Team:KEYSTONE/Partnership
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Please also refer to: https://2020.igem.org/Team:BUCT-China/Partnership.
Throughout the course of the competition, we have worked closely with an undergraduate team BUCT-China, whose project aims to degrade PE plastic, another predominant kind of plastic that is harder to break down and recycle than PET.
We got in touch with BUCT-China on a virtual meet up where we both presented our projects and noticed the parallel. We decided to become partners as we thought we could benefit from each other’s projects and expertise. Following the meet-up, we had meaningful discussions on our respective projects and how we can work together to benefit from each other.
Since the instructor of BUCT-China, prof. Luo Liu, is authoritative in the field of biological degradation of plastic, we conducted an interview with him which provided significant information and advices that guided our further experiments, business plan and hardware, such as:
- PET plastic becomes amorphous in high temperatures which increases the rate of reaction, therefore, plastic degradation should be conducted in a much higher temperature than for protein expression (70-80˚C).
- LCC enzyme has highly stable activity, therefore, the enzyme in our bin can be reused for multiple times after collecting the remains of the degradation instead of making E.coli produce it continuously. This inspired us to add a cultivation bin on our hardware, that promotes bacteria growth. Some bacteria will then be transformed into a degradation bin which heats to 72 degrees that kills the bacteria and releases the enzymes, entering the degradation process, without needing to introduce new bacteria frequently.
- The products of LCC degradation, EG and TPA, as materials of PET, are more expensive than the PET themselves. Based on this point and his suggestions that we should go beyond just degrading the materials themselves, we developed a business plan [hyperlink] that helps locals and people in remote areas to take benefit from the degraded EG and TPA, which becomes a closed circle that would benefit both the environment and the indigenous people.
- In the industrial recycling processes, PET is heated to become amorphous, then stretched to become fabrics. The more times the PET is recycled, its properties are also reduced. Therefore, degradation by biological means is a better method, which maintains the plastic performance. This is taken into consideration in our business plan.
See Integrated HP section for an elaborated summary.
BUCT-China lacked capable artists in their team. In exchange to the help they have provided us, the artist from our team created the cartoon demonstration of BUCT-China’s project which was used in their promotion which helped to explain the contents in a clearer and more eye-catching way.
Since both our projects are used to solve similar needs—waste disposal, they would have similar applications in alike scenarios. Where PET is disposed, PE is likely to be disposed as well, since they are both common plastics used in many types of packaging. In fact, if our B.B.Bin is only capable of degrading PET plastics, it would have significant limitations as the PE plastic wastes generated by tourists also creates a great threat for the environment and is even harder to recycle than PET. On the other hand, while BUCT-China did not focus on the application and hardware of their project, KEYSTONE has designed a functional hardware for independent plastic degradation, which can also be served as an application of BUCT-China’s PE degrading bacteria. Therefore, we enhanced our current hardware 3D model and added an extra shredding and degradation section dedicated to PE degradation. This combined model would be able to degrade both types of plastic and have a wider range of applications other than natural tourist attractions, since PE degradation is also difficult in urban areas.
When KEYSTONE’s experiments with LCC proceeds to the degradation part, our team members had a meeting with members of BUCT-China to discuss and use their professional knowledge as undergraduates and experience on biological degradation of plastic to give advices on how we can enhance our experiments, and potential ways of collaborating on degradation experiments in their lab. The suggestions we received from the discussion include:
- Conduct enzyme activity assays after ultrasonication based on different buffers, pH values (7, 8, 9) and temperatures
- Conduct assays approximately once every three hours in the process of degradation experiments and produce mathematical models that would suggest the appropriate frequencies of renewing LCC in the bin
- Since the efficiency of binding of our enzyme with PET would be hindered by the hydrophilic nature of LCC and hydrophobic nature of PET, we may consider two solutions:
- Make PET hydrophilic
- Redesign our plasmid and link a hydrophobic protein with LCC
- MHET is produce in the process of PET degradation which hinders the efficiency of degradation. Therefore, we may consider also producing MHETase, which can hydrolyze MHET into TPA and EG.
- Negative control:
- When doing SDS-PAGE, resuspend the precipitant of cell ultrasonication in distilled water to serve as negative control to the supernatant.
- Since PET undergoes degradation to a certain extent in natural environments, therefore, a controlled sample of PET in the same buffer without LCC should also be tested in our degradation experiments, to demonstrate the true effect of LCC.
Due to time and COVID regulations constraints, we weren’t able to carry out all the suggested improvements or collaborate in labs, however, some of these suggestions did inspire us into enhancing our experiments:
- We added the ultrasonication precipitant into our SDS-PAGE as negative control---[which BUFFER better]
- We added a sample of 100mg PET powder in 49ml 100 mM potassium phosphate buffer pH 8 and 1ml 20 mM Tris-HCl, pH 8, 300 mM NaCl (same as the other groups) but without LCC to be degraded in the same condition as the others (70˚C and 72˚C under agitation for 24h) as negative control [AND FOUND]
- As we researched about MHETase, we found a study published in September 2020 where scientists from NREL and University of Portsmouth were able to physically link MHETase with PETase, which is another PET-degrading enzyme, and increased the rate of degradation by three times1. Therefore, we included linking MHETase to LCC as part of our design for future experimental work.
In regards of MHET, which is also a product of PET degradation by LCC, can potentially be made useful by BUCT-China’s parts (Part:BBa_K3664005, Part:BBa_K3664006 and Part:BBa_K3664007), which is a polymerization system comprised of CoA ligase and acyltransferase. This system can cause esterification of the hydroxyl and carboxyl that make up MHET and form new materials that may be potentially useful in areas such as 3D printing. BUCT-China has also provided us with standard samples of TPA for us to conduct gas chromatography.
As the end of iGEM approached, BUCT-China was invited to the second synbio fair that KEYSTONE hosted (see public engagement section for more information), and we were able to meet in person, share and discuss our results so far. Since members from BUCT-China have participated in iGEM in previous years, they were also able to share their experience on other aspects of the competition, including wiki, presentation, etc.
Just like our collaboration on BUCT-China’s promotion video, KEYSTONE once again shared our expertise in art and recreated the animation used in BUCT-China’s final presentation video to explain their project.
