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This protocol enables the preparation of culture medium suitable for Escherichia coli.
Table 1. Composition of liquid LB medium
| Tryptone | 10 g |
| Dried yeast | 5 g |
| NaCl | 10 g |
| DW | 1 L |
Table 2. Composition of solid LB medium
| Tryptone | 10 g |
| Dried yeast | 5 g |
| NaCl | 10 g |
| DW | 1 L |
| Agar | 15 g |
Autoclave the solution.
Allow the medium to cool at room temperature (or cool in a water bath).
If antibiotics are required, add them at this step. For kanamycin, add 1000 μL/L of a 50 mg/mL solution.
Collect and store the medium at room temperature.
This technique efficiently introduces insert fragments into linearized vectors to generate vector solutions for transformation.
Table 3. Composition of reaction mixture
| Vector fragment (pET28a) | 0.5 μL |
| Insert fragment | 1.5 μL |
| In-Fusion snap | 0.5 μL |
Incubate at 50°C for 15 minutes.
Use the incubated solution as the vector solution and follow the “Transformation” protocol.
This protocol enables transformation of E. coli using vector solutions by heat shock.
Add 0.5 μL of vector solution to a microcentrifuge tube.
Add 20 μL of competent E. coli cells (JM109 or BL21 strain) to the tube containing the vector solution.
Incubate the tube containing cells on ice for 5 minutes.
Heat shock the tube at 42°C for 45 seconds.
Immediately after heat shock, incubate on ice for 2 minutes.
Add 100 μL of liquid LB medium or liquid SOC medium without antibiotics to the tube (5 times the volume of competent cells added).
Incubate at 37°C for 1 hour.
Plate onto solid LB plates using aseptic technique.
Incubate overnight at 37°C.
Check for colony formation the following day.
If colonies are observed, store at room temperature.
This protocol enables extraction of vectors and follows the Pure Yield™ Plasmid Miniprep System protocol.
Pick a single colony from the recovered plate and inoculate into 5 mL of liquid LB supplemented with 50 mg/L kanamycin.
Culture overnight at 37°C with shaking.
Harvest the sample the following day.
Centrifuge the culture at 11,000 g.
Remove the supernatant.
Resuspend the pellet in 1000 μL of MilliQ water.
Extract the plasmid following the Miniprep protocol.
Store the extracted DNA at 4°C.
This protocol performs colony PCR to confirm the presence of the insert.
Table 4. Composition of reaction mixture
| 2× Gflex PCR Buffer | 5 μL |
| Primer ① (T7 promoter primer) | 0.2 μL |
| Primer ② (T7 terminator primer) | 0.2 μL |
| Tks Gflex DNA Polymerase | 0.2 μL |
| MilliQ water | 4.4 μL |
| Total | 10 μL × number needed |
Gently touch a colony with a pipette tip and resuspend in the PCR reaction mixture.
Perform PCR using the following thermal cycler settings:
94°C for 10 minutes → (98°C for 10 seconds → 55°C for 15 seconds → 68°C for 1 minute) × 35 cycles
Add 2 μL of 10× Loading Buffer and 8 μL of MilliQ water to each sample.
Load 5 μL of marker and 10 μL of each sample onto a 1% agarose gel.
Perform electrophoresis at 100 V for 30 minutes.
Expose under UV light and photograph.
This protocol quantifies protein concentration in solutions according to the TaKaRa Bradford Protein Assay Kit standard protocol.
Table
| 2 mg/mL BSA Standard (μL) | Diluent (μL) | Final BSA concentration (μg/mL) |
| 50 | 50 | 1000 |
| 30 | 50 | 750 |
| 20 | 60 | 500 |
| 20 | 140 | 250 |
| 10 | 150 | 125 |
| 5 | 395 | 25 |
| 0 | 100 | 0 (Blank) |
Dispense 20 μL of each dilution of BSA standard solution prepared in step 1 and samples (prepare dilution series as necessary) into 1.5 mL microcentrifuge tubes. Measure each dilution and sample in duplicate (n = 2) or more.
Add 1 mL of Bradford Dye Reagent to each tube and mix well. Incubate for 5 minutes in a 25°C water bath or at room temperature approximately 25°C.
Measure absorbance at 595 nm. (Absorbance measurement should be performed within 1 hour after the reaction.)
After subtracting the blank value, calculate the mean of duplicate (or more) measurements for each sample. Determine sample concentration using the standard curve generated from the BSA standard dilution series.
This protocol enables extraction and purification of fluorescent proteins.
Add 1 mL of sonication buffer to the pellet, resuspend thoroughly, and transfer to a 1.5 mL tube.
Sonicate for 4 minutes, alternating 30 seconds of sonication with 30-second rest periods.
Add 0.3 mL of 5 M NaCl at pH 7.8 and 2.33 mL of ammonium sulfate to the supernatant.
Immediately mix the entire suspension with 1.2 mL of 96% ethanol and shake vigorously for 30 seconds.
Centrifuge at 3000 g for 7 minutes.
Collect the upper ethanol layer and transfer to a 15 mL tube.
Add n-butanol (one-quarter the volume of the ethanol layer) and shake vigorously for 30 seconds.
Centrifuge at 3000 g for 7 minutes.
Collect the lower layer and transfer to a 15 mL tube.
Add 100% ammonium sulfate at one-quarter the volume of the collected solution.
Let stand at room temperature for 2 hours.
Centrifuge at 2000 g for 5 minutes.
Remove the upper organic layer and lower aqueous layer, and collect the precipitate at the interface.
Resuspend and dissolve in 1 mL of sonication buffer.
This protocol determines whether protein purification was successful.
| Purified protein solution | 20 μL |
| 5× Western Blotting buffer | 20 μL |
| 1% SDS | 75 μL |
| 2-Mercaptoethanol | 5 μL |
Heat the above solution in a heat block set at 100°C for 10 minutes.
Set the pre-cast gel in the electrophoresis tank and add running buffer.
Remove the comb and load 4 μL of marker and 20 μL of each sample into the lanes. Samples T02_01, 08, and 28 were used.
Remove the gel, replace with distilled water, and wash twice for 5 minutes each.
Add enough CBB staining solution to cover the gel and stain for 1 hour.
Discard the staining solution, change DW, and wash twice for 5 minutes each.
This protocol obtains data necessary for preparing calibration curves for bioassays.
Pick three colonies from E. coli transformed the previous day and dissolve in 450 mL of MilliQ water.
Prepare 100 mg/mL ampicillin diluted 16-fold, 32-fold, 64-fold, 128-fold, 256-fold, and 512-fold.
Prepare two plates with 200 μL of E. coli suspension added dropwise.
Create sections with 16-fold, 32-fold, and 64-fold diluted ampicillin on one plate and sections with 128-fold, 256-fold, and 512-fold dilutions on the other, and add dropwise.
Incubate both plates at 37°C.
This protocol prepares resin for His-tag purification.
This protocol purifies proteins from cell-free reaction mixtures.
Thaw the cell-free reaction mixture on ice.
Pipette and aliquot 2 μL into a separate tube.
Label the aliquoted tube as GFP-total.
Store GFP-total in the freezer.
Centrifuge the remaining reaction mixture at 18,000 g for 20 minutes.
Aliquot 2 μL of the supernatant into a separate tube.
Label as GFP-sol and store in the freezer.
Briefly spin down the remaining reaction mixture.
Collect 12 μL of supernatant and aliquot into a separate tube.
Dilute the 12 μL supernatant with 80 μL of Binding Buffer.
Add the diluted sample to the equilibrated resin and gently pipette.
Incubate on ice for 1 minute.
Mix by inversion for 1 hour in a cold room using an inverter.
Briefly centrifuge (approximately 5 seconds) to pellet the resin and collect the supernatant (save as FT fraction).
Wash the resin twice with 50 μL of Binding Buffer, centrifuge, and collect the supernatant (save as W1 and W2 fractions).
Wash the resin three times with 50 μL of Wash Buffer, centrifuge, and collect the supernatant (save as W3, W4, and W5 fractions).
Wash the resin three times with 50 μL of Elution Buffer, centrifuge, and collect the supernatant (save as E1, E2, and E3 fractions).
Store in the freezer.
This protocol enables protein expression using a cell-free system, following the PUREfrex protocol.
Warm Solution I, Cysteine, and GSH at room temperature to 37°C for approximately 5 minutes to completely dissolve, then keep at room temperature.
Thaw Solution II and Solution III on ice.
Briefly vortex the thawed Solution I, Solution II, Solution III, Cysteine, and GSH, then centrifuge to collect contents at the bottom of the tube.
Prepare the reaction mixture as follows. Add DNA to a final concentration of 0.5–3 ng/μL per kbp.
| Water | 7~X μL |
| Solution I | 8 μL |
| Cysteine | 1 μL |
| GSH | 1 μL |
| Solution II | 1 μL |
| Solution III | 2 μL |
| Template DNA | X μL |
| Total | 20 μL |
Incubate at 37°C for 4–6 hours to synthesize protein.
Freeze in liquid nitrogen and store at −20°C.
This protocol measures excitation and fluorescence spectra as well as brightness using a fluorescence spectrophotometer. This allows determination of the excitation and fluorescence wavelengths of each protein and the fluorescence brightness compared to a standard fluorescent protein.
First, apply excitation light at approximately 300 nm and acquire the fluorescence spectrum.
Based on the spectrum from step 1, identify the peak wavelength and apply that wavelength to acquire the excitation spectrum.
Based on the spectrum from step 2, identify the peak at the shortest wavelength and apply that excitation wavelength to acquire the fluorescence spectrum again.
Next, measure brightness. Acquire the fluorescence spectrum of a standard fluorescent protein (we used avGFP) and adjust the sensor sensitivity appropriately.
Acquire fluorescence spectra of all samples using the sensor sensitivity from step 4. Dilute samples appropriately if necessary. The excitation wavelength used for each sample should be adjusted according to each protein.
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