"Let our advance worrying become advance thinking and planning."
~ Winston Churchill
The methodology of real-life implementation forms the cornerstone of any well-thought scientific project to make it to the real world from the laboratory. It is a well-researched and carefully framed plan of action that determines the fate of whether an idea can be implemented in accordance with the theoretical ambitions it presents once laid out on paper. Our proposition for implementation is a dyad of ‘Disease control implementation’ and ‘Product implementation’. Our main aim is to deliver our product in the “safest” way to our end-users. Through consultation with stakeholders and experts who have experience in the field of disease detection and control, we were able to ideate a realistic implementation of our solution. Understanding the basic needs, requirements, and expectations of our stakeholders from our solution has allowed us to ensure that our project is sound and responsible towards the world.
Team iGEM IISER Kolkata went on in-person interviews with local dairy farmers based in Asansol, a city in West Bengal. Upon discussing with them, we came to know about the current scenario revolving around the current detection techniques and treatment methods involved with subclinical bovine mastitis disease. Focussing on the current detection methods, the conventional ones are California Mastitis Test (CMT) and Somatic Cell Count (SCC). Both these methods have some major drawbacks. CMT is not sensitive and as a result, it is hardly used by them for detection. SCC, on the other hand, is very time consuming and costly rendering to major losses faced by the farmers. They were even looking forward to a new detection method which is cheap, fast and easy-to-use. Regarding treatment, the indiscriminate use of antibiotics has posed a serious problem which was also evident from our conversation with the farmers. We planned on an antibiotic-free approach for the cure of this disease. The reason behind choosing an intramammary injection to release our GMO was the reluctance depicted by the cows when provided with oral supplements. From our human practices interactions we came to know that injections are in maximum use. Dairy farmers are also trained by the government regularly about how to apply intramammary injections. Therefore we also plan to implement our therapeutic through intramammary injection. Upon contacting the officials at Haringhata Dairy Farm, based in Kalyani, we realised that the prevention and detection techniques play a major role in such industries and consequently, the implementation of our detection methods allows it to be more sound and approachable.
Our project is a mini toolbox to keep Bovine mastitis at bay and be of help to the dairy industry where this disease has been a problem for a long time. We propose two independent products. The first product is a biomarker-based colorimetric detection kit to identify the disease at the subclinical stage. Whereas the second product is a single dose GMO-based medicine to cure the deadly bovine disease.
The Colorimetric Detection kit
We are working on the development of a rapid, efficient, portable, and cost-effective real-time diagnostic paper-based kit. Our main aim while developing this product was to ensure that the final output exhibited from our test kit can be easily inferred by the general mass without any prior requirement of scientific and technical knowledge. Hence we propose to use a microfluidic paper-based detection kit nature µPAD to visualize the results under the naked eye. µPAD is a 5-layered wax strip. It is made of very low-cost materials and is designed to be used in a low-resource environment. The pre-incubation mixture does not contain any hazardous material/chemicals/microorganisms, hence its release in the environment will not pose any threat. We plan to use a blue coloured dye for the visual readout so that colourblind people can visualise the results on micro-pad at their ease. To make our kit accessible to individuals with complete colorblindness (Anomalous trichromacy) we plan to use the Coloursence - an program proposed by Balraj Rathod, an alumni of IISER Kolkata. The program would detect color and produce unique sounds to help the user listen to colors. Moreover, the wax-based strip is eco-friendly ultimately rendering the whole detection kit the property of being safe to use.
Mode of use - The kit has been designed such that it can be used effortlessly at any time. One has to take some amount of milk sample and add it to the tube containing the pre-incubation mixture provided in the kit. Then the mixture has to be incubated at room temperature for a particular amount of time. Post incubation some amount has to be added to the µPAD. If the sample milk is mastitis positive then the wax strip will render a visible color, if the sample is not mastitis positive no color will be visible (details).
Antibiotic-free GMO based cure -
Post-detection if the cow tests positive, then we propose the use of our second product. Unlike the present broad-spectrum antibiotic-based treatment, this medicine is antibiotic-free and uses a combination of narrow-spectrum pathogen-specific bacteriocins and endonuclease for the killing of the pathogen in its biofilm directly. These treatment-inducing molecules will be delivered via a probiotic strain, Lactococcus lactis LMG7930 (details). The treatment will be available as a single-dose use and has to be injected in the infected udder of the cow through the teat canal as an intramammary injection. The use of this product will require a basic level of training and knowledge of injecting medicines. This medicine will be available in glass vials and has to be handled with care as improper handling can cause cuts and tears. Risks associated with the handling of GMOs are minimal as two independent highly efficient kill switches have been incorporated to avoid any kind of accidental release or if it remains unused. As the lifetime of the GMOs is dependent on the concentration of thymidine provided initially (details) in the vials. Hence shelf life of this is yet to be figured out. This product is recommended to be stored at 4℃ to retain its maximum effectiveness.
Mode of use - This medicine has to be injected into the mammary glands of infected cows by intramammary injection by a trained individual.
The aforementioned products shall be sold separately to our end-users. Now, whom do we consider as our end-users? Well, they are the ones who will be directly or indirectly involved in the implementation of our product into the actuality. Primarily, our end users will be small-scale, large-scale dairy farmers and dairy farms who can avail our products directly from the authorized pharmaceuticals and/or veterinary hospitals. Looking into this matter, we shall also have a secondary set of end-users for our product, namely national/state government and veterinary hospitals, who shall not only help us deliver our product(s) to our concerned primary end-users but will also enable us to send our detection and genetically-engineered kit to treat subclinical bovine mastitis.
We have talked with several veterinary doctors, scientists and other stakeholders of our project to understand the customer base of our product involved in this field. The details can be found in Integrated Human Practices. Our product was designed and planned on the same footnotes. Upon discussing with some farmers based in and around Bengal, we came to realize that they usually believe in using broad-spectrum antibiotics which have been the primary cause of AMR in the current world. Consequently, we went on an antibiotic-free approach to our eco-friendly, time-saving, and cost-effective product(s).
We plan to sell our products separately to our primary end-users. The virtue behind it is to allow the customer to choose between the two products as per need and demand. Our secondary end-users, being national/state government and veterinary hospitals shall help us distribute our concerned product to the primary end-user in an effective and efficient manner. As our motive behind generating these unique cost-effective products is to stop the increasing negative impacts of the overuse of antibiotics we believe the government will acknowledge the application of synthetic biology with proper safety precautions into a real-world problem.
Verification of the Proof of Concept
Our team wants to ensure that our proposed implementation is socially, morally, and ethically responsible. In this limited time frame, we so far have been able to partially verify the proof of concepts. So we now propose to test the detection and therapeutic components of our project completely to ensure that our products based on these concepts are efficient and fully functional. For the colorimetric detection kit, we plan initially to test that the pre-incubation mixture along with the μPAD can detect the targeted miRNAs effectively and sensitively. Whereas, for the bacteriocin-induced therapeutics we plan to test if our GMO can effectively sense the externally provided AIP molecules and post sensing is able to express Nisin PV, DNaseI, and Lysis E7 as per our designed genetic circuit. We also propose to explicitly check if these therapeutics-inducing molecules are being released out of the GMO in its working surrounding.
Post conductance and successful completion of the proof of concepts for the fundamentals of our products, we propose to proceed to seek permission for the lab trials for our products. The detection kit will not require any clearances from any regulatory authorities. But, the lab trials for therapeutics will initially require clearance from the Institutional biosafety committee. We have already proceeded officially with our project and yet to receive clearance from the institutional level biosafety regulatory authorities. Then we plan to approach the Institutional animal ethics committee for further clearance to continue and conduct our trials on animals.
We plan to verify the sensitivity of our detection kit with respect to the results of the Somatic cell count test performed upon a herd of cows both infected and uninfected with subclinical bovine mastitis. Quite similarly we also plan the lab trials for our therapeutics by conducting tests on a herd of cows suffering from different stages of bovine mastitis. During the conduct of this trial we will need to ensure that the infected cows are kept in isolation and under continuous surveillance by veterinary doctors. We will also perform an analysis of the effectiveness of our curative.
After successful completion and favorable outcomes of the lab trials, we will initiate to contact concerned authorities to seek permission for conducting clinical trials on a large scale. The results will be obtained and analyzed to understand the efficacy and efficiency of our detection kit and curative. The final results will be submitted for approval to The Central Drugs Standard Control Organisation(CDSCO), Ministry of Health & Family Welfare, Government of India for marketing and public usage of our products.
A supply chain is the activities required by the organization to acquire raw materials and deliver finished products to the consumer. Let’s take a look at our plan of acquiring the raw materials, turning them into finished products, and delivering them to our end users.
The pre-incubation mixture of our colorimetric detection kit consists of ssRNA crosslinkers(it maybe part of hydrogel depending upon the material of the hydrogel), Cas13a enzyme, csm6 molecule which all can be ordered to produce in bulk from any biotechnology company. Whereas, the RNA sequence with protecting groups will be ordered from IDT. The wax-based strips, dyes, and buffers will also have to be ordered separately in bulk. Post acquiring all the required components the RNA hydrogel has to be made and then all of them have to be assembled to make the μPAD. Whereas for the production of our therapeutics we can order our desired GMO by specifying our chassis organism and gene circuit in bulk from any commercially GMO producing company. Finally, we have added the desired amount of thymidine and store it in vials at 4℃.
Now we plan on distributing our products to distributors and big dairy farms following our Marketing and sales management plans for further distribution to our customers.
- ssRNA crosslinkers
- Cas13a enzyme
- Csm6 enzyme
- RNA sequence with protecting group
- Chromatographic filter paper
- Colour dyes
- Glass Vials
- Fully designed GMO
We through our project have accomplished the following sustainable development goals. 
SDG 3: Good health and well being
By helping farmers to keep a check on cases of bovine mastitis in their herds, we are ensuring the delivery and consumption of quality dairy products by delivering them access to a user friendly detection kit and an antibiotic-free drug that will treat Bovine mastitis. We are utilising the benefits of scientific research to develop a medicine for a communicable disease among cows that causes significant losses to the dairy industry, specifically small scale dairy farmers. Among the indicators of this goal, is the proportion of the target population covered by the vaccines or drugs. Our treatment method, being user-friendly and very simple to use, is expected to reach a significant proportion of our target population, which includes large and small scale dairy industries, with the small scale dairy farmers forming a major chunk of the target consumer base of our product. Our product aims to revolutionise the veterinary drug market by developing an efficient drug with no absolute ramifications, directly or indirectly, on the environment in general and livestock in particular. Our drug can also be utilised as a foundational template for the development of other antibiotic-free drugs for the different sectors of the animal drug market which would incrementally revolutionise the state of animal husbandry in low and middle-income countries from the perspective of animal health.
SDG 4: Quality Education
Sustainable Goal 4 , i.e. the delivery of quality education, has been a cornerstone of the various initiatives that our team undertook in the human practices component of our project. We have undertaken several activities to impart education to different sections of society. We have worked towards realising the various targets underscored under SDG 4 that translate to free and equal access to quality education without any discriminatory barriers. Our various activities have been directed towards not just children who have not been able to get access to quality education but also to people who have not been fortunate enough to get proper education throughout their lives. To promote the participation of girl students in STEM, we conducted a mini summer school for girl students from various schools. We also did the same for children from Bengali-medium schools, thereby addressing the issue of inclusivity from both the perspectives of gender and language. We visited a local orphanage and an old age home to distribute educational informative material which would be of help to the people there. We have tried to reach out to every community possible to educate them regarding science and synthetic biology in particular in the spirit of the sustainable development goal to deliver universal quality education.
SDG 8: Decent work and Economic Growth
Sustainable Goal 8 pertains to the fostering of sustainable and inclusive economic growth with full and productive employment for all. Our project aims to strive towards higher levels of economic growth and productivity by designing a product that could be a shot in the arm for the dairy industry. In a dairy-intensive country like India, a majority of the milk produced comes from small scale dairy farmers who have negligible knowledge of the scientific details of bovine mastitis and the adverse ramifications of antibiotic overuse in response to bovine mastitis cases. By designing a complete user-friendly product for the average small scale dairy farmer, we aim to eliminate the losses caused by bovine mastitis to the dairy industry. The large scale production and distribution of our product will help dairy farmers upscale their production levels without compromising with the quality and quantity of their output. The indicator for this goal being the growth rate of GDP per capita, is expected to significantly increase following the usage of our product. As dairy farmers use our kit to keep a check on mastitis cases in their herds and treat current mastitis cases, it is possible to maintain steady production and prevent losses, leading to greater earnings and an improved quality of life. This would translate to full and productive employment and contribute to a sustainable and inclusive economy as an absence of losses would enhance the dairy sector, paving the way for an increase in the size of the workforce.
SDG 9: Industry, Innovation, and Infrastructure
SDG 9 pertains to the development of resilient infrastructure, sustainable industrialisation and the fostering of innovation to achieve the same. Crucial to the achievement and delivery of the goals pertaining to earlier SDGs in order of precedence such as those aiming for zero hunger and poverty, SDG 9 dives into the need of resuscitating the global pattern of how economies work and operate, thereby ensuring that in the long run, peoples’ livelihoods are not compromised due to the risks presented by the current system and patterns of industrial activity. Our project seeks to upgrade industrial technology by revolutionising the way we look and respond to Bovine mastitis, a disease which has plagued the dairy industry for a long time and which has been particularly aggravated in the recent past due to the development of antibiotic resistant strains. The problems caused due to bovine mastitis are particularly higher in middle and low income countries where awareness of people regarding good hygiene practices in dairy farms as well as scientific awareness regarding the disease are less. In such a scenario, it is indispensable to come up with devices and treatment methods for such a widespread issue that can breach the wall of lack of education and access to information, which is why our detection kit and treatment drug are designed to be extremely simple and user-friendly and cause no environmental ramifications at all. They can be smartly used by the average small scale dairy farmer without any issues and will help in resuscitating the dairy sector from the grassroots. In this way, we are utilising scientific research to meet industrial needs of sustainable and resilient upgrade via a sustainable approach. By achieving this, we will also be able to improve the economic situation of farmers and contribute to the sustainable development goals of zero poverty and no hunger.
SDG 12: Responsible production and consumption
SDG 12 seeks to ensure sustainable patterns of consumption and production which would translate to a better and more efficient use of resources, greater access to services and a better quality of life for all. Our project seeks to fulfill this sustainable development goal by providing an alternative for antibiotic mediated treatment methods for bovine mastitis. The continuous and rampant overuse of antibiotics for the treatment for Bovine mastitis leads to the accumulation of antibiotics not just into the cow’s body but also in the human food chain through the dairy products prepared from cow’s milk. By achieving a gradual elimination of antibiotics from the dairy sector, we aim to ensure a pattern of production and consumption in the dairy sector which is sustainable and improves the quality of life of all directly or indirectly related to the consumption of dairy products. This will play a crucial role in averting the further development of antibiotic resistance in bacteria which is already causing a number of issues. The kit that we have designed and our drug for bovine mastitis are completely free of hazardous substances which could harm the environment. Our product is totally safe and biodegradable and is a step towards sustainable production of drugs for the animal drug market.
To ensure that our product is safe and eco-friendly, we incorporated a Lysis E7 gene into our chassis organism such that when it comes in contact with the pathogens, LysisE7 gets released leading to the lyse of our chassis GMO and releasing the pathogen killing enzymes. In case it doesn't come in contact with the pathogens, we have equipped a kill switch, i.e. mutating the thyA gene for lysing our chassis organism as the thyA mutants are unable to survive in low thymidine. This has allowed us to ensure that even if the GMO is released into the atmosphere, it won’t be able to survive and thrive in natural conditions which have diminished any possible threats. We are waiting for the clearance of the Institutional Biosafety Committee (IBSC) at IISER Kolkata on the chassis organism that we have planned to use. The details on the same have been mentioned in the Safety section. In the near future, we also plan to get approval from the Animal Ethics Committee who will check the safety-related issues of the cow for injecting the GMO into its udder. After receiving approvals and clearances at the institutional level we will proceed for clearances at the national level. 
From the viewpoint of environmental safety we will seek clearance from the Genetic Engineering Approval Committee (GEAC), which is responsible for providing clearance for large-scale field trials, industrial application, and the commercial cultivation of GMOs in India. The GEAC can approve or prohibit GMOs used for import, export, transfer, manufacture, processing, use or sale of GMOs. 
For social licensing, we have communicated and discussed with a number of farmers who were completely in agreement with the idea of a new detection method as well as the administration of a GMO into the teats of the udder. Administration of a new detection method won’t be much of a problem as we have incorporated an easy and efficient approach to using it for the general public. They also quoted that they were completely fine with the execution of a GMO if it showed positive results. At the end of the day, they were fine with application of a new technique when prescribed by the veterinary doctors.
For patenting our detection method and treatment procedure respectively, we have talked to several officials and took their useful suggestions on the same. We will start contacting the concerned people to ask permission for conducting clinical trials on a bigger scale and the final results when analyzed will be submitted for approval to the CDSCO, Ministry of Health & Family Welfare, Government of India who shall help us to market for the public use of our products. However, iGEM being an open-source registry of biological parts, this intellectual property is off-patent. To make our product patentable we can introduce new modifications in the gene circuit that was submitted to iGEM parts registry.
Limitations faced due to the pandemic
We had to face a lot of challenges due to the outbreak of COVID-19, primarily starting the wet lab work very late. The outbreak of the second wave in India posed a major hurdle on our way of joining the labs. Fortunately, we could start wet lab work in mid August and give our best. Due to COVID-19 guidelines issued by the Government of West Bengal, India, there were several restrictions in our country. Unfortunately, we were unable to conduct awareness programmes in groups, but we made efforts to organise in-person interviews and make science accessible to the general public. The details of which are mentioned in the Education section. We also conducted several interviews online to meet our stakeholders and discuss various aspects of our project.
Ethical and Social Concerns
In a diverse country like India and other parts of the world, people will have different opinions and mindsets. There are people who do feel that GMOs are not naturally built and as a result, they might be harmful to them and the environment. Despite the growing world of synthetic biology, we have tried to make people aware of the implications and importance it plays in our lives. We also want to make sure that the chassis organism we are using will obtain a green signal by the IBSC of IISER Kolkata and in the near future, we shall clear all the rules and regulations determined by the Animal Ethics Committee and make sure to pass all the trials in order to release our product in the market.
There has not been much focus on an antibiotic free approach to tackle subclinical bovine mastitis in India. People were more accustomed to using antibiotics for a long time and have found comfort in the same. Consequently, the upbringing of a newer approach to tackle this problem using synthetic biology might face some resistance from the general public. We have been creating awareness regarding the importance of an antibiotic free synbio approach to tackle the problems of AMR and shall continue doing the same in the future too.