About 4.5 trillion cigarette butts are deposited somewhere in the environment every year. Out of the waste collected by coastal cleanups every year, cigarette butts comprise the largest percentage of this, which account for approximately 19 - 38 % of total waste products by count. Nicotine from cigarette butts negatively affect marine environment. Pseudomonas putida S16 can metabolite nicotine. We are planning to introduce this pathway into Escherichia coli. We expect that Escherichia coli gets ability to degrade nicotine. Through the pathway, nicotine is changed to 2,5-dihydroxypyridine. 2,5-dihydroxypyridine is a precursor of 5-aminolevulinic acid which is the main material of fertilizer and pharmaceuticals. We hope that our project helps stop littering cigarettes. And we hope that living things are more safely and peacefully.
Introduction
If cigarette butts were valuable, would people litter with them?
We use the power of synthetic biology to transform cigarette butts into something "worthwhile".
By doing this, we hope to create a "Nico-friendly" society in which nicotine is not artificially released into the environment, thus protecting the ecosystem.
Inspiration
We have learned that the nicotine being released into the environment is having a negative impact on the ecosystem. Nicotine is an endocrine disruptor that acts on sex determination and other factors. Nevertheless, some have calculated that there are between 0.6 and 32 μg of nicotine per liter in the environment. This is enough to affect the reproduction and growth of Daphnia magna.
Problem
Where does the nicotine released into the environment come from? The answer is "littered cigarette butts": according to the WHO, about 4.5 trillion cigarettes are thrown away irresponsibly. We wondered how to prevent cigarettes from being littered. So we decided that we could use the power of synthetic biology to make cigarette butts "valuable".
fig.1 littered cigarette butt
Idea
Nicotine has a negative impact on the environment, and it is difficult to break down nicotine by chemical pathways. Therefore, we focused on Pseudomonas putida S16, a bacterium that can degrade nicotine. We have created a bacterium that can degrade nicotine using synthetic biology and converted the degradation product, 2,5-dihydroxypyridine, into 5-aminolevulinic acid by a chemical pathway. 5-Aminolevulinic acid is a "high-value" chemical that is useful in pharmaceuticals and fertilizers, and therefore We believe that if we create this pathway, we can make the nicotine-containing cigarette butts "worth it".
Engineering
Since nicotine is known to solve in organic layer, we will extract it. (Qayyum et al., 2018) After the extraction, we will convert it to 2,5-dihydroxypyridine using genes from Pseudomonas Putida S16. After the conversion, 2,5-dihydroxypyridine will be converted to 5-aminolevulinic acid chemically.
1,Extraction of Nicotine from Tobacco Waste
Shake with NaOH solution
→Filtration
→Add water and filter again.
→Dissolve in organic solvent and extract the organic layer three times.
→Potassium carbonate was added and filtered.
→Evaporate the diethyl ether in a water bath form.
→Measured by infrared radiation spectroscopy.
fig.1 The extraction method of nicotine. Solve the tobacco waste into 5% NaOH and filter them. Add diethyl ether and evaporate the organic layer.
2,Nicotine is biologically converted to 2,5-dihydroxypyridine.
There are two methods. One method is to use Escherichia coli as a chassis and the other is to use Pseudomonas putida S16 (P. putida S16) as a chassis.
More details will be presented in the next section.
fig.2 PlanA,PlanB.
3,Conversion of 2,5-hydroxypyridine to 5-aminolevulinic acid
After recovering 2,5-dihydroxypyridine, 5-aminolevulinic acid will be produced. It is known that this can be done chemically, as shown in Figure 3. (Herdeis and Dimmerling, 1984) 2,5-dihydroxypyridine is in equilibrium with 5-hydroxy-2-pyridone. It is a known lactam-lactime tautomer. It is converted to 2,5-piperidinedione by heating 30°C for 5 hours with palladium on carbon. This is reacted with hydrochloric acid for 12 hours to produce the final product, 5-aminolevulinic acid.
fig.3 2.5-dihydryoxypyridine is converted to 5-aminolevulinic acid. 2,5-dihydroxypyridine is equilibrium with 5-hydroxy-2-pyridine due to lactam-lactim tautomer. With palladium on carbon, 2,5-piperidinedione is made. Adding HCl to the product, 5-aminolevulinic acid is produced.
PlanA and PlanB
PlanA:Using E. coli as a chassis
As shown in figure 1, genes related to cell communication and nicotine degradation will be inserted to E. coli. There are two devices. The first device which consists of constitutive promoter, luxI, and luxR is responsible for regulating the second device. The second device which consists of nicotine degrading genes form P. putida S16 and a molybdenum cofactor gene(molybdopterin cytidylyltransferase, mocA: is responsible for degrading nicotine and producing 2,5-dihydroxypyridine.
fig.1 The genes introduced to E. coli. a: The regulating device consists of constitutive promoter, luxI, and luxR. The nicotine degrading device consists of nicotine degrading genes and mocA. b and c: First, the lux promoter is not activated. If the cells reach to certain density, the lux promoter will be activated by quorum sensing. d: By activating of lux promoter, nicotine degrading genes and mocA will be transcribed.
PlanB:Using Pseudomonas putida S16 as a chassis
The second way for converting nicotine into 2.5-dihydroxypyridine is done using P. putida S16 as a chassis. A gram-negative bacterium P. putida S16 has known to degrade nicotine.(Liu et al., 2015) As Tang et al. did in 2012(figure 2) , by knocking out 2,5-dihydroxypyridine dioxygenase, 2,5-dihydroxypyridine can be obtained.(Tang et al., 2012)
More yield could be achieved by quorum sensing as shown in figure 3. Two devices, regulating device and hpo complement device, are introduced to hpo knockout mutant of P. putida S16. The regulating device consists of constitutive promoter, luxI , and luxRi. The hpo complement device consists of tet promoter, tetR, tet promoter, and hpo. First, lux promoter is not activated and tet promoter is activated, meaning transcription of tetRis suppressed and hpo . is promoted. hpo . from plasmid is complementing deleted genome hpo.. By mechanism explained above, if the cells reach to certain density, lux promoter is activated and tetR . is transcribed. TetR is the repressor of tet promoter and inactivates it, which suppress transcription of hpo .. Both plasmid and genome hpo . is suppressed.
fig.2 a: The regulating device consists of constitutive promoter, luxI, and luxR. The hpo complement device consists of lux promoter, tetR, tet promoter, and hpo. b: First, lux promoter is not activated and tet promoter is activated. This means tetR is not transcribed and hpo is transcribed. hpo inserted in plasmid is complementing the deleted hpo of the genome. c: If the cells reach to certain density, the lux promoter will be activated by quorum sensing. d: By activating of lux promoter, TetR will suppress tet promoter and hpo transcription. Hence, both plasmid and genome hpo is suppressed.
Hardware
When we try to make our project real, a bioreactor is practical. We developed our hardware based on best hardware nominee NCKU Tainan 2018 team. We focused on the pH change. Our device will sense the pH change and when the pH reaches to the threshold, pH adjustment solution will be added.By adding this hardware to the NCKU Tainan 2018 hardware, it will be a step closer to the industrial use.
fig.1 Left and Upright: The overview of our device. An Arduino, pH sensor, water cock, and servo motor is used. Downright: Close up view of the device. The servo motor is attached to the water cock attached to the container for pH adjustment solution. The pH sensor is attached to the container for liquid medium.
Circuit diagram is shown on figure 2. Since our servo motor did not work using 5V from Arduino, we used an adaptor that has more current. By using ceramic capacitor, electrolytic capacitor, and three-terminal regulator, electric potential of the adaptor is down to 5V. By mixing the 5V from Arduino and 5V from the adaptor with 2-bit voltage translator, servo motor can receive the signal from Arduino. The servo motor turns with the change in pH measured by the pH sensor. Not only that, but also signal from switch can turn the servo motor. Arduino code and materials we used is shown on figure 3.s
fig.2 Circuit diagram of the device. The signal from switch or pH sensor can turn the servo motor.
Human and Practice
1, Communication with the local government
We assumed that the source of the nicotine being released into the environment would be leftover buds and remnants from tobacco farmers, pollutants and waste from tobacco production factories, cigarette butts, and water in smoking areas. So, we first contacted Kagoshima Prefecture in Japan, a municipality with a large number of tobacco leaf-growing farmers. We recognized that the tobacco industry is one of the most important industries facing the municipality and that it was necessary to reduce the negative environmental impact of nicotine without making the tobacco industry obsolete.
fig.1Kagoshima Prefecture in Japan
2, Contact with farmers
A person from Kagoshima Prefecture introduced us to someone who manages a tobacco farm and we got in touch with him. We asked him if it was possible to get the tobacco farmers to donate their surpluses, and he replied that it was possible. So, we came up with a project to take tobacco waste from tobacco farmers and use it to fertilize them with the help of synthetic biology and return it to the tobacco farmers.
3, Liaison with tobacco production companies
We then spoke with Mr. Mochizuki of JAPAN TOBACCO INC. which is the only company in Japan licensed to produce and sell tobacco. He was very interested in our project. At the meeting, we were told that Japanese tobacco factories do not produce nicotine-contaminated waste, that the arbors and remnants of tobacco discarded by farmers do not contain much nicotine, and that the collection of arbors and remnants is a burden to the farmer and is expensive to transport and store. . Mr. Mochizuki advised us to use cigarette butts and we decided to look at them as the actual nicotine released into the environment. It was a very meaningful meeting that changed the direction of our project.
4, Contact with four volunteer organizations that picks up cigarette butts
To find out more about the current state of littering, we contacted four volunteer organizations that picks up cigarette butts. We found out that some volunteer teams collected 500 cigarette butts in an hour, and that many of them would be able to donate cigarette butts if they had a system to collect them. This suggests that if we set up a system to collect cigarette butts, we can expect to collect a lot of cigarette butts.
Thus, we contacted organizations at all stages of tobacco involvement for advice and to identify and enhance the need and feasibility of the project.
Collaboration
・hosted iGEM Japan Online Meetup on 3/30
Japan meetup was held on March 30th. We had ZOOM discussion with participants from Gifu University, Waseda University, Kyoto Sangyo University, University of Tokyo, Tokyo Institute of Technology, Kyushu University, Tokyo Metropolitan University and other universities. Ambassadors Ryo Niwa, Dorothy Chan and Kaavya Ashok were also invited to the event. We discussed the team's plans for this year, feedback on the project, how it should be run, the impact of COVID-19, and future activities. This meetup has helped me stay positive and engaged in the project. I also thought that many of the teams will be holding online meetups for the first time this year, so I thought I would create a report on the experience and post it on my website to help other teams.
fig.2 iGEM Japan Meetup on 3/30
・participated iGEM Japan Summer Meetup on 8/28
・joined video collaboration by iGEM TU Delft
Results
Results
Due to the pandemic of SARS-CoV-2, we could not get into the lab. However we have materials needed for experiments and are ready for it. For the hardware, we were able to solve the problem of the hardware of NCKU 2018 team.
Qayyum, I., Fazal-ur-Rehman, M., & Ibrahim, M. S. (2018). Extraction of Nicotine (3-(1-methyl-2-pyrrolidinyl) pyridine) from Tobacco Leaves Separated from Gold Live Classic BrandTM Cigarettes by Solvent Extraction Approach and Characterization via IR Analysis. Biosciences Biotechnology Research Asia, 15(4), 799–804. https://doi.org/10.13005/bbra/2688
Proposed Implementation
♦Put the created bacteria in the ashtray in the smoking area.
We put bacteria which we created in the ashtray. The bacteria converted nicotine contained in cigarette butts into 2,5-dihydroxypyridine. The big merit of this system is that 2,5-dihydroxypyridine is generated constantly. However, there are restrictions on taking the GMO out of the system.
fig.1Put the created bacteria in the ashtray in the smoking area. ♦Turning cigarette butts into recyclable waste
Currently, cigarette butts are incinerated. However, cigarette butts have the potential to produce useful substances. If this system is realized, it will change people's attitudes. They will start to think of cigarette butts as recyclable waste.
♦To prevent people from littering.
Littering of cigarette butts is bad for the environment. We would like to think about how to prevent the littering of cigarettes. We will make posters to inform people about the usefulness of cigarette butts and display them. We believe that if people know the usefulness of cigarette butts, they will stop littering. In addition, we would like to work with volunteer organizations to collect cigarette butts that have been thrown away and utilize them. In fact, we have talked to several volunteer organizations and they have responded positively to the idea of donating the collected cigarette butts. I think the more people know about it, the more active we will be.
Science Communication
Science Communication on Youtube
As part of our science communication activities, we have created and uploaded some videos.
One of this is explain our project. We tried to give an overview of the project using the analogy of an urban mine, with as few technical terms as possible.
We also made videos about “Fundamentals of Genetic Recombination Technology and Its Use, Enlightening Synthetic Biology”. Specifically, we took up the following contents.
・the difference between genes and DNA
・genetic recombination technology and what we are actually doing with it in simple
fig.1 Youtube Channel for Science Cmmunication
RIDAISAI (held in Tokyo University of Science)
We held a workshop to make DNA-shaped charms and sold butterfly pea tea, which changes color by pH
Press Release Reading Club
At this club, by sharing and discussing the various press releases of research related to the interests of participants, we managed to cultivate a better understanding of each article and broaden our horizon. Some of the participants did not usually have access to information about research, they gained a certain familiarity with science in the form of knowing the actual research results through this meeting.
Genetic Modification Study Session
At the session, we learned about the domestic Law that establishes genetic modification and biodiversity and how genetic modification are used. We also researched and discussed that genetic modification is how to be regulated and how people feel this technology in each country.
fig.2 Genetic Modification Study session announcement poster
Attending the club thinking of SC
We gave a presentation on a workshop on genetic modification for elementary school students. We also discussed with various science communication circles on how to attract and advertise online and how to practice science communication. We thought attending an hour-long meeting is a hurdle but watching a three-minute video is easy. So, we decided to work on making videos.
fig.3 Group photo of the club thinking of SC
science
What did your team achieve? What do you plan to work on moving forward?
References and Acknowledgements
PI
Toshiki Furuya (Junior Associate Professor of Tokyo University of Science)
Project adviser
Kazuyuki Kuchitsu (Professor of Tokyo University of Science), Kouji Kuramochi (Professor of Tokyo University of Science), Takeshi Wada (Professor of Tokyo University of Science), Kengo Morohashi (Visiting Professor of Michigan State University), Masahiro Nakajima (Associate Professor of Tokyo University of Science), So Maezawa (Associate Professor of Tokyo University of Science), Yoshikazu Nakamura (Associate Professor of Tokyo University of Science)
Project Supporter
Daichi Mochizuki and other members of Japan Tobacco Inc., members of Promega Corporation of Japan, members of Leave a Nest Co., Ltd., members who belong to our past team
Reference
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Iobbi-Nivol, C., & Leimkühler, S. (2013). Molybdenum enzymes, their maturation and molybdenum cofactor biosynthesis in Escherichia coli. In Biochimica et Biophysica Acta - Bioenergetics (Vol. 1827, Issues 8–9, pp. 1086–1101). Elsevier B.V. https://doi.org/10.1016/j.bbabio.2012.11.007
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Qayyum, I., Fazal-ur-Rehman, M., & Ibrahim, M. S. (2018). Extraction of Nicotine (3-(1-methyl-2-pyrrolidinyl) pyridine) from Tobacco Leaves Separated from Gold Live Classic BrandTM Cigarettes by Solvent Extraction Approach and Characterization via IR Analysis. Biosciences Biotechnology Research Asia, 15(4), 799–804. https://doi.org/10.13005/bbra/2688
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