Team:Mingdao/Poster
Poster: Mingdao
Introduction
TOOTH DECAYS are quite common among children in Taiwan. We genetically modified a probiotic strain of E. coli Nissle (GM EcN) that can eliminate Streptococcus mutans, the main cause of dental caries.
GM EcN were equipped with pyruvate oxidase (SpxB) gene to generate H2O2, aquaporin (AQP) gene to enhance H2O2 permeation, and catalase (KatG) gene to survive from oxidative stress by decomposing H2O2 into oxygen and water.
CLEANDY® is a lollipop-shaped candy we made with features to clean teeth. The Cleandy® is composed of sugar alcohols and GM EcN, which was demonstrated in H2O2 production assay, growth inhibition and antagonistic competition test against the growth of S. mutans.
GM EcN were equipped with pyruvate oxidase (SpxB) gene to generate H2O2, aquaporin (AQP) gene to enhance H2O2 permeation, and catalase (KatG) gene to survive from oxidative stress by decomposing H2O2 into oxygen and water.
CLEANDY® is a lollipop-shaped candy we made with features to clean teeth. The Cleandy® is composed of sugar alcohols and GM EcN, which was demonstrated in H2O2 production assay, growth inhibition and antagonistic competition test against the growth of S. mutans.
CLEANDY® – A candy that cleans your teeth
Presented by Team Mingdao 2020
Team leader: Jui-Hung Yeh & Yu-Lin Wu
Experimental design: Chu-Hsuan Shen, Sheng-Fong Li & Chiang-Yu Chen
Wet lab: Bo-Che Wu, Yu-Ze Huang, Kai-Yuan Tu, Cheng-Wei Lin
Model: Yi-Kai Liao, Chih-Fang Chen
Hardware: Yi-Syun Liao, Shao-Yuan Tien, Tai-Jan Tsao & Shih-Shiuan Peng
Human practices: Ya-Zhu Jhan, Yi-Hsuan Yu, Chun-Jui Tien, Pin-Hsun Lin & Zi-Yi Kung
GMO debate: Shu-Han Liu
Product design: Zi-Xuan Chen & Wan-Hua Chang
Enterprise: Kuan-Yu Huang & Jie-Ni Liu
Board game: Kuang-Yu Lo
Sustainable development goals: Ting-Yun Liao
Art: Pei-Tzu Chen & Hsin-Hsin Wu
Wiki design: Yung-Hsiang Yang
(Listed above as main attributes)
Team leader: Jui-Hung Yeh & Yu-Lin Wu
Experimental design: Chu-Hsuan Shen, Sheng-Fong Li & Chiang-Yu Chen
Wet lab: Bo-Che Wu, Yu-Ze Huang, Kai-Yuan Tu, Cheng-Wei Lin
Model: Yi-Kai Liao, Chih-Fang Chen
Hardware: Yi-Syun Liao, Shao-Yuan Tien, Tai-Jan Tsao & Shih-Shiuan Peng
Human practices: Ya-Zhu Jhan, Yi-Hsuan Yu, Chun-Jui Tien, Pin-Hsun Lin & Zi-Yi Kung
GMO debate: Shu-Han Liu
Product design: Zi-Xuan Chen & Wan-Hua Chang
Enterprise: Kuan-Yu Huang & Jie-Ni Liu
Board game: Kuang-Yu Lo
Sustainable development goals: Ting-Yun Liao
Art: Pei-Tzu Chen & Hsin-Hsin Wu
Wiki design: Yung-Hsiang Yang
(Listed above as main attributes)
Research
Antagonistic relationship between S. mutans and S. sanguinis
Microbiome imbalance in the oral environment results in tooth decays. S. mutans causes dental cavities by producing lactic acid. S. sanguinis contributes to plaque alkalinization and prevent the cariogenic microbiota. The PCR results showed that all saliva of 7 volunteers of our team members were tested positive for S. sanguinis but at different levels of S. mutans.
The growth of S. mutans was inhibited by H2O2.
Researches indicated that H2O2 production by S. sanguinis is a key effector to inhibit the growth rate of S. mutans. Our experiment confirmed the results showing that significant growth inhibition of S. mutans by H2O2 in a dose-dependent manner.
Microbiome imbalance in the oral environment results in tooth decays. S. mutans causes dental cavities by producing lactic acid. S. sanguinis contributes to plaque alkalinization and prevent the cariogenic microbiota. The PCR results showed that all saliva of 7 volunteers of our team members were tested positive for S. sanguinis but at different levels of S. mutans.
The growth of S. mutans was inhibited by H2O2.
Researches indicated that H2O2 production by S. sanguinis is a key effector to inhibit the growth rate of S. mutans. Our experiment confirmed the results showing that significant growth inhibition of S. mutans by H2O2 in a dose-dependent manner.
Research
Antagonistic relationship between S. mutans and S. sanguinis
Microbiome imbalance in the oral environment results in tooth decays. S. mutans causes dental cavities by producing lactic acid. S. sanguinis contributes to plaque alkalinization and prevent the cariogenic microbiota. The PCR results showed that all saliva of 7 volunteers of our team members were tested positive for S. sanguinis but at different levels of S. mutans.
The growth of S. mutans was inhibited by H2O2.
Researches indicated that H2O2 production by S. sanguinis is a key effector to inhibit the growth rate of S. mutans. Our experiment confirmed the results showing that significant growth inhibition of S. mutans by H2O2 in a dose-dependent manner.
Microbiome imbalance in the oral environment results in tooth decays. S. mutans causes dental cavities by producing lactic acid. S. sanguinis contributes to plaque alkalinization and prevent the cariogenic microbiota. The PCR results showed that all saliva of 7 volunteers of our team members were tested positive for S. sanguinis but at different levels of S. mutans.
The growth of S. mutans was inhibited by H2O2.
Researches indicated that H2O2 production by S. sanguinis is a key effector to inhibit the growth rate of S. mutans. Our experiment confirmed the results showing that significant growth inhibition of S. mutans by H2O2 in a dose-dependent manner.
Promoter I
Constitutive promoter
Lactate dehydrogenase promoter (ldhp) is a strong and constitutive promoter in S. mutans used and well-documented in many studies. Lactate dehydrogenase catalyzes the fermentation of pyruvate to lactic acid. The promoter plays a pivotal role in the regulation of glucose metabolism.
GM EcN carrying ldhp-GFP-Tr/pSB1C3 was cultured overnight and the GFP was measured at Ex/Em = 483/513. The data indicated ldhp has high level of gene expression, compared the limited activity of tpxp.
Toxin
H2O2 production
Pyruvate oxidase (SpxB) of S. sanguinis catalyzes the reaction between pyruvate and oxygen to generate acetyl phosphate and H2O2. It plays a role in an antagonistic relationship against S. mutans.
GM EcN carrying ldhp-AQP-RBS-SpxB-Tr/pSB1C3 was cultured in LB media with 5% or 10% glucose. The supernatants of overnight culture were measured 2.15 mM and 1.84 mM of H2O2 production, respectively.
Anti-toxin
H2O2 decompositionKatG is bifunctional enzyme with catalase and peroxidase activity. It catalyzed the H2O2 reduction to H2O and O2, and plays a role in E. coli to prevent DNA damage caused by an oxidative stress.
The growth of GM EcN carrying ldhp-AQP-RBS-SpxB/pSB1C3 was severely retarded due to H2O2 production. The growth rate of GM EcN carrying ldhp-AQP-RBS-SpxB-tpxp-KatG-Tr/pSB1C3 was recovered to the levels as control groups, indicated that KatG is capable of decomposing H2O2 and releasing cells from the stress.
Design
In order to equip probiotics to attack S. mutans, we genetically engineered E. coli Nissle (EcN) with toxin, anti-toxin and transporter genes to produce H2O2.
Toxin gene
Pyruvate oxidase (SpxB) gene was used by S. sanguinis to generate H2O2 to antagonize S. mutans.
Anti-toxin gene
Catalase (KatG) gene was expressed by E. coli to decompose H2O2 to oxygen and water.
Transporter
Aquaporin (AQP) gene was utilized by S. cristatus to facilitate H2O2 transport across cell membrane.
Constitutive promoter
Lactate dehydrogenase promoter (ldhp) is a constitutive and strong promoter in S. mutans. We used ldhp to drive toxin (SpxB) and transporter (AQP) genes.
Regulated promoter
Thiol peroxidase promoter (tpxp) is a stress-related promoter in S. mutans, which is induced by the presence of H2O2. We used tpxp to drive anti-toxin gene (KatG).
Regulated promoter
Thiol peroxidase promoter (tpxp) is a stress-related promoter in S. mutans, which is induced by the presence of H2O2. We used tpxp to drive anti-toxin gene (KatG).
Reference
1. B L Triggs-Raine, B W Doble, M R Mulvey, P A Sorby, and P C Loewen. J Bacteriol. 1988; 170(9): 4415–4419.
2. Lan-yan Zheng, Andreas Itzek, Zhi-yun Chen, and Jens Kreth. Int J Oral Sci. 2011; 3(2): 82–89.
3. Jessica K Kajfasz, Tridib Ganguly, Emily L Hardin, Jacqueline Abranches, José A Lemos. Sci Rep 2017; 7(1):16018.
4. Huichun Tong, Xinhui Wang, Yuzhu Dong, Qingqing Hu, Ziyi Zhao, Yun Zhu, Linxuan Dong, Fan Bai, and Xiuzhu Dong. J Biol Chem. 2019; 294(12): 4583–4595.
Promoter II
Inducible promoter
Thiol peroxidase promoter (tpxp) is a H2O2-responsive promoter in S. mutans. Thiol peroxidase catalyzes the reduction of hydrogen peroxide to water. The promoter activity is highly correlated to the concentration of H2O2 under oxidative stress environment.
GM EcN carrying ldhp-AQP-Tr-tpxp-GFP-Tr/pSB1C3 was cultured in the various concentrations of H2O2. Compared to basal level of tpxp activity, GFP intensity was enhanced with increasing concentrations of H2O2 in a dose-dependent manner.
Transporter
H2O2 TransporterH2O2 permeability across the cell membrane is limited. The aquaporin (AQP) from Streptococcus cristatus facilitates bidirectional transmembrane H2O2 transport.
GM EcN carrying ldhp-AQP-Tr-tpxp-GFP-Tr/pSB1C3 was cultured without or with 3 mM of H2O2. Compared to tpxp-GFP, AQP facilitated H2O2 transportation which resulted in significant enhanced GFP intensity.
Device
The best composite part – H2O2 production machine
The protein expressions of GM EcN carrying ldhp-AQP-RBS-SpxB-Tr-tpxp-KatG-Tr/pSB1C3 were analyzed in SDS-PAGE by Coomassie blue staining. The device is able to produce H2O2 in the presence of various concentrations of glucose.
Antagonism test
There’s no antagonistic effect between S. mutans and WT EcN control. The GM EcN gained a strong ability to inhibit the growth of S. mutans as shown in the cell shape and size on the agar plates, and even better than S. sanguinis. The results significantly indicated that the H2O2 produced by the device in GM EcN may be capable of efficiently eliminating S. mutans.
Model
Objective 1: What are the effects on dental caries when consuming different types of sugar?
Objective 2: How long does it take for dental caries to develop after eating candy?
Acid generation capability model
Based on our NMR data for the metabolites of S. mutans in 1% of glucose, we applied the equation of theoretical chemical shift of acetate to build a model for acid production in S. mutans. Then, pH values of carbon sources (i.e., sucrose, erythritol and xylitol) consumed by S. mutans could be derived directly from the model.
Dental caries model
Stephan curve is a description of the change in pH value caused by acid-producing oral bacteria in the response to sugars, showing a critical value at pH=5.5 for tooth decay formation. Based on the equation and acid production capability of various carbon sources, we developed a time-course model to depict the dental caries formation.
Reference
1. Stephan RM, Miller BF. J Dent Res. 1943;22;45-51.
2. Ye L, De Iorio M, Ebbels TMD. Metabolomics. 2018;14(5):56.
Objective 2: How long does it take for dental caries to develop after eating candy?
Acid generation capability model
Based on our NMR data for the metabolites of S. mutans in 1% of glucose, we applied the equation of theoretical chemical shift of acetate to build a model for acid production in S. mutans. Then, pH values of carbon sources (i.e., sucrose, erythritol and xylitol) consumed by S. mutans could be derived directly from the model.
Dental caries model
Stephan curve is a description of the change in pH value caused by acid-producing oral bacteria in the response to sugars, showing a critical value at pH=5.5 for tooth decay formation. Based on the equation and acid production capability of various carbon sources, we developed a time-course model to depict the dental caries formation.
Reference
1. Stephan RM, Miller BF. J Dent Res. 1943;22;45-51.
2. Ye L, De Iorio M, Ebbels TMD. Metabolomics. 2018;14(5):56.
Model
Objective 1: What are the effects on dental caries when consuming different types of sugar?
Objective 2: How long does it take for dental caries to develop after eating candy?
Acid generation capability model
Based on our NMR data for the metabolites of S. mutans in 1% of glucose, we applied the equation of theoretical chemical shift of acetate to build a model for acid production in S. mutans. Then, pH values of carbon sources (i.e., sucrose, erythritol and xylitol) consumed by S. mutans could be derived directly from the model.
Dental caries model
Stephan curve is a description of the change in pH value caused by acid-producing oral bacteria in the response to sugars, showing a critical value at pH=5.5 for tooth decay formation. Based on the equation and acid production capability of various carbon sources, we developed a time-course model to depict the dental caries formation.
Reference
1. Stephan RM, Miller BF. J Dent Res. 1943;22;45-51.
2. Ye L, De Iorio M, Ebbels TMD. Metabolomics. 2018;14(5):56.
Objective 2: How long does it take for dental caries to develop after eating candy?
Acid generation capability model
Based on our NMR data for the metabolites of S. mutans in 1% of glucose, we applied the equation of theoretical chemical shift of acetate to build a model for acid production in S. mutans. Then, pH values of carbon sources (i.e., sucrose, erythritol and xylitol) consumed by S. mutans could be derived directly from the model.
Dental caries model
Stephan curve is a description of the change in pH value caused by acid-producing oral bacteria in the response to sugars, showing a critical value at pH=5.5 for tooth decay formation. Based on the equation and acid production capability of various carbon sources, we developed a time-course model to depict the dental caries formation.
Reference
1. Stephan RM, Miller BF. J Dent Res. 1943;22;45-51.
2. Ye L, De Iorio M, Ebbels TMD. Metabolomics. 2018;14(5):56.
Prototype
Proof-of-concept
Cleandy is a homemade probiotic candy with H2O2-producing GM EcN. We made lozenges to verify the protein expression and H2O2 production. As shown in the figures, GFP was glowing sharply and 0.58 mM of H2O2 could be measured from our AQP-SpxB-KatG device.
Safety
Food stays in the stomach at pH between 1.5 – 3.5 for 4 – 6 hrs. We incubated 104 cells of GM EcN in the LB media at a pH of 2 or 7 to test the survival and growth rates. The results showed that zero CFU detected after 5 min post-treatment and ~70% growth inhibition of the overnight culture.
Cleandy is a homemade probiotic candy with H2O2-producing GM EcN. We made lozenges to verify the protein expression and H2O2 production. As shown in the figures, GFP was glowing sharply and 0.58 mM of H2O2 could be measured from our AQP-SpxB-KatG device.
Safety
Food stays in the stomach at pH between 1.5 – 3.5 for 4 – 6 hrs. We incubated 104 cells of GM EcN in the LB media at a pH of 2 or 7 to test the survival and growth rates. The results showed that zero CFU detected after 5 min post-treatment and ~70% growth inhibition of the overnight culture.
Enterprise
Customers and market analysis
We made a lollipop-shaped candy and looked for potential customers across ages with surveys and found that most people (77%) were satisfied with Cleandy® and were willing to buy the products. Based on our positioning map analysis, we got conclusion that we could become competitive brand-new product with lower price and similar satisfaction level.
SWOT analysis and the stakeholders
After SWOT analysis, we understood our strength, weakness, opportunities and threats. According to our competitors not all of biotech companies, this become advantage for us in skills and capability to improve our product. And most importantly, we have stakeholders of dental clinics to support us for product development.
Outreach
Experts and biotech companies
Integrated Human Practices is a process of constantly improving our work with the feedback we receive. We visited China Medical University and Chung Shan Medical University to meet professors and college students at the Department of Dentistry. Also, we consulted with experts at SEPOM Technology Ltd. and Genmont Biotechnology Inc. to improve the functional probiotics.
iGEM meetups and collaboration
We met up with NCKU_Tainan, NCTU_Formosa and got the E. coli probiotic strain, Nissle from TAS_Taipei. We further collaborated with CCU_Taiwan and become partnership with CSMU_Taiwan. We also participated in Taiwan iGEM conference to present our project.
Outreach
Experts and biotech companies
Integrated Human Practices is a process of constantly improving our work with the feedback we receive. We visited China Medical University and Chung Shan Medical University to meet professors and college students at the Department of Dentistry. Also, we consulted with experts at SEPOM Technology Ltd. and Genmont Biotechnology Inc. to improve the functional probiotics.
iGEM meetups and collaboration
We met up with NCKU_Tainan, NCTU_Formosa and got the E. coli probiotic strain, Nissle from TAS_Taipei. We further collaborated with CCU_Taiwan and become partnership with CSMU_Taiwan. We also participated in Taiwan iGEM conference to present our project.
Science Communication
Board Game – Teeth Battle®
Teeth Battle® is designed to introduce the ecology of the human oral cavity in an entertaining manner. Scientific terms are simplified without taking away its concept. Our board game provides an educational platform to help young schoolchildren understand oral health and have fun along the way.
Education
We visited with young schoolchildren at Chiao-Jen Elementary School to raise awareness about oral hygiene and also taught them the oral microbiology through our board game. The kids were enthusiastic about learning and proactive in the game.
Campus
STEAM Fair
The STEAM Fair is an annual science exhibition with interactive workshops and lectures. We hosted an iGEM special session to demonstrate the application of synthetic biology and taught participants with lab skills and the concept of the BioBricks.
GM food debate
The debate was conducted in a civil manner with participants of opposite blocs, who should politely respond to questions asked and provide logical arguments in support of their stance. This event allowed us to practice several skills such as critical thinking, effective communication and awareness of global issues.
Intramural project exhibition
We held the annual iGEM exhibition in our campus. Classmates, teachers and parents were invited to join to see what we learned through this iGEM season. We took the opportunity to ask for first impressions, questions, and feedback from the attendees.
SDGs
MDHS SDGs Summit
The SDGs summit held in Mingdao High School (MDHS) was our starting point, leading us to explore more about how we could make connection between synthetic biology and society issues. We presented our project as Goals 3,4,10 &12. Goals 3 and 12 focused on tangible influences, the other two were aimed to reduce the gap of wealth and education.
Interactions between Goals
We conducted a survey with our classmates to discover the relationship between our project and the seventeen SDGs. The chart indicated that besides the four goals we’ve addressed, most remained as neutral, while negative impacts were inspected significantly on Goals 1 & 8.
Conclusion
In the lab, we successfully genetically engineered a probiotic strain E. coli Nissle (GM EcN). The function of the device was confirmed by protein expression and H2O2 production assay. A proof-of-concept prototype was demonstrated by attacking etiological S. mutans in the antagonistic competition test.
For the product, firstly we built a dental caries mathematical model to describe the tooth decay formation caused by various carbohydrates. As a result, CLEANDY® was made of sugar alcohol with GM EcN. The product was going to Enterprise by customer discovery, surveys, market and SWOT analyses. We also have implementation plans and entrepreneurship supported by dental clinics.
Through human practices, we connected all the work to the society. We met up with experts, biotech companies and senior iGEM teams in Taiwan. We communicated with primary school children in science through our educational board game. In our campus, we promoted iGEM by hosting STEAM Fair and intramural exhibition. We reached out to the world by engaging in SDGs summit held in Mingdao High School.
In conclusion, CLEANDY® and Mingdao are promised to promote oral health and wellness for the people.
Acknowledgement
Instructor
Expert and advisor
Sponsor
Mingdao High School
GenScript Biotech
GenMont Biotech Incorporation
Sepoms Technology CO., LTD.
HiNew Dental Clinic / 高新牙醫診所
Guang Qun Dental Clinic / 光群牙醫診所
Shine Dental Clinic / 晶采牙醫診所
Dr. PEI-HONG CHEN
Mrs. CHIA-LE MENG
Expert and advisor
Dr. CHUN-CHENG CHEN
Dr. MING-TA LI
Dr. MING-SHIOU JAN
Dr. Eugene Lin
Dr. MING-TA LI
Dr. MING-SHIOU JAN
Dr. Eugene Lin
Dr. WEN-YING SHEN
Dr. CHIEN-FAN LI
Dr. MING-SHIOU JAN
Dr. Yuqing Li
Dr. CHIEN-FAN LI
Dr. MING-SHIOU JAN
Dr. Yuqing Li
Sponsor
Mingdao High School
GenScript Biotech
GenMont Biotech Incorporation
Sepoms Technology CO., LTD.
HiNew Dental Clinic / 高新牙醫診所
Guang Qun Dental Clinic / 光群牙醫診所
Shine Dental Clinic / 晶采牙醫診所
