115
further tenfold and add 100 μL of the Lipofectamine- DNA
mixture to each well and allow to stand for 3–6 h at 37 °C and
5% CO 2 .
8. Optional step: Exchange the medium after 4–6 h to remove
the transfection reagent.
9. 24–48 h after transfection, label the sensor by replacing the
medium with HBSS supplemented with 10 mg/mL BSA and
2 μM BG-based and 10 μM BC-based fluorophore-ligand
label. In order to avoid using large amounts of the synthetic
compounds, the glass coverslips can be transferred from the
12-well plate onto a sheet of parafilm and 100 μL of the labeling solution carefully pipetted onto the edge of the coverslip.
10. After 10 min of labeling at room temperature, aspirate the
labeling solution and wash the glass coverslip four times with
HBSS.
11. For perfusion experiments, transfer the coverslip into a flow
cell. We use a Warner imaging chamber RC-20 and gravity-fed
perfusion at a flow rate of 0.5 mL/min. If no perfusion system
is available, the analyte can be added to the cells by pipetting
(see Note 6).
12. Image the sensor using a standard wide-field or confocal fluorescence microscope.
4 Notes
1. The polyproline region can be difficult to clone, as it is highly
repetitive. We have cloned the region by annealing 5′-phosphrylated oligonucleotides and ligating them into a restrictiondigested plasmid.
2. We use pET vector expression systems that are induced by
IPTG (0.5–1 mM final concentration) and expressed at 16 °C
overnight.
3. You can follow the sensor in the different fractions by labeling
it with a fluorescent substrate and doing an in-gel fluorescent
scan.
4. We recommend keeping the DMSO concentration below 5%,
as higher concentrations may denature the protein. The duration of labeling depends on the intramolecular ligand, but we
find that the reaction is finished after 1 h.
5. The kinetics of opening may differ between sensors, depending on the affinity of the intermolecular tether for the binding
protein.
6. We find that measuring the half-life of t opening and t closing on cell
surfaces is often limited by the perfusion speed of the perfusion
chamber.
SNIFITS and LUCIDs: Semi-Synthetic Modular Biosensors
further tenfold and add 100 μL of the Lipofectamine- DNA
mixture to each well and allow to stand for 3–6 h at 37 °C and
5% CO 2 .
8. Optional step: Exchange the medium after 4–6 h to remove
the transfection reagent.
9. 24–48 h after transfection, label the sensor by replacing the
medium with HBSS supplemented with 10 mg/mL BSA and
2 μM BG-based and 10 μM BC-based fluorophore-ligand
label. In order to avoid using large amounts of the synthetic
compounds, the glass coverslips can be transferred from the
12-well plate onto a sheet of parafilm and 100 μL of the labeling solution carefully pipetted onto the edge of the coverslip.
10. After 10 min of labeling at room temperature, aspirate the
labeling solution and wash the glass coverslip four times with
HBSS.
11. For perfusion experiments, transfer the coverslip into a flow
cell. We use a Warner imaging chamber RC-20 and gravity-fed
perfusion at a flow rate of 0.5 mL/min. If no perfusion system
is available, the analyte can be added to the cells by pipetting
(see Note 6).
12. Image the sensor using a standard wide-field or confocal fluorescence microscope.
4 Notes
1. The polyproline region can be difficult to clone, as it is highly
repetitive. We have cloned the region by annealing 5′-phosphrylated oligonucleotides and ligating them into a restrictiondigested plasmid.
2. We use pET vector expression systems that are induced by
IPTG (0.5–1 mM final concentration) and expressed at 16 °C
overnight.
3. You can follow the sensor in the different fractions by labeling
it with a fluorescent substrate and doing an in-gel fluorescent
scan.
4. We recommend keeping the DMSO concentration below 5%,
as higher concentrations may denature the protein. The duration of labeling depends on the intramolecular ligand, but we
find that the reaction is finished after 1 h.
5. The kinetics of opening may differ between sensors, depending on the affinity of the intermolecular tether for the binding
protein.
6. We find that measuring the half-life of t opening and t closing on cell
surfaces is often limited by the perfusion speed of the perfusion
chamber.
SNIFITS and LUCIDs: Semi-Synthetic Modular Biosensors