333
The following protocol is designed for the identification of mating
pathway gene variants that enable a strong response to the peptide
pheromone α-factor. For instance, the gene STE2, which encodes
the pheromone receptor, can be mutated to identify changes that
lead to mating pathway induction in the presence of a pheromone
from the related species Kluyveromyces lactis [14]. In this system, a
ste2Δ yeast strain which expresses green fluorescent protein (GFP)
from a mating-inducible promoter is transformed with a mutant
STE2 library and used to select active Ste2 variants.
1. Transform yeast cells with the plasmid library of mutant
DNA. We recommend using the high-efficiency lithium acetate transformation method [15]. This method can be expected
to yield 15,000 colonies from an initial 5 mL of log-phase
yeast culture and 1 μg of a 5–10 kbp DNA plasmid.
2. Pick 100 colonies or more and assay the mating pathway
response of each. This pre-sorting screen will reveal the ratio of
active to inactive mutants as well as the diversity of the active
mutants’ phenotypes (Fig. 2a).
3. Place individual mutant colonies in 2 mL of drop-out medium.
Also inoculate 2 mL volumes with a negative control, such as
cells transformed with an empty vector, and a positive control,
such as cells expressing wild-type STE2. Grow overnight at
30 °C in a 225 RPM shaker incubator.
4. Transfer 40 μL of the overnight culture to 2 mL of drop-out
medium. Grow this dilution to an optical density at 600 nm
(OD 600 ) of 0.4 to 0.6 (early log-phase).
5. Add α-factor pheromone to each culture to a final concentration
of 100 nM. Cultures can also be split into two to measure the
pathway response in the absence of pheromone. Grow for 2 h.
6. Add cycloheximide to a final concentration of 10 μg/mL to
each culture to arrest protein expression, including GFP.
7. Sonicate each log-phase culture briefly to break large cell
aggregates. This typically requires two sonication pulses at the
lowest setting.
8. Run each culture in a flow cytometer to measure GFP fluorescence. This requires a 488 nm laser and a 525/50 nm filter.
9. To proceed with cell sorting, combine the mutant library into
a single liquid culture. For this, dispense 5 mL of drop-out
medium onto each plate of transformed yeast cells and scrape
off the colonies using a plating stick. Aspirate the mixed colonies and add them to a tube on ice.
10. Vortex the colony mixture on a low setting for 30 s.
11. Inoculate 50 mL of drop-out medium with 50 μL of the colony mixture. Also inoculate 2 mL volumes with the negative
and positive controls. Grow overnight.
3.3 Yeast-Based
Sorting of Rewired
Pathway Interactions
Rewiring Signaling Networks
The following protocol is designed for the identification of mating
pathway gene variants that enable a strong response to the peptide
pheromone α-factor. For instance, the gene STE2, which encodes
the pheromone receptor, can be mutated to identify changes that
lead to mating pathway induction in the presence of a pheromone
from the related species Kluyveromyces lactis [14]. In this system, a
ste2Δ yeast strain which expresses green fluorescent protein (GFP)
from a mating-inducible promoter is transformed with a mutant
STE2 library and used to select active Ste2 variants.
1. Transform yeast cells with the plasmid library of mutant
DNA. We recommend using the high-efficiency lithium acetate transformation method [15]. This method can be expected
to yield 15,000 colonies from an initial 5 mL of log-phase
yeast culture and 1 μg of a 5–10 kbp DNA plasmid.
2. Pick 100 colonies or more and assay the mating pathway
response of each. This pre-sorting screen will reveal the ratio of
active to inactive mutants as well as the diversity of the active
mutants’ phenotypes (Fig. 2a).
3. Place individual mutant colonies in 2 mL of drop-out medium.
Also inoculate 2 mL volumes with a negative control, such as
cells transformed with an empty vector, and a positive control,
such as cells expressing wild-type STE2. Grow overnight at
30 °C in a 225 RPM shaker incubator.
4. Transfer 40 μL of the overnight culture to 2 mL of drop-out
medium. Grow this dilution to an optical density at 600 nm
(OD 600 ) of 0.4 to 0.6 (early log-phase).
5. Add α-factor pheromone to each culture to a final concentration
of 100 nM. Cultures can also be split into two to measure the
pathway response in the absence of pheromone. Grow for 2 h.
6. Add cycloheximide to a final concentration of 10 μg/mL to
each culture to arrest protein expression, including GFP.
7. Sonicate each log-phase culture briefly to break large cell
aggregates. This typically requires two sonication pulses at the
lowest setting.
8. Run each culture in a flow cytometer to measure GFP fluorescence. This requires a 488 nm laser and a 525/50 nm filter.
9. To proceed with cell sorting, combine the mutant library into
a single liquid culture. For this, dispense 5 mL of drop-out
medium onto each plate of transformed yeast cells and scrape
off the colonies using a plating stick. Aspirate the mixed colonies and add them to a tube on ice.
10. Vortex the colony mixture on a low setting for 30 s.
11. Inoculate 50 mL of drop-out medium with 50 μL of the colony mixture. Also inoculate 2 mL volumes with the negative
and positive controls. Grow overnight.
3.3 Yeast-Based
Sorting of Rewired
Pathway Interactions
Rewiring Signaling Networks