336
20. Run a screen as done in steps 2–8 in order to confirm that the
sorted mutants have the desired phenotype (see Fig. 2c for an
example with our data on S. cerevisiae Ste2 mutants capable of
responding to a pheromone from a distantly related yeast species). Interesting mutants can be isolated and their plasmid
extracted using the Zymoprep™ Yeast Plasmid Miniprep II to
be sequenced.
21. Optional: Perform iterative rounds of sorting on the sorted
library to isolate an ever-greater proportion of desired mutants.
4 Notes
1. DNA primers must contain AarI restriction sites, or alternative
endonuclease sites appropriate for inserting the product into the
desired acceptor plasmid, followed by 18–20 bp annealing to the
Target DNA. This annealing sequence will not be mutated.
2. Listed values are similar to those suggested by the manufacturer, although several conditions can be tested simultaneously
to achieve the desired mutation frequency. In general, using
less Template DNA and more cycles will increase the mutation
frequency.
3. If the plasmid library will be transformed into yeast, a plasmid
DNA concentration of 1 μg/μL is recommended. The DNA
concentration can be increased by standard precipitation with
sodium acetate and ethanol.
Acknowledgments
Supporting Grant Information: This work was funded by an
NSERC (National Science and Engineering Research Council,
Canada) Discovery Grant 418467-2012 (S.G.P), a CFI- ORF
(S.G.P.), an Early Research Award from the Province of Ontario
(S.G.P.), a Boehringer-Ingelheim Young Researcher Award
(S.G.P.), an NSERC Canadian Graduate Scholarship (R.B.D.),
and an Ontario Graduate Scholarship (B.S.).
References
1. Good MC, Zalatan JG, Lim WA (2011)
Scaffold proteins: hubs for controlling the flow
of cellular information. Science 332(6030):680–
686. doi:10.1126/science.1198701
2. Peisajovich SG (2012) Evolutionary synthetic
biology. ACS Synth Biol 1(6):199–210.
doi:10.1021/sb300012g
3. Romero PA, Arnold FH (2009) Exploring protein fitness landscapes by directed evolution.
Nat Rev Mol Cell Biol 10(12):866–876.
doi:10.1038/Nrm2805
4. Leung DW, Chen E, Goeddel DV (1989) A
method for random mutagenesis of a defined
DNA segment using a modified polymerase
chain reaction. Technique 1(1):11–15
5. Stemmer WPC (1994) DNA shuffling by random fragmentation and reassembly - in-vitro
recombination for molecular evolution. Proc
Raphaël B. Di Roberto et al.
20. Run a screen as done in steps 2–8 in order to confirm that the
sorted mutants have the desired phenotype (see Fig. 2c for an
example with our data on S. cerevisiae Ste2 mutants capable of
responding to a pheromone from a distantly related yeast species). Interesting mutants can be isolated and their plasmid
extracted using the Zymoprep™ Yeast Plasmid Miniprep II to
be sequenced.
21. Optional: Perform iterative rounds of sorting on the sorted
library to isolate an ever-greater proportion of desired mutants.
4 Notes
1. DNA primers must contain AarI restriction sites, or alternative
endonuclease sites appropriate for inserting the product into the
desired acceptor plasmid, followed by 18–20 bp annealing to the
Target DNA. This annealing sequence will not be mutated.
2. Listed values are similar to those suggested by the manufacturer, although several conditions can be tested simultaneously
to achieve the desired mutation frequency. In general, using
less Template DNA and more cycles will increase the mutation
frequency.
3. If the plasmid library will be transformed into yeast, a plasmid
DNA concentration of 1 μg/μL is recommended. The DNA
concentration can be increased by standard precipitation with
sodium acetate and ethanol.
Acknowledgments
Supporting Grant Information: This work was funded by an
NSERC (National Science and Engineering Research Council,
Canada) Discovery Grant 418467-2012 (S.G.P), a CFI- ORF
(S.G.P.), an Early Research Award from the Province of Ontario
(S.G.P.), a Boehringer-Ingelheim Young Researcher Award
(S.G.P.), an NSERC Canadian Graduate Scholarship (R.B.D.),
and an Ontario Graduate Scholarship (B.S.).
References
1. Good MC, Zalatan JG, Lim WA (2011)
Scaffold proteins: hubs for controlling the flow
of cellular information. Science 332(6030):680–
686. doi:10.1126/science.1198701
2. Peisajovich SG (2012) Evolutionary synthetic
biology. ACS Synth Biol 1(6):199–210.
doi:10.1021/sb300012g
3. Romero PA, Arnold FH (2009) Exploring protein fitness landscapes by directed evolution.
Nat Rev Mol Cell Biol 10(12):866–876.
doi:10.1038/Nrm2805
4. Leung DW, Chen E, Goeddel DV (1989) A
method for random mutagenesis of a defined
DNA segment using a modified polymerase
chain reaction. Technique 1(1):11–15
5. Stemmer WPC (1994) DNA shuffling by random fragmentation and reassembly - in-vitro
recombination for molecular evolution. Proc
Raphaël B. Di Roberto et al.