273
tested for differential yeast growth in light/dark. Channel variants
showing improved complementation ability, compared to the
parental channel, were selected for further electrophysiological
characterization of the channel currents.
2 Materials
1. LOV2 domain: aminoacids 404–546 of Avena sativa
Phototropin 1 (NPH1-1) (GenBank: AAC05083.1).
2. Kcv: aminoacids 2–94 of Paramecium bursaria Chlorella virus 1
potassium ion channel protein (PBCV-1-Kcv) (NP_048599.1).
1. The pYES2-Met25 expression vector [6] was used for protein
expression in Saccharomyces cerevisiae.
1. Escherichia coli (DH5α) for cloning procedures and plasmid
DNA amplification.
2. Saccharomyces cerevisiae Δtrk1 Δtrk2 mutant (SGY1528) for
functional complementation by light-driven K
+
ion channels.
1. LB medium: add 10 g Tryptone, 5 g Yeast Extract, 10 g NaCl
to 800 mL ultrapure water. Mix and adjust pH to 7.0 with 1 M
NaOH. Make up to 1 L with water. Autoclave and store at
RT. Before use, add selective antibiotics. For solid LB medium
proceed as previous step and add 15 g/L agar. After sterilization allow the medium to cool down, add selective antibiotics,
and then pour medium into petri dishes. Seal solid plates with
parafilm and store in the dark at +4 °C (see Note 1).
2. Ampicillin: 50 mg/mL stock solution. Weigh 500 mg
Ampicillin sodium salt and dissolve in 10 mL ultrapure water.
Make aliquots and store in the dark at −20 °C (see Note 2).
3. Gentamycin: 20 mg/mL stock solution. Weigh 200 mg
Gentamycin sulfate; prepare a 10 mL solution and aliquot as in
the previous step (see Note 2).
1. Complete Yeast Medium supplemented with 100 mM KCl
(YPDA + 100 KCl): weigh 20 g Peptone, 20 g D-Glucose,
10 g Yeast Extract, 50 mg Adenine hemisulfate, 7.4 g KCl and
add to 800 mL ultrapure water. Mix and adjust pH to 5.6 with
1 M HCl; make up to 1 L with water and autoclave. To obtain
solid medium, add 20 g Agar (Plant cell culture tested, SigmaAldrich) before autoclaving. Pour agar-containing medium
into petri dishes while still molten and seal with parafilm when
cooled. Store liquid medium at RT and plates at +4 °C.
2. Minimal SD medium without Uracil and supplemented with
Adenine and 100 mM KCl (SD-U + 100 KCl): Weigh 26.7 g
2.1 Coding
Sequences
for the LOV2 Domain
and the Kcv Channel
2.2 Expression
Vector
2.3 Cloning
and Expression
Organisms
2.4 E. coli
Growth Media
2.5 Yeast
Growth Media
Engineering Light-Regulated K
+
Channels
tested for differential yeast growth in light/dark. Channel variants
showing improved complementation ability, compared to the
parental channel, were selected for further electrophysiological
characterization of the channel currents.
2 Materials
1. LOV2 domain: aminoacids 404–546 of Avena sativa
Phototropin 1 (NPH1-1) (GenBank: AAC05083.1).
2. Kcv: aminoacids 2–94 of Paramecium bursaria Chlorella virus 1
potassium ion channel protein (PBCV-1-Kcv) (NP_048599.1).
1. The pYES2-Met25 expression vector [6] was used for protein
expression in Saccharomyces cerevisiae.
1. Escherichia coli (DH5α) for cloning procedures and plasmid
DNA amplification.
2. Saccharomyces cerevisiae Δtrk1 Δtrk2 mutant (SGY1528) for
functional complementation by light-driven K
+
ion channels.
1. LB medium: add 10 g Tryptone, 5 g Yeast Extract, 10 g NaCl
to 800 mL ultrapure water. Mix and adjust pH to 7.0 with 1 M
NaOH. Make up to 1 L with water. Autoclave and store at
RT. Before use, add selective antibiotics. For solid LB medium
proceed as previous step and add 15 g/L agar. After sterilization allow the medium to cool down, add selective antibiotics,
and then pour medium into petri dishes. Seal solid plates with
parafilm and store in the dark at +4 °C (see Note 1).
2. Ampicillin: 50 mg/mL stock solution. Weigh 500 mg
Ampicillin sodium salt and dissolve in 10 mL ultrapure water.
Make aliquots and store in the dark at −20 °C (see Note 2).
3. Gentamycin: 20 mg/mL stock solution. Weigh 200 mg
Gentamycin sulfate; prepare a 10 mL solution and aliquot as in
the previous step (see Note 2).
1. Complete Yeast Medium supplemented with 100 mM KCl
(YPDA + 100 KCl): weigh 20 g Peptone, 20 g D-Glucose,
10 g Yeast Extract, 50 mg Adenine hemisulfate, 7.4 g KCl and
add to 800 mL ultrapure water. Mix and adjust pH to 5.6 with
1 M HCl; make up to 1 L with water and autoclave. To obtain
solid medium, add 20 g Agar (Plant cell culture tested, SigmaAldrich) before autoclaving. Pour agar-containing medium
into petri dishes while still molten and seal with parafilm when
cooled. Store liquid medium at RT and plates at +4 °C.
2. Minimal SD medium without Uracil and supplemented with
Adenine and 100 mM KCl (SD-U + 100 KCl): Weigh 26.7 g
2.1 Coding
Sequences
for the LOV2 Domain
and the Kcv Channel
2.2 Expression
Vector
2.3 Cloning
and Expression
Organisms
2.4 E. coli
Growth Media
2.5 Yeast
Growth Media
Engineering Light-Regulated K
+
Channels