281
1. To prepare the vector for a gap repair reaction, prepare a
restriction digest of the plasmid by mixing in a tube 10 μg of
circular pYES2-Met25 expression plasmid, 50 U of each
restriction enzyme, 1× reaction buffer according to restriction
enzyme manufacturer’s instructions. Make final volume up to
50 μL with water (see Note 12).
2. Incubate at the appropriate temperature following restriction
enzyme manufacturer’s instructions for more than 1 h 30 min.
3. Isolate digested fragments running the reaction on 1% Agarose
gel and purify the linearized plasmid with any commercially
available DNA gel extraction kit.
4. To perform a gap repair reaction, prepare into a 2 mL tube a
mixture of 500 ng EP-PCR randomly mutated amplicons with
1 μg of cut yeast expression vector.
5. Transform the mixture into 50 μL SGY1528 competent cells
using any commercially available yeast transformation kit, providing high transforming rate. When transforming yeast cells
with Frozen-EZ yeast transformation II kit, eight transformation reactions are enough to plate cells onto 36 ∅ 150 mm
petri dishes (see Note 13).
6. Dilute transformed cells with MilliQ water up to 6.5 mL and mix.
7. Plate 180 μL of diluted cells on 36 ∅ 150 mm petri dishes
containing SD-U + 100 mM KCl
8. Number the plates and provide each with a mark reporting the
orientation for the following replica plating step.
9. Incubate at 30 °C in the dark up to 4 days.
The yeast mutant strain SGY1528 (see Note 14) is suitable for
functional complementation tests of light-gated synthetic potassium channels inserted in pYES2-Met25 vector. Functional complementation is performed on SEL-U-M medium supplemented
with KCl at a selective concentration. The differential growth
between light and dark exposed colonies determines which clones
are functional. Light-driven functional complementation can either
be performed in low throughput by means of a drop-test assay or
in high-throughput.
The drop test method allows fine screening of tens of colonies on
a single plate by functional complementation. Tenfold serial dilutions of liquid cell culture are spotted onto solid selective medium
and grown under light irradiation or in the dark. The differential
growth rate of the same colony, at the same dilution, at the two
different conditions, reflects a different ability in complementing
the defective yeast phenotype.
3.4 Light-Driven
Functional
Complementation
3.4.1 Low-Throughput
Screening by Drop-Test
Engineering Light-Regulated K
+
Channels
1. To prepare the vector for a gap repair reaction, prepare a
restriction digest of the plasmid by mixing in a tube 10 μg of
circular pYES2-Met25 expression plasmid, 50 U of each
restriction enzyme, 1× reaction buffer according to restriction
enzyme manufacturer’s instructions. Make final volume up to
50 μL with water (see Note 12).
2. Incubate at the appropriate temperature following restriction
enzyme manufacturer’s instructions for more than 1 h 30 min.
3. Isolate digested fragments running the reaction on 1% Agarose
gel and purify the linearized plasmid with any commercially
available DNA gel extraction kit.
4. To perform a gap repair reaction, prepare into a 2 mL tube a
mixture of 500 ng EP-PCR randomly mutated amplicons with
1 μg of cut yeast expression vector.
5. Transform the mixture into 50 μL SGY1528 competent cells
using any commercially available yeast transformation kit, providing high transforming rate. When transforming yeast cells
with Frozen-EZ yeast transformation II kit, eight transformation reactions are enough to plate cells onto 36 ∅ 150 mm
petri dishes (see Note 13).
6. Dilute transformed cells with MilliQ water up to 6.5 mL and mix.
7. Plate 180 μL of diluted cells on 36 ∅ 150 mm petri dishes
containing SD-U + 100 mM KCl
8. Number the plates and provide each with a mark reporting the
orientation for the following replica plating step.
9. Incubate at 30 °C in the dark up to 4 days.
The yeast mutant strain SGY1528 (see Note 14) is suitable for
functional complementation tests of light-gated synthetic potassium channels inserted in pYES2-Met25 vector. Functional complementation is performed on SEL-U-M medium supplemented
with KCl at a selective concentration. The differential growth
between light and dark exposed colonies determines which clones
are functional. Light-driven functional complementation can either
be performed in low throughput by means of a drop-test assay or
in high-throughput.
The drop test method allows fine screening of tens of colonies on
a single plate by functional complementation. Tenfold serial dilutions of liquid cell culture are spotted onto solid selective medium
and grown under light irradiation or in the dark. The differential
growth rate of the same colony, at the same dilution, at the two
different conditions, reflects a different ability in complementing
the defective yeast phenotype.
3.4 Light-Driven
Functional
Complementation
3.4.1 Low-Throughput
Screening by Drop-Test
Engineering Light-Regulated K
+
Channels