95
5. The PCRs using these primer sets should result in two fragments,
with one fragment overlapping with the T7 promoter region
and the mutagenic region, and the other fragment overlapping
with the mutagenic region and the T7 terminator region. The
mutagenic regions of these fragments will overlap and anneal
during Gibson assembly. Gel purification is highly recommended to improve cloning efficiency, even if the agarose gel
electrophoresis of the PCR product shows a single clear band.
6. Linear vector for use in the Gibson assembly reaction can be
made through PCR using complementary primers of the T7
regions. Specifically, a T7 terminator forward primer and a T7
promoter reverse primer should be used. The linearized vector
produced from this PCR reaction will have T7 regions that can
overlap with the T7 regions of the gene fragments for the
Gibson assembly reaction.
7. The PCR fragments (for both the sensor and vector) can then
be combined into the Gibson master mix (in equimolar ratios).
The typical incubation time for the master mix is 50 °C for 1 h.
8. The Gibson assembly mixture should then be transformed into
competent cells that are designed for DNA cloning (e.g.,
TOP10 cells). If electrocompetent cells are used, the Gibson
mix can be diluted with water to prevent arcing.
9. After confirming that the cloning is successful through
sequencing, the gene should be transformed into an expression
cell type (e.g., BL21DE3).
10. If the sensor construct contains a histidine tag, it can then be
purified through nickel affinity chromatography.
1. To a volume of 849 μL buffer add 100 μL of the concentrated
protein solution (assuming the concentration of the protein
solution is 500 μM) and 1 μL of the TCEP solution. Wait
approximately for 5 min to allow this solution to equilibrate to
room temperature and for any disulfides to be reduced. This
reaction can be performed in an Eppendorf tube or an equivalent (see Note 14).
2. To the reaction mixture add 50 μL of the dye stock. The final
composition of the reaction mixture should contain approximately 50 μM of protein, 500 μM TCEP, and 500 μM dye.
The reaction should be allowed to proceed overnight (16 h) at
4 °C in the dark with constant agitation (see Note 15).
3. After the reaction period, centrifuge the reaction mixture at high
speed (18,000 × g, 5 min) to separate out any precipitated dye
or protein (see Note 16).
4. The mixture should then be purified through gel filtration,
with a desalting column usually being sufficient (see Note 17).
3.3 Dye Labeling
and Purification
Synthetic-Dye Fluorescent Protein FRET Sensors
5. The PCRs using these primer sets should result in two fragments,
with one fragment overlapping with the T7 promoter region
and the mutagenic region, and the other fragment overlapping
with the mutagenic region and the T7 terminator region. The
mutagenic regions of these fragments will overlap and anneal
during Gibson assembly. Gel purification is highly recommended to improve cloning efficiency, even if the agarose gel
electrophoresis of the PCR product shows a single clear band.
6. Linear vector for use in the Gibson assembly reaction can be
made through PCR using complementary primers of the T7
regions. Specifically, a T7 terminator forward primer and a T7
promoter reverse primer should be used. The linearized vector
produced from this PCR reaction will have T7 regions that can
overlap with the T7 regions of the gene fragments for the
Gibson assembly reaction.
7. The PCR fragments (for both the sensor and vector) can then
be combined into the Gibson master mix (in equimolar ratios).
The typical incubation time for the master mix is 50 °C for 1 h.
8. The Gibson assembly mixture should then be transformed into
competent cells that are designed for DNA cloning (e.g.,
TOP10 cells). If electrocompetent cells are used, the Gibson
mix can be diluted with water to prevent arcing.
9. After confirming that the cloning is successful through
sequencing, the gene should be transformed into an expression
cell type (e.g., BL21DE3).
10. If the sensor construct contains a histidine tag, it can then be
purified through nickel affinity chromatography.
1. To a volume of 849 μL buffer add 100 μL of the concentrated
protein solution (assuming the concentration of the protein
solution is 500 μM) and 1 μL of the TCEP solution. Wait
approximately for 5 min to allow this solution to equilibrate to
room temperature and for any disulfides to be reduced. This
reaction can be performed in an Eppendorf tube or an equivalent (see Note 14).
2. To the reaction mixture add 50 μL of the dye stock. The final
composition of the reaction mixture should contain approximately 50 μM of protein, 500 μM TCEP, and 500 μM dye.
The reaction should be allowed to proceed overnight (16 h) at
4 °C in the dark with constant agitation (see Note 15).
3. After the reaction period, centrifuge the reaction mixture at high
speed (18,000 × g, 5 min) to separate out any precipitated dye
or protein (see Note 16).
4. The mixture should then be purified through gel filtration,
with a desalting column usually being sufficient (see Note 17).
3.3 Dye Labeling
and Purification
Synthetic-Dye Fluorescent Protein FRET Sensors