditions (darkness) gives a measure
for the depletion efficiency and the overall performance of the
psd module variant. Ongoing target protein synthesis will lead
to minimal amounts of target protein even after prolonged
exposure to blue light (see Note 6). The overall performance
of a psd module variant can be assessed with mutants, in which
the degron or the photoreceptor is inactivated (see Note 7).
4 Notes
1. Homologous recombination offers high flexibility for vector
design. The illustration shows the most simplified case, in yeast
ligation of a single gene or gene fragment into an existing vector (Fig. 2). However, with the same approach, several fragments can be ligated together in one step. To do so, it is
necessary to generate DNA fragments with flanking homologous sequences. The same strategy as depicted for AtLOV2
can be used to combine several genes or gene fragments, e.g.,
one- step- fusion of a photoreceptor with a degron sequence.
Also the vector backbone can be produced by homologous
recombination of PCR fragments [19].
2. To generate photoreceptor variants, the PCR to amplify the
AtLOV2 domain can be performed with a polymerase lacking
proofreading activity like the Taq polymerase. Furthermore,
3.10 Blue Light
Illumination of Yeast
Christof Taxis