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− | 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]. | + | Methylmercury is a toxic, organometallic compound with the primary source of exposure to humans being consumption of food (primarily but not limited to fishes) contaminated with it [15]. |
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− | 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. Thus the end consumer can be severely affected [5]. | + | 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 then consumed by big fishes. At each level, the amount of toxic methylmercury dangerously increases, resulting in biomagnification. Thus the end consumer can be severely affected [5]. |
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− | Officially discovered in Minamata City, Japan in the year 1956, the Minamata disease was caused due to release of industrial wastewater which contained mercury, by the Chisso Corporation’s chemical factory. This began in the year 1932 and went on till 1968. The local population consumed shellfish and fishes from the Minamata Bay and the Shiranui Sea, which contained this highly toxic chemical due to bio-accumation, resulting in mercury poisoning. [6] | + | Officially discovered in Minamata City, Japan in the year 1956, the Minamata disease was caused due to release of industrial wastewater which contained mercury, by the Chisso Corporation’s chemical factory. This began in the year 1932 and went on till 1968. The local population consumed shellfish and fishes from the Minamata Bay and the Shiranui Sea, which contained this highly toxic chemical due to bio-accumulation, resulting in mercury poisoning. [6] |
| <br> | | <br> |
| 2,265 victims have been officially recognised as of March 2001, 1,784 of whom had died. Chisso has provided financial compensation to over 10,000 persons. [16] | | 2,265 victims have been officially recognised as of March 2001, 1,784 of whom had died. Chisso has provided financial compensation to over 10,000 persons. [16] |
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− | 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% fatalities of the reported cases. Many children were affected, which affected the CNS and caused slower development. [2][12] | + | In 1971, a large outbreak of methylmercury poisoning took place in Iraq due to consumption of grain that was treated with a methylmercury based fungicide. Nearly one-sixth of the rural population was admitted to hospitals for cases of methylmercury poisoning due to bio-accumulation in the grains. Pregnant females were at high risk with 45% fatality of the reported cases. Many children were affected, which affected the CNS and caused slower development [2][12]. |
| </div> | | </div> |
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| <center><img src="https://static.igem.org/mediawiki/2020/6/6b/T--MIT_MAHE--Poster_comic.png" style="width:600px;height:200px;"></center> | | <center><img src="https://static.igem.org/mediawiki/2020/6/6b/T--MIT_MAHE--Poster_comic.png" style="width:600px;height:200px;"></center> |
| <br><br> | | <br><br> |
− | 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. | + | 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 (MeHg) poisoning, an issue of global importance. As most of the 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. |
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| <center><img src="https://static.igem.org/mediawiki/2020/e/e9/T--MIT_MAHE--Poster_compare.png" style="width:300px;height:200px;"></center> | | <center><img src="https://static.igem.org/mediawiki/2020/e/e9/T--MIT_MAHE--Poster_compare.png" style="width:300px;height:200px;"></center> |
− | 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] | + | When methylmercury is ingested, nearly 95% of it gets absorbed by the gastrointestinal tract. It binds with proteins that contain cysteine and the complex thus formed is recognized by the body as methionine. This allows the methylmercuric-cysteine complex to pass through many biological barriers including the blood-brain barrier and the blood-placenta barrier, where it is absorbed by the developing foetus. [1] |
| <br><br> | | <br><br> |
| 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] | | 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] |
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− | A dual plasmid system is proposed as the bacteria's weapon 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 <i>Escherichia coli</i> Nissle 1917. | + | A dual plasmid system is proposed as the bacteria's weapon to break the carbon-mercury bond and prevent this toxic chemical to be absorbed by the gut. The probiotic bacteria we intend to use is genetically engineered <i>Escherichia coli</i> Nissle 1917. |
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| <center><img src="https://static.igem.org/mediawiki/2020/5/5f/T--MIT_MAHE--Poster_plasmids.png" style="width:600px;height:262px;"></center> | | <center><img src="https://static.igem.org/mediawiki/2020/5/5f/T--MIT_MAHE--Poster_plasmids.png" style="width:600px;height:262px;"></center> |
− | 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]. | + | In the absence of mercury, a repressor molecule encoded by the MerR gene is produced which binds to the PmerT promoter, and prevents further 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 genes downstream to it [3]. |
| <br> | | <br> |
− | MerT, MerP, MerE and MerC encode proteins which help in the efficient transport of methylmercury inside the cell[13][14]. | + | MerT, MerP, MerE and MerC encode proteins which help in the efficient transport of methylmercury inside the cell [13][14]. |
| <br> | | <br> |
− | 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 mercuric ions to elemental mercury [8][11]. This elemental mercury is eliminated out of the body with faeces. | + | A dual enzyme system is proposed as the working element to convert methylmercury to less toxic elemental mercury. MerB encodes organomercurial lyase which breaks the carbon-mercury bond and MerA encodes mercuric reductase which reduces mercuric ions to elemental mercury [8][11]. This elemental mercury is eliminated out of the body with faeces. |
| <center><img src="https://static.igem.org/mediawiki/2020/8/8a/T--MIT_MAHE--Poster_collage1.png" style="width:600px;height:200px;"></center> | | <center><img src="https://static.igem.org/mediawiki/2020/8/8a/T--MIT_MAHE--Poster_collage1.png" style="width:600px;height:200px;"></center> |
− | The release of elemental mercury has the potential to disturb the gut microbiota and induce inflammations. To tackle this issue, Composite Bio-Brick 2 is proposed. SoxR produced by Bio-Brick 1 controls the transcription of Bio-Brick 2 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, a type II secretion system, containing HlyB, HlyD and TolC is proposed to transport the Interleukin-10 (IL-10) fused to HlyA (a secretion signal peptide) outside the cell [4]. | + | The release of elemental mercury has the potential to disturb the gut microbiota and induce inflammations. To tackle this issue, composite Bio-Brick 2 is proposed. SoxR produced by composite Bio-Brick 1 controls the transcription of composite Bio-Brick 2 ensuring that it is dormant in the absence of mercury. Only in the simultaneous presence of mercury and Nitric oxide (a proinflammatory signal), will SoxR activate the SoxS promoter which initiates the transcription of genes in composite Bio-Brick 2 [7]. Hemolysin secretion system, a type II secretion system containing HlyB, HlyD and TolC, is proposed to transport the Interleukin-10 (IL-10) fused to HlyA (a secretion signal peptide) outside the cell [4]. |
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| <center><img src="https://static.igem.org/mediawiki/2020/c/c3/T--MIT_MAHE--Poster_collage2.png" style="width:600px;height:200px;"></center> | | <center><img src="https://static.igem.org/mediawiki/2020/c/c3/T--MIT_MAHE--Poster_collage2.png" style="width:600px;height:200px;"></center> |
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| <center><img src="https://static.igem.org/mediawiki/2020/1/18/T--MIT_MAHE--Poster_textbiobrick1.png" style="width:600px;height:337.5px;"></center> | | <center><img src="https://static.igem.org/mediawiki/2020/1/18/T--MIT_MAHE--Poster_textbiobrick1.png" style="width:600px;height:337.5px;"></center> |
| <br><br> | | <br><br> |
− | 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. | + | In order to validate different aspects of our proposed system, we have designed several experiments to ensure that the chassis and the two composite Bio-Bricks are working as theorized. |
| <br><br> | | <br><br> |
| We have designed several experiments to | | We have designed several experiments to |
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| <center><img src="https://static.igem.org/mediawiki/2020/e/ee/T--MIT_MAHE--Poster_textbiobrick2.png" style="width:600px;height:337.5px;"></center> | | <center><img src="https://static.igem.org/mediawiki/2020/e/ee/T--MIT_MAHE--Poster_textbiobrick2.png" style="width:600px;height:337.5px;"></center> |
| <br><br> | | <br><br> |
− | To ensure the safety and efficacy of Bio-Brick 2, we have designed several experiments to check | + | To ensure the safety and efficacy of composite Bio-Brick 2, we have designed several experiments to check |
| <ul> | | <ul> |
− | <li>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.</li> | + | <li>The relationship between the amount of oxidative stress and rate of transcription of the genes in response to Paraquat (methylviologen dichloride hydrate) which acts as an inducer, using a GFP reporter.</li> |
| <li>The amount of oxidative stress that is required to activate SoxR and in turn activate SoxS promoter.</li> | | <li>The amount of oxidative stress that is required to activate SoxR and in turn activate SoxS promoter.</li> |
| <li>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.</li> | | <li>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.</li> |
− | <li>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.</li> | + | <li>The efficiency of the type II secretion 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.</li> |
| <li>The overall working of Plasmid 2 and demonstrate its specificity to just mercury-induced inflammations.</li> | | <li>The overall working of Plasmid 2 and demonstrate its specificity to just mercury-induced inflammations.</li> |
| </ul> | | </ul> |
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− | 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. | + | 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 taking the product to the consumer. |
| <br><br> | | <br><br> |
− | 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 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 other upstream processes that are of importance prior to scale up. |
| <br><br> | | <br><br> |
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| <b>Model - 1</b> | | <b>Model - 1</b> |
| <br> | | <br> |
− | 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. | + | 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. |
| <br> | | <br> |
| Result: Optimal order of enzyme concentration to be 10<sup>-4</sup> (reduction rate - 6-7 hours) | | Result: Optimal order of enzyme concentration to be 10<sup>-4</sup> (reduction rate - 6-7 hours) |
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| <center><img src="https://static.igem.org/mediawiki/2020/c/c5/T--MIT_MAHE--Poster_equations.png" style="width:600px;height:200px;"></center> | | <center><img src="https://static.igem.org/mediawiki/2020/c/c5/T--MIT_MAHE--Poster_equations.png" style="width:600px;height:200px;"></center> |
| <br> | | <br> |
− | But due to the lack of information about the activities of the protein further analysis could not be performed. | + | But due to the lack of information about the activities of the protein, further analysis could not be performed. |
| <br><br> | | <br><br> |
| <b>VMD</b> | | <b>VMD</b> |
| <br> | | <br> |
− | 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. | + | 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 were used for the mercuric ion and cysteine (sulphur) interactions. |
| <br><br> | | <br><br> |
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| <i>Escherichia coli</i> DH5α and <i>Escherichia coli</i> Nissle 1917 - require Biosafety Level 1 (BSL-1) laboratory [17][18]. None of the experiments are expected to increase the pathogenicity of the bacteria. | | <i>Escherichia coli</i> DH5α and <i>Escherichia coli</i> Nissle 1917 - require Biosafety Level 1 (BSL-1) laboratory [17][18]. None of the experiments are expected to increase the pathogenicity of the bacteria. |
| <br><br> | | <br><br> |
− | To ensure safety, PPE (personal protective equipment) must always be worn while experimenting. (lab-coats, gas masks and Specialized gloves) [19]. | + | 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. |
− | Toxic chemicals must be disposed of safely in accordance with the guidelines. | + | |
| <br><br> | | <br><br> |
| <center><img src="https://static.igem.org/mediawiki/2020/7/7c/T--MIT_MAHE--Poster_safetyeqp.png" style="width:200px;height:200px;"></center> | | <center><img src="https://static.igem.org/mediawiki/2020/7/7c/T--MIT_MAHE--Poster_safetyeqp.png" style="width:200px;height:200px;"></center> |
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| 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. | | 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. |
| <br> | | <br> |
− | 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. | + | Extensive study has been conducted with respect to the safety considerations of the implementation of our project, including powder design, plant and personnel hygiene, cleaning, process design, packaging, waste management and recycling. |
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| </div> | | </div> |