Overview

“Local people solving local problems” is a phrase that describes AMPifin accurately. Following a value-centric approach, we noticed that Manipal being a coastal town is home to a massive aquaculture industry setup. As our conversations with various stakeholders and communities developed, we came across Mr. Kaushik Alike, who introduced us to Vibrio parahaemolyticus and the diseases it causes. This bacteria piqued our curiosity, and through this interaction, we fixated on our problem statement and decided to build on it systematically.

The food-chain supply for seafood consists of hatcheries, farms, markets, restaurants, and finally, consumers. To understand the impact of vibriosis, we interacted with various stakeholders from in and around India in Maharashtra, Karnataka, Hyderabad, Tamil Nadu and Kerala, and internationally, in Dubai. These interactions helped augment our project and accelerated its growth.

Interaction with Canares Aquaculture LPP

On 21st November 2022, we contacted Kaushik Alike, one of the founders of Canares Aquaculture. He spoke to us about various topics, including parasites and viruses, as well as feed and water purification. We learnt about the major diseases in fish and the present methods of prevention and cure. He was the one who first mentioned Vibrio parahaemolyticus and Oxytetracycline to us. We decided to work on the bacteria Vibrio parahaemolyticus owing to the insightful conversation we had with him.An aspect he emphasised was the significance of not entirely eradicating all microorganisms from the water body. He also discussed several strategies for distributing feed and raising fish.

Government Shrimp and Fish Hatchery

To gain more knowledge of areas outside of our locality, we decided to approach a government hatchery to get more information about the prevalence of vibriosis.

On 1st July 2022, we visited a Government Shrimp and Fish Hatchery in Azhikode, Kerala, India . We interacted with their association members—Assistant Fisheries Extension Officers—Mrs. Merit Kurian, Mrs. Devi Chandran, and Mr. Vinu Jacob. The primary focus of this institution is to hatch shrimp and fish for 20–25 days using biofloc technology and then sell them to various fish farmers.

They mentioned that the main types of fish that are hatched are Etroplus and Pompano. They elaborated on the primary local diseases observed in their farm. It astounded us to hear that there are multiple species responsible for various levels and types of pathogenicity. The fisherfolk mostly look for cuts or bruises found on the scales and the fish's skin for early detection of any diseases. To avoid necrosis, a significant symptom of vibriosis, pretreatment methods are employed immediately after the seeds are hatched. In the case of an infection, antibiotics are used as a treatment method. They emphasised on how the dosage has seen a surge over the years. Furthermore, they mentioned that detection is primarily carried out via PCR. Their hatchery consists of massive units, maintained at a temperature varying from 25-28 degrees celcius using regulated heaters, and the salinity of the water is maintained at 20 ppm. Through this visit, we learnt about the various methods to identify the levels of pathogenicity and how fisherfolk differentiate between diseased and healthy fish. We also learned more about methods by which they prevent or cure bacterial infections in fish.

When we explained AMPifin to them, they were interested in our solution's approach. In particular, they were impressed with our focus on sustainability. In particular, they were keen on exploring a solution that has no known side effects and is unlikely to induce antibiotic resistance. Through this visit, we comprehended the large-scale working of fish hatcheries and developed an understanding of how AMPifin can be stitched toward being a more operational solution. The data we received from them was valuable and helped improve the interpretation of our literature surveys. Furthermore, we gained confidence in our sustainable approach and focused on making AMPifin more understandable to our main stakeholders.

Laxmipuri Fish Market

Fish markets are the hub for fish trade and team AMPifin decided to interact with them to understand the food-supply chain and their methods of identification for diseased fish. Furthermore, the platform served as a location to interact with consumers, too.

On 6th July 2022, we visited Laxmipuri Fish Market located in Kolhapur, Maharashtra, India. It is owned by Mr. Ashish, Mr. Kiran, and Mr. Ashok—fish sellers and consumers from a local fish market. They mentioned that their fish market receives over a hundred varieties of fish from the Ratnagiri district of Maharashtra. The fish supply is stored and sold for two to three days until freshness is retained. Furthermore, in case any spoilage is detected, they discard the fish immediately in a safe manner. As we conversed with them, we realised that the methods of prevention of diseases in most seafood shops were nebulous, especially for the wild-caught ones. Since most of them were sellers and not pisciculture farmers, they did not have much knowledge on the various medicines used in fish hatcheries. We explained our project to them and they provided us with their opinion on selling or consuming seafood that has been treated with our peptide. Since most were sellers and not pisciculture farmers, they did not know much about the various medicines used in fish hatcheries. Furthermore, they were encouraging of more such sustainable solutions. From this visit, we understood the importance of consulting secondary stakeholders, who are a critical part of the food supply chain.

Visit to Shrimp and Fish Farm

On 8th July 2022, we visited Mr. Sampath's shrimp and fish farm in Chennai, India. While showing us around his farm, he elaborated on the two main species of shrimps grown—giant Tiger prawns (Penaeus monodon) and Whiteleg shrimps ( Litopenaeus vannamei). The salinity range maintained in his farm roughly accounts for about 25%, along with a pH range of 8.5, and the breeding period is usually carried out for four months. He mentioned how these conditions were applicable for fish as well.

For fish, any disease is detected by visual cues—cuts, scars, bleeding on the skin, and necrosis. The detection of such diseases is carried out by testing the water with a calcium soda mix. Since AMPifin could potentially be a broad-spectrum solution for all gram-negative pathogens, we were delighted to see such accepting nature toward the utilisation of our solution.

ICAR-CIBA Institute, Chennai, India

On 8th July 2022, we visited the Indian Council of Agricultural Research—Central Institute of Brackishwater Research (ICAR-CIBA) located in Chennai. We met with Dr. M Shashi Shekar and his team consisting of Dr. Ashok, Dr. Raymond, Dr. Sujeet and Dr. Patil who are currently working on vibriosis in fish. We felt that the information they had would benefit our project’s geographical statistics and research-related data. There was a brief discussion about the types of projects they were working on for the research-oriented communities and looking for potential areas in which they could be of great help to us. Dr. Shekar introduced us to his lab and team. The exchange of scientific data and information was fruitful to our project's advancement.

Al Fahlain Aquaculture and Agricultural LLC

We visited The Al Fahlain fish farm in Dubai, UAE, headed by Mr. Liju, to check the prevalence of vibriosis in locations outside of India. Initially, he exchanged data about the food they usually feed the fish, which were mostly floating pellets that they import from Saudi Arabia and sinking pellets which are rich sources of protein imported from Turkey.

They indicated that vibriosis is a common disease in fish in that area, but due to restricted lab facilities, it is not usually tested.

From our visit, we observed that they use antibiotics to treat these infected fish and the most commonly used one is Oxytetracycline (OTC). OTC is a broad-spectrum antibiotic and is hence helpful in most common diseases. Erythromycin was used previously along with OTC but isn’t effective for many diseases anymore, and hence, it has been banned in many countries, including India and Dubai. In cases where they cannot detect the disease in the fish, they usually flush the column with salt water if freshwater fish are being grown and vice versa. They spend approximately 1% of their income treating these fish (approximately 500 AED—i.e., 136 USD). To reduce the number of infected fish imported, they usually run a PCR test from the country of import. This has reduced the mortality rate of fish by 20%.

Apart from these diseases in fish, the main cause of diseases in this fish farm was the high-density culturing of fish, due to which the oxygen level and space for each fish reduces. They have external liquid oxygen that is usually pumped into the fish columns depending on the need and necessity.

This visit helped us analyse the prevalence of vibriosis outside of India as well. Furthermore, we distributed large-size pamphlets on the safe usage of antibiotics, and explained AMPifin to the fish farm owners.

Aquatic Biosystems

We visited Aquatic Biosystems in Mangalore, owned by Mr. Ronald D’Souza. He set up Aquatic Biosystems in 2006, which is currently the largest ornamental fish farm in the state. He began by talking to us about the history and origin of his biosystem. As he showed us around his fish farm he spoke about the major diseases they face. He mentioned that viral and bacterial infections are quite common. If there are any symptoms of bacterial infections, they isolate the fish and send a sample to the lab at the Fisheries Institute, Mangalore. The common bacterial species they have to deal with are Aeromonas species, Vibrio species and Pseudomonas species. The common treatment methods for these are Oxytetracycline (OTC) and Enrofloxacin (ENR). Of these, OTC works better than ENR. However, if OTC does not work, they use Seromycin followed by lab testing.

Every year, they face a loss percentage of 5–10% due to different types of diseases. On explaining our project to him, he was eager to see the result. He explained how the pipe fittings and the connections were formed in order to incorporate various water filters. This paved the route for our delivery mechanism to find an appropriate real-life application. Given the description of the Recirculatory Aquaculture System (RAS) in his farm, we made our implementation design to increase the feasibility of integrating AMPifin in fish farms. He displayed eagerness toward our project and said that solutions like AMPifin are required owing to antibiotic resistance becoming a rising concern.

Thimmapa Restaurant

To understand the impact of vibriosis on our secondary stakeholders—i.e., seafood restaurant chefs and owners, we decided to visit a renowned restaurant in the Udupi region of Karnataka. We wanted to observe the various procurement, cleaning, sanitisation, and cooking practices carried out by the hotel administrators. Furthermore, we wanted to understand the impact and significance of our solution at this stage of the food supply chain.

On October 3rd 2022, a few of our team members visited Hotel Thimappa—a seafood restaurant in Udupi, Karnataka. We spoke with the owner about how the fish is stored and prepared at their restaurant. He explained that the monsoon season around July to October is the best time of the year for fishing. For the past 40 years, the restaurant has purchased its fish from the same vendor located in and around the coast of Malpe. When we enquired if they receive diseased or rotten fish from the vendors, they replied that most of the time they sort out all the fish right after they are caught, so that the disease spread is contained. However, during the months of May and June, they may have more probability of receiving rotten fish as they are less available at this time and refrigerated for longer durations. To gain more insight on the issues that restaurants face with diseased fish, we enquired about the different ways by which they recognise diseased fish. The chefs told us that if it is a rotten fish, it can be distinguished while cleaning due to the difference in texture and smell. However, if it is a diseased fish, it is hard to detect while cleaning. These fish are identified during the cooking stage.

We introduced them to our project—AMPifin and they were inquisitive about our delivery mechanism. From this visit, we understood the effects of diseased/rotten fish on different stages of consumption. We also learned the methods of segregating rotten or diseased fish at a seafood restaurant. We ended this visit by trying two of their famous seafood dishes. The dishes were absolutely delicious, and highlighted how these cuisines form the backbone of our rich Indian culture. This visit helped us broaden our stakeholder interaction spectrum and understand the prevalence of vibriosis at various stages of the food-supply chain.

Podcast with The Astronomy Club, Manipal.

On 7th October 2022, we collaborated with the Astronomy Club, Manipal to talk about synthetic biology and its applications in the space industry. The podcast comprised two members of the Astronomy Club and two members of Manipal BioMachines. The podcast started off with an introduction to the respective members and iGEM. The discussion was based on how synthetic biology could be used to enhance and use the extreme environments of space to our advantage. For example—extremophiles for the elimination of biowaste and synthesis of biomaterial, self healing bio-compounds for integrity of the space shuttle, and nutrient-rich-space-conserving food given the limitation on resources that can be carried. There were talks about previous iGEM teams’ projects that built on the motive of improving space travel quality for the travellers by integrating pure synthetic biology based components.

This podcast was an opportunity for us to collaborate with astrophiles and understand their areas of interest. By engaging with the astrophile community, we were able to spread the idea of synthetic biology to various groups. Astrobiology is a vast field and has developed for itself avid learners. Through this podcast, our aim was to introduce them to a novel field under the same branch of science. Refer to our collaboration section to listen to the podcast.

Consumer Survey

To understand the perception of the general public on the pisciculture industry, we rolled out an exhaustive consumer survey. This survey helped us understand the opinion of the general public on sustainable solutions based on synthetic biology. The responses brought a broader perspective to our project, and it was interesting to see the level of awareness amongst various seafood consumers on diseases in fish and their prevalence. Attached below is a detailed consolidation and summarised inference:

• Mr. Kaushik Alike

  Outreach

  November 2021

• Dr. Ramesh Sistala

  Company

  April 2022

• Dr. Nithin M.S.

  Academia

  May 2022

• Mr. M Madan

  Academia

  May 2022

• Dr. Seetha Krishnan

  Company

  May 2022

• Dr. Mamatha Ballal

  Doctor

  June 2022

• Dr. Ramesh Sistala

  Company

  June 2022

• Dr. Kannan Ramaswamy

  Doctor

  July 2022

• Dr. M Shashi Shekar

  Academia

  July 2022

• ICAR-CIBA Team

  Academia

  August 2022

• Dr. Manickam G

  Company

  August 2022

• Reiss Jones

  Entrepreneur

  August 2022

• Ronald Kevin D’Souza

  Outreach

  August 2022

• Miroslav Gasparek

  Entrepreneur

  September 2022

• Dr. Vinod Jyothikumar

  Company

  September 2022

Dr. Ramesh Sistala

Dr. Ramesh Sistala, Founder and Managing Director at thinkMolecular Technologies Pvt. Ltd., Bangalore, India.

THEIR INPUT

We approached Dr. Ramesh Sistala on 13th April 2022 during the initial stages of our project to receive help on the dry lab facet of our project. He gave us some ground rules for finding a template to use for our protein study. Initially, he recommended we use BLAST for the available information in databases, and find homologous protein structures. Based on his suggestions, we built a homology model, and utilised Alphafold for structure prediction. Furthermore, he suggested exploring the available peptide libraries to comprehend the protein and the interaction between MAM7 and Fibronectin. Finally, he asked us to make sure that the hypothesis we produce is functional by carrying out a trial run.

OUR INFERENCE

The ideas and suggestions he provided laid the foundation for our Dry Lab work. He was crucial in helping us find the most appropriate template to work with. We explored the two possibilities he mentioned about generating our desired sequence. Otherwise, there was another possibility where we could find an average sequence by using Alphafold for MAM7 and then utilising it to generate the fibronectin structure. Based on that, we decided to run the molecular dynamics simulation (MDS) of MAM7, and MAM7 with fibronectin.

Dr. Nithin M.S.

Dr. Nitin Muliya Sankappa, Ph. D. researcher in Fisheries Microbiology, Department of Aquatic Animal Health Management at College of Fisheries, Karnataka Veterinary Animal and Fisheries Sciences University, Karnataka, India.

THEIR INPUT

Dr. Nithin M.S. is proficient in the field of using bacteriophages to work on Vibriosis. We approached him on 28th May 2022 to gather data on the prevalence of vibriosis in and around Manipal. We explained how MAM7 is an adhesion molecule involved in the early stages of the infection cycle. We wanted to imply that it was the most appropriate target in order to inhibit adhesion and thus prevent the infection itself. During the discussion on vibriosis, he indicated the increasing demand for solutions such as AMPifin given the disease's prominence in fish. He also suggested ways to deliver the peptide, and recommended using bioinformatics tools to test our solution's efficacy.

OUR INFERENCE

Via this interaction, we identified the statistical significance of vibriosis and its prevalence. We understood the plausible impact that our project could have on society and the impact AMPifin—a sustainable solution, could have on the fisherfolk of our country.

Mr. M Madan

Mr. M Madan, Feedmill Operator at ICAR-CIBA, Chennai, India.

THEIR INPUT

We contacted Mr. Madhan on 29th May 2022 to get data on the basic feeding and cultivation practices of fish. He told us that vibriosis is quite prevalent in fish breeds such as Mullet and Milkfish. Being well-versed with the different diagnostic methods used for various ecosystems such as freshwater, saline, marine, brackish water, etc., he mentioned the commonly utilised medicines or antibiotics, which are fed to the fish in the system. According to his experience, vibriosis is prevalent due to the changes in various environmental factors, weather, climatic change, and the water’s pH and contaminant levels, collectively termed as “stress-factors”. He concluded that the effect of vibriosis in humans is dependent on each individual’s immune system and how their body reacts to it. Furthermore, he elaborated on the various early detection methods such as PCR, western blotting, kit-based methods, and microscopic analysis.

OUR INFERENCE

This interaction shed light on the feeding mechanisms practised in the fish farms. The nutritional value of the fish can only be maintained by providing a balanced supply of the diet. Furthermore, in order to incorporate our project as a practical approach, our formula will vary for various fish species with different intake capacities. His input pushed us to study the current methods used for on-field fish testing. We found out that diseased fish are usually isolated in the fish ponds or taken to the labs for further testing in an artificial ecosystem. He mentioned that the usual practice of the local fish farmers is to subject the fishes to various antibiotics, chemicals, such as KMnO4, NaCl, and soda lime, etc to get rid of the disease in the farm. This helped us brainstorm ideas to spread awareness on the safe usage of antibiotics.

Dr. Seetha Krishnan

THEIR INPUT

We interacted with Dr. Seetha Krishnan on 30th May 2022. She was intrigued by our project idea and its potential impact on the pisciculture industry. Based on the stability of our peptide when released into a pisciculture system, she highlighted the possibilities of our peptide undergoing degradation and high dilution. She spoke about lab-scale testing of our peptide for interaction studies. To overcome such challenges, she suggested we look into methods like encapsulation and check the integrity properties in accordance with the release of the peptide into the aquaculture set-up. She also told us to look into our solution's economic viability, upscaling its production and food safety factors. Additionally, she conveyed her concerns regarding the peptide purification aspects of our project. She recommended we use the HPLC system for regular purification, which would be a practical approach for our experiments due to its cost-effectiveness.

OUR INFERENCE

Dr. Seetha laid the foundation for our delivery system. Our interaction with her made us consider the factors involving the cost-effectiveness of our solution. Through this, we decided Escherichia coli was the best host for our peptide production. She helped us look into our idea's biosafety and bioethical concerns. Her recommendations also made us look at CRISPR-based systems for plasmid designing. The papers she sent helped us with inputs to design our delivery mechanism—i.e. chitosan nanoparticle encapsulation. Towards the end of the meeting, we accounted for her varied insights on the ethical issues revolving around our project. This made us analyse and work on an effective approach for the society and the environment.

Dr. Mamatha Ballal

THEIR INPUT

We approached Dr. Mamatha Ballal on 2nd June 2022 to get solutions for our research-based queries and information on the prevalence of vibriosis. Since vibriosis is a cause of concern in humans as well, her expertise area made her the most appropriate person to reach out to. We learnt the symptoms and treatment methods of vibriosis in humans. Additionally, we gained more information on the pathogenicity and virulence of V. parahaemolyticus. Commenting on the frequency of people suffering from vibriosis in our locality, she mentioned that V. parahaemolyticus is pervasive in people who consume non-vegetarian food. According to her, the severity of the infection is majorly caused due to poor sanitation and hygiene habits followed. Though vibriosis is a devastating disease, it is most commonly prevalent in rural areas as compared to urban areas. She mentioned that people suffering from vibriosis usually suffer from diarrhoea which leads to higher chances of mortality, especially among immunocompromised people. She gave her suggestions for reducing the risks associated with Vibrio spp. and also said that V. parahaemolyticus is a common food pathogen. She mentioned how temperature change could also affect the pathogenicity factors of vibriosis. She also mentioned that stool samples are collected to analyse and detect vibriosis in humans. Furthermore, she elaborated on the culture-based methods for V. parahaemolyticus—i.e., using blood agar and nutrient agar.

OUR INFERENCE

The interaction with Dr. Ballal helped us gain more statistical data on the reported cases concerning vibriosis. This helped us build a core understanding of the importance of following good hygiene practices by taking small steps to avoid such diseases. We also learnt about the various detection methods used. She also demonstrated lab techniques that could benefit our future implementation studies.

Dr. Ramesh Sistala

THEIR INPUT

We had our second interaction with Dr. Ramesh on 28th June 2022 to discuss the structure prediction of MAM7. We explained the adhesion mechanism and how we used ClusPro for docking. To design our peptide at this stage, we tried to integrate the concept of peptidomimetics. He mentioned that fibronectin is the apparent binding site with five repeating units, and the repeating modules are used for the initial peptide design. Dr. Ramesh suggested that we could use a computational alanine scan to refine the binding sites and molecular dynamic simulations. If the receptor is prominent, we cannot do it on the 40 amino acids and would have find the local binding regions. To inhibit the reaction, the five continuous domains of MAM7 are required to interact. In this case, the peptide acts as an inhibitor, and the peptide docking is done on the receptor. If the receptor is big, a hypothesis should be formulated that interacts with MAM7. The model can be verified by profiling the interaction. The relevant residues can be docked by using only specific residues and changing the others. Furthermore, he elaborated that five or six types of amino acids are required, and all 20 amino acids need not be required. He further elaborated more on the Build option in Pymol that could be used for visualising the protein structure.

OUR INFERENCE

In this meeting, Dr. Ramesh provided us with the technique to generate a pool of peptides. This is where we decided our peptide to act as an inhibitor for our target molecule—MAM7. Furthermore, the alanine scan test he suggested was the basis for our modification cycles, including our software contribution.

Dr. Kannan Ramaswamy

Dr. Kannan Ramaswamy is a Gastroenterologist at Bhagwan Mahaveer Jain Hospital, Bangalore, and Sagar Hospitals in Jayanagar, Bangalore. He is a member of the Indian Medical Association (IMA) and Karnataka Medical Council.

THEIR INPUT

We approached Dr. Kannan on 3rd July 2022 to get information on the effect of vibriosis on human beings. Based on his research, he indicated that vibriosis involves a sporadic outbreak that is not as prevalent in urban areas since they consume filtered water. With many years of experience, he mentioned that an epidemic is more noticeable around river bed regions due to direct river water consumption and spreads as a communicable disease. The severity of these infections is often treatable by following the appropriate hydration measures as the patients are observed for an incubation period of three days for the development of any symptoms like vomiting, loose motions, or rice water diarrhoea. According to him, the major source of vibriosis infection in humans is untreated water. Furthermore, he mentioned that vaccines are administered to reduce a local epidemic risk. However, he also mentioned that often, vaccines don't find their way to rural areas, leading to large-scale epidemics that could be lethal beyond a point.

OUR INFERENCE

The interaction with Dr. Kannan gave us an insight into statistics associated with reported cases of vibriosis in India and worldwide. This made us consider the ethical aspects concerning AMPifin and the treatment plans followed when such cases are reported. We could also gauge the vicious cycle of administration of antibiotics that could be harmful to an individual or be redundant for the Vibrio species due to the development of antibiotic resistance.

Dr. M Shashi Shekar

Dr. M. Shashi Shekar, Principal Scientist & SIC, Nutrition, Genetics and Biotechnology Division, working since 1994 in ICAR-CIBA, Chennai, India.

THEIR INPUT

We visited the Indian Council of Agricultural Research-Central Institute of Brackishwater Research (ICAR- CIBA) institute located in Chennai on 8th July 2022. Dr. M Shashi Shekar and his team consisting of Dr. Ashok, Dr. Raymond, Dr. Sujeet and Dr. Patil who were working on the prevalence of vibriosis in fish. We felt that the information they had would benefit our project’s geographical statistics and research-related data. There was a brief discussion about the types of projects they were working on for the research-oriented communities and looking for potential areas in which they could assist us with. Dr. Shashi Shekar introduced us to his lab and his team which would help us in the future with our project.

OUR INFERENCE

The visit to their institute assisted us in gathering more information on the field studies of their testing is carried out, which are similar to our project. We utilised this to bring out the efficacy of our peptide and the disease. Furthermore, they also gave data on the products they manufactured and their current ongoing research. They also provided specific lab protocols that were useful for our future implementation. After this meeting, we decided to have another meeting with them to further exchange data on the research carried out.

ICAR-CIBA

ICAR-CIBA (Dr. M Shashi Shekar & team), Chennai, India.

THEIR INPUT

We approached Dr. Shashi Shekhar and his team from ICAR-CIBA on 10th August 2022 after meeting them at their institute. He introduced us to Dr. Ashok, well-versed in bioinformatics, who discussed our dry lab aspects. Dr. Raymond, who holds good knowledge of aquaculture operations, helped us gather the parameters necessary for the fish tank dimensions. Likewise, Dr. Patil and Dr. Sujeet were the two microbiologists who helped us collect data for our research. They provided relevant data about the various bacteria present in Vibrio species that affect fish and shrimps. We further interacted with them about the various other bacterial and viral diseases that cause economic losses to the fisherfolk. They provided more data on parameters, such as pH value range and ammonia concentration that are to be maintained to prevent such outbreaks of diseases in the fish farm. Furthermore, they appreciated our idea of targeting initial adhesion and discussed current treatment methods.

OUR INFERENCE

It was due to Dr. Raymond that we could formulate a design system for our mesh that would fit at the inlet pipe. His input on the numbers corresponding to the tank and pipe dimensions helped us get a clear idea of our design.

Dr. Manickam G

THEIR INPUT

We interacted with Dr. Manickam on 12th August 2022, to receive inputs on the economic viability and large-scale production of our peptide. He was impressed with our usage of chitosan nanoparticles as a delivery system since it is a natural polymer with immunomodulatory properties and is biodegradable, too. He suggested that we increment the length of our peptide that would help boost our large-scale production. Through this, the purification cost would decrease and the ideal stability conditions and formulation required for the same would be at a more convenient level. This drastically decreases the volume, making our solution more convenient for biological maintenance. He mentioned that the current methods include the biological-based drugs and proteins that are orally administered to animals. On the aspects of bioavailability, the carriers or linkers do not have that much of an impact at a particular site. With regards to the shelf-life of storing the peptide, he suggested that it could be in the form or texture of salt which has durability for almost a week. Towards the end, he suggested focusing on the ethical issues associated with the disposal of the residue, if any, in the environment and also highlighting the impact being visible on human beings, the end consumers of the food chain.

OUR INFERENCE

The interaction with Dr. Manickam gave us a better understanding of implementation of appropriate purification methods for commercial production and for the isolation of our peptide using modern techniques. This helped us formulate the best fit peptide production using a synthetic approach. It also made us consider the peptide stability condition through salt formation at room temperature or an elevated temperature.

Reiss Jones

Reiss is a sustainability-based entrepreneur and has previously co-founded two startups.

THEIR INPUT

We approached Reiss Jones on 16th August 2022 to understand the possible entrepreneurial aspects we could incorporate in our project. He was quite intrigued with our team's approach for the peptide design and he mentioned the skyrocketing demand for sustainable solutions in the industry. We explained to him that the fusion protein we intend to have is to increase the stability of the short peptide sequence given their instability. We also gave him a gist on our plan of performing interaction studies with MAM7 and our peptide. After arriving at a proper understanding of our project, he helped us by connecting with various synthetic biology professors and other experts in the field of marine biotechnology.

OUR INFERENCE

He assisted us in navigating the kind of companies we must reach out to and the aquaculture specialists we can approach for better insight on our project and shared a few helpful contacts. Furthermore, he assisted us in reaching out to other experts who could be beneficial to AMPifin.

Ronald Kevin D’Souza

THEIR INPUT

We visited Aquatic Biosystems in Mangalore, owned by Mr. Ronald D’Souza on 22nd August 2022. He set up Aquatic Biosystems in 2006, which is currently the largest ornamental fish farm in the state of Karnataka and the country’s most scientifically developed ornamental fish farm. He explained his journey of setting up the farm and why he chose to use intensive systems, despite the general preference of using extensive methods for traditional fish farming. He conducts imports and exports from countries like Germany and Netherlands. He mentioned the procedures involved in the Recirculatory Aquaculture System (RAS) filter system for aeration using filters. However, a common problem their farm faces is the slow stream of water due to electric power issues in India. The general protocol they follow involves quarantining the fish to identify and prevent any diseases in the hatchery for the breeding methods. Additionally, he mentioned that 5 grams per 100 litres of tetracycline is added to the farming unit. The feed added includes fresh blanched meat and dry granular feed. Fish feeding schedule at the larval stage is five times per day, and in the adult stage, the fish are fed two times per day. The monsoon season enhances the production rate with higher export value, simultaneously increasing susceptibility to diseases. The optimum pH maintained in their breeding unit ranges from 6.5 to 7.2. In case of any increased pH condition, alum is added to balance it out, and in case of any decreased pH range, the oyster shells are utilised as a buffer.

OUR INFERENCE

We were delighted to observe the application of filters in the fish farm. This gave us the confidence to propose a future implementation of our project in the form of a mesh that the inlet of the fish tank would be subjected to. The parameters provided by him formed a basis for our factors into consideration when searching for an appropriate delivery mechanism.

Miroslav Gasparek

Mr. Miroslav Gasparek is a Bioengineer, Entrepreneur, and Researcher. He is also a Co-founder at Stealth Biotech Startup and a Co-Managing Director at Nucleate UK.

THEIR INPUT

We interacted with Miroslav Gasparek on 26th September 2022, who provided our team with insights on the biosafety and biosecurity aspects revolving around our project and synthetic biology as well. The interaction with him led to have major conversations on how Genetically Modified Organisms (GMOs) play a crucial role in the current emerging field of synthetic biology

OUR INFERENCE

We were pleasantly surprised by the immediate conceptual grasp he exhibited regarding our project. We incurred valuable insight on the various aspects of biosafety, good lab practices and biosecurity that play a crucial role in the field of synthetic biology. Given his area of expertise, he questioned the gravity of our problem. This helped us string our research-work toward a more impact-oriented approach, while also ensuring a safe and sustainable workflow of AMPifin.

Dr. Vinod Jyothikumar

Dr. Vinod Jyothikumar, currently working as a Operations Management Consultant in a consulting firm.

THEIR INPUT

We interacted with Dr. Vinod Jyothikumar on 28th September 2022 to receive inputs on the biosafety aspects associated with our project and how we have integrated synthetic biology aspects to make it a viable practical application. He suggested we use PubMed graphs for tracking the horizontal transfer of pathogenicity of the Vibrio species which turned out to be milder. Since Vibrio parahaemolyticus is an important human pathogen according to WHO guidelines, Dr. Vinod suggested we analyse the risk assessment factors associated with it. We mentioned that for further testing, since our project involves a BSL2 pathogen, our approach is to express Multivalent Adhesion Molecule 7 (MAM7) in Escherichia coli. He highlighted the biochemistry and biophysics aspects involved in making our peptide more commercially realistic

He spoke about how we could include a visual signal for our binding by making use of a GFP. He asked us about the parameters that affect the pathogenicity of Vibrio parahaemolyticus, such that we could derive an ideal condition that renders this bacteria harmless. For the His-tag pull-down mechanism, he spoke about using quantum dots with Nickel coating. These conditions can include yellow or green wavelength along with Nickel, and once the carrier is ready, it could be treated in the lab to observe the quantum dots under the slide microscope where it glows. He urged us to include a visual proof of the MAM7 and peptide interaction. The major application of visualising, would involve methods of integration of quantum dots.

OUR INFERENCE

The interaction with Dr. Vinod helped us look for a more realistic approach of synthetic-biology to our project for the binding. As seafood is a major staple diet of South-East tropical parts of Asia, Dr. Vinod's suggestions assisted us in browsing through how its effects in the market considering the socio-economic element such as Environmental, Social, and Governance (ESG) indices.