Team:SMS Shenzhen/Measurement
Project-Measurement
Measurement
We need 2 fundamental data: photoswitches efficiency and HpaBC efficiency. To determine the photoswitches efficiency, we use the primary plasmid pET28b-sfGFP-7*His, a plasmid that contains GFP that is controlled by T7 promoter. The fluorescence intensity is measured to represent switches' efficiencies in proportion.To determine the enzyme efficiency and how photoswitches regulate the system, we measure the yielding, which is the levodopa concentration in the solution.
Measure photoswitches efficiency
In the photoswitches test, we obtain bacteria culture samples that are cultured in different environments. Therefore, we need to measure a relative GFP expression of each cell. By using a fluorescence intensity reader, we derived fluorescent intensity value and absorbance under OD 600nm. To get a relative value, we divide the intensity by absorbance under OD 600nm. This value indicates the relative amount of GFP expressed in each cell. This leads to determined photoswitches efficiency.
Detailed processes
1. Take 1mL cell culture for each measurement.
2. harvest the cells by centrifuge. Discard the supernatant and add 1mL PBS. Blow the bacteria to dissolve.
3. Repeat step 2 for three times, to exclude the deviations influenced by the medium.
4. Take 100μL cell culture and mix it with 900μL PBS. Sufficiently mix.
5. Transferred 200μL of it to a 96 well plate.
6. Measure absorbance under OD 600 nm and the fluorescent intensity by a microplate reader. The excitation light is 485nm, and emission light is 535nm.
Measure Levodopa concentration
Levodopa is a diphenol. It can be spontaneously oxidized by oxygen to dopaquinone in standard conditions. Dopaquinone can spontaneously aggregate to melanin, an important pigment that contributes to dark color in human skin. Known as the protecter of our DNA from UV light, it has a high ultraviolet absorbance. According to Beer’s law, the light absorbance is positively proportional to the concentration of dopaquinone. And this is equal to the original levodopa concentration. Therefore, we can determine the concentration of levodopa by measuring OD 400. However, the reaction is slow. Therefore, NaIO4, a transparent and highly oxidative salt, is added to accelerate the reaction.
Detailed processes
1. Take 1mL cell culture for each measurement.
2. centrifuge for 12000 rpm and 5 minutes to ensure the deposition of bacteria.
3. 180μL supernatant was transferred to a well of 96 wells plate, repeat this three times (if applicable).
4. Add 20μL 100mM NaIO4 to each well.
5. place the plate in still for 45 minutes.
6.After the reaction, measure the absorbance under OD 400nm by a microplate reader, and derive an average value.
7. Use the standard curve to transform absorbance into the concentration of levodopa.
Determine the standard curve of levodopa
As we have mentioned in levodopa measurement, we need a standard curve to help us transfer the measured value of absorbance under OD 400nm into the levodopa concentration. We prepare 4mM levodopa for the measurement. We then add 0μL, 22.5μL, 45μL, 67.5μL, 90μL, 112.5μL, 135μL, 157.5μL, and 180μL of levodopa to a well in a 96-wells plate respectively. Repeat this three times. Supply each well with water to 180μL. Therefore, we prepared a series of solution with a leveled concentration of 0.5mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM and 4mM. Each well is then supplied with 20μL 100mM NaIO4 to help coloration. We take measurements at (nearly) 0 minutes (the coloration is rapid in the beginning), 40 minutes, 60 minutes, 80 minutes, 120 minutes, and 150 minutes. An average number of each concentration is taken, and the results are graphed below.
[1]: Yao, J., He, Y., Su, N., Bharath, S., Tao, Y., Jin, J., Chen, W., Song, H. and Tang, S.
2020. Developing a highly efficient hydroxytyrosol whole-cell catalyst by de-bottlenecking
rate-limiting steps. Nature Communications, 11(1).
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