iGEM 2020 — Team Mingdao

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# Team:Mingdao/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

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# Team:Mingdao/Engineering

# Mingdao

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# Team:Mingdao/Experiments

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# Mingdao

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## PROJECT

### DESCRIPTION

#### According to statistics, almost 50% of preschool children in Taiwan are now having tooth decays, which is five times more than the normal prevalence rate of dental caries suggested by WHO. In addition, resources in rural areas around the world are scarce and hard to obtain, leading to the problem of dental cavities in remote places. As a result, we hope to maintain the oral health of children and adults around the globe in an easy, feasible and sustainable way.

### RESEARCH

#### Microbiome imbalance in the oral environment results in caries. Dental cavities are mainly caused by the lactic acid produced by acidogenic microorganisms (e.g, Streptococcus mutans). Less aciduric commensal residents (e.g., Streptococcus sanguinis) contribute to plaque alkalinization and prevent the cariogenic microbiota. Researchers proposed the ratio between S. mutans and S. sanguinis is an indicator of the risk for caries.     Our team members were wondering their oral microbiome. They spat some saliva and tested S. mutans and S. sanguinis by PCR with the extracted bacterial gDNA samples. PCR was performed by two pairs of specific primers to S. mutans and S. sanguinis, respectively. The results showed in Fig. 1 that all tested samples were positive for S. sanguinis, while there existed different levels of S. mutans. However, who tested as S. mutans-positive are not willing to tell us whether tooth decay they have and shut their mouth before us.

![](https://static.igem.org/mediawiki/2020/d/d4/T--Mingdao--experiment_fig_1.jpeg)

#### Fig. 1. The test of gDNA of S. mutans and S. sanguinis. Seven team members (A-G) were volunteered to test the existence of oral microbiome. Primers specific for Streptococcus strains were designed according to Seow’s research (Int J Paediatr Dent. 2009 Nov;19(6):406-11.)

### INSPIRATION

#### The current treatment options of cavities are fluoride treatments, fillings, crowns, root canals, and tooth extractions. Fluoride treatments can kill pathogens and are successful cavity prevention agents. However, 80% of fluoride toxicity caused by ingestion of fluoride-containing products is seen in children. This may progress to detrimental effects in severe cases. In addition, fluoride-containing products aren’t natural to our oral environment and are a threat to many oral species. Thus, we hope to use something more natural to eliminate dental cavities.     Probiotics are considered live microorganisms that are similar to beneficial microorganisms which are usually found natural in the human body. Nevertheless, the commercial products said to be effective at preventing oral cavities are mostly constituted with dead microorganisms or not with natural oral microbiota. As a result, we came up with making “real probiotics” with live microorganisms that can prevent tooth decays.     In nature, S. sanguinis is a key factor in maintaining healthy oral microbiome and has an antagonistic ability against etiological S. mutans. Jens Kreth, et al. thoroughly researched on the Streptococcal antagonism in oral biofilms, finding that S. sanguinis inhibited the growth of S. mutans on the aerobic culture agar plate. The inhibition had disappeared by adding catalase, implying the H2O2 production by S. sanguinis plays an essential role.     To determine the H2O2 production by S. sanguinis in our lab, we used the Fluorimetric Hydrogen Peroxide Assay Kit (SIGMA-ALDRICH) to gain infra-red (IR) fluorescence signal which is corresponding to the level of H2O2 concentration by the reaction of horseradish peroxidase. Firstly, we made a calibration curve through 0.01, 0.03, 0.1, 0.3, 1, 3 to 10 mM of H2O2 (Fig. 2) according to the manufacture’s instruction.

![](https://static.igem.org/mediawiki/2020/f/f4/T--Mingdao--experiment_fig_2.jpeg)

#### Fig. 2. The calibration curve of H2O2 concentration at 0, 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 mM

#### The aerobic or anaerobic overnight culture media of S. sanguinis were subjected to measuring H2O2 concentration. As shown in Fig. 3, S. sanguinis produced 2.82 mM aerobically and 0.92 mM anaerobically, giving clues to antagonistic effect against S. mutans.

![](https://static.igem.org/mediawiki/2020/1/14/T--Mingdao--experiment_fig_3.jpeg)

#### Fig. 3. The production of H2O2 by S. sanguinis in an overnight growth medium of brain heart infusion (BHI).

#### To verify the H2O2 effect on S. mutans growth, S. mutans was cultured in a BHI broth with a 10-fold serial dilution of 3M of H2O2. The results in Fig. 4 showed 51% and 57% growth inhibition of S. mutans at the concentrations of 0.3 mM and 3 mM of H2O2 for 24 hr, respectively.

![](https://static.igem.org/mediawiki/2020/1/14/T--Mingdao--experiment_fig_4.jpeg)

#### Fig. 4. The growth rate of overnight S. mutans culture at the indicated concentrations of H2O2. The growth rate was set as 100% in the media without H2O2. And the growth inhibition was calculated by subtracting from 100%.

#### Taken together, S. sanguinis produces obvious amounts of H2O2 aerobically and weak amounts of H2O2 anaerobically against S. mutans. S. sanguinis plays an important role in maintaining oral microbiome balance and might be beneficial to oral health.

### GOAL

#### In our project, we want to enhance the ability of health-associated oral commensal bacteria or probiotics to prevent colonization of S. mutans producing lactic acid. H2O2, which is naturally generated by various oral microbiota including S. sanguinis, has an antagonistic effect on oral pathogen such as S. mutans and maintains the balance of oral microbiome for oral health. H2O2 is also prescribed for the treatment of periodontal disease by avoiding overuse of antibiotics in the concern of antimicrobial resistance. Therefore, H2O2 production by a GM probiotic or oral commensal bacteria was focused on the design of our engineering.

## ENGINEERING

### CHASSIS & DESIGN

#### The chassis to be engineered may be considered in the oral environment. S. mutans, a major etiological pathogen, causes dental caries by the production of lactic acid and developing acidic condition at the critical pH value of 5.5. We tested acidic tolerance of two predominant oral residents, hostile S. mutans and benign S. sanguinis, and also one probiotic E. coli strain Nissle (EcN).

![](https://static.igem.org/mediawiki/2020/5/5d/T--Mingdao--experiment_fig_5.jpeg)

#### Fig. 5. The growth curve of (A) S. mutans (B) S. sanguinis (C) E. coli Nissle at pH values of 2, 4, 6, 7, 8. The pH value of the original medium is around 7, and others were adjusted to the indicated level. The growth of bacteria was measured at OD600 periodically every 20 min over a period of 16 hr

#### Our data in Fig. 5 showed that all bacteria tested can’t grow at pH values of 2 and 4 and grew well at pH values of 7 and 8. At pH of 6 compared to pH of 7, E. coli Nissle grew best, S. mutans comes second, and S. sanguinis retards. Acid-resistant E. coli Nissle is consistent with the finding by Chelliah Ramachandran, et al. S. mutans produces lactate and is adapted itself to acidic oral environment, where S. sanguinis is hard to compete. Notably, S. sanguinis increases exponentially at pH of 8 compared to pH of 7, suggesting oral health with alkaline pH prefers the growth of S. sanguinis. Bin Zhu, et al. also reported that S. sanguinis utilizes arginine deiminase system (ADS) to maintain pH homeostasis and protect against caries caused by S. mutans.     In conclusion, to survive in harsh acidic oral environment (pH = 4.5 ~ 6.5) created by S. mutans, the acid-sensitive S. sanguinis was not considered. The acid-resistant E. coli Nissle was a candidate but not perfect because it is not a commensal strain in the oral environment. The etiological S. mutans was chosen as ideal oral strain if not producing the major caries causal factor, lactic acid.     Dr. Jeffrey D. Hillman, a dentist, published paper in 2002 and 2007 describing genetically modified S. mutans for the prevention of dental carries, in which lactic acid production was eliminated by replacing the gene of lactate dehydrogenase with alcohol dehydrogenase. He defined the replacement therapy by using this effector strain, and also founded Oragenics. Inc. for clinical trials. We’re encouraged and inspired, as well as wondering if giving this GM strain a weapon equipped with H2O2 production mechanism.     The blueprint for genetically engineering (i.e., constitutive and regulated promoters, functional genes, etc.) was designed based on ideal strain of S. mutans. However, E. coli Nissle was firstly chosen as our proof-of-concept chassis because of easily being genetically modified. In addition, Dr. Divya Pandya also mentioned in a review paper and pointed out E. coli Nissle species is potential used as probiotics in oral cavity with a non-lactic acid producing feature. This application is also inspired by the project of iGEM team TAS-Taipei in 2018, where they contained the probiotic E. coli Nissle in their prototype for oral treatment. Then, we got the E. coli strain Nissle from team TAS-Taipei and have collaborated with them for experiences in transforming the strain and designing the product.     E. coli Nissle was streaked on LB agar plate and incubated at 37 °C for 16 hr. Four single colonies were picked up for plasmid extraction. There are two endogenous plasmids (pMut1 and pMut2) in the cells of E. coli Nissle. The plasmids were checked by HindIII for pMut1 and EcoRI for pMut2, given the 3250-bp and 5591-bp bands, respectively (Fig. 6).

![](https://static.igem.org/mediawiki/2020/2/26/T--Mingdao--experiment_fig_6.jpeg)

#### Fig. 6. The endogenous plasmids of E. coli Nissle were checked by HindIII and EcoRI. The gel data showed 3250-bp and 5591-bp bands.

#### We performed E. coli Nissle transformation with BTX™ Gemini X2 Electroporation System in our lab. Preparation of electro-competent E. coli was following the manufacture’s protocol for E. coli. Electroporation was conducted in a 2-mm gap cuvette by a single electric pulse of 5ms with settings of 2.5 kV, 25 µF, 200Ω. The transformants with GFP reporter (ldhp-GFP-Tr/pSB1C3 [BBa\_K3376002]) were selected on a LB agar plate supplemented with 20 µg/ml of chloramphenicol (Fig. 7)

![](https://static.igem.org/mediawiki/2020/5/52/T--Mingdao--experiment_fig_7.jpeg)

#### Fig. 7. E. coli Nissle was transformed with pSB1C3-based GFP reporter plasmid by electroporation.

#### All the research and our preliminary experiment results told us that we are ready to go further for engineering!

### BLUEPRINT

#### S. mutans as a chassis is designed to be genetically modified with pyruvate oxidase (SpxB) gene to generate H2O2, aquaporin (AQP) gene to facilitate H2O2 transport, and catalase (KatG) gene to revive from oxidative stress by decomposing H2O2 to oxygen and water. The SpxB gene is driven under the constitutive and strong promoter of lactate dehydrogenase (ldhp). The KatG gene is regulated under the promoter of thiol peroxidase (tpxp) which is induced by H2O2. Therefore, catalase is expressed in the presence of H2O2 and detoxify the H2O2. Furthermore, the endogenous gene of lactate dehydrogenase is broken to eliminate the production of lactic acid.

![](https://static.igem.org/mediawiki/2020/e/e1/T--Mingdao--experiment_fig_8.jpeg)

#### Fig. 8. The schematic diagram of genetically engineered S. mutans. (A) GM S. mutans carries the genes of pyruvate oxidase from S. sanguinis, catalase from E. coli and aquaporin from S. cristatus. (B) the biobrick composite part construction on pSB1C3. (C) the legend of basic part element and gene name.

### BUILD & TEST – THE PARTS

#### Promoters

#### First of all, we need two kinds of promoters (i.e., a constitutive strong promoter and a regulated promoter) to drive gene expression in various situation. We made two basic parts of lactate dehydrogenase promoter (ldhp) and thiol peroxidase promoter (tpxp) from gDNA of S. mutans by PCR, both including the endogenous RBS. Ldhp is a strong and constitutive promoter in S. mutans used and well-documented in many studies. Thiol peroxidase is a stress-related gene in S. mutans and its promoter activity is induced by the presence of H2O2. The promoters were assembled with a GFP reporter and a terminator, respectively. The gene expression was measured as GFP level with overnight culture, showing a strong activity of ldhp and a limited expression of tpxp. (Fig. 9)

![](https://static.igem.org/mediawiki/2020/2/2b/T--Mingdao--experiment_fig_9.jpeg)

#### Fig. 9. Promoters in this study. The GFP was detected at Ex/Em = 483/513. (A) Basic part: ldhp/pSB1C3 (BBa\_K3376000), Composite part: ldhp-GFP-Tr/pSB1C3 (BBa\_K3376002), (B) Basic part: tpxp/pSB1C3 (BBa\_K3376003), Composite part: tpxp-GFP-Tr/pSB1C3 (BBa\_K3376004), (C) GFP was expressed at different levels with the indicated promoters. Inset plot: GFP fluorescence under a blue LED box.

#### H2O2 transporter

#### H2O2 permeability across the cell membrane is limited. The aquaporin (AQP) from Streptococcus cristatus facilitates bidirectional transmembrane H2O2 transport. Huichun Tong, et al. demonstrated that E. coli transformed with the gene of AQP from S. cristatus has increased H2O2 uptake and facilitated H2O2 permeation.     We made the basic part of AQP of S. cristatus and tested the function with the H2O2-inducible GFP reporter under the tpxp promoter. As shown in Fig. 10, GFP expression was enhanced significantly in the presence of AQP and H2O2, indicating AQP functions as H2O2 transporter.

![](https://static.igem.org/mediawiki/2020/f/f0/T--Mingdao--experiment_fig_10.jpeg)

#### Fig. 10. AQP facilitates H2O2 permeation. (A) Basic par: AQP/pSB1C3 (BBa\_K3376005) and Composite part: ldhp-AQP-Tr-tpxp-GFP-Tr/pSB1C3 (BBa\_K3376007), (B) GFP expression level was increased with AQP in the presence of H2O2 compared to the control without AQP.

#### Furthermore, the GFP intensity was enhanced in response to the increasing concentrations of H2O2 in a dose-dependent manner, suggesting the promoter activity of tpxp was regulated by H2O2 (Fig. 11).

![](https://static.igem.org/mediawiki/2020/1/10/T--Mingdao--experiment_fig_11.jpeg)

#### Fig. 11. The promoter activity of tpxp was increased dose-dependently with H2O2 concentration in the presence of AQP.

#### 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 antagonistic relationship against S. mutans. We constructed the basic part with IDT synthesized gene based on the gene sequence of S. sanguinis. The part with a RBS (BBa\_B0034) was assembled behind AQP using the same promoter of ldhp.     The lozenge with a probiotic will be our demonstration prototype, which contains 3% - 10% glucose. We tested the transformed E. coli Nissle carrying ldhp-AQP-RBS-SpxB-Tr/pSB1C3 (GM EcN) in the culture medium of LB supplemented with 5% or 10% glucose. The supernatants of overnight culture were subjected to H2O2 production measurement. Fig. 12 pointed out that 2.15 mM and 1.84 mM of H2O2 per OD600 were produced by GM EcN cultured in 5% and 10% glucose, respectively, compared to the basal level of H2O2 production (i.e., 0.33 mM) by WT EcN.

![](https://static.igem.org/mediawiki/2020/7/7e/T--Mingdao--experiment_fig_12.jpeg)

#### Fig. 12. SpxB produces H2O2. The H2O2 was measured with a Fluorimetric Hydrogen Peroxide Assay Kit (SIGMA-ALDRICH). (A) Basic part: SpxB/pSB1C3 (BBa\_K3376008) and Composite part: ldhp-AQP-RBS-SpxB-Tr/pSB1C3 (BBa\_K3376010), (B) The H2O2 was produced by the transformants expressing AQP and SpxB.

#### This result demonstrated the function of SpxB in the transformed cells. However, the bacterial growth was restricted, possibly resulting from the oxidative stress of H2O2 (Fig. 13, blue dots).

#### H2O2 decomposition

#### Catalase converts H2O2 to O2 and H2O preventing cells from oxidative stress. We used an existing BioBrick part of catalase gene (KatG) from E. coli (BBa\_S04059). The function of KatG was characterized by assembling with the promoter of tpxp. The growth defect of transformed E. coli Nissle with AQP and SpxB (ldhp-AQP-SpxB, blue dots in Fig. 13) was recovered by supplement of KatG (ldhp-AQP-SpxB-tpxp-KatG, red dots in Fig. 13), indicating H2O2 decomposition by the function of catalase.

![](https://static.igem.org/mediawiki/2020/6/63/T--Mingdao--experiment_fig_13.jpeg)

#### Fig. 13. Growth recovery and the function of KatG. (A) An existing BioBrick part of KatG-Tr/pSB1AK3 (BBa\_S04059) (B) Growth rate of E. coli Nissle transformed with the indicated plasmid was measured at OD600. The cells were cultured in LB broth supplemented with 1%, 5% or 10% glucose for 5 hr.

#### H2O2-producing device

#### To test the function of our final composite device (ldhp-AQP-RBS-SpxB-Tr-tpxp-KatG-Tr/pSB1C3, BBa\_K3376012), the supernatants of 5-hr culture in LB broth supplemented with 1%, 5%, 10% glucose were subjected to H2O2 production assay. As shown in Fig. 14, the amounts of H2O2 production were increased dose-dependently in response to the concentration of glucose. According to the above analyses of H2O2 transportation, H2O2-regulated promoter activity, the growth recovery by catalase, and H2O2 production, the results demonstrated the functionality of our composite BioBrick device.

![](https://static.igem.org/mediawiki/2020/1/18/T--Mingdao--experiment_fig_14.jpeg)

#### Fig. 14. The final components and functionality of our composite device in this study (A) Composite part: ldhp-AQP-RBS-SpxB-Tr-tpxp-KatG-Tr/pSB1C3 (BBa\_K3376012) (B) H2O2 production of E. coli Nissle transformed with the indicated plasmid were measured and normalized by the values of OD600. The cells were cultured in LB broth supplemented with 1%, 5% or 10% glucose for 5 hr.

#### Protein expression

#### To confirm the gene expression in our device, we conducted SDS-PAGE with Coomassie blue staining for aquaporin (AQP), pyruvate oxidase (SpxB) and catalase (KatG) with lysates of the transformed E. coli Nissle carrying the indicated gene. When overexpression under the promoter of ldhp, KatG (80kDa) and AQP (17kDa) have sharp band on the PAGE (Fig. 15, A). However, the predicted 65-kDa of SpxB was not detected, possibly because of the oxidative stress induced by the production of H2O2. The phenomenon is consistent with the result of growth inhibition shown in Fig. 13.     In the presence of KatG, SpxB was shown up along with AQP, though expressed at a week level (Fig. 15, B). In addition, the expression of tpxp-driven KatG induced by H2O2 was further confirmed. Taken together, our device is able to not only produce H2O2 by expressing SpxB (pyruvate oxidase), which permeates across cell membrane by H2O2 transporter, AQP (aquaporin), but also relive the oxidative stress by KatG (catalase).

![](https://static.igem.org/mediawiki/2020/a/aa/T--Mingdao--experiment_fig_15.jpeg)

#### Fig. 15. Protein expression with Coomassie blue staining on 4 – 20% gradient SDS-PAGE. E. coli Nissle carrying the indicated plasmid vector was cultured in LB supplemented with 34 µg/ml of chloramphenicol. The lysates were harvested from overnight culture and 5 µg of them was subjected to SDS-PAGE.

### IMPROVE – FUTURE WORK

#### However, we designed the genetic blueprint based on S. mutans as a probiotic. We believed a probiotic is not only defined as non-toxic or benefit, but also an advantage in residing and even occupying the space where the pathogens live. Therefore, we were transforming oral S. mutans with the device and also knocking down the gene of lactate dehydrogenase which produces lactic acid to cause tooth cavities.

#### Chassis S. mutans and the gDNA check

#### We got the S. mutans from Prof. Ming-Shiou Jan, who helped us manipulate the oral bacteria of S. mutans and S. sanguinis, and also guided us to design the related experiments. We learned how to culture bacteria anaerobically and extract the genomic DNAs followed by checking with PCR (Fig. 16).

![](https://static.igem.org/mediawiki/2020/5/56/T--Mingdao--experiment_fig_16.jpeg)

#### Fig. 16. The 100-bp fluorescent DNA ladder and PCR check for gDNA of S. sanguins and S. mutans. S. sanguinis specific primers and PCR product size: san-F (5'-CAAAATTGTTGCAAATCCAAAGG-3') and san-R (5'-GCTATCGCTCCCTGTCTTTGA-3'), 74 bp. S. mutans specific primers: mut-F (5'-GCCTACAGCTCAGAGATGCTATTCT-3’) and mut-R (5'-GCCATACACCACTCATGAATTGA -3’), 111 bp.

#### pDL278, a E. coli/Streptococcus shuttle vector

#### pDL278 is a shuttle vector between E. coli and Gram-positive bacteria esp. for Streptococcus spp., which was created by Donald J. LeBlanc, et al. in 1992. It could be applied for cloning vectors in E. coli and transforming S. mutans with a spectinomycin resistance cassette and the origin of replication of pBR322 for E. coli and ori (+) for Gram(+) bacteria, respectively. Ori (+) is from S. aureus with an ORF encoding an undefined protein possibly for plasmid replication (Fig. 17, A). We got the plasmid from the lab of Dr. Yuqing Li at Sichuan University in China. Firstly, we made the pDL278 compatible to BioBrick assembly system (i.e., EcoRI-XbaI-insert-SpeI-PstI). We amplified the backbone by PCR and did 2 rounds of site-directed mutagenesis with the primers listed in Fig. 17, C. The resulting plasmid was checked by restriction enzymes (Fig. 17, B) and further confirmed by sequencing.

![](https://static.igem.org/mediawiki/2020/6/62/T--Mingdao--experiment_fig_17.jpeg)

#### Fig. 17. BioBrick compatible pDL278 E. coli/Streptococcus shuttle vector. (A) Key features in pDL278 (B) Restriction enzyme check with a 1kb DNA ladder (C) Primers for site-directed mutagenesis and sequencing.

#### Transformation of E. coli DH5alpha

#### E. coli DH5alpha competent cells can be efficiently transformed using the traditional heat-shock method. The transformants carrying the BBa\_J04450/pDL278 vector can be selected with 50 ug/ml of spectinomycin and express a red color in the colonies by the RFP coding device (BBa\_J04450) (Fig. 18). The mini-prep of plasmid concentration we usually got from overnight culture is around 200 ng/ul (260/280 = ~1.8, 260/230 > 4).

![](https://static.igem.org/mediawiki/2020/9/95/T--Mingdao--experiment_fig_18.jpeg)

#### Fig. 18. Transformation of E. coli DH5alpha competent cell with the plasmid of J04450/pDL278.

#### Transformation of S. mutans by electroporation

#### We tested transforming S. mutans by electroporation based on Vuokko Loimaranta’s protocol, briefly described as follows. Preparation of the electrocompetent cells:   ↓Cultivate S. mutans in BHI to OD600 of 0.6   ↓Wash twice with ice-cold buffer (10mM HEPES (pH 7.0), 15% glycerol)   ↓Resuspend in the electroporation buffer (5% sucrose, 15% glycerol) Electroporation in BTX™ Gemini X2 Electroporation System:   ↓40 µl of ice-cold electrocompetent cells in a 1-mm gap cuvette   ↓1 µl of pDL278-based plasmid DNA (~10ng)   ↓A single electric pulse of 4.5 ms (setting: 1.25 kV, 25 µF, 200µ)   ↓Immediately add fresh 960 µl of BHI broth   ↓After 1 hr, plate the cells onto BHI agar plate supplemented with 1mg/ml of spectinomycin   ↓Grown for 2 days at 37°C, check the colony by PCR.     The gel data shown in Fig. 19 indicated the successful transformation of S. mutans by colony PCR with primer sets against the plasmid vector (pDL278-F/R) and gDNA of S. mutans (Mut-F/R). Currently we’re transforming S. mutans with our device “ldhp-AQP-RBS-SpxB-Tr-tpxp-KatG-Tr/pSB1C3 (BBa\_K3376012)” and going to test the antagonistic effect against the WT S. mutans.

![](https://static.igem.org/mediawiki/2020/4/46/T--Mingdao--experiment_fig_19.jpeg)

#### Fig. 19. PCR check for the transformed colonies of S. mutans. (A) Colonies 1-4 were checked by pDL278-specific primers with a 1321-bp size of PCR product. (B) Colonies 1-4 were checked with primers against gDNA of S. mutans (111 bp). (C) Control colonies 1-2 of WT S. mutans were checked by S. mutans (111 bp) and pDL278 (1321 bp) specific primers, respectively.

#### CRISPR-mediated gene knockout of Lactate dehydrogenase

#### The final version of our probiotic S. mutans must lose the ability of producing lactic acid which causes caries. Based on the research of genome editing in Streptococcus mutans by Prof. Yuqing Li, the gene of S. mutans can be broken down by a synthetic gRNA against the 30-bp target sequence upstream of the PAM (i.e., TGG) through the intrinsic CRISPR-Cas 9 system. We’ve examined the sequence of lactate dehydrogenase in S. mutans and targeted 6 elements with the end connected to TGG (Fig. 20). We designed the 6 30-bp spacers and synthesized the DNA by Integrated DNA Technologies (IDT), Inc. The editing template was made by assembling two DNA fragments which were amplified 1000-bp upstream and downstream of the target sequence by PCR, respectively. The editing templates on pSB1C3 will be used for editing genome by recombination into the targeted locus. We’re excited and expect to transform S. mutans as a perfect probiotic to equip H2O2 and lack producing lactic acid.

![](https://static.igem.org/mediawiki/2020/1/14/T--Mingdao--experiment_fig_20.jpeg)

#### Fig. 20. CRISPR-Cas9 system in S. mutans and the designed synthetic gRNA with an editing template. The sequences of the 6 spacers on the gRNAs were shown. The gRNA sequences were designed based on the research published on Mol Oral Microbiol. 2018, 33(6):440-449.

## PROOF-OF-CONCEPT

### ANTAGONISM

#### In the natural environment, S. sanguinis and S. mutans are antagonists which can inhibit the growth of each other. As a result, they are usually used as a model to probe the competition between different species that occupy the same ecological niche. To test the competition between wild-type S. mutans and genetically modified E. coli Nissle (GM EcN) expressing AQP, SpxB and induced KatG, we conducted the experiments of antagonism test between S. mutans and S. sanguinis, S. mutans and WT EcN, S. mutans and GM EcN. 8 µL of the overnight culture of both species was adjusted to an optical density at OD600 of 0.5 in LB broth (WT EcN, GM EcN) or BHI broth (S. sanguinis, S. mutans), followed by being inoculated on LB agar plates at the same time beside each other. The cell shape and size on agar plate in Fig. 21 demonstrated that GM EcN has a strong ability to inhibit the growth of S. mutans, and even better than S. sanguinis (Fig. 21). There’s no antagonistic effect between S. mutans and WT EcN control. The results significantly indicated that the H2O2 produced by the device in GM EcN may be capable of efficiently eliminating S. mutans.

![](https://static.igem.org/mediawiki/2020/1/16/T--Mingdao--experiment_fig_21.jpeg)

#### Fig. 21. Antagonism test between S. mutans and S. sanguinis (A), S. mutans and WT EcN (B), S. mutans and GM EcN (C). 8 µL of the overnight cultures adjusted to an OD600 of ~0.5 were inoculated on LB agar plates beside each other. The data showed two repeats observed after 24-hr culture.

### PROTOTYPE

#### The handmade lozenge we created is a sweet candy made of 5% sugar alcohols (i.e, xylitol and erythritol) and weighs 1 gram. It contains approximately 3 x 108 CFU (colony forming unit) of EcN (E. coli Nissle) per tablet. To determine the protein expression in our prototype, we made three kinds of lozenges containing GM EcN, which carries the empty vector, the GFP expression device and the AQP-SpxB-KatG expression device. When exposed to blue LED light, EcN with GFP glowed brightly compared to the controls (Fig. 22, A), indicating that GM EcN can express proteins even made into a candy. Moreover, to test the functionality of GM EcN with AQP-SpxB-KatG, we examined the production of H2O2 in the lozenges. The saliva production in the mouth will reach to 3 – 5 ml per minute during eating, chewing or other stimulating activities. Therefore, we dissolved the lozenge in the 3 ml of ddH2O for 5 min and measured H2O2 concentration. GM EcN with AQP-SpxB-KatG can produce 0.58 mM per OD600 compared to the background level (0.21 mM/OD600) by the vector-only control (Fig.22, B). Taken together, the candy as a prototype we made with GM EcN not only carries live probiotics expressing proteins, but also produces H2O2, implying an effective product against S. mutans for maintenance of oral health.

![](https://static.igem.org/mediawiki/2020/f/f7/T--Mingdao--experiment_fig_22.jpeg)

#### Fig. 22. The handmade lozenges contain GM EcN with the empty vector as control, GFP expression device, or AQP-SpkB-KatG expression device. (A) GFP was observed in a blue LED box. (B) The lozenges were dissolved in the ddH2O and tested for H2O2 production.

### SAFETY

#### Image the application in the real world, the candy we made contains E. coli Nissle cells, which will be released when dissolved in the mouth. Prior to reaching to the intestine and be absorbed by tissues, they have to pass through the stomach and incubate at the acidic environment at pH of 1.5 - 3.5 for 4 – 6 hours. Then, we tested the growth of Nissle strains in the medium at pH of 2 compared to pH of 7. The results in Fig. 23 indicated that the bacterial growth was dramatically inhibited and no bacteria survived on agar plates after 5-min treatment in media at pH of 2, suggesting a safe usage of the candy with probiotics in the real world.

![](https://static.igem.org/mediawiki/2020/e/eb/T--Mingdao--experiment_fig_23.jpeg)

#### Fig. 23. The growth inhibition and survival rate of E. coli Nissle in LB medium and on agar plate at pH of 2. (A) The values of overnight culture were measured at OD600. The percentage of growth inhibition was calculated by dividing OD600 at pH = 7. (B) 104 bacterial cells were treated in LB broth at pH of 2 for the indicated time, followed by spreading onto the agar plate. The survival rates were ~10%, 0%, 0% for 1 min, 5 min, 30 min, respectively.

### CONCLUSION

#### Jens Kreth, et al. studied the Streptococcal antagonism in oral biofilms showing S. sanguinis is capable of antagonize the growth of S. mutans. Osnat Feuerstein, et al. found that H2O2 has antibacterial effect on S. mutans. Probiotic strain, E. coli Nissle, was mentioned by Divya Pandya to treat dental caries.     Herein, we proposed a model of probiotic H2O2-producing system against S. mutans. We successfully genetically modified a probiotic strain E. coli Nissle (GM EcN) with aquaporin (AQP) and pyruvate oxidase (SpxB) genes driven under a strong promoter of lactate dehydrogenase (ldhp), and catalase (KatG) gene regulated by a H2O2-inducible thiol peroxidase promoter (tpxp). We’ve significantly demonstrated the function of the device producing H2O2 and attacking etiological S. mutans in the experiments of H2O2 production assay and antagonistic competition test.     Finally, we produced a handmade lozenge as our prototype, which carries GM EcN, and also demonstrated the protein expression and the functionality of H2O2 production in the candy. In summary, the candy that can clean your teeth, we named Cleandy, is promised to be useful in maintaining oral health in the future.

## REFERENCE

#### * Izabela Struzycka. The oral microbiome in dental caries. Pol J Microbiol 2014;63(2):127-35. MID: 25115106. * Reyes E, Martin J, Moncada G, Neira M, Palma P, Gordan V, Oyarzo JF, Yevenes I. Caries-free subjects have high levels of urease and arginine deiminase activity. J Appl Oral Sci. 2014 Jun;22(3):235-40. doi: 10.1590/1678-775720130591. * Ge Y, Caufield PW, Fisch GS, Li Y. Streptococcus mutans and Streptococcus sanguinis colonization correlated with caries experience in children. Caries Res. 2008;42(6):444-8. doi: 10.1159/000159608. * Seow WK, Lam JH, Tsang AK, Holcombe T, Bird PS. Oral Streptococcus species in pre-term and full-term children - a longitudinal study. Int J Paediatr Dent. 2009 Nov;19(6):406-11. doi: 10.1111/j.1365-263X.2009.01003.x. * Kreth J, Zhang Y, Herzberg MC. Streptococcal antagonism in oral biofilms: Streptococcus sanguinis and Streptococcus gordonii interference with Streptococcus mutans. J Bacteriol. 2008 Jul;190(13):4632-40. doi: 10.1128/JB.00276-08. * Barnard JP, Stinson MW. Influence of environmental conditions on hydrogen peroxide formation by Streptococcus gordonii. Infect Immun. 1999 Dec;67(12):6558-64. PMID: 10569775 * Dunlap T. Prescribing Hydrogen Peroxide in the Treatment of Periodontal Disease. Oral Health 2016;Dec:64-68. * Lussi A, Carvalho TS. Erosive tooth wear: a multifactorial condition of growing concern and increasing knowledge. Monogr Oral Sci. 2014;25:1-15. doi: 10.1159/000360380. * Chelliah Ramachandran, Ramakrishnan Sudha Rani and Antony Usha. Evaluation of safety, antimicrobial activity and probiotic properties of Escherichia coli Nissle 1917 isolated from Idli batter. Research Journal of Biotechnology. 2016 July; 11(7):42-49 * Zhu B, Macleod LC, Kitten T, Xu P. Streptococcus sanguinis biofilm formation & interaction with oral pathogens. Future Microbiol. 2018 Jun 1;13(8):915-932. doi: 10.2217/fmb-2018-0043. * Harper DS, Loesche WJ. Inhibition of acid production from oral bacteria by fluorapatite-derived fluoride. J Dent Res. 1986 Jan;65(1):30-3. doi: 10.1177/00220345860650010401. * Hillman JD. Genetically modified Streptococcus mutans for the prevention of dental caries. Antonie Van Leeuwenhoek. 2002 Aug;82(1-4):361-6. PMID: 12369203. * Hillman JD, Mo J, McDonell E, Cvitkovitch D, Hillman CH. Modification of an effector strain for replacement therapy of dental caries to enable clinical safety trials. J Appl Microbiol. 2007 May;102(5):1209-19. doi: 10.1111/j.1365-2672.2007.03316.x. * Pandya, Divya. Benefits of Probiotics in Oral cavity - A Detailed Review. Annals of International medical and Dental Research. 2016 2(5)10-17 doi: 10.21276/aimdr.2016.2.5.DE3. * Jessica K Kajfasz, Tridib Ganguly, Emily L Hardin, Jacqueline Abranches, José A Lemos. Transcriptome responses of Streptococcus mutans to peroxide stress: identification of novel antioxidant pathways regulated by Spx. Sci Rep 2017 Nov 22;7(1):16018. doi: 10.1038/s41598-017-16367-5. * Huichun Tong, Xinhui Wang, Yuzhu Dong, Qingqing Hu, Ziyi Zhao, Yun Zhu, Linxuan Dong, Fan Bai, and Xiuzhu Dong. A Streptococcus aquaporin acts as peroxiporin for efflux of cellular hydrogen peroxide and alleviation of oxidative stress. J Biol Chem. 2019 Mar 22; 294(12): 4583–4595. doi: 10.1074/jbc.RA118.006877 * Lan-yan Zheng, Andreas Itzek, Zhi-yun Chen, and Jens Kreth. Oxygen dependent pyruvate oxidase expression and production in Streptococcus sanguinis. Int J Oral Sci. 2011 Apr; 3(2): 82–89. doi: 10.4248/IJOS11030 * B L Triggs-Raine, B W Doble, M R Mulvey, P A Sorby, and P C Loewen. Nucleotide sequence of katG, encoding catalase HPI of Escherichia coli. J Bacteriol. 1988 Sep; 170(9): 4415–4419. doi: 10.1128/jb.170.9.4415-4419.1988 * LeBlanc DJ, Lee LN, Abu-Al-Jaibat A. Molecular, genetic, and functional analysis of the basic replicon of pVA380-1, a plasmid of oral streptococcal origin. Plasmid. 1992 Sep;28(2):130-45. doi: 10.1016/0147-619x(92)90044-b. * Loimaranta V, Tenovuo J, Koivisto L, Karp M. Generation of bioluminescent Streptococcus mutans and its usage in rapid analysis of the efficacy of antimicrobial compounds. Antimicrob Agents Chemother. 1998 Aug;42(8):1906-10. doi: 10.1128/AAC.42.8.1906. * Gong T, Tang B, Zhou X, Zeng J, Lu M, Guo X, Peng X, Lei L, Gong B, Li Y. Genome editing in Streptococcus mutans through self-targeting CRISPR arrays. Mol Oral Microbiol. 2018 Dec;33(6):440-449. doi: 10.1111/omi.12247. * Kreth J, Zhang Y, Herzberg MC. Streptococcal antagonism in oral biofilms: Streptococcus sanguinis and Streptococcus gordonii interference with Streptococcus mutans. J Bacteriol. 2008 Jul;190(13):4632-40. doi: 10.1128/JB.00276-08. * Feuerstein O, Moreinos D, Steinberg D. Synergic antibacterial effect between visible light and hydrogen peroxide on Streptococcus mutans. J Antimicrob Chemother. 2006 May;57(5):872-6. doi: 10.1093/jac/dkl070.

1. [PROJECT](#PROJECT)
   * [DESCRIPTION](#DESCRIPTION)
   * [RESEARCH](#RESEARCH)
   * [INSPIRATION](#INSPIRATION)
   * [GOAL](#GOAL)
2. [ENGINEERING](#ENGINEERING)
   * [CHASSIS & DESIGN](#CHASSISDESIGN)
   * [BLUEPRINT](#BLUEPRINT)
   * [BUILD & TEST – THE PARTS](#BUILDTESTTHEPARTS)
   * [IMPROVE – FUTURE WORK](#IMPROVEFUTUREWORK)
3. [PROOF-OF-CONCEPT](#PROOF-OF-CONCEPT)
   * [ANTAGONISM](#ANTAGONISM)
   * [PROTOTYPE](#PROTOTYPE)
   * [SAFETY](#SAFETY)
   * [CONCLUSION](#CONCLUSION)
4. [REFERENCE](#REFERENCE)

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# Team:Mingdao/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

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### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

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### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)