B.B.Bin is an abbreviation for Beach Bacteria Bin.
Team: KEYSTONE 2020
Highschool
Beijing, China
Team:KEYSTONE/Poster
Poster: KEYSTONE
B.B.Bin
B.B.Bin is an abbreviation for Beach Bacteria Bin.
Team: KEYSTONE 2020
Highschool
Beijing, China
Introduction
Beach: The problem
PET plastic is easily recycled and made into other products in urban areas. However, remote natural tourist attractions such as beaches in Bali are still under the threat of PET pollution created by tourists and local people due to the deficiency or absence of local plastic recycling systems, poor waste management and environmental conditions. In such places, plastic waste easily enters the environment and become difficult to collect.
Bacteria: A synbio approach
The leaf compost cutinase (LCC) is a type of enzyme that degrades PET polymer into TPA and EG, which are valuable materials for PET production. It is cheap, effective, and thermostable. Comparing to chemical recycling processes, this biological degradation preserves the properties and, therefore, the value of the plastic by depolymerizing it into its substrates. Consequently, the commercial value of TPA and EG will encourage the bin to be maintained by local people.
Bin: The application
Our bacteria and enzyme will be contained in a bin comprised of three parts: the shredder, the power source, and the decomposer. This bin is powered by solar panels and prevents PET plastic from entering the environment in places not covered by modern recycling systems. Linalool will also be produced by our bacteria to enhance the experience of tourists.
Inspiration
Our iGEM project is mainly inspired by several daily experiences that were brought up during the meeting:
1. Our school’s plastic-free policy: Our school encourages plastic-free living style. There are no plastic shopping bags provided at the school shop. This informed us to focus on plastic pollution.
2. Beijing’s new trash sorting policy in 2020: Beijing began a new and more strict trash sorting system in 2020. Inspired by this idea, we began thinking about contributing our effort to manage the trash that has been collected, and we believe a trash can that can help degrade plastic right on site would help.
3. Our daily “trash” experiences: while discussing about trash sorting, our team reached a consensus that trash cans smell bad. Thus, we decided to explore the ways that synthetic biology could be used to produce fragrance and make trash cans smell more pleasant.
1. Our school’s plastic-free policy: Our school encourages plastic-free living style. There are no plastic shopping bags provided at the school shop. This informed us to focus on plastic pollution.
2. Beijing’s new trash sorting policy in 2020: Beijing began a new and more strict trash sorting system in 2020. Inspired by this idea, we began thinking about contributing our effort to manage the trash that has been collected, and we believe a trash can that can help degrade plastic right on site would help.
3. Our daily “trash” experiences: while discussing about trash sorting, our team reached a consensus that trash cans smell bad. Thus, we decided to explore the ways that synthetic biology could be used to produce fragrance and make trash cans smell more pleasant.
Goal & Research
The goal of our project is to develop a PET-degrading trash bin that could be implemented into remote areas. This would assist in eliminating the plastic pollution problems that are unmanaged in distant tourist sites. We have done various interviews with experts in the field of PET recycling, plastic managing, and PET degradation as well as public surveys to find this issue, aim our project, and improve our project in order to better tackle it.
Our project research is divided into four sectors, discover, aim, verify, and improve. We discovered, through talking to the secretary in the Plastic Reuse and Recycle Association, that PET recycling has evolved into a comprehensive and complete cycle within urban areas, with no need for PET degradation mechanisms, though the plastic pollution problem remains unsolved in places where the urban PET recycling cycle cannot reach. By talking to various environmental NGOs working to eliminate PET littering, we aimed our project to the remote tourist sites, where collecting and recycling the plastic is not possible or economically desirable, therefore creating pollutions. We located three places where our project could be used: the Bali Islands, the Great Wall, and the Nanshan mountain. After disseminating surveys and receiving over 280 responses, we confirmed that our project is necessary in the public’s opinion and that the idea of synthetic biology is accepted. Eventually, implementing suggestions we received from Dr. Liu, we improved the degradation efficiency of our LCC enzyme.
Sustainable Development
The entire goal of our project mainly targets the SDG goal 12 of "responsible consumption and production".
Moreover, our B.B.Bin also helps to achieve SDGs goals including 11 and 14, which are respectively "sustainable cities and communities" and "life below water".
Engineering
LCC Production
We aimed to produce the leaf-branch compost cutinase (LCC), which is an enzyme capable of Polyethylene terephthalate (PET) degradation that outperforms PET hydrolase and BTA while having a high thermostability. The mutation LCC-ICCG used in our experiment is created by Tournier et al. in 2020 and was among 25 out of 209 that has 75% or more increased activity compared with the original wild-type LCC. The terephthalic acid (TPA) and ethylene glycol (EG) formed as a result of PET degradation can be used to make new PET plastic with similar properties to the PET plastic from factories [1].
The LCC-ICCG gene was coded onto the standard expression vector pET28a(+) with a 6xHis-tag on both side of N-terminal and C-terminal for more effective purification on Ni-NTA column.
Linalool Synthesis
We also aimed to produce linalool, which is a monoterpene, for fragrance. This is achieved by a plasmid with linalool synthase and the MVA pathway that is originally constructed to produce limonene. Through the MVA pathway, simple sugars are made into Acetyl-CoA through glycolysis. Through varies reactions, geranyl pyrophosphate (GPP) is synthesized from dimethylallyl pyrophosphate by the GPP synthase [2]. Then, the original article successfully produces limonene with the addition of a LS (limonene synthase) gene.
In our project, we applied the pathway to a new fragrance synthase. The linalool synthase used in our experiments is the linalool synthase from Streptromyces clavuligerus that is called bLIS. It is a very effective linalool synthase which produces linalool from GPP. The synthesis of linalool can also be increased by the optimization of the fusion tags and the ribosome binding sites of the protein [3].
We constructed 2 plasmids for linalool production.
The first plasmid produces DMAPP, which is the substrate of GPPS. In the second plasmid, GPPS and bLIS express GPPS and linalool synthase that synthesizes linalool. Therefore, the two plasmids can work together to achieve linalool synthesis.
References
[1] Tournier, V., Topham, C.M., Gilles, A. et al (2020). An engineered PET depolymerase to break down and recycle plastic bottles. Nature 580, pp.216–219. https://doi.org/10.1038/s41586-020-2149-4.
[2] Alonso-Gutierrez, J., Chan, R., Batth, T. S. et al (2013). Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production. Metabolic Engineering, volume 19, pp.33-41. https://doi.org/10.1016/j.ymben.2013.05.004.
[3] Xun, W., Jing, W., Jiaming, C. et al (2019). Efficient biosynthesis of R-(-)-linalool through adjusting expression strategy and increasing GPP supply in Escherichia coli. https://doi.org/10.21203/rs.2.18761/v1.
Results-LCC
Production & Purification
Out of all conditions tested, we were able to produce an optimal yield of 27.3mg/L of LCC with 300µM IPTG induction and purification with 20mM Tris-HCL, 300mM NaCl buffer.
Figure 1: Protein gel electrophoresis of LCC expression with 300µM IPTG and purification with 20mM Tris-HCL, 300mM NaCl buffer, showing a molecular mass of 28kDa as expected.
PET Degradation
We repeated the degradation experiment by Tournier et al. with the LCC proteins that we produced, using 1 ml of LCC solutioin in 20 mM Tris-HCl, pH 8, 300 mM NaCl, and combined with 100 mg PET powder with 49ml of 100 mM potassium phosphate buffer pH 8 in a 100 ml flask for degradation in a 72˚C environment under agitation.
Figure 2: Gas chromatography test for ethylene glycol in LCC degradation supernatant with different LCC concentrations after 24h.
The standard sample used in the GC test was 20mg/ml EG. In our sample of 100mg PET in 50ml buffer, the maximum concentration of EG that can be obtained is 0.54mg/ml, which would produce a peak with an area of 0.27% of the area of the standard peak. Therefore, although difficult to obtain a precise calculation due to the low concentration, it can be seen that the amount of PET degraded by 5µM and 10µM of LCC was fairly significant, and a distinct difference with the no LCC controlled sample can be seen.
Out of all conditions tested, we were able to produce an optimal yield of 27.3mg/L of LCC with 300µM IPTG induction and purification with 20mM Tris-HCL, 300mM NaCl buffer.
Figure 1: Protein gel electrophoresis of LCC expression with 300µM IPTG and purification with 20mM Tris-HCL, 300mM NaCl buffer, showing a molecular mass of 28kDa as expected.
PET Degradation
We repeated the degradation experiment by Tournier et al. with the LCC proteins that we produced, using 1 ml of LCC solutioin in 20 mM Tris-HCl, pH 8, 300 mM NaCl, and combined with 100 mg PET powder with 49ml of 100 mM potassium phosphate buffer pH 8 in a 100 ml flask for degradation in a 72˚C environment under agitation.
Figure 2: Gas chromatography test for ethylene glycol in LCC degradation supernatant with different LCC concentrations after 24h.
The standard sample used in the GC test was 20mg/ml EG. In our sample of 100mg PET in 50ml buffer, the maximum concentration of EG that can be obtained is 0.54mg/ml, which would produce a peak with an area of 0.27% of the area of the standard peak. Therefore, although difficult to obtain a precise calculation due to the low concentration, it can be seen that the amount of PET degraded by 5µM and 10µM of LCC was fairly significant, and a distinct difference with the no LCC controlled sample can be seen.
Result - Linalool
In linalool synthesis, we used Gibson assembly to construct the plasmids, pR6K-ptac-GPPS-LIS and p15A-MVA from their fragments.
We transformed plasmid pR6K-ptac-GPPS-LIS to E. Coli DH5α, and performed a colony PCR. (figure 1 A). the result of it shows that the plasmid has been successfully constructed, Besides, the E. Coli can also produce linalool synthase when induced with IPTG, which further confirms our success in plasmid construction. (figure 1B)
(we put the incorrect annotated picture in our wiki page, this picture is the correctly annotated one)
Figure 1: (A) DNA gel electrophoresis for plasmid pR4K-ptac-GPPS-LIS. (B) protein gel electrophoresis for linalool synthase
For p15A-Lac UV5-atoB-HMGS-HMGR-Trc-MK-PMK-PMD-idi, we sent the transformed bacteria to a biotech company that helped us to perform a gene sequencing. The result of the sequencing indicates that we successfully constructed plasmid. (figure2 A)
Figure 2: Result of gene sequencing
We cotransformed the two plasmids to bacteria DH5 α. After inducing it with IPTG, we extracted linalool by adding n-hexane to the sample. The transparent layer in the picture is the extracted linalool. we can smell a strong aroma from the sample, which covers the smell of E. Coli. the fragrance in our sample have a peppery smell which is close to the smell of the standard sample of linalool, indicating that we have successfully produced linalool. However, due to Covid-19, we do not have access to gas chromatography, which may help us further confirm our result. (figure 4)
Figure 3: the result of linalool extract. Groups induced with the addition of glucose produced a significantly stronger scent.
*Don't miss the animation below ⬇️
We transformed plasmid pR6K-ptac-GPPS-LIS to E. Coli DH5α, and performed a colony PCR. (figure 1 A). the result of it shows that the plasmid has been successfully constructed, Besides, the E. Coli can also produce linalool synthase when induced with IPTG, which further confirms our success in plasmid construction. (figure 1B)
(we put the incorrect annotated picture in our wiki page, this picture is the correctly annotated one)Figure 1: (A) DNA gel electrophoresis for plasmid pR4K-ptac-GPPS-LIS. (B) protein gel electrophoresis for linalool synthase
For p15A-Lac UV5-atoB-HMGS-HMGR-Trc-MK-PMK-PMD-idi, we sent the transformed bacteria to a biotech company that helped us to perform a gene sequencing. The result of the sequencing indicates that we successfully constructed plasmid. (figure2 A)
Figure 2: Result of gene sequencing
We cotransformed the two plasmids to bacteria DH5 α. After inducing it with IPTG, we extracted linalool by adding n-hexane to the sample. The transparent layer in the picture is the extracted linalool. we can smell a strong aroma from the sample, which covers the smell of E. Coli. the fragrance in our sample have a peppery smell which is close to the smell of the standard sample of linalool, indicating that we have successfully produced linalool. However, due to Covid-19, we do not have access to gas chromatography, which may help us further confirm our result. (figure 4)
Figure 3: the result of linalool extract. Groups induced with the addition of glucose produced a significantly stronger scent. *Don't miss the animation below ⬇️
Hardware
To convert the goal and purpose of our project into practice, we designed a “trash can” in order to contribute to the solution.
Tough, it is a trash can, its ability is far beyond containing garbage. In fact, we actually made it into a mini trash processing center in which plastic can be broken-down and recycled.
The uniqueness of this hardware is the ability of individually maintain its functionality under harsh natural conditions.
The functional regions of this machine are compartmentalized into multiple modules by their roles. They are the shredder, the decomposer and the power source.
To enable our hardware the ability of engaging the society, promotion quotes regarding environmental protection and advertisements regarding the business plan will be established.
Future work
Due to technical limitations, many of our vision for the product could not be realized and need to be improved. For example, in the interview with prof. Liu, he pointed out that the current degradation equipment is not optimized for this reaction. A better way is to replace the current degradation can with a tubular reactor that allows the fluid to have a greater surface area.
Tough, it is a trash can, its ability is far beyond containing garbage. In fact, we actually made it into a mini trash processing center in which plastic can be broken-down and recycled.
The uniqueness of this hardware is the ability of individually maintain its functionality under harsh natural conditions.
The functional regions of this machine are compartmentalized into multiple modules by their roles. They are the shredder, the decomposer and the power source.
To enable our hardware the ability of engaging the society, promotion quotes regarding environmental protection and advertisements regarding the business plan will be established.
Future work
Due to technical limitations, many of our vision for the product could not be realized and need to be improved. For example, in the interview with prof. Liu, he pointed out that the current degradation equipment is not optimized for this reaction. A better way is to replace the current degradation can with a tubular reactor that allows the fluid to have a greater surface area.
Education
We designed and carried out a variety of public education activities related to synthetic biology and environmental awareness instructed by our relevant survey, the two most effortful ones being:
1. We created an educational game which teaches students about different concepts of synthetic biology. The idea behind this project is to develop a method of education that is fun, innovative, engaging and makes people interested in synbio. The end product is a bilingual chaptered Keynote game created based on a continuous script, collectively written by a number of teams. The nature of the game and script allows it to be played by individuals or in classrooms, can be freely shared, accessed, edited, and new chapters can be freely added.
We let many students try learning about synbio with our game, and received a lot of positive feedback.
2. We run a series of Synbio Fairs where teams can introduce and demonstrate their projects to people in an interactive way, and sell their merchandises under strict COVID-prevention measures. Since hosting in only one school would only reach a limited audience, we moved the same fair to different host schools. We chose to host these fairs in middle schools because we thought we may be able to invoke their interest in synthetic biology and consider pursuing this subject in the future.
The first fair was hosted in Keystone Academy, and the second fair was hosted in Tsinghua University Highschool.
Inclusivity
The inclusivity of synthetic biology throughout our school community, the local community, and even the international community was promoted by our education activities—our activities are designed and promoted to be understandable, accessible and meaningful for audiences across all ages and backgrounds in terms of education, socioeconomics and culture.
1. We created an educational game which teaches students about different concepts of synthetic biology. The idea behind this project is to develop a method of education that is fun, innovative, engaging and makes people interested in synbio. The end product is a bilingual chaptered Keynote game created based on a continuous script, collectively written by a number of teams. The nature of the game and script allows it to be played by individuals or in classrooms, can be freely shared, accessed, edited, and new chapters can be freely added.
We let many students try learning about synbio with our game, and received a lot of positive feedback.
2. We run a series of Synbio Fairs where teams can introduce and demonstrate their projects to people in an interactive way, and sell their merchandises under strict COVID-prevention measures. Since hosting in only one school would only reach a limited audience, we moved the same fair to different host schools. We chose to host these fairs in middle schools because we thought we may be able to invoke their interest in synthetic biology and consider pursuing this subject in the future.
The first fair was hosted in Keystone Academy, and the second fair was hosted in Tsinghua University Highschool.
Inclusivity
The inclusivity of synthetic biology throughout our school community, the local community, and even the international community was promoted by our education activities—our activities are designed and promoted to be understandable, accessible and meaningful for audiences across all ages and backgrounds in terms of education, socioeconomics and culture.
Future Work
Due to COVID-19, many of our plans have to change. Here are some works that we would like to continue in the future to enhance our project.
In terms of our experiments with LCC, we would like to:
In terms of linalool synthesis, we want to:
In terms of Hardware:
In terms of Education:
Acknowledgements
Supported by:
Keystone Academy;
Zeno Academy;
Link Spider;
Snapgene;
Benchling;
and Insentec.
Special Thanks:
Li Xin for help and instructions in WIki development
Zhang Chaofeng for help and instructions in Hardware development
Wang Xiaohui for help and instructions in Hardware development
Yang Qihui for help and instructions in Hardware development
Bibliography
Alonso-Gutierrez, J., Chan, R., Batth, T. S. et al (2013). Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production. Metabolic Engineering, volume 19, pp.33-41. https://doi.org/10.1016/j.ymben.2013.05.004.
Greatbay_China (2018). Demonstration. Retrieved Oct 22 2020, from https://2018.igem.org/Team:GreatBay_China/Demonstrate.
Luo, L. (2020). Personal Interview.
The Association of Plastic Recyclers. (Nov. 15, 2018). Report on postconsumer PET container recycling activity in 2017. https://www.epa.gov/facts-and-figures-about-
Ritchie, Hannah, and Max Roser. "Plastic Pollution." Our World in Data, Sept. 2018, ourworldindata.org/plastic-pollution.
Siddharta, Amanda Tazkia, and Photographs by Nyimas Laula. “Bali Fights for Its Beautiful Beaches by Rethinking Waste, Plastic Trash.” National Geographic, 14 Oct. 2019, www.nationalgeographic.com/science/2019/10/bali-fights-for-its-beautiful-beaches-by-rethinking-waste-plastic-trash/.
Tournier, V., Topham, C.M., Gilles, A. et al. An engineered PET depolymerase to break down and recycle plastic bottles. Nature 580, 216–219 (2020). https://doi.org/10.1038/s41586-020-2149- 4
Xun, W., Jing, W., Jiaming, C. et al (2019). Efficient Biosynthesis of R-(-)-linalool through Adjusting Expression Strategy and Increasing GPP Supply in Escherichia coli. https://doi.org/10.21203/rs.2.18761/v1.
Keystone Academy;
Zeno Academy;
Link Spider;
Snapgene;
Benchling;
and Insentec.
Special Thanks:
Li Xin for help and instructions in WIki development
Zhang Chaofeng for help and instructions in Hardware development
Wang Xiaohui for help and instructions in Hardware development
Yang Qihui for help and instructions in Hardware development
Bibliography
Alonso-Gutierrez, J., Chan, R., Batth, T. S. et al (2013). Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production. Metabolic Engineering, volume 19, pp.33-41. https://doi.org/10.1016/j.ymben.2013.05.004.
Greatbay_China (2018). Demonstration. Retrieved Oct 22 2020, from https://2018.igem.org/Team:GreatBay_China/Demonstrate.
Luo, L. (2020). Personal Interview.
The Association of Plastic Recyclers. (Nov. 15, 2018). Report on postconsumer PET container recycling activity in 2017. https://www.epa.gov/facts-and-figures-about-
Ritchie, Hannah, and Max Roser. "Plastic Pollution." Our World in Data, Sept. 2018, ourworldindata.org/plastic-pollution.
Siddharta, Amanda Tazkia, and Photographs by Nyimas Laula. “Bali Fights for Its Beautiful Beaches by Rethinking Waste, Plastic Trash.” National Geographic, 14 Oct. 2019, www.nationalgeographic.com/science/2019/10/bali-fights-for-its-beautiful-beaches-by-rethinking-waste-plastic-trash/.
Tournier, V., Topham, C.M., Gilles, A. et al. An engineered PET depolymerase to break down and recycle plastic bottles. Nature 580, 216–219 (2020). https://doi.org/10.1038/s41586-020-2149- 4
Xun, W., Jing, W., Jiaming, C. et al (2019). Efficient Biosynthesis of R-(-)-linalool through Adjusting Expression Strategy and Increasing GPP Supply in Escherichia coli. https://doi.org/10.21203/rs.2.18761/v1.