Team:BUCT-China/Partnership
In the field of cooperation, we have worked closely with a high school team (KEYSTONE) and carried out a lot of exchanges.On the project, we all chose the biodegradation-related topics of plastics. There are many common places. KEYSTONE's project is about PET degradation. We hope that we can help them through our studies. Of course, we also harvested many creative ideas from them.
| 1st stage |
Getting in touch
We learned about KEYSTONE's project at the online meeting, and we decided to be a partner because we had very similar project ideas and goals. We hope that we can harvest new ideas from each other's projects and continue to improve our projects through exchanges.After the meeting, we had meaningful discussions on our projects and how to work together to benefit each other.
further discussion（to KEYSTONE）
Professor Liu Luo, the primary PI of our team, has rich research experience in the field of biodegradation of plastics. At the beginning of the project, KEYSTONE team members interviewed Professor Liu Luo. Professor Liu Luo gave them many suggestions on experimental design and provided ideas on hardware design. We collected some simple records of KEYSTONE team:
1.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).
2.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.
3.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.
4.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.
1.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).
2.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.
3.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.
4.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.
Promotion video （Provided by KEYSTONE）
In the early stage of the project, we also got great help from KEYSTONE.Our team has been relatively deficient in art design.How to clearly describe our project in the video has been troubling us.KEYSTONE's painters provided us with this Promotion video.The cartoon presentation process, which is used for promotion, helps explain content in a clearer and more compelling way.KEYSTONE's work is very satisfactory and our team likes it very much.
| 2nd stage |
After many online discussions, our discussions are more in-depth, and we hope that our work can influence each other and benefit each other from exchanges and discussions, thus making our projects more complete.We further discussed the details of the experiment and reached the intention of cooperation in hardware.All this makes us better.
Hardware Supply（Provided by KEYSTONE）
Both of our teams are committed to solving the problems caused by plastic waste, and our team has been seeking an effective way or appropriate occasion to apply our experimental results.In the middle of the project, KEYSTONE described their hardware system to us, and in their original design, they only considered the degradation of PET.In our communication process, we proposed our demand for hardware equipment, and we soon reached the intention of cooperation. KEYSTONE added PE cutting and degradation modules to our original basis.Therefore, we have found a suitable place for our project.This modular hardware system can degrade both PET and PE plastics and can be applied to a wider range of occasions.We are very grateful for the hardware from KEYSTONE to make our project more complete.
This is a very good way out for our team and has a great impact on the integrity of our project.Our project concept has always been focused on environmental protection. Such a hardware device integrated with recycling and resynthesis system perfectly fits our project concept
This is a very good way out for our team and has a great impact on the integrity of our project.Our project concept has always been focused on environmental protection. Such a hardware device integrated with recycling and resynthesis system perfectly fits our project concept
Experimental enhancements（to KEYSTONE）
In addition to online discussions, we also conducted offline exchanges to further enhance our cooperative relationship. During the communication activities, KEYSTONE presented to us the problems they encountered during the experiment, and the enzyme (LCC) they used was less efficient in degrading PET.Based on our knowledge and experience in plastic degradation, we have provided KEYSTONE with some experimental suggestions that can be improved, as well as some research that can be continued in the future. Here are some notes made by KEYSTONE and their improvement work:
1. Conduct enzyme activity assays after ultrasonication based on different buffers, pH values (7, 8, 9) and temperatures
2. 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
3. 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:
a. Make PET hydrophilic
b. Redesign our plasmid and link a hydrophobic protein with LCC
4. 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.
5. Negative control:
a. When doing SDS-PAGE, resuspend the precipitant of cell ultrasonication in distilled water to serve as negative control to the supernatant.
b. 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.
Because of the impact of the new coronavirus, this has had a certain impact on our entry into the laboratory to complete our work.We learned that KEYSTONE was also troubled by this problem, but fortunately they also completed part of the improvement work in the laboratory after receiving our suggestions, which we would like to see.We obtained a description of their experimental work from KEYSTONE:
-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.
1. Conduct enzyme activity assays after ultrasonication based on different buffers, pH values (7, 8, 9) and temperatures
2. 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
3. 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:
a. Make PET hydrophilic
b. Redesign our plasmid and link a hydrophobic protein with LCC
4. 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.
5. Negative control:
a. When doing SDS-PAGE, resuspend the precipitant of cell ultrasonication in distilled water to serve as negative control to the supernatant.
b. 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.
Because of the impact of the new coronavirus, this has had a certain impact on our entry into the laboratory to complete our work.We learned that KEYSTONE was also troubled by this problem, but fortunately they also completed part of the improvement work in the laboratory after receiving our suggestions, which we would like to see.We obtained a description of their experimental work from KEYSTONE:
-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.
| 3rd stage |
Synbio fair（invited by KYESTONE & to KEYSTONE）
Later in the project, we were invited by KEYSTONE to participate in the synbio Fair sponsored by them, and we had project exchanges with many excellent high school teams.We are very happy to share our project and participation experience. We have shared our experimental progress and some experimental methods with KEYSTONE and other teams. We hope that our sharing will stimulate their interest in synthetic biology and scientific research.Our sharing has received a lot of attention from them, and we enjoy the process very much and feel a sense of achievement.In addition to scientific research, based on our previous experience in iGEM, we described our participation process and Jamboree's feelings. We shared our experience in Wiki production and poster production.We all look forward to meeting Jamboree again in the future.
Presentation video（Provided by KEYSTONE）
As with our Promotion videos, we are very satisfied with the hand-drawing process provided by KEYSTONE, and in Presentation videos, we still sought the help of KEYSTONE.We believe that such help is valuable, not only to make our videos more vivid, but also to make our descriptions more understandable.
In conclusion, form a partnership with KEYSTONE is a very interesting experience.Our two teams have established an unusual partnership, which is the first partnership with a high school team for us, and the first partnership with a university team for KEYSTONE.We cherish this partnership very much. As a university team, we feel that it is a very meaningful thing to be able to cooperate with the high school team in the project, and we can share our experience really great.We believe that it is in the spirit of iGEM competition to be able to disseminate knowledge about synthetic biology and share the good experience of participating in iGEM with other teams and that's what we've been doing
.