247
3. Clear plastic cell culture flasks equipped with a ventilated cap
for illumination of yeast cultures growing in liquid medium.
4. A lightproof box to grow yeast cells in darkness.
5. A box with LEDs attached to the lid, spaced evenly to ensure
uniform illumination. Covering the interior of the box with
reflective material optimizes illumination. The box should be of
sufficient size to host several Petri dishes or cell culture flasks.
6. Lightproof, heat shrinkable tubing adjusted to a diameter
which is slightly bigger than a test tube to protect small yeast
cultures from ambient light. To create the tubing, test tubes
were covered with aluminum foil and placed in heat shrinkable tubing, which was subsequently adjusted in size with a
heat gun. After cooling, the aluminum foil was removed with
forceps.
3 Methods
Generation of shuttle vectors for yeast and Escherichia coli by
homologous recombination has been described previously [19].
The method can be adapted in many ways to generate gene fusions
(Fig. 2).
1. Design primers for amplification of the gene of interest. The
primers have two parts: for the 3′-end, sequences that allow
AtLOV2 amplification are chosen. At the 5′-end, sequences
derived from the vector are added, which will be used for the
homologous recombination step in yeast that generates the
final vector.
2. Perform PCR to generate the DNA fragment with the LOV2
photoreceptor domain (see Note 1). To generate photoreceptor variants, a PCR using mutagenic conditions can be performed (see Note 2).
3. Linearize the target vector by restriction enzymes. The
sequences for homologous recombination should flank the gap.
4. Cotransform the fragments into frozen-competent S. cerevisiae
cells.
5. Rescue the plasmid from yeast into E. coli cells, isolate it and
verify the construct by enzymatic digest and sequencing.
Target gene-specific primers contain sequences that are homologous to the target gene and sequences homologous to the vector
sequence, which are added to the 5′ ends (Fig. 2). The resulting
PCR product contains the target gene (e.g., LOV2 photoreceptor)
flanked by sequences for homologous recombination. In the same
way, the vector backbone can be generated with one or several
oligonucleotide pairs (see Note 1).
3.1 Cloning
by Homologous
Recombination
in Yeast
3.2 Design
of Primers for Cloning
by Homologous
Recombination
Controling Protein Stability with Light
3. Clear plastic cell culture flasks equipped with a ventilated cap
for illumination of yeast cultures growing in liquid medium.
4. A lightproof box to grow yeast cells in darkness.
5. A box with LEDs attached to the lid, spaced evenly to ensure
uniform illumination. Covering the interior of the box with
reflective material optimizes illumination. The box should be of
sufficient size to host several Petri dishes or cell culture flasks.
6. Lightproof, heat shrinkable tubing adjusted to a diameter
which is slightly bigger than a test tube to protect small yeast
cultures from ambient light. To create the tubing, test tubes
were covered with aluminum foil and placed in heat shrinkable tubing, which was subsequently adjusted in size with a
heat gun. After cooling, the aluminum foil was removed with
forceps.
3 Methods
Generation of shuttle vectors for yeast and Escherichia coli by
homologous recombination has been described previously [19].
The method can be adapted in many ways to generate gene fusions
(Fig. 2).
1. Design primers for amplification of the gene of interest. The
primers have two parts: for the 3′-end, sequences that allow
AtLOV2 amplification are chosen. At the 5′-end, sequences
derived from the vector are added, which will be used for the
homologous recombination step in yeast that generates the
final vector.
2. Perform PCR to generate the DNA fragment with the LOV2
photoreceptor domain (see Note 1). To generate photoreceptor variants, a PCR using mutagenic conditions can be performed (see Note 2).
3. Linearize the target vector by restriction enzymes. The
sequences for homologous recombination should flank the gap.
4. Cotransform the fragments into frozen-competent S. cerevisiae
cells.
5. Rescue the plasmid from yeast into E. coli cells, isolate it and
verify the construct by enzymatic digest and sequencing.
Target gene-specific primers contain sequences that are homologous to the target gene and sequences homologous to the vector
sequence, which are added to the 5′ ends (Fig. 2). The resulting
PCR product contains the target gene (e.g., LOV2 photoreceptor)
flanked by sequences for homologous recombination. In the same
way, the vector backbone can be generated with one or several
oligonucleotide pairs (see Note 1).
3.1 Cloning
by Homologous
Recombination
in Yeast
3.2 Design
of Primers for Cloning
by Homologous
Recombination
Controling Protein Stability with Light