80
3. Theoretically construct the circularly permuted SBP sequence
by concatenating the three protein fragments: the N*-terminus
to C-terminus fragment, the flexible linker, and the N-terminus
to C*-terminus fragment.
4. Back-translate the circularly permuted SBP sequence to obtain
a nucleotide sequence codon-optimized for expression in
E. coli, and synthesize the gene (see Note 16).
5. Design primers to amplify the gene with flanking SapI sites for
insertion into pDOTS4 or pDOTS10. The forward primer
contains the sequence 5′-GCTCTTCAATC-3′ followed by the
first 10–15 nucleotides of the circularly permuted gene. The
reverse primer contains the sequence 5′-GCTCTTCCGAG-3′
followed by the first 10–15 nucleotides of the reverse complement of the circularly permuted gene (see Note 17). The
length of the primers should be chosen such that the melting
temperatures of the primers are approximately 65 °C.
Carry out all procedures at room temperature unless otherwise
specified.
1. Using standard PCR with a high-fidelity DNA polymerase
(e.g., Phusion Hot Start II), amplify the SBP gene using the
cloning primers from Subheading 3.7. Purify the PCR product using a PCR purification kit, according to the manufacturer’s instructions.
2. In a thin-walled PCR tube, chilled on ice, mix 2 μL of 10× T4
DNA Ligase buffer, 1 μL 400 U/μL T4 DNA ligase, 1 μL
10 U/μL SapI, 100 ng pDOTS4 or pDOTS10, a fivefold
molar excess of the purified PCR product from the previous
step, and water to give a final volume of 20 μL.
3. Perform the Golden Gate assembly reaction using the following thermocycling protocol: 33 cycles of (37 °C for 2 min,
then 16 °C for 3 min); 55 °C for 10 min; 80 °C for 5 min;
then hold at 4 °C.
4. Transform one aliquot of E. coli TOP10 cells with 2.5 μL of
the Golden Gate assembly reaction mixture by electroporation
(see Note 18). After electroporation, resuspend the cells in
1 mL YenB and incubate with shaking at 37 °C for 1 h. Plate
100 μL of the culture on an LB/ampicillin agar plate and
incubate at 37 °C overnight (see Note 19).
5. Use colony PCR and Sanger sequencing of the resulting PCR
products to confirm correct insertion of the gene into the vector (see Note 20).
After expression of the FRET sensor in E. coli and subsequent purification (see Notes 21 and 22), the dynamic range of the sensor
and the affinity of the sensor for its ligand should be measured
3.8 Cloning
into a FRET Sensor
Construct
3.9 Characterization
of the FRET Sensors
In Vitro and In Situ
Ben E. Clifton et al.
3. Theoretically construct the circularly permuted SBP sequence
by concatenating the three protein fragments: the N*-terminus
to C-terminus fragment, the flexible linker, and the N-terminus
to C*-terminus fragment.
4. Back-translate the circularly permuted SBP sequence to obtain
a nucleotide sequence codon-optimized for expression in
E. coli, and synthesize the gene (see Note 16).
5. Design primers to amplify the gene with flanking SapI sites for
insertion into pDOTS4 or pDOTS10. The forward primer
contains the sequence 5′-GCTCTTCAATC-3′ followed by the
first 10–15 nucleotides of the circularly permuted gene. The
reverse primer contains the sequence 5′-GCTCTTCCGAG-3′
followed by the first 10–15 nucleotides of the reverse complement of the circularly permuted gene (see Note 17). The
length of the primers should be chosen such that the melting
temperatures of the primers are approximately 65 °C.
Carry out all procedures at room temperature unless otherwise
specified.
1. Using standard PCR with a high-fidelity DNA polymerase
(e.g., Phusion Hot Start II), amplify the SBP gene using the
cloning primers from Subheading 3.7. Purify the PCR product using a PCR purification kit, according to the manufacturer’s instructions.
2. In a thin-walled PCR tube, chilled on ice, mix 2 μL of 10× T4
DNA Ligase buffer, 1 μL 400 U/μL T4 DNA ligase, 1 μL
10 U/μL SapI, 100 ng pDOTS4 or pDOTS10, a fivefold
molar excess of the purified PCR product from the previous
step, and water to give a final volume of 20 μL.
3. Perform the Golden Gate assembly reaction using the following thermocycling protocol: 33 cycles of (37 °C for 2 min,
then 16 °C for 3 min); 55 °C for 10 min; 80 °C for 5 min;
then hold at 4 °C.
4. Transform one aliquot of E. coli TOP10 cells with 2.5 μL of
the Golden Gate assembly reaction mixture by electroporation
(see Note 18). After electroporation, resuspend the cells in
1 mL YenB and incubate with shaking at 37 °C for 1 h. Plate
100 μL of the culture on an LB/ampicillin agar plate and
incubate at 37 °C overnight (see Note 19).
5. Use colony PCR and Sanger sequencing of the resulting PCR
products to confirm correct insertion of the gene into the vector (see Note 20).
After expression of the FRET sensor in E. coli and subsequent purification (see Notes 21 and 22), the dynamic range of the sensor
and the affinity of the sensor for its ligand should be measured
3.8 Cloning
into a FRET Sensor
Construct
3.9 Characterization
of the FRET Sensors
In Vitro and In Situ
Ben E. Clifton et al.