277
homogenous radiation distribution over the table surface.
LED assemblies are grouped in series and groups are in parallel with each other, according to power supply specifications.
This connection system allows easy removal of a single LED
in case of technical maintenance (Fig. 2b). White panels
reflecting irradiated light are placed around the table, in order
to homogeneously distribute light over the plate surface. A
space between the panels and the table was left to allow air
circulation. Moreover, the open conformation of the scaffold
ensures homogenous temperature conditions all over the
chamber.
2. Dark condition is obtained by means of a dark cardboard and
a black cloth covering the plates and allowing air circulation.
3 Methods
The selection assay is based on a S. cerevisiae mutant Δtrk1 Δtrk2
(SGY1528) that cannot grow in low external K
+
. Expression of
heterologous K
+
transport proteins restores growth in low K
+
.
Functional synthetic channels were identified by their ability to
complement the yeast growth phenotype in low external K
+
.
1. Grow SGY1528 cells on YPDA + 100 mM KCl solid medium
up to 3 days at 30 °C.
2. Inoculate a single colony in 3 mL YPDA + 100 mM KCl broth
and grow overnight in 30 °C shaking incubator (see Note 7).
3. Dilute cell culture 1:50 in YPDA + 100 mM KCl broth and
grow in 30 °C shaking incubator until OD 600 is between 0.8
and 1.0 (see Note 8).
4. Centrifuge cells at 500 × g for 4 min, and then discard
supernatant.
5. Commercially available kits such as the Frozen-EZ yeast transformation II provide reagents and protocols to obtain competent
yeast cells with high transformation efficiency.
6. Aliquot cells in small volumes and store at −80 °C (see Note 9).
Commercially available kits such as the Frozen-EZ yeast transformation II allow for a high yield in yeast transformation
procedures.
1. Follow manufacturer’s instruction to transform cells (see
Note 10).
2. Plate up to 5 μL cells over 1/3 of a minimal SD medium plate.
3. Incubate plates up to 3–4 days at 30 °C.
3.1 Preparation
of S. cerevisiae
Competent Cells
3.2 Transformation
of S. cerevisiae Cells
Engineering Light-Regulated K
+
Channels
homogenous radiation distribution over the table surface.
LED assemblies are grouped in series and groups are in parallel with each other, according to power supply specifications.
This connection system allows easy removal of a single LED
in case of technical maintenance (Fig. 2b). White panels
reflecting irradiated light are placed around the table, in order
to homogeneously distribute light over the plate surface. A
space between the panels and the table was left to allow air
circulation. Moreover, the open conformation of the scaffold
ensures homogenous temperature conditions all over the
chamber.
2. Dark condition is obtained by means of a dark cardboard and
a black cloth covering the plates and allowing air circulation.
3 Methods
The selection assay is based on a S. cerevisiae mutant Δtrk1 Δtrk2
(SGY1528) that cannot grow in low external K
+
. Expression of
heterologous K
+
transport proteins restores growth in low K
+
.
Functional synthetic channels were identified by their ability to
complement the yeast growth phenotype in low external K
+
.
1. Grow SGY1528 cells on YPDA + 100 mM KCl solid medium
up to 3 days at 30 °C.
2. Inoculate a single colony in 3 mL YPDA + 100 mM KCl broth
and grow overnight in 30 °C shaking incubator (see Note 7).
3. Dilute cell culture 1:50 in YPDA + 100 mM KCl broth and
grow in 30 °C shaking incubator until OD 600 is between 0.8
and 1.0 (see Note 8).
4. Centrifuge cells at 500 × g for 4 min, and then discard
supernatant.
5. Commercially available kits such as the Frozen-EZ yeast transformation II provide reagents and protocols to obtain competent
yeast cells with high transformation efficiency.
6. Aliquot cells in small volumes and store at −80 °C (see Note 9).
Commercially available kits such as the Frozen-EZ yeast transformation II allow for a high yield in yeast transformation
procedures.
1. Follow manufacturer’s instruction to transform cells (see
Note 10).
2. Plate up to 5 μL cells over 1/3 of a minimal SD medium plate.
3. Incubate plates up to 3–4 days at 30 °C.
3.1 Preparation
of S. cerevisiae
Competent Cells
3.2 Transformation
of S. cerevisiae Cells
Engineering Light-Regulated K
+
Channels