249
1. Standard PCR conditions can be used to generate the products
used for homologous recombination. For cloning purposes, a
kit containing a high-fidelity polymerase (e.g., Phusion, KOD,
or Herculase) is advisable to reduce the introduction of errors
during the reaction.
2. For a directed evolution approach, error-prone PCR followed
by in yeast ligation generates a library of clones [16, 20].
3. PCR is performed with Taq polymerase under conditions that
increase the error rate of the enzyme which is achieved by the
presence of different concentrations of MnCl 2 (0, 0.62, and
1.25 mM) and a twofold excess of dCTP and dTTP (500 μM
each in the final PCR reaction).
4. If necessary, the template vector can be destroyed after the
PCR step by enzymatic digest with DpnI, which cleaves
methylated DNA. Products obtained in this way are used
without further manipulations for homologous recombination in yeast.
The QuickChange protocol can be followed to change single positions in the psd module by site-directed mutagenesis, which may
result in psd modules with distinct characteristics. Mutations
should be selected based on previous studies examining the same
photoreceptor domain or a homologous one. Furthermore,
changes that have been found in different variants obtained by random mutagenesis can be combined in a single construct by this
method.
1. The oligonucleotides are designed as follows: the changed
codon is flanked by 15 bases matching the vector sequence.
Both forward and reverse primers are the reverse complement
of each other. At each end, a cytosine or guanidine is preferred
due to increased binding stability.
2. A whole vector PCR is performed with the template plasmid
using standard conditions. A low number of cycles is recommended (<20).
3. The template plasmid is digested with DpnI that cleaves specifically methylated DNA.
4. The product of the PCR is transformed into E. coli by a standard method [21].
The protocol for yeast transformation is based on the lithium acetate method [22] and has been described previously [23–26].
1. Yeast cells are inoculated from an overnight preculture (approx.
1:50 dilution) and grown to an optical density (A 600 ) of 0.8–1.0
at 30 °C in 50 ml of YPD medium.
3.3 High-Fidelity
and Error-Prone PCR
to Generate DNA
for Homologous
Recombination
3.4 Site-Directed
Mutagenesis to Create
Specific psd Module
Variants
3.5 Yeast
Transformation
Controling Protein Stability with Light
1. Standard PCR conditions can be used to generate the products
used for homologous recombination. For cloning purposes, a
kit containing a high-fidelity polymerase (e.g., Phusion, KOD,
or Herculase) is advisable to reduce the introduction of errors
during the reaction.
2. For a directed evolution approach, error-prone PCR followed
by in yeast ligation generates a library of clones [16, 20].
3. PCR is performed with Taq polymerase under conditions that
increase the error rate of the enzyme which is achieved by the
presence of different concentrations of MnCl 2 (0, 0.62, and
1.25 mM) and a twofold excess of dCTP and dTTP (500 μM
each in the final PCR reaction).
4. If necessary, the template vector can be destroyed after the
PCR step by enzymatic digest with DpnI, which cleaves
methylated DNA. Products obtained in this way are used
without further manipulations for homologous recombination in yeast.
The QuickChange protocol can be followed to change single positions in the psd module by site-directed mutagenesis, which may
result in psd modules with distinct characteristics. Mutations
should be selected based on previous studies examining the same
photoreceptor domain or a homologous one. Furthermore,
changes that have been found in different variants obtained by random mutagenesis can be combined in a single construct by this
method.
1. The oligonucleotides are designed as follows: the changed
codon is flanked by 15 bases matching the vector sequence.
Both forward and reverse primers are the reverse complement
of each other. At each end, a cytosine or guanidine is preferred
due to increased binding stability.
2. A whole vector PCR is performed with the template plasmid
using standard conditions. A low number of cycles is recommended (<20).
3. The template plasmid is digested with DpnI that cleaves specifically methylated DNA.
4. The product of the PCR is transformed into E. coli by a standard method [21].
The protocol for yeast transformation is based on the lithium acetate method [22] and has been described previously [23–26].
1. Yeast cells are inoculated from an overnight preculture (approx.
1:50 dilution) and grown to an optical density (A 600 ) of 0.8–1.0
at 30 °C in 50 ml of YPD medium.
3.3 High-Fidelity
and Error-Prone PCR
to Generate DNA
for Homologous
Recombination
3.4 Site-Directed
Mutagenesis to Create
Specific psd Module
Variants
3.5 Yeast
Transformation
Controling Protein Stability with Light