Team:MIT MAHE/Poster

Breaking Bond

Abstract

Pollution is a direct result of man's ever-increasing need for natural resources and energy, one of the notorious pollutants being – mercury. Mercury in water bodies is converted by anaerobic microbes into methylmercury. This toxic compound, gets Biomagnified and Bioaccumulated through the aquatic trophic levels. At the level of humans, it can be dangerously high. It is no coincidence that this was responsible for one of the world's worst environmental disasters, i.e. the Minamata Disease. Previous projects attempted to solve the problem of methylmercury poisoning using bioremediation - converting free methylmercury (95% absorption) to elemental mercury (<0.01% absorption). However, these don't account for the methylmercury already inside millions of fish. We wish to develop a probiotic bacterium with similar capabilities, this exploits the fact that Hg is inert in the gut and prevents absorption of organic mercury. We will also have a system that will help in reducing mercury-induced inflammations.

Mentors

Principal Investigator - Dr. Ritu Raval
Secondary PI - Dr. Praveen Kumar
iGEM Mentor - Ugne Baronaite

Team

Aayush Panday, Adithi Somayaji, Aniket Naik, Anushka Jain, Apoorva Kapadia, Bhumika Narang, Lavanya Karinje, Naman Kapoor, Niyati Sanghvi, Pavithra Sathyan, Pradyumna Iyengar, Raksha Nayak, Sanya Lakhotia, Shameshwar Muralidarran, Shravan Balasubramaniam, Soumodeep Sarkar, Urvi Gupta, Utkarsh Mohan, Vaishnavi Chavan, Varun Udaya Kumar.
Project Introduction

Methylmercury is a toxic, organometallic compound with the primary source of exposure being consumption of food (primarily but not limited to fishes) contaminated with it [15].


Mercury finds its way into water bodies through a variety of sources such as improper disposal by industries, mining, volcanic activity etc. This mercury is methylated by anaerobic microbes found in these water bodies. In this form, it becomes bioavailable to phytoplanktons. As methylmercury is fat soluble, it gets bio-accumulated through the food chain as these phytoplanktons are consumed in large quantities by zooplanktons which are food for small fishes which are consumed by big fishes. At each level, the amount of toxic methylmercury dangerously increases resulting in biomagnification, and thus the end consumer can be severely affected [5].








Ingestion can lead to mental retardation, seizure disorders, cerebral palsy, cardiovascular malfunctions, blindness and deafness with paralysis, coma, insanity, and death occurring in extreme cases [10]. Moreover, owing to its capability of crossing the blood placenta barrier, it can even affect foetuses regardless of whether the mother is symptomatic [1].
Case Studies
Methylmercury was responsible for two of the worst environmental disasters, namely, the Minamata Disaster and the Basra grain poisoning disaster of Iraq.





Minamata disease was first officially discovered in Minamata, Kumamoto prefecture, Japan in 1956. It was caused by the release of methylmercury in the industrial wastewater from the Chisso Corporation's chemical factory, which continued from 1932 to 1968. This highly toxic chemical bio-accumulated in shellfish and fishes in Minamata Bay and the Shiranui Sea, which when eaten by the local populace resulted in mercury poisoning. The disease owes its name to this disaster. [6]
As of March 2001, 2,265 victims had been officially recognised (1,784 of whom had died) and over 10,000 had received financial compensation from Chisso. [16]


In 1971, a large outbreak of methylmercury poisoning took place in Iraq due to consumption of grain that was treated with methylmercury based fungicide. Nearly one-sixth of the rural population was admitted to hospitals for cases of methylmercury poisoning due to bioaccumulation in the grains. Pregnant females were at high risk with 45% case fatalities of the reported cases. Many children were affected, which affected the CNS and caused slower development. [2][12]


Our Proposal


The richness and versatility of biological systems and the technological developments of synthetic biology make them the ideal candidates for solving humanity's most pressing challenges. Our project 'Breaking Bond' aims to utilize synthetic biology to provide a solution to combat Methylmercury poisoning, an issue of global importance. As methylmercury absorption occurs through the gut, we have proposed a novel engineered probiotic bacterium, capable of converting methylmercury to elemental mercury (a less toxic form), as an addition to the gut microbiome.


This exploits the fact that elemental mercury is comparatively less toxic in the gut than MeHg, hence providing a basis to the use of engineered probiotic bacteria as a therapeutic intervention to prevent mercury poisoning and its allied health effects in humans. This can serve as a long term solution that can be made accessible to many people, both in terms of cost as well as usability. Our project also deals with the issue of Hg induced inflammation in the gut so that gut microbiota is not disturbed.

Concept
When methylmercury is ingested, nearly 95% of it gets absorbed by the gastrointestinal tract. It binds with amino acids that contain cysteine and the complex thus formed is recognised by amino acid transporting proteins in the body as methionine, another essential amino acid. This allows the methyl mercuric-cysteine complex to pass through many biological barriers including the blood brain barrier and the placental barrier, where it is absorbed by the developing foetus. [1]

Elemental mercury on the other hand, is relatively stable inside the human gut. Less than 0.01% of the total is absorbed by the gastrointestinal tract. Due to lack of charges, it cannot form any complexes, and cannot cross any biological barrier. [9]

Hence methylmercury is far more dangerous than elemental mercury. By converting it to elemental mercury, we will reduce its toxicity, which serves as an effective solution to methylmercury poisoning).
Design
A dual plasmid system is proposed as the bacteria's weapon is to break the methylmercury bond and prevent this toxic chemical to be absorbed by the gut. The probiotic bacteria we intend to use is genetically engineered Escherichia coli Nissle 1917.

In the absence of mercury, a repressor molecule encoded by the MerR gene is produced which binds to the PmerT promoter and prevents transcription. In the presence of mercury, MerR binds to mercury instead of the PmerT promoter changing its conformation, and thus, allowing RNA polymerase to freely transcribe the downstream genes[3].
MerT, MerP, MerE and MerC encode proteins which help in the efficient transport of methylmercury inside the cell[13][14].
A dual enzyme system is proposed as the working elements to convert methylmercury to the less toxic elemental form. MerB encodes organomercurial lyase which breaks the carbon mercury bond and MerA encodes mercuric reductase which reduces mercury ions to elemental mercury. This elemental mercury is eliminated out of the body via faeces.
The release of elemental mercury has the potential to disturb the gut microbiota and induce inflammations. To tackle this issue, Composite Bio-Brick II is proposed. SoxR produced by Bio-Brick-I controls the transcription of Bio-Brick-II ensuring that it is dormant in the absence of mercury. Only in the presence of mercury and Nitric oxide (a proinflammatory signal), will SoxR activate the SoxS promoter which initiates the transcription of downstream genes [7]. Hemolysin secretion system which contains HlyB, HlyD and TolC is proposed to transport the IL-10 fused to HlyA, a secretion signal peptide outside the cell[4].

Composite Bio-Brick 1


In order to validate different aspects of our proposed system, we have designed several experiments to ensure that the chassis and the two Bio-Bricks are working as theorized.

We have designed several experiments to
  • Check the threshold and tolerance of mercury concentration required by the transformed bacteria to generate a response (i.e. both low and high concentrations of methylmercury).
  • Characterization experiments for determining the most efficient combination of transport genes to ensure least genetic burden.
  • Characterize MerA and MerB to observe the increase in Mer spectrum with the introduction of these genes. We expect to conclude that the addition of the two genes confers better resistance to methylmercury.
  • Check the activity of the enzymes to find the optimal CFU/mL to be used.
Composite Bio-Brick 2


To ensure safety and efficiency of the Composite Bio-Brick II we have proposed to design, where Escherichia coli Nissle 1917 would be transformed with different circuits  to analyze

We have designed several experiments to
  • The relationship between the amount of oxidative stress and rate of transcription of the genes in response to Paraquat (methyl viologen dichloride hydrate) which acts as an inducer, using a GFP reporter.
  • The amount of oxidative stress that is required to activate SoxR and in turn activate SoxS promoter.
  • The basal level of transcription of the SoxS  promoter so that unwanted anti-inflammatory substances do not get released in the absence of oxidative stress.
  • The efficiency of the transport system consisting of HlyB, HlyD, TolC and HlyA against a control with the genes absent. The HlyA + GFP transported outside the cells, HlyA + GFP remaining inside the cells and the total HlyA + GFP produced with absorbance vs time graphs to show the efficiency in getting the anti-inflammatory cytokines outside the cell to the target site.
  • To test the overall working of Plasmid 2 and demonstrate its specificity to just mercury-induced inflammations.
Hardware
Systematic Analyzer of Underlying Limitations (SAUL) is a bioreactor that aims to imitate the complex dynamic environment of the small intestine and to determine the viability of probiotic drug components in it.


A semi permeable dialysis tube is used as the apical chamber of the intestine. Imitation of peristalsis is achieved through periodic compression of the dialysis tube by three ball bearings. The bearings are actuated using a DC motor. The dialysis tube is fitted into a 3D printed case, which mimics the basolateral chamber.
Structural analysis of the dialysis tube was performed on ANSYS to determine the appropriate tube diameter that would incur shear stresses as close as possible to the stresses experienced in the intestine.



A comprehensive set of experiments was formulated to check the effectiveness of the probiotic in a variable dynamic environment and to optimize the capsule parameters for appropriate delivery.

Total estimated cost of making = 84 USD.
Implementation
The implementation plan is formulated so as to guide the individuals involved in the production of the therapeutic probiotic and to give the stakeholders a rough idea of making the product marketable.

Prior to implementation we have designed several experiments that would ensure that the strain to be used has optimum capabilities. We have worked on optimization of the growth medium, capsule formulation, capsule testing, excipient selection and determination of the active pharmaceutical ingredient and other upstream processes that are of importance prior to scale up.

We have also designed three bioreactors of different sizes (100L, 1000L, 10000L) depending on fish consumption in different states of India ensuring minimal wastage of resources. We have also devised methods for separation, purification, manufacturing, cryo and lyo protection, freeze-drying and handling of waste production along with appropriate techniques used to package our product.


We intend to have 2 variants of the probiotic enteric tablet, for children and adults respectively. To ensure efficiency in terms of the quantity and quality produced, automated capsule filler is proposed. Quality check and recycling of waste have been kept into consideration for every step of our proposal.
Integrated Human Practices
To shape our project from a problem statement to a solution, we consulted many experts.




  • Dr. Abdul Ajees - Associate Professor, Department of Atomic and Molecular Physics, Manipal Academy of Higher Education.
  • Dr. Keyur Raval - Associate Professor, Department of Chemical Engineering, National Institute of Technology, Karnataka.
  • Dr. Saran Kumar - Postdoctoral Fellow, Faculty of Medicine, Hebrew University of Jerusalem, Israel.
  • Dr. Sudha Somayaji - Gynaecologist and Obstetrician practicing in Mangalore, Karnataka.
  • Dr. Vytla Ramachandra Murty - Professor, Department of Biotechnology, Manipal Institute of Technology.
  • Dr. Subbalaxmi Selvaraj - Assistant Professor - Senior Scale, Department of Biotechnology, Manipal Institute of Technology.
  • Ms. Archana Mahadev Rao - Assistant Professor, Department of Biotechnology, Manipal Institute of Technology.
Model
The functional analysis was modelled using MATLAB – SimBiology.

Model - 1
It deals with three graphs – mercury concentration vs time, enzyme concentration vs activity and no. of cells vs enzyme concentration; with the help of which we can estimate the optimal amount of enzyme required to be produced for optimal activity and thus, estimate the optimum CFU/mL to be used to get maximum activity. We have used Michaelis Menten kinetics to simulate the kinetics of our two enzymes, alkylmercury lyase and mercuric reductase.
Result: Optimal order of enzyme concentration to be 10-4 (reduction rate - 6-7 hours)



Model - 2
This deals with the transport system activity. We’ve assumed that the production of the proteins has already started due to a small amount of CH3Hg having diffused inside the cell before and bound with MerR, initiating the transcription and the amount of protein within the cell will remain constant. Solving the chemical equations modelled we will get –


But due to the lack of information about the activities of the protein further analysis could not be performed.

VMD
The structural analysis was carried out using Visual Molecular Dynamics, a molecular visualization program to model the proteins alkylmercury lyase and mercuric reductase. A CHARMM force-field with additional entries in the topology and parameter files was used for the mercury ion and cysteine (sulphur) interactions.

Mercuric reductase
Alkylmercury lyase
Safety
Safety is a very important aspect to be kept in mind as the project requires the usage of toxic chemicals like methylmercury. Material Safety Data Sheet (MSDS) for all the reagents must be documented to ensure good lab practice. Potential safety and security issues have been identified, and we have designed procedures and practices that are context-specific, responsive, flexible and principle-based.

Escherichia coli DH5α and Escherichia coli Nissle 1917 - require Biosafety Level 1 (BSL-1) laboratory. None of the experiments are expected to increase the pathogenicity of the bacteria.

To ensure safety, PPE (personal protective equipment) must always be worn while experimenting. (lab-coats, gas masks and Specialized gloves) [19]. Toxic chemicals must be disposed of safely in accordance with the guidelines.


While handling SAUL - isolation of electronic components from conducting media, proper insulation of connecting wires, thorough cleaning and discarding of dialysis tubes once used with toxic chemicals etc. must be ensured.
Extensive study has been conducted with respect to the safety considerations of the implementation of our project, whose aspects include powder design, plant and personnel hygiene, cleaning, process design, packaging, waste management and recycling.
References
[1] Aaseth, J., Wallace, D. R., Vejrup, K., & Alexander, J. (2020). Methylmercury and developmental neurotoxicity: A global concern. Current Opinion in Toxicology, 19(January), 80–87. https://doi.org/10.1016/j.cotox.2020.01.005
[2] Bakir, F., Damluji, S. F., Amin-Zaki, L., Murtadha, M., Khalidi, A., al-Rawi, N. Y., Tikriti, S., Dahahir, H. I., Clarkson, T. W., Smith, J. C., & Doherty, R. A. (1973). Methylmercury poisoning in Iraq. Science (New York, N.Y.), 181(4096), 230–241. https://doi.org/10.1126/science.181.4096.230
[3] Brown, N. L., Stoyanov, J. V., Kidd, S. P., & Hobman, J. L. (2003). The MerR family of transcriptional regulators. FEMS Microbiology Reviews, 27(2–3), 145–163. https://doi.org/10.1016/S0168-6445(03)00051-2
[4] Gentschev, I., Dietrich, G., & Goebel, W. (2002). The E. coli α-hemolysin secretion system and its use in vaccine development. Trends in Microbiology, 10(1), 39–45. https://doi.org/10.1016/S0966-842X(01)02259-4
[5] Gilmour, C., & Riedel, G. (2009). Biogeochemistry of Trace Metals and Mettaloids. Encyclopedia of Inland Waters, 7–15. https://doi.org/10.1016/B978-012370626-3.00095-8
[6] Harada M. (1995). Minamata disease: methylmercury poisoning in Japan caused by environmental pollution. Critical reviews in toxicology, 25(1), 1–24. https://doi.org/10.3109/10408449509089885
[7] Hidalgo, E., Leautaud, V., & Demple, B. (1998). The redox-regulated SoxR protein acts from a single DNA site as a repressor and an allosteric activator. EMBO Journal, 17(9), 2629–2636. https://doi.org/10.1093/emboj/17.9.2629
[8] Mathema, V. B., Thakuri, B. C., & Sillanpää, M. (2011). Bacterial mer operonmediated detoxification of mercurial compounds: A short review. Archives of Microbiology, 193(12), 837–844.
[9] National Research Council. (2000). Toxicological Effects of Methylmercury. In Toxicological Effects of Methylmercury. National Academies Press. https://doi.org/10.17226/9899
[10] Nogara, P. A., Oliveira, C. S., Schmitz, G. L., Piquini, P. C., Farina, M., Aschner, M., & Rocha, J. B. T. (2019). Methylmercury’s chemistry: From the environment to the mammalian brain. Biochimica et Biophysica Acta - General Subjects, 1863(12), 129284. https://doi.org/10.1016/j.bbagen.2019.01.006
[11] Parks, J. M., Guo, H., Momany, C., Liang, L., Miller, S. M., Summers, A. O., & Smith, J. C. (2009). Mechanism of Hg-C protonolysis in the organomercurial lyase MerB. Journal of the American Chemical Society, 131(37), 13278–13285. https://doi.org/10.1021/ja9016123
[12] Skerfving, S. B., & Copplestone, J. F. (1976). Poisoning caused by the consumption of organomercury-dressed seed in Iraq. Bulletin of the World Health Organization, 54(1), 101–112.
[13] Sone, Y., Nakamura, R., Pan-Hou, H., Itoh, T., & Kiyono, M. (2013). Role of MerC, MerE, MerF, MerT, and/or MerP in resistance to mercurials and the transport of mercurials in escherichia coli. Biological and Pharmaceutical Bulletin, 36(11), 1835–1841. https://doi.org/10.1248/bpb.b13-00554
[14] Sone, Y., Nakamura, R., Pan-Hou, H., Sato, M. H., Itoh, T., & Kiyono, M. (2013). Increase methylmercury accumulation in arabidopsis thaliana expressing bacterial broad-spectrum mercury transporter MerE. AMB Express, 3(1), 1–8. https://doi.org/10.1186/2191-0855-3-52
[15] Thackray, C. P., & Sunderland, E. M. (2019). Seafood methylmercury in a changing ocean. In A. M. Cisneros-Montemayor, W. W. L. Cheung, & Y. B. T.-P. F. O. Ota (Eds.), Predicting Future Oceans: Sustainability of Ocean and Human Systems Amidst Global Environmental Change (pp. 61–68). Elsevier. https://doi.org/10.1016/B978-0-12-817945-1.00006-X
[16] https://en.wikipedia.org/wiki/Minamata_disease
[17] https://www.phe.gov/s3/BioriskManagement/biosafety/Pages/Biosafety-Levels.aspx
[18] https://consteril.com/biosafety-levels-difference/
[19] https://www.concordia.ca/content/dam/concordia/services/safety/docs/EHS-DOC-112_MercuryGuidelines.pdf
Sponsors
We thank the following organizations for their financial and material support, without which this project could not have come to fruition.