Team:ZJU-China/Safety

Our project aims to detect the injury based on a cell-free device with three connected enzymes which fixed on a biofilm scaffold. We use blood specimens with biochemical analysis to calibrate our device and help us with our machine learning modeling to set a diagnosis criterion.

Project Safety Biofilm Scaffold

Our biofilm scaffold consists of curli which is the protein found on the surface of E.Coli. Curli may facilitate bacteria’s invasion into host cells and activate corresponding cytokines and inflammatory mediators in plasma. But the system we wish to produce will be ultimately cell-free and no living organisms will be included. Besides, curli will be immobilized and covered by Nafion on the electrode, so contact with curli will not happen. Therefore, biofilm scaffold is relatively safe.

Blood Samples

We have gotten approval from our university authority and collected some blood samples from SIR RUN RUN SHAW HOSPITAL, which is classified as a Grade 3 Class A hospital by Chinese government. The procedure of blood sampling was done by medical personnel using ideal blood collection equipment. Besides, our experiment was conducted under professional doctors' supervision and waste was collected by the specialties in the hospital. (It is also an essential part of Integrated Human Practice.)

Calibration

We used the plate reader Synergy Neo2 for all the measurements and we used black 96 well plates with flat, transparent bottom.

• OD600 Reference Point

Add 100 µl LUDOX into wells A1, B1, C1, D1 and 100 µl of H2O into wells A2, B2, C2, D2. Then measure absorbance at 600 nm of all samples in all standard measurement modes in instrument and turn off the pathlength correction at the same time. The temperature setting was 26.6°C. Record the data.

• Particle Standard Curve

Obtain the tube labeled “Silica Beads“ from the InterLab test kit and vortex vigorously for 30 seconds. Then immediately pipet 96 µL microspheres into a 1.5 mL eppendorf tube. Add 904 µL of ddH2O to the microspheres and vortex well.

Prepare the serial dilution of microspheres as shown below. Set 4 copies.

Fig.1 Dilution of microspheres ^[1]

Measure the plate in plate reader, the excitation filter was set to 485nm/10nm and the emission filter was set to 525nm/10nm. Pathlength correction was turned off. The gain setting was 50. Fluorescence was from the top. The temperature setting was 26.6°C. Record the data.

• Fluorescence standard curve

Spin down fluorescein stock tube. Prepare 10x fluorescein stock solution (100 µM) by resuspending fluorescein in 1 mL of 1xPBS. Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration 10 μM.

Prepare the serial dilutions of fluorescein as shown below. Set 4 copies.

Fig.2 Dilution of fluorescein ^[1]

Measure the plate in plate reader, the excitation filter was set to 485nm/10nm and the emission filter was set to 525nm/10nm. Pathlength correction was turned off. The gain setting was 50. Fluorescence was from the top. The temperature setting was 26.6°C. Record the data.

Cell measurement

Make a 1:10 dilution of of the overnight cultures prepared after colony selection in LB medium + Chloramphenicol and measure Abs 600 of these 1:10 diluted cultures. Then dilute the cultures further to a target Abs600 of 0.02 in a final volume of 12 ml LB medium + Chloramphenicol in 50 mL falcon tube (amber or covered with foil to block light). Incubate the cultures at 37°C and 220 rpm. Take 500 µL samples of the cultures from each of the 8 devices, two colonies per device, at 0 and 6 hours of incubation and add them into 96 well plates as shown below. Place samples on ice before measurements.

Fig.3 Loading samples ^[1]

Measure the samples (Abs 600 and fluorescence measurement). The cell measurement was under the same condition with the particle standard curve and the fluorescence standard curve, using the same plate.

Counting colony-forming units (CFUs)

Measure the OD600 of cell cultures, making sure to dilute to the linear detection range of the plate reader. Then dilute the overnight culture to OD600 = 0.1 in 1 mL of LB + Cam media. Do this in triplicate for each culture and check the OD600 to make sure it is 0.1.

Do the following serial dilutions as blow.

Fig.4 Dilutions ^[1]

Count the colonies on each plate with fewer than 300 colonies. And multiple the colony count by the final dilution factor on each plate to get the colony forming units (CFU) per 1mL of an OD600 = 0.1 culture.

Results OD600 Reference point

Tab.1 OD600 reference point

Particle Standard Curve

Fig.5 Particle standard curve 1 | Fig.6 Particle standard curve 2

Fluorescence standard curve

Fig.7 Fluorescence standard curve 1 | Fig.8 Fluorescence standard curve 2

Cell measurement

Tab.2 Raw plate reader measurements of fluorescence raw at 0 Hour

Tab.3 Raw plate reader measurements of fluorescence raw at 6 Hour

Tab.4 Raw data of Abs600 measurement at 0 hour

Tab.5 Raw data of Abs600 measurement at 6 hour

The results of the CFUs and the flow cytometry data have been submitted in time by online forms and a zip file.

Discussion

Our experiments have got a great result, showing that the protocol is rather detailed and easy to operate. We are pleased to share our data with other teams around the world and we sincerely hope the Interlab study this year goes well.

References

[1] https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf

[2] https://2018.igem.org/Measurement/InterLab

[3] https://2018.igem.org/Measurement/InterLab/Plate_Reader

[4] https://2018.igem.org/Measurement/InterLab/Flow_Cytometry