Team:IISER-Kolkata
BacMan
A probiotic bacterial BATMAN to protect the citizens of Gotham from Arsenic Poisoning.
Though several remediation and decontamination techniques exist to remove Arsenic form water, yet Arsenic poisoning remains a severe ailment in highly affected regions such as West Bengal, India (the province of our Institute) and Bangladesh.
These cases of Arsenic toxicity arise due to heavy intake of the metal through food (rice, other cereals and vegetables) raised in Arsenic laden soil and water paddy. There are no preventive measures to these 'vectors' of the 'disease'.
We, Team IISER-Kolkata plan to design a probiotic bacteria that can efficiently intake and sequester Arsenic in the physicochemical conditions existing in the human gut.
Therefore, it will enable us to shield people highly sensitive to Arsenic from its poisoning using low cost but effective result yielding probiotic pill prescription.
We aim to show the proof of concept of our project in Escherichia coli and later move on to probiotic strains such as Lactobacillus acidophilus.
We also plan to design safe techniques to deliver the probiotic pill into the gastrointestinal tract where the bateria will compete with GI tract epithelium for Arsenic absorption.
Description:
Starting with analyzing and surveying of severe challanges in our locality but also of global concern, we came across the issue of Arsenic contamination of groundwater as the project problem. We are in the process of performing ground surveys, social awareness visits and interviews of experts to obtain meaningful data on this problem.
However, upon preliminary examination it has come to our notice that though water filtration and decontamination plants exist to remove Arsenic from potable water, yet use of untreated water for irrigation of food crops (rice) and/or raising fishes etc. leads to inclusion of the heavy metal into the staple diet of the locals. Arsenic ingestion through food causes severe poisoning and several long term effects even leading to cancer.
Present purification methods do not cover this side of the problem leaving people open to the risk of Arsenic toxicity. Team iGEM 2018 IISER Kolkata therefore plans to put synthetic biology to use to combat Arsenic toxicity mainly caused by heavy metal laden ingested food.
Our aim is to design a probiotic Arsenic sequestering bacteria which will be administered as an inexpensive pill to colonize gut and eliminate the toxic heavy metal entering through diet (mostly food).
We will first test the idea and establish proof of concept in the easy to work with gram negative E. coli species often called the lab rat of molecular biology and only then move on to working with gram positive Lactobacillus (probiotic species).
Significance:
1. Novel prevention based approach as a long term solution to Arsenic Poisoning in severely affected regions of the world.
2. The problem is severe and relatively undealt with. It is of particular importance in the province of our Institute (West Bengal, India), but is also present in other parts of the globe.
3. Our approach is innovative, cheap and effective.
Details:
Arsenic is present mostly in its toxic inorganic +3 and +5 ionic forms in water and food that manifest into the poisoning symptoms when ingested. These As+3 and As+5 ions naturally permeate into the bacterial cell through its Phosphate transporters. Most bacteria including E. coli and Lactobacillus however contain ars Operon that confers Arsenic tolerance to it principally by exporting back to the exterior the unintentionally intaken Arsenic ions.
This in built resistance mechanism of the bacterium is hampering to our plan as we intend to make the bacteria intake and sequester Arsenic within. Therfore, we will first try to delete arsA and arsB genes of the chromosomally located ars Operon using a gene deletion kit.
To sequester Arsenic within the bacteria, a Synthetic Phytochelatin under a strong T7 promoter will be used. However, to keep the entire circuit under the control of Arsenic exposure to the cell, the T7 RNA polymerase required for Phytochelatin gene transcription will itself be under arsR the promoter operator that is turned on by deinhibition of ars promoter by binding of As(III) and/or Ars(IV) to the ArsR (repressor).
Along with phytochelatin, we have planned to include a gene encoding HMT (heavy metal tolerance protein) from S. pombe and another encoding AOX (Arsenic Oxidase) from Thermus thermophilus to make an Arsenic Responsive Operon. HMT is known to transport Arsenic bound Phytochelatin to the periplasm in gram negative bacteria.
This will help reduce the cytosolic concentration of arsenic as well as phytochelatin driving the equilibrium to cause more Arsenic intake and Phytochelatin production. AOX is an outer membrane protein in gram negative bacteria which converts external As(III) to As(V). This will help to reduce the more toxic As(III) present in the gut into a relatively docile As(V) form.
Finally, we plan to design an effecient probiotic pill delivery system so that the bacteria can be accurately targetted to the small intestine where majority of the Arsenic absorption occurs. Through out the course of the project we will incorporate the data obtained through "Human Practices exercise" thus integrating newly obtained information with ongoing lab work for a more realistic achievement of our goals.
While bacman is gearing up to protect people from arsenic , we decided to pay a visit to the gfpexpressing bacteria. It s no doubt that measuring flourescen sounds exciting but even more appealing was the opportunity to be a part of a large community and share data.
So we prepared ourselves to answer the Intelab question : Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?
And then we started :
Day 1 : Transforming E coli DH5 alpha cells with the required biobricks.
Day 2: Checking plates . Some biobricks didn’t give enough colonies.
Day 3 : Transforming bacteria with 1.5 uL of biobricks. Checking plates. We had beautiful and ample colonies this time.
Day 4 : Performing all calibration protocols with ludox , silica beads , fluorescein.
Day 5 : Performed the cell measurements.
Day 6 : Repeated cell measurements.
Day 7 : Performing CFU protocol.
Day 8 : Counting colonies and updating excel files.
Fluorescence Raw Readings:
| Hour 0: | Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) |
|---|---|---|---|---|---|---|---|---|---|
| Colony 1, Replicate 1 | 3172 | 4940 | 7332 | 4129 | 3103 | 11501 | 9596 | 3409 | 3096 |
| Colony 1, Replicate 2 | 3052 | 4405 | 6672 | 4129 | 3096 | 11512 | 11568 | 3705 | 3093 |
| Colony 1, Replicate 3 | 3062 | 4513 | 8277 | 4535 | 3313 | 13552 | 9811 | 3612 | 3561 |
| Colony 1, Replicate 4 | 2939 | 4559 | 8165 | 4393 | 3302 | 12578 | 10198 | 3595 | 3501 |
| Colony 2, Replicate 1 | 2694 | 4122 | 9256 | 4855 | 3459 | 11239 | 7266 | 3883 | 3138 |
| Colony 2, Replicate 2 | 2569 | 3211 | 8933 | 4591 | 3636 | 11017 | 6864 | 3606 | 3429 |
| Colony 2, Replicate 3 | 3512 | 3431 | 9091 | 4728 | 3668 | 10965 | 7255 | 3981 | 3510 |
| Colony 2, Replicate 4 | 1739 | 3625 | 8312 | 4983 | 3606 | 11556 | 7160 | 3682 | 3439 |
| Hour 6: | Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) |
|---|---|---|---|---|---|---|---|---|---|
| Colony 1, Replicate 1 | 3452 | 52293 | 20259 | 33054 | 3866 | 30129 | 19804 | 11665 | 3751 |
| Colony 1, Replicate 2 | 3411 | 48522 | 18326 | 31173 | 3667 | 27049 | 14295 | 12347 | 3677 |
| Colony 1, Replicate 3 | 3662 | 48774 | 18739 | 32604 | 3646 | 26488 | 16048 | 11921 | 3409 |
| Colony 1, Replicate 4 | 3646 | 56393 | 21454 | 31670 | 3704 | 28766 | 16617 | 10720 | 3600 |
| Colony 2, Replicate 1 | 3618 | 35310 | 19407 | 67038 | 3680 | 21001 | 20176 | 11145 | 3641 |
| Colony 2, Replicate 2 | 4077 | 34070 | 21004 | 69337 | 4237 | 18555 | 19423 | 10521 | 3911 |
| Colony 2, Replicate 3 | 3509 | 34721 | 20843 | 62524 | 3723 | 17359 | 16708 | 11012 | 3134 |
| Colony 2, Replicate 4 | 3466 | 36496 | 17450 | 61077 | 3757 | 18339 | 16887 | 10866 | 3084 |
Abs600 Raw Readings:
| Hour 0: | Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) |
|---|---|---|---|---|---|---|---|---|---|
| Colony 1, Replicate 1 | 0.085 | 0.088 | 0.085 | 0.087 | 0.08 | 0.087 | 0.084 | 0.08 | 0.073 |
| Colony 1, Replicate 2 | 0.086 | 0.082 | 0.087 | 0.09 | 0.09 | 0.088 | 0.088 | 0.088 | 0.079 |
| Colony 1, Replicate 3 | 0.08 | 0.08 | 0.078 | 0.084 | 0.083 | 0.078 | 0.081 | 0.085 | 0.068 |
| Colony 1, Replicate 4 | 0.082 | 0.085 | 0.079 | 0.078 | 0.085 | 0.08 | 0.077 | 0.087 | 0.069 |
| Colony 2, Replicate 1 | 0.081 | 0.082 | 0.076 | 0.076 | 0.08 | 0.078 | 0.081 | 0.079 | 0.068 |
| Colony 2, Replicate 2 | 0.08 | 0.074 | 0.076 | 0.079 | 0.077 | 0.076 | 0.076 | 0.077 | 0.063 |
| Colony 2, Replicate 3 | 0.067 | 0.075 | 0.08 | 0.083 | 0.076 | 0.078 | 0.077 | 0.072 | 0.069 |
| Colony 2, Replicate 4 | 0.072 | 0.069 | 0.068 | 0.07 | 0.075 | 0.072 | 0.074 | 0.077 | 0.071 |
| Hour 6: | Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 | LB + Chlor (blank) |
|---|---|---|---|---|---|---|---|---|---|
| Colony 1, Replicate 1 | 0.325 | 0.334 | 0.092 | 0.393 | 0.39 | 0.162 | 0.098 | 0.352 | 0.064 |
| Colony 1, Replicate 2 | 0.319 | 0.316 | 0.091 | 0.382 | 0.383 | 0.152 | 0.081 | 0.372 | 0.062 |
| Colony 1, Replicate 3 | 0.346 | 0.329 | 0.09 | 0.388 | 0.382 | 0.145 | 0.087 | 0.368 | 0.062 |
| Colony 1, Replicate 4 | 0.344 | 0.349 | 0.096 | 0.386 | 0.388 | 0.155 | 0.082 | 0.341 | 0.059 |
| Colony 2, Replicate 1 | 0.353 | 0.318 | 0.076 | 0.531 | 0.379 | 0.137 | 0.129 | 0.346 | 0.056 |
| Colony 2, Replicate 2 | 0.368 | 0.311 | 0.082 | 0.533 | 0.409 | 0.133 | 0.132 | 0.334 | 0.059 |
| Colony 2, Replicate 3 | 0.338 | 0.312 | 0.076 | 0.527 | 0.385 | 0.149 | 0.14 | 0.355 | 0.076 |
| Colony 2, Replicate 4 | 0.338 | 0.328 | 0.084 | 0.514 | 0.387 | 0.156 | 0.137 | 0.349 | 0.076 |
Dr. Partho Sarothi Ray
Primary PI
Dr. Arnab Gupta
Secondary PI
Mr. Debabrato Sutradhar
Project Instructor
Paresh Priyadarshan Rana
The Team Leader
Animish Nilesh Vaze
The Skeptical Experimentalist
Diptatanu Das
The Skilled Microbiologist
Pallab Datta
The Intelligent Artist
Anmol Kumar
The Cheerful Professionalist
Ankit Amrutkar
The Righteous Physicist
Hrishika Rai
The Starved Biologist
Madhavi Andhari
The Confused Hardworker
Souvik Ghosh
The Drunk Craftsman
Rajalaxmi Saha
The Ghost
Pavan Vynatheya
describe yourself
Prajwal Padmanabha
describe yourself
Department of Biotechnology
Selected and Awarded Funds through iBEC (pre-iGEM India level competition)
GeneBridges
Company producing and distributing products and kits for use in Recombinant DNA technology
Integrated DNA Technologies
Official sponsor for all iGEM teams supplying customized synthesized gBlocks for upto 20kb genes
IISER Kolkata
Home Institute
Providing financial assistance to the team
New England Biolabs
Official sponsor for all iGEM teams supplying required Mol Bio Kits and chemicals
SnapGene
Company producing and distributing softwares for Molecular Biology
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Near Harringhata Farm
Mohanpur, Dist: Nadia
West Bengal, India-741246
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Dr. Partho Sarothi Ray
Primary PI
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Dr. Arnab Gupta
Secondary PI
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Paresh Priyadarshan Rana
Team Leader
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