Description

"The chronic arsenic poisoning in West Bengal represents the single largest environmental health problem the world has ever seen, other than that associated with the Chernobyl disaster"
                              - D. Chakraborti et. al., Elsevier, May 2002

Introduction

Arsenic has been called “The king of poisons” and “The poison of the kings” owing to its use by the ruling class to murder one another. A Group-A carcinogen on WHO list, arsenic in all forms is also ranked at the top of the list of hazardous substances by U.S. Agency for Toxic Substances and Diseases Registry. Now imagine if your food and drinking water had been contaminated with arsenic enough to kill you but slowly by gradual accumulation in your body over years, what a miserable living that would be! This is the plight of the millions of inhabitants in West Bengal, India and Bangladesh.

Origin of Arsenic in Bengal Alluvial Plains and Underground aquifers

The sedimentation of Arsenic on the plains of Bengal started 15,000 to 12,000 years ago with the melting of glaciers at the end of the Pleistocene epoch. The water on the Himalayan slopes eroded the exposed arsenic laden rocks from the Indus ophiolite belt and the high grade gneisses solvating the arsenic minerals and carrying them downstream along the Ganga and Brahmaputra rivers. Upon reaching the flat lands of Bengal, the river waters start deloading their carries depositing heavy alluvial sediments along with high concentration of eroded arsenic salts on these plains. When rain and accumulated water seeps into the ground through such arsenic contaminated soil layers, it solvates the inorganic salts on its way down to the aquifers. This in turn leads to arsenic pollution of groundwater aquifers with contamination increasing steadily over years as more deposits are laid upon by the rivers and more water seeps through the soil reaching the underground stores.

Problem severity in West Bengal

WHO allowed levels of Arsenic in drinking water for it to be deemed safe is 10μg/L, while the government national standards for India and Bangladesh are 50μg/L. In comparison to these recommended figures, let us compare the average arsenic concentrations in groundwater in the six most badly affected districts of West Bengal.

Ref: D. Das et. al., Arsenic in groundwater in six districts of West Bengal, India, Env. Geochem. And Health, 1996

District in West Bengal	Average Arsenic conc. In groundwater (in μg/L)	Range of Arsenic concentrations in samples (in μg/L)
North 24 Parganas	737	50 to 1250
South 24 Parganas	278	50 to 3700
Murshidabad	257	50 to 950
Nadia	241	50 to 1180
Malda	210	50 to 930
Bardhaman	193	50 to 640

The above chart is sufficient to paint a realistic and drastic picture of the severity of the arsenic contamination problem in West Bengal. It is also worth noting that our Institute IISER Kolkata is situated in the district Nadia, one of the most severely impacted regions in West Bengal.

Problem severity across the globe

Though of particular importance in India and Bangladesh, contamination of ground water by arsenic is a global issue with its presence being felt in over 66 countries of the world spanning all continents. Argentina, Chile, Mexico, USA and China are among the other major sufferers of the crisis along with India and Bangladesh. Our collaboration with iGEM TecCEM Mexico team has helped us get a hold on the arsenic contamination data in Mexico and Latin America improving our insights regarding the problem.

Ref: S. Shankar et.al., Arsenic contamination of groundwater, The Scientific World Journal, 2014

Country	Range of Arsenic concentrations in groundwater aquifers
Mexico	8 to 620 μg/L
China	50 to 4440 μg/L
USA	Upto 2600 μg/L
Brazil	0.4 to 350 μg/L
Vietnam, Thailand	1 to 5000 μg/L
Greece	Upto 10,000 μg/L

Data obtained from collaborator team TecCEM Mexico:

• Alluvial aquifers in northern and central mexico show highest levels of contamination.
• These regions are also amongst the most populated regions of Mexico leaving a large population vulnerable to water extracted from these polluted groundwater stores.
• The Arsenic concentration in these Mexican aquifers varies from 3 to 443 μg/L while more than 50% samples had arsenic levels above the national standard of 50μg/L.

Fixating on the Problem

Therefore, team IISER Kolkata chose to work on the problem of “Arsenic contamination of groundwater and resulting health crisis due to poisoning” due to its high relevance and importance especially in the region where our institute is located as well as in other parts of the world. In this problem we could see the exciting challenge of putting our knowledge and skills to use in solving something really pressing for the common people around us while by large, still staying committed to the needs of a global community facing the same issue.

Existing solutions and their shortcomings

Although arsenic contamination is still a pertinent problem, people have been trying to address it in the ways listed below. Also described are the shortcomings of each of the previously existing solutions to counter this issue:

Arsenic Removal Technique	Description	Shortcomings
Chemical Oxidation	Process is complex and produces difficult to dispose As rich residue.	Oxidise highly toxic and difficult to separate As(III) form to docile and easily separable As(V) form followed by further treatment, filtration or flocculation.
Coagulation and Flocculation	Leads to secondary pollution of the environment due to problems involved in disposal of Arsenic laden sludge whose management is difficult and expensive.	Addition of coagulants like FeCl3 agglutinates colloidal suspension of Arsenic ions in water and further flocculation by adding substances like alum produces an easily separable sludge.
Selective Filtration	Membrane filters need constant pH maintenance and reagent refilling. Also they are slow due to long time needed for conditioning.	Several filtration techniques make use of sieves and membranes that filter out arsenic based on size and/or charge. Diatomaceous Earth matrix is also used as a filter.
Adsorption on Surfaces	Specificity in action of these units is low and have problems with regeneration of the adsorbent material after repeated uses.	Certain material surfaces such as that of Coconut Shell Carbon etc. can be activated to bind and adsorb Arsenic ions due to their chemical affinity.

Apart from the above mentioned drawbacks of each of the existing techniques there are certain shortcomings common to all:

• Expensive Kits not affordable to the majority of suffering households in West Bengal and Bangladesh.
• Kits and Technologies are mostly developed in U.S. or abroad and therefore do not suit the specific requirements of India and Bangladesh. Indigenously researched technologies for Arsenic removal are mostly in the process of development and scaling up to be released as products.

Ref: Technologies for Arsenic Removal from Water, Nicomel N. R., et. al., Int. Journal of Environmental Research and Public Health, 2016

Arsenicosis

Consumption of arsenic contaminated food and water over long term of 5 to 20 years leads to accumulation of the heavy metal in body tissues starting from hair to skin epidermis causing several dreadful symptoms such as:

1. Hair loss
2. Hyperpigmentation (darkening of the skin)
3. Bowen’s lesions
4. Peripheral neuropathy
5. Diabetes
6. Cardiovascular diseases
7. Still-births to affected pregnant women
8. Expensive Kits not affordable to the majority of suffering households in West Bengal and Bangladesh.
9. Cancers of skin, bladder, kidney and lung

Now one can filter groundwater before drinking. However, substantially high amount of As is also ingested through food. When contaminated groundwater is used to irrigate fields of crop plants such as rice or to fill ponds to rear fishes, the heavy metal enters their living tissues and starts getting accumulated therein. Later, when these cereals or fishes enter the human food chain they lead start being absorbed into the human body and getting accumulated in human tissues which later manifest into the symptoms of arsenicosis.

Based on the study conducted in high As contamination Nadia district of West Bengal, India:

Ref: Dietary Arsenic Exposure in West Bengal

Food Item	Mean Arsenic Content (in μg/Kg)
Raw rice grains	315
Cooked Rice	97
Cooked Fish	96
Cooked Egg	191
Chapati (Wheat Tortilla)	171
Fruit	20
Milk	60
Cooked Meat	29

Ref: Dietary Arsenic Exposure and Biomarkers

Arsenic Intake	Mean Value
Daily intake through diet	164 μg/day
Daily intake through water and diet	349 μg/day
Total As dose through diet	3.37 μg/Kg/day
Total As through water and diet	6.93 μg/Kg/day

From the above tabular representation of the data, it is clear that food is a major contributor of As ingestion along with contaminated water. Currently, no techniques exist to combat this pathway of As entry into human body due to lack of feasibility of filtering out As from large amounts of water used to raise food crops and livestock. Also, it is very alarming that Rice and Fish both of which form the staple food of a large population in Bengal, have the highest levels of Arsenic accumulation among all food items and hence pose a great risk to the health of these people.
Assuming on average 450g of cooked rice and 4L of water is consumed daily by an adult in West Bengal, 550 ppb and 110 ppb of As gets ingested daily through these respective sources.

Ref: What are Safe Levels of Arsenic in Food and Soils?, Duxbury and Zavala, Cornell Univ

The median excess internal cancer risk parameter is a measure of the likelihood of cancer in a population. This value is 0.7 per 10,000 Italians as compared to 22 per 10,000 for residents of Bangladesh.

Ref: Arsenic and Food Chain

Therefore, Team IISER Kolkata wanted to develop a novel and prevention based approach to battle Arsenicosis by targeting its intake from diet as well as water by engineering a probiotic bacterial system to compete with gut epithelium to uptake ingested Arsenic.

<section class="subpage" id="problem">

Problem

"The chronic arsenic poisoning in West Bengal represents the single largest environmental health problem the world has ever seen, other than that associated with the Chernobyl disaster"</br>                               - D. Chakraborti et. al., Elsevier, May 2002

Introduction

Arsenic has been called “The king of poisons” and “The poison of the kings” owing to its use by the ruling class to murder one another. A Group-A carcinogen on WHO list, arsenic in all forms is also ranked at the top of the list of hazardous substances by U.S. Agency for Toxic Substances and Diseases Registry. Now imagine if your food and drinking water had been contaminated with arsenic enough to kill you but slowly by gradual accumulation in your body over years, what a miserable living that would be! This is the plight of the millions of inhabitants in West Bengal, India and Bangladesh.

Origin of Arsenic in Bengal Alluvial Plains and Underground aquifers

The sedimentation of Arsenic on the plains of Bengal started 15,000 to 12,000 years ago with the melting of glaciers at the end of the Pleistocene epoch. The water on the Himalayan slopes eroded the exposed arsenic laden rocks from the Indus ophiolite belt and the high grade gneisses solvating the arsenic minerals and carrying them downstream along the Ganga and Brahmaputra rivers. Upon reaching the flat lands of Bengal, the river waters start deloading their carries depositing heavy alluvial sediments along with high concentration of eroded arsenic salts on these plains. When rain and accumulated water seeps into the ground through such arsenic contaminated soil layers, it solvates the inorganic salts on its way down to the aquifers. This in turn leads to arsenic pollution of groundwater aquifers with contamination increasing steadily over years as more deposits are laid upon by the rivers and more water seeps through the soil reaching the underground stores.

Problem severity in West Bengal

WHO allowed levels of Arsenic in drinking water for it to be deemed safe is 10μg/L, while the government national standards for India and Bangladesh are 50μg/L. In comparison to these recommended figures, let us compare the average arsenic concentrations in groundwater in the six most badly affected districts of West Bengal.

<thead>

</thead> <tbody>

</tbody>

Ref: D. Das et. al., Arsenic in groundwater in six districts of West Bengal, India, Env. Geochem. And Health, 1996

District in West Bengal	Average Arsenic conc. In groundwater (in μg/L)	Range of Arsenic concentrations in samples (in μg/L)
North 24 Parganas	737	50 to 1250
South 24 Parganas	278	50 to 3700
Murshidabad	257	50 to 950
Nadia	241	50 to 1180
Malda	210	50 to 930
Bardhaman	193	50 to 640

The above chart is sufficient to paint a realistic and drastic picture of the severity of the arsenic contamination problem in West Bengal. It is also worth noting that our Institute IISER Kolkata is situated in the district Nadia, one of the most severely impacted regions in West Bengal.

Problem severity across the globe

Though of particular importance in India and Bangladesh, contamination of ground water by arsenic is a global issue with its presence being felt in over 66 countries of the world spanning all continents. Argentina, Chile, Mexico, USA and China are among the other major sufferers of the crisis along with India and Bangladesh. Our collaboration with iGEM TecCEM Mexico team has helped us get a hold on the arsenic contamination data in Mexico and Latin America improving our insights regarding the problem.

<thead>

</thead> <tbody>

</tbody>

Ref: S. Shankar et.al., Arsenic contamination of groundwater, The Scientific World Journal, 2014

Country	Range of Arsenic concentrations in groundwater aquifers
Mexico	8 to 620 μg/L
China	50 to 4440 μg/L
USA	Upto 2600 μg/L
Brazil	0.4 to 350 μg/L
Vietnam, Thailand	1 to 5000 μg/L
Greece	Upto 10,000 μg/L

Data obtained from collaborator team TecCEM Mexico:

• Alluvial aquifers in northern and central mexico show highest levels of contamination.
• These regions are also amongst the most populated regions of Mexico leaving a large population vulnerable to water extracted from these polluted groundwater stores.
• The Arsenic concentration in these Mexican aquifers varies from 3 to 443 μg/L while more than 50% samples had arsenic levels above the national standard of 50μg/L.

Fixating on the Problem

Therefore, team IISER Kolkata chose to work on the problem of “Arsenic contamination of groundwater and resulting health crisis due to poisoning” due to its high relevance and importance especially in the region where our institute is located as well as in other parts of the world. In this problem we could see the exciting challenge of putting our knowledge and skills to use in solving something really pressing for the common people around us while by large, still staying committed to the needs of a global community facing the same issue.

Existing solutions and their shortcomings

Although arsenic contamination is still a pertinent problem, people have been trying to address it in the ways listed below. Also described are the shortcomings of each of the previously existing solutions to counter this issue:

<thead>

</thead> <tbody>

</tbody>

Arsenic Removal Technique	Description	Shortcomings
Chemical Oxidation	Process is complex and produces difficult to dispose As rich residue.	Oxidise highly toxic and difficult to separate As(III) form to docile and easily separable As(V) form followed by further treatment, filtration or flocculation.
Coagulation and Flocculation	Leads to secondary pollution of the environment due to problems involved in disposal of Arsenic laden sludge whose management is difficult and expensive.	Addition of coagulants like FeCl3 agglutinates colloidal suspension of Arsenic ions in water and further flocculation by adding substances like alum produces an easily separable sludge.
Selective Filtration	Membrane filters need constant pH maintenance and reagent refilling. Also they are slow due to long time needed for conditioning.	Several filtration techniques make use of sieves and membranes that filter out arsenic based on size and/or charge. Diatomaceous Earth matrix is also used as a filter.
Adsorption on Surfaces	Specificity in action of these units is low and have problems with regeneration of the adsorbent material after repeated uses.	Certain material surfaces such as that of Coconut Shell Carbon etc. can be activated to bind and adsorb Arsenic ions due to their chemical affinity.

Apart from the above mentioned drawbacks of each of the existing techniques there are certain shortcomings common to all:

• Expensive Kits not affordable to the majority of suffering households in West Bengal and Bangladesh.
• Kits and Technologies are mostly developed in U.S. or abroad and therefore do not suit the specific requirements of India and Bangladesh. Indigenously researched technologies for Arsenic removal are mostly in the process of development and scaling up to be released as products.

Ref: Technologies for Arsenic Removal from Water, Nicomel N. R., et. al., Int. Journal of Environmental Research and Public Health, 2016

Arsenicosis

Consumption of arsenic contaminated food and water over long term of 5 to 20 years leads to accumulation of the heavy metal in body tissues starting from hair to skin epidermis causing several dreadful symptoms such as:

1. Hair loss
2. Hyperpigmentation (darkening of the skin)
3. Bowen’s lesions
4. Peripheral neuropathy
5. Diabetes
6. Cardiovascular diseases
7. Still-births to affected pregnant women
8. Expensive Kits not affordable to the majority of suffering households in West Bengal and Bangladesh.
9. Cancers of skin, bladder, kidney and lung

Now one can filter groundwater before drinking. However, substantially high amount of As is also ingested through food. When contaminated groundwater is used to irrigate fields of crop plants such as rice or to fill ponds to rear fishes, the heavy metal enters their living tissues and starts getting accumulated therein. Later, when these cereals or fishes enter the human food chain they lead start being absorbed into the human body and getting accumulated in human tissues which later manifest into the symptoms of arsenicosis.

Based on the study conducted in high As contamination Nadia district of West Bengal, India:

<thead>

</thead> <tbody>

</tbody>

Ref: Dietary Arsenic Exposure in West Bengal

Food Item	Mean Arsenic Content (in μg/Kg)
Raw rice grains	315
Cooked Rice	97
Cooked Fish	96
Cooked Egg	191
Chapati (Wheat Tortilla)	171
Fruit	20
Milk	60
Cooked Meat	29

<thead>

</thead> <tbody>

</tbody>

Ref: Dietary Arsenic Exposure and Biomarkers

Arsenic Intake	Mean Value
Daily intake through diet	164 μg/day
Daily intake through water and diet	349 μg/day
Total As dose through diet	3.37 μg/Kg/day
Total As through water and diet	6.93 μg/Kg/day

From the above tabular representation of the data, it is clear that food is a major contributor of As ingestion along with contaminated water. Currently, no techniques exist to combat this pathway of As entry into human body due to lack of feasibility of filtering out As from large amounts of water used to raise food crops and livestock. Also, it is very alarming that Rice and Fish both of which form the staple food of a large population in Bengal, have the highest levels of Arsenic accumulation among all food items and hence pose a great risk to the health of these people.
Assuming on average 450g of cooked rice and 4L of water is consumed daily by an adult in West Bengal, 550 ppb and 110 ppb of As gets ingested daily through these respective sources.

Ref: What are Safe Levels of Arsenic in Food and Soils?, Duxbury and Zavala, Cornell Univ

The median excess internal cancer risk parameter is a measure of the likelihood of cancer in a population. This value is 0.7 per 10,000 Italians as compared to 22 per 10,000 for residents of Bangladesh.

Ref: Arsenic and Food Chain

Therefore, Team IISER Kolkata wanted to develop a novel and prevention based approach to battle Arsenicosis by targeting its intake from diet as well as water by engineering a probiotic bacterial system to compete with gut epithelium to uptake ingested Arsenic.

</section>

<section class="subpage" id="bacman">

BacMan

<img id="baclogo" src=""/>

Introducing to you BacMan, protagonist of Team IISER Kolkata’s project. BacMan is an engineered bacterium designed to uptake and sequester arsenic from the ingested food inside the gut at the prevailing physiological conditions. The bacteria will be delivered through a probiotic pill into the GI tract. Probiotic treatment of Arsenicosis with engineered bacteria beneficial and suitable to colonize the gut presents a novel prevention based approach to combat the disease. The bacteria upon serving their purpose will be flushed out from the body by the innate excretory system along with faeces.

The primary aim of Team IISER Kolkata is to establish the proof of concept in the easy to work with model organism E. coli and then proceed to translate the results to a probiotic species such as Lactobacillus spp.

BacMan’s Superpower and Toolset

In order to successfully accomplish the desired purpose, BacMan has to have a set of diverse tools:

1. Innate Arsenic Resistance Mechanism of the Bacteria
   Most bacterial species have an arsenic efflux system that makes them resistant to it as all of the uptaken ions are actively transported out of the cell. Allowing wild type functioning of this efflux system is discouraging to our ambitions of designing a bacteria that can uptake and keep the arsenic ions sequestered within. Hence, the genes coding for this efflux pathway need to be deleted.
2. Arsenic sensing unit
   The bacterium must be able to sense to Arsenic concentration outside and inside the cell so that it can launch a response to uptake and sequester it only when substantially high amounts of the toxic ion are present. A constitutively on response from the bacteria will be an extra stress for the cellular metabolism of the microbe.
3. Arsenic responsive unit
   Upon sensing arsenic concentrations beyond threshold, the bacteria must be able to switch on its response circuit.
4. Arsenic uptake channels and transporters
   The bacterium must be able uptake arsenic once ample amounts are sensed in the surrounding.
5. Arsenic sequestration response
   Once the arsenic starts pouring into the bacterial cell and it’s response circuitry is switched on, an arsenic sequestering protein must be produced to chelate the heavy metal ions.
6. Bacterial recovery unit
   Binding of ligand to any protein is a reversible process and as the sequestering proteins start getting saturated within the cell, the backward reaction will be favored and chelated arsenate and arsenite ions will dissociate and diffuse freely within the cell. These ions are toxic as they bind nonspecifically to several important enzymes and proteins involved in bacterial metabolism thereby disrupting their natural function. Therefore, there has to be a system within the bacterial cell that will recover it from the saturation reached and keep pushing the equilibrium towards the forward direction making the sequestration biphasic.

Detailed explanation of the components of the BacMan

1. Verifying presence of and thereafter deleting ars Operon
   Most bacteria have a chromosomal or plasmid encoded arsenic resistance operon arsRDABC. arsC converts As(V) in the cell to As(III). Genes arsA and arsB of the operon code for an ATP driven arsenite pump that then extrude As(III) out of the cell. arsR and arsD are regulatory proteins that control the expression of the operon.
   Ref: The ars Operon of Escherichia coli Confers Arsenical and Antimonial Resistance, Carlin et. al., Journal of Bacteriology, 1995
   This resistance system encoded by the operon effluxes out the arsenic ions and is hence undesirable in a bacterial strain being designed to to sequester arsenite and arsenate. Therefore, the primary requirement is to verify the presence of ars operon and if present, delete it.
2. Arsenic channels and transporters
   Arsenic exists in two oxidation states As(III) and As(V). Pentavalent As(V) exists as an oxyanion AsO4 3- which is chemically very similar to phosphate and hence is taken up nonspecifically through Pst and Pit and phosphate transporters in bacterial cells. Trivalent As(III) is found as neutral hydroxide As(OH)3 which structurally mimics Glycerol. Thus AQP channels such as GlpF membrane protein that are actually conduits with large pores that facilitate bidirectional diffusion of water, glycerol and other neutral solutes also allow As(OH)3 through them.
   Ref: Pathways of Arsenic Uptake and Efflux, H.C. Yang et. al., Curr. Topics in Membranes, 2015 <img class="" src=""/> The above image depicts the mechanism of action of ars Operon which imparts arsenic resistance to the bacterial cell by causing its efflux. Also shown are the pathways of arsenate and arsenite uptake by the cell through phosphate channels and GlpF aquaporins.
3. Arsenic Sensing and Responsive Unit
   After we delete the innate efflux mechanism and enhance arsenic uptake through above mentioned pathways, we plan to create a genetic circuitry that will respond to the building internal concentration of arsenite and arsenate ions and produce proteins that will chelate and keep it sequestered. An overview of the design is nicely provided by the image below.
   <img class="" src=""/>
   1. pArsR
      The first element of the circuit will be a promoter that can respond to the arsenite as well as arsenate concentrations within the cell. BBa_J33201 is a well characterized biobrick that which provides for a promoter whose transcription activity responds to the concentrations of either of arsenate and arsenite with 1uM being sufficient to induce full expression of the downstream protein. ArsR codes for an autorepressor which gets inhibited by presence of Arsenic ions allowing transcription from the promoter to proceed.
   2. T7 RNA pol
      Our planned response circuit is a very long cassette and hence transcription of such a long operon would require a very processive polymerase enzyme. Therefore, instead of directly placing the response cassette under pArs, we have first placed expression of T7 RNA pol under pArs so that upon induction by arsenic presence the circuit will first produce T7 RNA polymerase capable to transcribe the downstream operon processively.
      Biobrick Bba_K145001 provides us with such a required gene.
   3. pT7
      As we are using T7 RNA polymerase to transcribe our response cassette genes, it is imperative that the entire response cassette be placed under the control of a strong T7 promoter.
   4. Synthetic Phytochelatin
      The first major protein produced from the device will be Synthetic phytochelatin. Phytochelatins are glutathione oligomers produced by an enzymatic cascade in algae and other plants to chelate and thereby detoxify heavy metals. iGEM Repository provides for a synthetic phytochelatin coding biobrick Bba_K1321101 expressible under pT7 which serves the desired purpose. The need of the enzymatic cascade is bypassed as the protein can now directly be produced from the processes of central dogma.
4. Bacterial Recovery Unit
   As mentioned previously, our mathematical modelling provided us with the insight that the system we have planned will soon saturate. Therefore, a need for a mechanism that can recover the bacterial system from this saturation and push the equilibrium towards uptake of more arsenic, production of more synPC and chelation of more arsenic was felt. Hence, we researched the literature and came across a protein known as HMT that can come to rescue as required. We therefore decided to incorporate HMT in our design.
   HMT1 (Heavy metal transferase 1) is a vacuolar membrane protein from yeast (S. cerevisiae) that is known to transport Cadmium bound synPC complexes from the cytoplasm to the vacuoles. Its heterologous expression in E. coli causes it to be sorted to the inner membrane of the cell wherein it functions to translocate synPC-heavy metal complex to the periplasmic space as no vacuoles are present in bacteria. Interacton of HMT1 with Arsenic bound PC is not yet studied. Our hypothesis is that HMT1 will also work to translocate synPC-As complexes similar what it does with synPC-Cd complexes. This hints at the possibility that HMT1 expression in our system will reduce the overall free synPC, free As ions and synPC-As complex concentration in the cytoplasm thereby pushing the equilibrium forward towards more uptake and more sequestration due to Le Chatelier’s principle. Our mathematical model confirms the same.
   Ref: Transport of Metal-binding Peptides by HMT1, a fission yeast ABC type vacuolar membrane protein, Ortiz et. al., JBC 1995
5. A gift in disguise
   The team realizes the limitations of its planned system in successfully sequestering the arsenic in the gut at substantial amounts to make a difference to the suffering patient. It is quite possible that the probiotic bacterial pill might not be able to uptake and sequester large amounts of arsenic from the gut due to saturation. Realizing this, we thought of incorporating in out design a protein that can at least leave the arsenic in the surrounding gut in a less docile form if not completely remove it by sequestration.
   Arsenite oxidase (aox) is an outer membrane protein found in several B-Proteobacterial species that oxidizes the ions in the surrounding media from As(III) form to As(V) form. It is well known that in humans As(III) is highly toxic as compared to As(V). Also, the rate of absorption at the GI epithelium is also greater for As(III) than As(V). Therefore, the greater responsibility of causing arsenicosis is on As(III) than on As(V). Hence, presence of outer membrane protein aox in our system will help to reduce the net concentration of As(III) in the gut by converting it to less toxic As(V) form.
   Ref: Arsenite Oxidase (aox) genes from metal resistant B-Proteobacterium, Muller et. al., Journal of Bacteriology, 2003
   Note: HMT1 and aox will be in the same operon as synPC all being expressed undet pT7. HMT1 and aox will be IISER Kolkata’s new contributions to the repository.

</section>

<section class="subpage" id="mathMod">

Math modelling

</section>