278
Two main strategies were followed to build a synthetic light-driven
ion channel: first, a rational approach was pursued, which consisted
in connecting the sensor to the pore module in different positions
guided by what is known on the gating mechanism of the channel.
This was followed by a random mutagenesis approach aiming to
improve the properties of a promising candidate retrieved from the
previous approach. Rational mutagenesis was performed using a
combination of different PCR amplification methods. The standard thermal cycler program in combination with Pfu DNA polymerase is as follows: step 1, 30 s at 95 °C; step 2 (15–35 cycles),
10 s at 95 °C, 20 s at T m , 1 min per kb of amplicon at 72 °C; step
3, 5–7 min at 72 °C; step 4, 7 °C for unlimited time.
TE-PCR extends both termini of a target sequence for further
sequence manipulation (e.g., restriction enzyme cloning; OE-PCR;
motif addiction; see Fig. 3a). To set up PCR parameters, only the
primer region annealing to the target sequence has to be taken into
account. The reaction is based on a standard PCR protocol based
on Pfu DNA polymerase, following the standard reagent concentration and the standard thermal cycler program as per manufacturer’s instructions.
The OE-PCR allows joining of regions originally separated in the
native sequence (see Fig. 3b). Two partially overlapping amplicons
are generated through terminal extension PCR.
OE-PCR occurs with an initial step using a mixture of these
amplicons as reciprocal primers; following this step, a standard
PCR protocol enriches the newly generated construct.
1. Generate partially overlapping amplicons through TE-PCR
(see Subheading 3.3.1) using a combination of one TE-PCR
primer and one OE-PCR primer: the resulting fragments will
have a common annealing region.
2. Isolate amplicons running the PCR reaction on 1% Agarose gel
and purify the desired fragment with any commercially available
DNA gel extraction kit.
3. First OE-PCR reaction: mix within a PCR tube 0.75 U Pfu
DNA polymerase, 1× Pfu polymerase buffer, 200 μM dNTPs,
40 ng of the longest and partially overlapping amplicon; 1:1
molar ratio of the shorter overlapping amplicon; make final
volume up to 50 μL with ultrapure water.
4. Set up standard PCR protocol, with following modifications:
Step 2, 15 cycles and T m at 55 °C; skip step 3, move directly
to step 4 (7 °C).
5. Second OE-PCR reaction: add 0.5 μL of 10 μM primers
(forward and reverse) to allow full amplification of the newly
generated construct.
3.3 Rational
and Random
Mutagenesis
Strategies
3.3.1 Rational
Mutagenesis: Terminal
Extension PCR (TE-PCR)
3.3.2 Rational
Mutagenesis: Overlap
Extension PCR (OE-PCR)
Cristian Cosentino et al.
Two main strategies were followed to build a synthetic light-driven
ion channel: first, a rational approach was pursued, which consisted
in connecting the sensor to the pore module in different positions
guided by what is known on the gating mechanism of the channel.
This was followed by a random mutagenesis approach aiming to
improve the properties of a promising candidate retrieved from the
previous approach. Rational mutagenesis was performed using a
combination of different PCR amplification methods. The standard thermal cycler program in combination with Pfu DNA polymerase is as follows: step 1, 30 s at 95 °C; step 2 (15–35 cycles),
10 s at 95 °C, 20 s at T m , 1 min per kb of amplicon at 72 °C; step
3, 5–7 min at 72 °C; step 4, 7 °C for unlimited time.
TE-PCR extends both termini of a target sequence for further
sequence manipulation (e.g., restriction enzyme cloning; OE-PCR;
motif addiction; see Fig. 3a). To set up PCR parameters, only the
primer region annealing to the target sequence has to be taken into
account. The reaction is based on a standard PCR protocol based
on Pfu DNA polymerase, following the standard reagent concentration and the standard thermal cycler program as per manufacturer’s instructions.
The OE-PCR allows joining of regions originally separated in the
native sequence (see Fig. 3b). Two partially overlapping amplicons
are generated through terminal extension PCR.
OE-PCR occurs with an initial step using a mixture of these
amplicons as reciprocal primers; following this step, a standard
PCR protocol enriches the newly generated construct.
1. Generate partially overlapping amplicons through TE-PCR
(see Subheading 3.3.1) using a combination of one TE-PCR
primer and one OE-PCR primer: the resulting fragments will
have a common annealing region.
2. Isolate amplicons running the PCR reaction on 1% Agarose gel
and purify the desired fragment with any commercially available
DNA gel extraction kit.
3. First OE-PCR reaction: mix within a PCR tube 0.75 U Pfu
DNA polymerase, 1× Pfu polymerase buffer, 200 μM dNTPs,
40 ng of the longest and partially overlapping amplicon; 1:1
molar ratio of the shorter overlapping amplicon; make final
volume up to 50 μL with ultrapure water.
4. Set up standard PCR protocol, with following modifications:
Step 2, 15 cycles and T m at 55 °C; skip step 3, move directly
to step 4 (7 °C).
5. Second OE-PCR reaction: add 0.5 μL of 10 μM primers
(forward and reverse) to allow full amplification of the newly
generated construct.
3.3 Rational
and Random
Mutagenesis
Strategies
3.3.1 Rational
Mutagenesis: Terminal
Extension PCR (TE-PCR)
3.3.2 Rational
Mutagenesis: Overlap
Extension PCR (OE-PCR)
Cristian Cosentino et al.