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PROJECT

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.

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.

    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.

    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.

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).

    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).

    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)

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.

BUILD & TEST – THE PARTS

    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)

    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.

    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).

    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.

    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.

    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.

    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).

IMPROVE – FUTURE WORK

    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).

    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.

    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).

    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.

    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.

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.

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.

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.

1. PROJECT
   • DESCRIPTION
   • RESEARCH
   • INSPIRATION
   • GOAL
2. ENGINEERING
   • CHASSIS & DESIGN
   • BLUEPRINT
   • BUILD & TEST – THE PARTS
   • IMPROVE – FUTURE WORK
3. PROOF-OF-CONCEPT
   • ANTAGONISM
   • PROTOTYPE
   • SAFETY
   • CONCLUSION
4. REFERENCE

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• Phone: +886-4-23341797
• Email: biolab@ms.mingdao.edu.tw