The overall goal of this protocol is to demonstrate the general applicability of the FRET analyzer under optimal conditions by quantifying the sugar content of commercially available beverages. This method can help answer key questions in the sensing and monitoring field of small molecules, such as sugar. The main advantage of this technique is that it can efficiently monitor a fresh signal responsing to the small molecules without expensive, high-end fluorometers.
Demonstrating the procedure will be Dr.Han from our laboratory. First, prepare the maltose or sucrose-specific cyan and yellow fluorescent biosensor plasmid. Then innoculate five milliliters of Luria broth with a single colony of DE3 E.Coli, and incubate the culture at 37 degrees Celsius in a shaking incubator for 16 hours.
Next, place one millileter of the culture into a flask containing 100 milliliters of LB.Incubate the flask at 37 degrees Celsius in a shaking incubator until the optical density at 600 nanometers is about 0.5. Centrifuge the culture for 20 minutes at four degrees Celsius. Quickly resuspend the pellet in 50 milliliters of ice-cold distilled water, and centrifuge for 20 minutes again.
Resuspend the pellet in 50 microliters of a 10 volume percent solution of glycerol and ice-cold distilled water. Gently swirl the mixture until the optical density at 600 nanometers reaches 100. In an ice-cold electroporation cuvette, combine the mixture with 10 nanograms of CMY plasmid.
Electroporate the mixture. In the cuvette, resuspend the cells in one millileter of SOC medium, and then transfer them to a 15 milliliter round-bottom tube. Recover the cells by gently shaking at 37 degrees for one hour.
Spread the cells on an LB agar plate with 100 micrograms per a milliliter ampicillin. Incubate the cells at 37 degrees for 12 hours. Using a loop, transfer a single colony to 10 milliliters of LB containing 100 micrograms per milliliter ampicillin.
Incubate the mixture at 37 degrees while shaking for 12 hours to create the seed culture. Transfer five milliliters of the seed culture to 500 milliliters of LB with ampicillin, and incubate the cells at 37 degrees in a shaking incubator. Once the optical density at 600 nanometers reaches 0.5, add IPTG to make a final concentration of 0.5 millimolar.
After incubating the culture for 24 hours, centrifuge the cells for 20 minutes. Carefully remove the supernatant and resuspend the pellet in five milliliters of binding buffer. Sonicate the cells on ice for 10 seconds, and then cool the cells for 10 seconds.
Repeat the sonication and cooling five more times. Centrifuge the lysate for 30 minutes. Transfer the supernatant to another collection tube.
Purify the supernatant on a nickel NTA affinity column. Fill a 10, 000 milliwatt concentrator with 20 milliliters of the purified sample. Centrifuge the sample for 10 minutes, and then refill the concentrator with 0.8%phosphate buffered saline.
Repeat this process with the rest of the sample in this way, 20 milliliters at a time, until all of the protein has been concentrated and desalted. Store the purified FRET biosensor at 80 degrees Celsius. Prepare a 0.2 micromolar solution of CMY biosensor protein and 0.8%PBS as the detection solution.
Turn on a handheld FRET analyzer. Set the analysis temperature to 53 degrees Celsius. Then set the instrument to calibration mode.
Fill a cuvette with 0.8%PBS as a background. Preheat the sample to 53 degrees Celsius in the analyzer, and then set the background. Next, fill a cuvette with detection solution.
Place the cuvette into the analyzer and set the baseline. Replace the cuvette with one containing detection solution and one millimolar sugar. Set the intensity at saturation to finish the calibration.
To begin preparing a beverage sample for sugar content analysis, using a 1.5 milliliter microcentrifuge tube, centrifuge one milliliter of the beverage. Remove the supernatant with a one milliliter syringe and filter the supernatant though a 0.2 micrometer syringe filter. Combine 0.1 milliliters of filtrate and 0.9 milliliters of 0.8%PBS in a 1.5 milliliter microcentrifuge tube.
Gently vortex the mixture to ensure complete dilution. Place five microliters of the diluted beverage sample and 495 microliters of detection solution in a cuvette. Place the cuvette in the analyzer and preheat the sample to 53 degrees Celsius.
Press the set button to measure the sugar content of the sample. In this method, the FRET efficiency is measured by the ratio of the emission intensity of the yellow fluorescent protein to the cyan fluorescent protein in the biosensor. The optimal measurement temperature for these CMY biosensors is between 50 and 55 degrees Celsius, as the difference in the emission intensity ratio between a control solution and a saturated solution of maltose is greatest.
The CMY-BII biosensor was used to evalute the maltose content of three beverages at 53 degrees. A dose response curve is generated from obtaining this ratio at various maltose concentrations at a given temperature. The emission ratio is related to the maltose concentration using the values obtained during calibration.
The measured maltose contents are shown with the total sugar contents reported by the beverage manufacturers. Beverage A contained maltose sources such as rice and barley. Correspondingly, the observed amount of maltose accounts for about half of the total reported sugar content.
Beverage C, a sports drink, had a very low maltose content. While attempting this procedure, it's important to remember to operate the device and FRET sensors between 50 and 55 degrees Celsius. After watching this video, you should have a good understanding of how to quantify such a small molecules with a custom-made handheld FRET analyzer.
