Team:Kyoto/Demonstrate
Demonstration
Overview
We demonstrated how our proteins could be applied to the real world. As a result, our protein device, “SONOBE” could precipitate fibers in the way described in Design.We also confirmed that this protein strongly and stably bound to PET fibers in realistic conditions. Furthermore, the optimum quantity of protein to put into laundry wastewater was calculated by Modeling,and we also made a Hardwareto introduce our protein solution into wastewater.
1.PET-binding proteins tightly bind to PET fibers
Firstly, we examined if our fluorescent plastic-binding proteins actually bind to fibers and stay even after they are washed. We put every purified protein on the PET cloth then washed it (see Result).Fig.1a is cloth before wash, and 1b is after wash. As shown in figures, although sfGFP was washed out, both plastic-binding proteins stuck strongly to PET cloth. According to our quantification, 65 to 75% of the fluorescence signal remained even after washing.
Fig.1a Cloth dot blot by fluorescent plastic-binding protein before washing
The dilution collection of each protein was dropped on PET cloth, then left for 20min. The protein fluorescent was imaged by LAS-3000. The exposure time was 10sec.
The dilution collection of each protein was dropped on PET cloth, then left for 20min. The protein fluorescent was imaged by LAS-3000. The exposure time was 10sec.
Fig.1b Cloth dot blot by fluorescent plastic-binding protein after washing
The dilution collection of each protein was dropped on PET cloth, then left for 20min. The cloth was washed in TBST for 5min x3, then protein fluorescent was imaged by LAS-3000. The exposure time was 10sec.
The dilution collection of each protein was dropped on PET cloth, then left for 20min. The cloth was washed in TBST for 5min x3, then protein fluorescent was imaged by LAS-3000. The exposure time was 10sec.
Fig.2 Fluorescent picture (our mascot “Konkon”) appears
We drew mascot “Konkon” with LCI KR-2 protein solution, then the cloth was soaked in the sfGFP solution. In this movie, the cloth is washed with tap water.
We drew mascot “Konkon” with LCI KR-2 protein solution, then the cloth was soaked in the sfGFP solution. In this movie, the cloth is washed with tap water.
2.We successfully created our device "SONOBE">
As shown in the Design page, our device "SONOBE" has three components: 1) Plastic-binding protein, 2) Encapsulin, and 3) SpyCatcher/SpyTag. As shown above, the first component works well. Also, the second component, Encapsulin, properly formed a protein capsule and third component, SpyCatcher/SpyTag, properly formed isopeptide bond between plastic-binding protein and Encapsulin (See section 2 and 3 of Result). Therefore, we succeed in creating protein complex "SONOBE": a protein capsule which has plastic-binding proteins on the surface.
3."SONOBE" makes microfibers aggregate
Next, we will show our device “SONOBE” actually makes microfibers precipitate. As shown in Fig.3, microfibers on the surface of the water precipitated when they are mixed with “SONOBE”.
Fig.3 Fibers on the water surface precipitated with "SONOBE"
Fibers are mixed with water. Some fibers float when they just mixed with water. (Left) When the device added, most of the fibers precipitated. (Right)
Fibers are mixed with water. Some fibers float when they just mixed with water. (Left) When the device added, most of the fibers precipitated. (Right)
In another experiment, we measured the microfibers’ particle size. In Fig.4, the frequency of each diameter particle was output. The blue line shows the result when only fibers were measured. The red line shows the result when the mixture of fibers and the device were measured. Compare to the blue line, particles more than 20 µm are increased in the red line. This result suggests some of 10 to 20 µm particles aggregate into larger particles by adding the device.
Fig.4 The sizes of microfiber particles increased
The distribution of microfibers’ particle size are shown. The blue line shows the result when only fibers were measured. The red line shows the result when the mixture of fibers and the device were measured. Microfibers’ particle size was measured with laser diffraction by Microtrak MT3000Ⅱ.
The distribution of microfibers’ particle size are shown. The blue line shows the result when only fibers were measured. The red line shows the result when the mixture of fibers and the device were measured. Microfibers’ particle size was measured with laser diffraction by Microtrak MT3000Ⅱ.
4.The behavior of “SONOBE” in practical conditions was simulated
As we showed above, we assessed the interaction between the device and microfibers in our experiment. But there are many kinds of compounds in wastewater, so they might interfere with our device. In the Modeling section, competitive inhibition by cellulose, which is abundant in wastewater was simulated. This simulation suggests our device can work in such a condition without being interfered. In advance, the required concentration of our device in wastewater was calculated, and this enabled us to calculate how much our device costs if we put into practice. Also, we tried to make hardware for the practical use of our device and we suggested the ideal model of hardware. It may help to realize our project.
Summary
Now we have the device “SONOBE” and confirmed that it worked as designed. To make our device work in a realistic world, we calculated the optimum protein quantity to put in the laundry wastewater.
We also designed hardware and checked its function by simulating the actual situation. We understand there are some points to improve our device (see Future Application) but we hope our idea to precipitate the fiber would be one possible approach for the microplastic problem.
