Team:HZAU-China/Poster

Poster: HZAU-China

Tooth Fairy

An automatic and personalized enamel repair scheme.

Presented by HZAU-China 2020.

Poster design: Lixiang Xu¹.
Content organization: Shengkun Tong¹, Ye Lu¹, Yv Han¹, Zichun Sun¹.
Advice: Binguang Ma², Ruonan Tian³

¹iGEM Student Team Menber, ²iGEM Team PI, ³iGEM Team Adviser. All authors are affiliated with Huazhong Agricultural University.

Abstract

Enamel is the hardest part in human body, which consist of ordered and tight hydroxyapatite. Nevertheless, it suffers from damages in our daily life due to many reasons such as physical knock, acid erosion and bacterial infection. In the early stage of enamel damage, it is usually difficult to notice changes on the teeth. However, in the later stage, when a tooth is decayed or ruptured, even doctors are unable to completely repair the damaged tooth. Our Tooth Fairy project could be an innovative solution to this problem. A Tooth Fairy contains several genetic circuits that enable her to provide automatic and personalized repair for the damaged tooth enamel. In addition, users can also know their oral status owing to the report ability of a Tooth Fairy.

Introduction

The goal of our project this year is to repair enamel damage in the early stage. Tooth Fairy was produced to achieve that. It can provide automatic and personalized repair for the damaged tooth enamel.

Like a real guardian fairy, Tooth Fairy will focus on the health of our teeth in real time and make corresponding responses through "Detection & Report" module. In addition, she can switch between two modules, "Sterilization" module which treats S. mutans infection, "Repair" module which repairs enamel. Considering the safety problem, we also add a "Suicide" module to Tooth Fairy.

Four matching products will be produced to put our Tooth Fairy into practical use. It includes: a "Teeth braces" to accommodate the Tooth Fairy, M9 agaroses medium and freeze-drying bacterial powder to easily use, a "Box" to active fluorescent protein, as well as an "App" to determine and report teeth bacterial infection situation based on color. These four products will encompass a complete set of usage processes.

Inspiration

Enamel is the hardest tissue in the human body.

However due to its growth characteristics, it will not be able to repair or regenerate itself when it is fully developed.

Physical wear, acid erosion, and bacterial infection in daily life have caused varying degrees of damage to the enamel of the public, which may increase the prevalence of some oral diseases.

In order to repair enamel and improve the public's oral health, we have introduced LARP as the reticular skeleton for repair of enamel.

Through a series of designs and experiments, after ten months, with the attention and participation of the public as well as the guidance and help of two professors in related fields, Tooth Fairy was finally presented to the world.

Design

The genetic circuit of our engineered bacteria can be divided into three main modules, "Detection & Report" module, "Sterilization" module and "Repair" module (Figure 1).

Figure 1. The outline of the genetic circuit.

"Detection & Report" module
The "Detection & Report" module can detect the amount of S. mutans by sensing a signal molecule, Competence-Stimulating Peptide (CSP), as well as lead to the production of fluorescent proteins with different colors to report conditions of users' teeth.

"Repair" module
The "Repair" module can repair the enamel by generating enamel repair protein LRAP. LRAP can self-assemble and serve as a framework for hydroxyapatite crystals, allowing the damaged site to reform enamel similar to its natural structure

"Sterilization" module
The "Sterilization" module can kill S. mutans by secreting ClyR. ClyR is a chimeric lysin with extended streptococcal host range and has been demonstrated high lytic activity against Streptococcal mutans.

These three modules were coupled by a signal process module, and the switch of "Sterilization" module and "Repair" module will be treated as an Exclusive-OR gate. Depending on the concentrations of S. mutans, the engineered bacteria will have three different reaction modes (Figure 2).

Figure 2. Three different respond patterns of the engineered bacteria. Safe: no detectable S. mutans. Solvable: low S. mutans concentrations. Dangerous: high S. mutans concentrations

Through regulations like that, we can achieve automatic and personalized enamel repair scheme.

"Suicide" module

To improve the safety of our project, we designed a "Suicide" module. This part was consisted of Fe²⁺ sensitive promoter P_fhuA1 and toxin MazF. The braces will be supplemented with Fe²⁺, once the engineered bacteria leakage to the environment, the "Suicide" module will open and kill the engineered bacteria (Figure 3).

Figure 3. The genetic circuit of the "Suicide" module.

Detection & Report

The "Detection & Report" module has two main functions:
1. Detect the concentration of S. mutans and transfer secondly signal to downstream module.
2. Feedback the detection result to the user by generating different fluorescent colors.

To achieve that, two promoters from S. mutans with different affinity to phosphorylated ComE were used. We have verified the function of the two promoters in E. coli (Figure 1). It means that the first function mention above was achieved.

Figure 3. The express conditions of mCherry after added different concentrations of sCSP. MCherry was controlled by P_nlmC.

In our project, we also hope that "Detection & Report" modules will show different fluorescence at different concentrations of S. mutans. To figure the feasibility of the design, we made an exploratory prediction by assuming different CSP concentrations correspond to different steady-state concentrations of phosphorylated ComE. The model indicates that the second function mention above is feasible. (Figure 2).

Figure 2. A schematic that proves our "solvable" pattern and "dangerous" pattern are distinguishable.

Exclusive-OR gate

The "Sterilization" module and "Repair" module are coupled by a signal process circuit, which make only one of the two modules open at a time.

It is a complex process, so we predicted its possible results through a mathematical model. The results show that, while the "Sterilization" module starts, the LRAP concentration is 3017 nM, which means the "Repair" module can’t be totally inhibited. So we plan to attach a LAA degradation tag to the LRAP protein. After adding a LAA degradation tag, LRAP concentration is decreased to about 300 nM, which means the repair circuit could be shut down. Thus, we considered whether to add a LAA tag in the actual experiment.

Figure 1. A-B. Changes in LRAP protein concentration over time in the case of Exclusive-OR gate. C-D. Changes in LRAP-LAA protein concentration over time in the case of Exclusive-OR gate. E.Changes in ClyR protein concentration over time in the case of Exclusive-OR gate.

To verify the prediction of mathematical model, we constructed a simplified circuit to replace LRAP with mCherry. As shown in Figure 2, after inducing the expression of TetR with IPTG, the expression of mCherry will be repressed. However, mCherry still has a background expression.

Figure 2. Fluorescent change of E. coli treated with different IPTG concentrations.

These results accord with the model's prediction, thus we decided to add a LAA degradation tag to the LRAP in the future work.

Sterilization and Repair

The "Sterilization" module and "Repair" module are two main output function modules.

"Sterilization" module
We have obtained ClyR protein from E. coli expression system and verified its lysis activity and secretion successfully (Figure 1).

Figure 1. Dose-dependent and time-dependent lytic activity of ClyR.

However, ClyR also has killing activity against some other streptococcus strains, thus we hope to adjust the expression of ClyR to achieve the best sterilization effect with lower ClyR concentration. A mathematical model based the data of experiments was built to help us achieve that (Figure 2).

According to the results of model, we indicated that 20 μg/mL is the best sterilization concentration, and chose RBS B0030 to achieve the target concentration.

"Repair" module

The repair function was implemented by LRAP. After adding the supernatant of lysed E. coli strain containing LRAP to the artificial saliva and pig tooth, we can demonstrate the nucleation and binding ability of LRAP.

Figure 3. Demostration of ClyR and LRAP. A. Effects of different supernatants on turbidity of artificial saliva. B. "Toothtern-blot" analysis of different supernatants. (a), (b) The same result in different exposures. (c) Picture of the teeth in reality.

Suicide

The "Suicide" module can restrict engineered bacteria to the braces, thus improve the safety of the project. We will add Fe²⁺ to the braces to inhibit the "Suicide" module. Once the engineered bacteria leakage to environment, "Suicide" module will open and kill the engineered bacteria. This process was demonstrated by experiments (Figure 1-2).

Figure 1. Bacterial growth of E. coli strain. A. Growth conditions of MazF- strain in different Fe²⁺ concentrations; B. Growth conditions of MazF+ strain in different Fe²⁺ concentrations.

Figure 2. Zone of growth test. The filter papers were soaked with 10 μL FeSO₄ (40 mM), and the same filter papers soaked with 10 μL double distilled water as control.

Methodology

This year, our team has used some novel measuring methods, and their protocols are shown below.

"Toothtern-blot"
To test the binding ability of LRAP to the tooth, we invented"Toothtern-blot".
1. The E. coli BL21(DE3) strain containing LRAP transformation is experimental group, the same strain without induction or without plasmid transformation as control.
2. Overnight cultured E. coli strains were lysed by ultrasonication, then centrifuge at 10000 rpm, 4℃. Take the supernatant for characterization.
3. Broke the teeth of domestic pigs into fragments with a side length of about 5 mm, and the fragments with more exposed sections are selected.
4. Take three pieces of pig teeth, and wash with PBS buffer. Place them in three 2 mL centrifuge tubes respectively, and then the three supernatants with uniform LRAP concentration are added respectively, incubated at 37℃ for 20 h. Centrifuge the tubes upside down for several times every 5 h.
5. Wash the incubated pig tooth fragments with PBS buffer solution by pipet-gun for 2-3 mins to remove the protein that is not bound to the teeth.
6. The rinsed pig teeth was placed in plastic petri dishes, and incubated with 50 mL 5 % skimmed milk 1:10000 diluted His-Tag Rabbit pAb (YEASEN Biotech,Shanghai, China) at room temperature for 2 h. Then wash with TBST buffer 3 times for 10 mins each.
7. Then the peroxidase-conjugated Goat anti-rabbit IgG (H+L) (YEASEN Biotech,Shanghai, China) of the same volume and dilution was added and incubated at room temperature for 2 h. Then wash the teeth with TBST buffer solution for 3 times, 10 mins each.
6. Use A Super ECL Detection Reagent ECL (YEASEN Biotech,Shanghai, China) to observed the results.

Zone of growth test
To verify the function of "Suicide" module, we did a growth zone test.
1. Take 50 μL seed culture of E. coli strain containing "Suicide" module, and add it into 5 ml LB medium containing 60 μg/mL ampicillin. Incubate for 8-9 hours at 37 ℃, 200 rpm.
2. Dilute the incubated bacterial fluid to 10-3 times respectively.
3. Take 10 μL bacterial fluid from the diluted group, spread it onto M9 medium containing 60 μg/mL ampicillin respectively.
4. Two filter papers soaked with 10 μL of FeSO₄ (40 mM), and the same filter papers soaked with 10 μL of double distilled are put onto each plate respectively. Blank controls are fresh M9 medium containing ampicillin (Amp, 60 μg/mL) with the engineered bacteria. Incubate them overnight at 37℃.

Hardware & Software

In order to put our Tooth Fairy into practical use, we have produced four matching products. It includes: a "Teeth braces" to accommodate the Tooth Fairy, Medium and bacterial powder to easily use, a "Box" to active fluorescent protein, as well as an "App" to determine and report teeth bacterial infection situation. These four products will encompass a complete set of usage processes.

Teeth braces
Our "Teeth braces" can completely wrap the user's teeth, and a cavity is designed on the occlusal surface of the braces for adding engineered bacteria (Figure 1).

Figure 1. Teeth braces in early generation.

Medium & bacterial powder
In future use, the products will also include M9 agaroses medium and freeze-drying bacterial powder (Figure 2). When using the braces, users should add them into braces.

Figure 2. The M9 agaroses medium and freeze-drying bacterial powder.

Box
In order to activate the fluorescence of fluorescent proteins, we designed the hardware "Box", which has a built-in excitation light for activating fluorescent proteins(Figure 3).

Figure 3. "Box" in early generation.

App
After activating the fluorescent proteins and taking a photo, users only need to import their photos into the "App", the "App" can automatically identify the fluorescent color, and report the condition of bacterial infection to users (Figure 4).

Figure 4. A. The user interface. B. The recognition processes and the report.

Human practices

Impact on us
Interviews with experts in the field of oral microbiology and oral clinics

After interviewing two experts：
1. Tooth Fairy's design feasibility and clinical value were recognized.
2. "Sterilization" module was added to Tooth Fairy.
3. The chassis of Tooth Fairy was changed to use the harmless E. coli Nissle 1917.
4. TPU was selected as the material of Tooth Fairy's the"Teeth braces".
5. Guide the setting of the switching threshold of Tooth Fairy's three working modules.

After the public survey：
1.We found that the public are unfamiliar with oral health knowledge.
2.We confirmed the public acceptance and requirements of Tooth Fairy.
3.We introduced a kill switch to Tooth Fairy to improve the biosafety of the product and simplified the internal structure of "Teeth braces" to improve its comfort.
4.In subsequent improvements, we would try to introduce a module with the function of producing fragrance into the engineered bacteria.

Impact on society
A series of multidisciplinary tweets featuring synthetic biology

HZAU-iGEM WeChat Official Account.

In the past two years, we have posted 60 tweets. And we have set up a special column for dental health this year.

Science Video series "Love Tooth Encyclopedia".

We also disseminate dental health knowledge through popular science videos

The Teeth Defense War

The Teeth Defense War.

Spread relevant knowledge through the way of entertaining and learning by card game.

Synthetic Biology and iGEM Sharing Session.

This year, we conducted several synthetic biology and iGEM sharing session with the form including interaction and experimental experience. In the end, we hope that readers can understand the synthetic biology and new technology from our previous tweets.

References & Acknowledgements

References

[1] Lemos J A, Palmer S R, Zeng L, et al. The biology of Streptococcus mutans[J]. Gram‐Positive Pathogens, 2019: 435-448.
[2] Liu T, Xue S, Cai W, et al. ComCED signal loop precisely regulates nlmC expression in Streptococcus mutans[J]. Annals of microbiology, 2014, 64(1): 31-38.
[3] van der Ploeg J R. Regulation of bacteriocin production in Streptococcus mutans by the quorum-sensing system required for development of genetic competence[J]. Journal of bacteriology, 2005, 187(12): 3980-3989.
[4] Xu J, Yang H, Bi Y, et al. Activity of the chimeric lysin ClyR against common Gram-positive oral microbes and its anticaries efficacy in rat models[J]. Viruses, 2018, 10(7): 380.
[5] Le Norcy E, Kwak S Y, Wiedemann-Bidlack F B, et al. Leucine-rich amelogenin peptides regulate mineralization in vitro[J]. Journal of dental research, 2011, 90(9): 1091-1097.
[6] Zhang Y, Zhang J, Hoeflich K P, et al. MazF cleaves cellular mRNAs specifically at ACA to block protein synthesis in Escherichia coli[J]. Molecular cell, 2003, 12(4): 913-923.
[7] Engelberg-Kulka H, Hazan R, Amitai S. mazEF: a chromosomal toxin-antitoxin module that triggers programmed cell death in bacteria[J]. Journal of cell science, 2005, 118(19): 4327-4332.
[8] Guan L, Liu Q, Li C, et al. Development of a Fur-dependent and tightly regulated expression system in Escherichia colifor toxic protein synthesis[J]. BMC biotechnology, 2013, 13(1): 25.

Acknowledgements

Prof. Ping Yin

Prof. Yingchun Qi

Prof. Jin He

Prof. Donghai Peng

Prof. Wenyuan Han

Prof. Binguang Ma

Sponsors