Team:IISc-Bangalore/Evaluate
Step 4: Evaluate and Iterate
It is important not just to take stakeholders into confidence while designing a solution but also to sustain stakeholder interactions at all stages of the project and understand the ways in which we could integrate their suggestions into our project. By engaging with other key players for our project, we could get our project evaluated through their lens and appreciate the various dimensions that our project could potentially have. The stakeholders whom we met in this regard are enlisted below:
Meeting Prof. T V Ramachandra
Prof. T V Ramachandra is a professor at the Centre of Ecological Sciences at IISc. At present, he is the Coordinator of Energy and Wetlands Research Group (EWRG) and the Convener of Environmental Information System (ENVIS) at Centre for Ecological Sciences (CES). During the past fifteen years he has established an active school of research in the area of energy and environment. He was a Member of Karnataka State level Environment Expert Appraisal Committee (2007-2010), appointed by the Ministry of Environment and Forests, Government of India and a member of Western Ghats task force appointed by the Government of Karnataka. TVR's research interests are in the area of energy systems, renewable energy, energy conservation, energy planning, aquatic ecosystems, biodiversity, EIA, valuation of ecosystems, environment auditing, ecological modelling, geoinformatics, urban planning, carbon footprint and environmental engineering education research.
He provided us with a fresh perspective on how we should design our project so that the end-result may be a scalable, affordable, and effective device, and not just restricted to a proof-of-concept (POC). He further gave us ideas on possible locations where a fully functioning model of our design could be installed.
Some key points of our discussion with him are enumerated below:
- Runoff is a distributed source of pollution. In monsoon, distributed runoff can't be channelized appropriately, and necessary residence time criteria might not be met. Thus, it is best to get a topological map of the region, identify large depressions where the runoff is most probable to collect and install our system there.
- It is best to not use commercially available enzymes.
- One should not be limited merely by the POC study. One must deliver science to the doorsteps of common people. We should thus focus on devising an effective, scalable yet low-cost solution.
Our interaction with Prof. Ramachandra provided us newer ideas on designing the filter and translating it to the real world. Based on his suggestions, we decided to try to design a prototype of the filter. Further details on our attempts at prototype designing have been enlisted below.
Meeting Mr. Amit Khurana
To understand the impact that organophosphates have had on society across India, we reached out to Dr. Sunita Narain, the Director of the Centre for Science and Environment (CSE). Centre for Science and Environment (CSE) is a public interest research and advocacy organisation based in New Delhi. CSE researches into, lobbies for and communicates the urgency of development that is both sustainable and equitable.
Dr. Narain connected us to Mr. Amit Khurana, the Program Director for Food Safety and Toxins. In a very illuminating conversation, Mr. Khurana talked to us about CSE's long-standing research on the toxic effects of pesticides and their impact on the rural populace of India. From Mr. Khurana, we learnt about the fact that the ban on some Class Ia organophosphates which we had read about was a draft notification and had ultimately been withdrawn. Mr. Khurana was also kind enough to share with us the reports of CSE's Pollution Monitoring Laboratory, an independent, analytical laboratory that monitors toxic contamination of the environment and uses the results of this monitoring to advocate for improved regulation of the use of toxins in the country.
CSE's Pollution Monitoring Laboratory had carried out Analysis of pesticide residues in blood samples from villages of Punjab in 2005. As discussed previously, Punjab was the "Ground Zero" of India's Green Revolution. PML scientists visited Punjab and collected blood samples from 20 randomly selected people from 4 different villages of Punjab - Mahi Nangal, Jajjal and Balloh in Bhatinda district and Dher in Ropar district. Among fourteen oragnophosphate pesticides analysed in whole blood samples four were commonly detected - monocrotophos, chlorpyrifos, malathion and phosphamidon. Monocrotophos was detected in 75% of the whole blood samples analysed from Punjab villages at mean levels of 0.0948 mg/L , with the highest detected level being 0.4915 mg/L. Similarly, chlorpyrifos was detected in 85% of the samples, while malathion and phosphamidon was detected in 70% of the samples.
The report also notes that mean levels of total organochlorines in whole blood samples from Punjab were 0.1424 mg/L while total organophosphorus was 0.2278 mg/L. Furthermore, the CSE PML's report notes that "Major contribution to total pesticide concentration in blood samples from Punjab is of organophosphorus pesticides. Presence of organophosphorus pesticides in blood means that they do persist in the body for good amount of time. It also indicates the presence in the body of the pesticide in its form as a primary compound." Furthermore, another CSE report found malathion in 29 out of 34 samples of bottled water and chlorpyrifos in 28 out of 34 samples of bottled water isolated from various parts of India.
Mr. Khurana made us aware of the policy aspects of our project. He suggested us to think about the threshold concentration of organophosphates which must be present in a sample of water so that there is a detectable reduction in toxin load. He also asked us to think about the potential application of our filter at the household level for obtaining clean drinking water.
The meeting with Mr. Khurana helped us understand about the dimensions of our project at the science-society-policy interface. It inspired us to try to model the minimum detectable concentration of pesticides which our filter would be able to reduce at a detectable level (say > 80%). His wholesome evaluation of our project also affirmed the impact of the problem we had chosen to tackle and made us surer about our objectives and goals.
Integrating advice: Wet Lab experts
Once we decided on our project and a rough plan for the constructs, we integrated the advice of umpteen number of experts for different aspects of our project.
We contacted Dr. Saravanan Palani for suggestions on fusion protein design. Dr. Palani is an expert in synthetic cell biology, and had joined the Dept. Of Biochemistry of IISc a few months back when we approached in. He gave us necessary inputs on how to keep the protein domains separate and how we could design our experiments to appropriately address the problem at hand. Given his enormous expertise in designing fusion proteins, we requested Dr. Palani to be a mentor for our team, a proposal to which he kindly consented.
Furthermore, we then met Prof. D.N. Rao, a Professor Emeritus at the Dept. of Biochemistry of IISc. Prof. Rao has been a protein biochemist all through his life. His lab studies DNA-protein interactions in various model systems. We approached Prof. Rao for his inputs. He suggested us to focus on how we could design assays to validate our claims experimentally, at least on a proof-of-concept level. Prof. Rao also put us in touch with Prof. Dayanand Siddavattam of the School of Life Sciences, University of Hyderabad. Prof. Dayanand has worked extensively on phosphotriesterases (PTEs) and provided us inputs on designing assays. We focussed on creating an assay which is cheap and simple to execute. Spectrophotometric assays were therefore chosen for our project.
Besides them, we have also sought the help of Dr. Jayanta Chatterjee (Associate Professor, Molecular Biophysics Unit, IISc) and Prof. Raghavan Varadarajan (Professor, Molecular Biophysics Unit, IISc). Both of them are structural biologists by practice, working on peptides and proteins. On hearing about the bottleneck in our project regarding C2 (see Results for details), Dr. Chatterjee suggested us to delete sfGFP as GFP has been shown to adversely impact the solubility of proteins in some cases. He also suggested us to perform assays with whole cell lysate before proceeding to purification. This suggestion was of great help to us as the whole cell lysate performed by us confirmed that some functional enzyme was really being produced and the bottleneck was being caused by issues concerning the protein solubility and stability.
On examining our experimental protocols, Prof. Varadarajan suggested us to delete the strongly hydrophobic signal sequence of OpdA from our construct. Reviewing corresponding scientific literature also pointed out the fact that the signal sequence was in fact deleted in cases where the protein had been isolated for crystallography. Based on his suggestion, we decided to model these fusion proteins using AlphaFold and investigate if there were issues with protein folding caused by the signal peptide. The structural predictions indeed confirmed this hypothesis. The signal peptide was found to be an IDR (intrinsically disordered region), which are known to be causes of protein aggregation and concomitant degradation in multiple cases. We integrated Prof. Varadarajan's suggestion by deleting the signal sequence and creating C2 (3.0), whose structural predictions were significantly satisfying. We are currently working on using C2 (3.0) for our filter.
In the meantime, as regards the production of bacterial cellulose, we contacted Dr. Mudrika Khandelwal (Associate Professor, Dept. of Materials Science and Engineering, IIT Hyderabad). She has extensively worked on bacterial cellulose, its properties and manipulating it for multifarious applications. Dr. Khandelwal connected us to Dr. Shivakalyani Adepu from her group, who gave us suggestions to optimise out protocol for bacterial cellulose sheet production. With her help and advice, we managed to figure out the relevant loopholes in the protocols used by us and decide the right set of conditions to suit our needs.
Furthermore, in early October, we met Prof. Mohan Balasubramaniam, a Professor at the Warwick Medical School of the University of Warwick. Prof. Balasubramaniam had come to IISc as a visitor in the lab of Dr. Palani. We presented our project to Prof. Balasubramaniam. He expressed his delight at the usage of synthetic biology in our project to address fundamental problems plaguing the health of the people in our local community. Furthermore, he suggested us to check for protein expression in cell-free systems in order to bypass issues associated with intracellular protein degradation. We are currently in the process of repeating our experiments with cell-free systems side-by-side with intracellular expression of C2 (3.0).
All these interactions with wetlab experts have been extensively illuminating for our team. We have been able to troubleshoot our project and repeatedly evaluate the feasibility of our solution through the perspectives of a diverse array of experts from a constellation of fields.
Integrating advice for suitable implementation strategies
Based on our interaction with Prof. T.V. Ramachandra, we decided to not be limited at the proof-of-concept stage but take our project beyond that. We therefore decided to try to design a hardware prototype for our filter. For this, we contacted Prof. Saptarshi Basu. He is a professor at the Dept. of Mechanical Engineering of IISc, and specialises in multiphase systems, microfluidics, and biofuels. We explained our project to Prof. Basu in a virtual interaction with him. He brought a fresh pulse of wind to our project owing to his completely different background as compared to the rest of us. This multidisciplinary understanding made it easier for us to model our project appropriately. In further interactions with him and his graduate student Mr. Omkar Hegde, we figured out that we would need micrometre-bore channels for our purpose instead of the conventionally used millimetre-sized channels. This is because in systems like ours with a very high flux rate (high Reynolds number system), if we were to use a millimetre-sized channel, there would be insufficient mixing of the bulk of the fluid and hence the OPs in the interior of the fluid channel would not encounter the enzymes coating the walls of the channel.
We also met Dr. Rajani Kanth Vangala in this regard. Dr. Vangala has more than 15 years of experience as scientist, entrepreneur, social worker and facilitator. His passion is to work with students and develop technologies in genomics, proteomics, nanotechnology, bioinformatics and engineering. He is currently the CEO of Neuome Technologies, aiming to develop healthcare solutions through integrative scientific technologies. Dr. Vangala explained to us the various stages through which a prototype must pass before being released to the market - Alpha, Beta and Gamma. He took us through the various stages of prototype design. In his opinion, scalability would not be problematic for our project once we were sure about the various components and design. Dr. Vangala advised us on the funding necessary for the execution of various stages of the project and the scientific milestones for every step. Our interaction with him illuminated us about the steps needed to take our product from the bench to the bedside, and helped us plan the road ahead.
Visit to Bangalore Bioinnovation Centre
While we have largely focussed on civil society activists and academia for our stakeholder interactions, we have been careful enough to also lend an ear to the end-users - the farmers. Furthermore, we have also been mindful to engage in a dialogue with the biotech entrepreneurial ecosystem. The strong biotech and synthetic biology-based product delivery ecosystem in and around Bengaluru was conducive to us for these interactions.
In this regard, we visited the Bangalore Bioinnovation Centre, which is a is a joint initiative of Department of Biotechnology, Government of India and Department of Electronics, IT, BT and S&T, Government of Karnataka. It works closely with Karnataka Innovation & Technology Society (KITS), a nodal centre for implementing schemes of the Department of Electronics, IT, BT and S&T, Government of Karnataka & its startup cell. It is a state-of-the-art translational research and entrepreneurship centre catering to all the needs of start-ups in life science.
We interacted with Mr. Malathesh, a Junior Scientific Officer at the Centre and Dr. Veeranna, a Senior Scientific Officer at the same place. During our interactions with them, the following points came up:
- The idea is readily applicable only to the hilly areas, where the farmers generally have pressure of creating a proper drainage system. In Karnataka, the farming culture is mostly different. Here the farmers let the soil soak in the water of field.
- We need to create the hardware so that it's easy to customise the module depending on the local agricultural practice, in a commercial scale.
- We could add a cheap and affordable water recycling module to our filter in order to reduce the headache of agriculturalists in drought-stricken areas.
- This device should be most successful in domestic application. Moreover, the success in domestic application can give us an edge to get in a good position to propose the idea for larger implementation. Hence, we should try to make the limit of detection of our filter sufficiently low to detect the organophosphate concentration in household water supply.
- Work on additional pre-filtration, temperature control and pH control module right within the device, to increase its optimum life period.
These interactions with the staff and scientific officers at Bangalore Bioinnovation Centre provided us new insights about the scalability of the project. We integrated these suggestions into refining our model of the limits of detection for our filter. Furthermore, we decided to work on optimising the various filter parameters for our device like pH and temperature, based on the stability of the proteins and the strength of the intermolecular interactions which form the citadel of our filter design. The work is still under progress and we hope to have some quantitative data on this soon.