Team:IISc-Bangalore/Design Solution
Step 3: Design a Solution
Keeping in mind Project Aqualose, we thought about using a bacterial cellulose based filter to combat organophosphate pollution. Extensive reviews of scientific literature led us to a cellulose-binding domain - dCBD and a broad-spectrum organophosphate hydrolase, OpdA. The kinetics and various parameters for the enzyme had been well mapped. We decided to make a fusion protein by combining the two domains.
At this point we thought about one of the key considerations that our stakeholders had asked us to keep in mind - cost and affordability. We had a discussion session with Dr. Samay Pande, Prof. Dipshikha Chakravortty, Prof. Utpal Nath and Dr. Sandeep M. Eswarappa, who are faculty members at the Division of Biological Sciences, IISc. They reaffirmed the lingering question in our minds - cost. In a country like India with a vast section of the rural populace living below poverty line, many excellent scientific solutions do not simply translate to the society because of economic inaccessibility.
A cursory examination of the costs involved in various stages of protein production led us to conclude that purification of proteins is one of the most expensive aspects of the process. Why not make the fusion protein reusable, we asked?
We then needed to identify the primary reason which would make the filter need to be replaced. After going through available literature and talking to our friends from Materials Engineering departments, we understood that the primary obstruction to this would be the We also focussed on making reusability not a compromise on the quality of our product. Therefore, we were focussing on two constructs - one carrying dCBD and one carrying OpdA. These two constructs were to be bound by some interaction which can be annihilated easily without denaturing the protein. We could then have the pure enzyme containing fraction. An enzymatic digestion of the cellulose sheet would then lead to the dCBD containing fraction, which might be contaminated by other
One of the interesting scientific developments in 2019 came to our rescue in this regard. We came across a report of SpyTag-SpyCatcher system and the SpyTag-SpyDock system. SpyTag binds to SpyCatcher via a covalent isopeptide bond and not SpyDock by a non-covalent bond. The affinity for SpyTag with SpyDock is also very high and can be abrogated by elution with imidazole. This was exactly what we needed! We therefore decided to pursue it further.
Having a rough idea in our minds, we decided to iterate through our process of identification of stakeholders. To do this, we first decided to be more specific in our definitions of stakeholders than our previous maiden attempt. This led to the following:
To systematically achieve this objective, we made the stakeholder map after taking help from the iGEMer's Guide to the Future. After thinking about the possible social influences of the stakeholders mentioned before, we categorized the stakeholders using the following stakeholder map:
We then decided to first focus on the key players for our project. To first decide on whether the solution proposed by us is feasible 'on paper', we decided to start by consultation with members of the academia.
Here are some of the background work we did in that context and the summaries of our interaction with stakeholders:
SWOT Analysis
SWOT analysis is a strategic planning technique used to help identify Strengths, Weaknesses, Opportunities and Threats related to a plan. A SWOT analysis for our project led to the following conclusions:
Interaction with Dr. Keeble
To fully understand the feasibility of application of SpyTag-SpyDock system in our project, we contacted Prof. Mark Howarth from the University of Oxford, the developer of the SpyTag-SpyDock system. He put us in touch with Dr. Anthony Keeble, post-doctoral associate in his lab who was the lead author in reports of the Spy&Go system. We sought his suggestions about our project implementation using SpyTag-SpyDock system. Dr. Keeble informed us of how shearing might cause detachment of the SpyTagged enzyme from the sheet and cause rapid protein loss, thereby requiring repeated functionalization of the sheet. Dr. Keeble suggested us to use SpyCatcher instead and provided us insights into how modularity in our "plug-n-play" platform provides a novel and feasible solution to the problem might indeed be, besides being a gamechanger for bioremediation efforts. Dr. Keeble also informed us that while the kinetic data for the interaction between SpyDock and SpyTag had been quantified, there had been no attempts to realistically see how it responds to high velocity flows.
No data was available then in literature about the half-life of the SpyTag-SpyDock interaction when subjected to shear by water flow. We decided therefore to fill this void by quantitatively modelling the interaction and following its kinetics. This would help us to understand the half-life of the SpyTag-SpyDock interaction, and thus make a more-informed choice for the DNA parts which we wanted to use. Our mathematical model indeed led to the conclusion that even a very small rate of dissociation of SpyTag from SpyDock would cause a rapid depletion of the SpyTag-SpyDock bound protein complex and a flushout of the SpyTagged enzyme. Thus, the interaction with Dr. Keeble contributed to our modelling efforts and led to a paradigm shift in our project idea (as described below).
Meeting Professor Padmanabhan Balaram
Once we were convinced about the unfeasibility of the usage of SpyDock, we decided to focus our attention to creating a modular filter, which would herald the revolution in state-of-the-art bioremediation strategies. Since our project required two fusion proteins, we sought the advice of Prof. P. Balaram, the former Director of IISc and a retired professor of the Molecular Biophysics Unit. Prof. Balaram's main area of research has been the investigation of the structure, conformation, and biological activity of designed and natural peptides. He has been a major contributor to the evaluation of factors influencing the folding and conformations of designed peptides and has investigated structural elements playing a key role in the formation of secondary structural motifs in proteins.
Our team met Prof. Balaram virtually to discuss about rational design of linkers for our fusion proteins. Based on our discussions with him, we could understand and appreciate the requirements which the desired fusion proteins would have for a successful implementation of the project:
- They should not influence the structure of the functional protein domains
- They should be flexible so as to allow ready access to the organophosphate substrates flooding into the pipes of the filter
These inputs were of significant value to us, based on which we could try to design the linkers appropriately. Furthermore, the issue of linker flexibility which Prof. Balaram mentioned to us motivated us to pursue structural predictions of our proteins of interest - a new dimension to our project that we have addressed under Structural Predictions of Proteins
Besides focussing on the "key players" for our project based on the stakeholder map created by us, we've also tried our best to focus on the end-users of our device - the stakeholders who occupy the lower right quadrant of the stakeholder map. We have tried to respectfully engage with those affected the most by the enormity of this toxicity. We have surveyed farmers from multiple places in India to be aware of their understanding of pesticide based pollution and organophosphate toxicity. Details of the survey have been documented below.