INTEGRATED HUMAN PRACTICES

Comprehending the project's influence on the community, obtaining input from stakeholders, and setting up a feedback mechanism are essential elements of integrated human practices. To facilitate this feedback process, we engaged with numerous individuals and organizations during the project's duration.

Step 1: Understanding the problem

1. Dr. Kutubuddin Molla

Senior scientist at ICAR- National Rice Research Institute.

In the realm of agriculture, synthetic biology offers exciting possibilities, especially when focusing on vital crops like rice. In our brainstorming phase, we had the privilege of attending a talk by Dr. Kutubuddin A Molla, an expert in genome engineering using CRISPR-Cas systems for rice plants. His talk sparked our interest in exploring CRISPR technology and its application to tackle various diseases affecting rice plants. Given our proximity to rice farming communities, we took the initiative to engage with local rice farmers and understand the challenges they face. We discovered alarming issues like pesticide misuse and the adverse effects of chemical pesticides on rice crops. Many of our team members have family members involved in farming, which deepened our understanding of the problems at hand. Through these conversations, we identified a concerning issue - bacterial blight, caused by the Xanthomonas oryzae pv oryzae bacteria, posing a significant threat to rice cultivation.

What did we learn from him?
 •He first introduced to us the disease of ‘Bacterial Leaf Blight’ of rice
 •He asked us to look into the specific genes which, upon introducing Double Strand Breaks in Xanthomonas, would completely stop the growth of bacteria.
 •He also reminded us to keep in mind the load of symbiotic bacteria a plant could handle while designing our experiments.

2. Farmers in Madandanga (West Bengal)

In our initial farmer survey, we encountered an elderly farmer Mr.Shamu from Madandanga who has devoted his entire life to cultivating a small piece of land. Lacking formal education, he has accumulated vast farming experience over the years. Our conversation revealed that he does indeed grapple with bacterial blight issues. However, it was evident that he faces multiple challenges, one of which is his inability to distinguish among the various agricultural problems. Additionally, due to limited financial resources and apprehension about adopting new technologies, he remains hesitant to explore innovative methods for combating bacterial blight. The primary barrier we identified in his case is a significant lack of awareness and knowledge, leading him to adhere to traditional and conventional farming practices.

3. Farmers in Medinipur (West Bengal)

In Medinipur, we interviewed Mr. Bola Duari first, a seasoned farmer with 30 years of hands-on experience in rice cultivation. Mr. Duari manages a 2 bigha plot of land and diligently produces rice twice a year during the summer and monsoon seasons.
He informed us that to safeguard his rice crops against bacterial blight, Mr. Duari follows a preventive approach. He uses a combination of fungicides, including folicur, bavistine, and ketazine, as well as antibiotics such as plantomycin and streptomycin. The dosage for both antibiotics is 15 grams in 15 litres of water, effectively countering potential outbreaks. But, the problem is he is actually not sure about the symptoms of bacterial light, and sometimes the addition of these biopesticides creates a problem for him, by magnifying the problems by many folds.
We next conversed with Mr. Haripada Das, an experienced farmer managing a plot of land spanning approximately 3-3.5 bighas. He diligently cultivates rice, yielding two harvests each year during the summer and monsoon seasons. His preferred rice variety is the well-known 'Lal Swarna.'
However, Mr. Das has to face several agricultural challenges. Some sections of his fields are susceptible to waterlogging issues, which have led to significant occurrences of bacterial blight in the past. To combat this disease, he has to rely on a combination of fungicides and antibiotics. Despite his efforts, Mr. Das expresses growing concerns about the rising expenses associated with fungicides and pesticides, which make up a considerable portion of his budget. He uses various brands of the fungicide 5% hexaconazole, each differing in price.

4. Farmers in Hili (West Bengal)

During the surveys in Hili, we encountered similar issues as those faced by people in Nadia: economic constraints and a lack of awareness. When asked about their preferences for a remedy, they expressed the need for portability, affordability, user-friendliness, and durability as essential features. Additionally, they emphasized the importance of achieving high productivity with minimal resource utilization. The pervasive lack of knowledge continues to be a significant obstacle for them.

5. Farmers in Sunderbans (West Bengal)

During our interactions with the farmers in the Sundarbans, we learned that while high salinity in the soil isn't a primary cause of bacterial blight, the rice plants in the region do indeed face issues referred to locally as 'Jhalsa Rog.' Despite their efforts to fortify the plants, the problem persists. The farmers often struggle to distinguish between bacterial blight and nutrient deficiency diseases and rarely conduct soil tests. Unfortunately, they receive limited support from NGOs, which seldom visit their communities. The root of the problem lies in their lack of modern agricultural knowledge and awareness, making it challenging for them to address these agricultural issues effectively.

6. Farmers in Birohi (West Bengal)

In Birohi, the level of awareness among farmers appears to be slightly higher compared to other regions we visited. While they possess some knowledge of bacterial and fungal problems and their respective treatments, they are only partially aware of bacterial blight and lack a comprehensive understanding of how to combat it effectively. From our extensive field surveys we got to know that the disease of Bacterial Leaf Blight is deeply ingrained in our locality and every year there are enormous losses faced due to this disease every year. The farmers survey form is given here: Pdf

7. Prof. Subrata Datta

Our campus is in the immediate vicinity of an Agricultural College, Bidhan Chandra Krishi Vidyalaya (BCKV). We talked to Prof. Subrata Datta of Plant Pathology Department. We discussed our observations in the fields and the problem of bacterial blight in rice with him.

What did we learn from him?
 •Two types of diseases create a severe havoc for rice cultivation in West Bengal. One is Bacterial Leaf Blight (called “Jholsha Rog” in local language) and the Sheath Blight. Both of them occur in two different seasons.
 •He informed us that farmers take up their crops to Pesticide Retailers and apply whatever the pesticide retailers sell to them.
 •Oftentimes, this is not very effective and farmers often buy incorrect medicines.
 •He suggested the idea to build a Detection Kit such that it can be used by farmers itself and does not require intervention of any other pesticide dealer to identify the problem.
 •Thus we built an early detection kit.

8. Mr Fozrul Hoque

Mr. Fozrul Hoque is a pesticide dealer in Madandanga, West Bengal. He mentioned that during the monsoon season, it's necessary to apply a small amount of a product called "tilt" to keep the plants upright. They assured that this particular product doesn't have significant side effects. Additionally, they recommended conducting soil tests to determine the specific requirements for your crops.
This application is typically done by spraying the pesticide, and it takes 3 to 4 days to take effect. The primary purpose of using this method is to prevent the spread of diseases or pests to other leaves. The dealer emphasized their interest in exploring new technologies that are more time-efficient and cost-effective. Some pesticides have the potential for biomagnification, making them harmful to the environment, while others do not pose this problem. However, plants are gradually adapting to these pesticides.
Farmers bring their plant leaves to the pesticide dealer for evaluation, and the dealer provides suggestions based on their observations. These pesticides are sometimes generic and can be used for various types of crops. In some cases, potash is also used, but it's not spread by sprinkling; rather, it's applied by hand.'

The key takeaways were:

After this the team had active brainstorming sessions trying to figure out an effective solution against the problem utilizing the tools of Synthetic biology and genetic engineering. We came up with two possible solutions. One is to use the technique of RNA interference or to use CRISPR gene editing techniques. Now we had to find a proper delivery vector for the same.

Step 2: Building up the Project

9. Dr. Amirul Islam Mallick

Dr. Mallick is professor at IISER Kolkata and a Bacterial OMV expert.

We discussed with him the problem of bacterial blight and discussed with him how we can try to find a unique and cost-effective solution to tackle the problem. He supported our proposal of utilizing bacterial nanovesicles as a delivery vector to carry CRISPR-Cas RNA complexes.

What did we learn from him?
 • We met with sir in the initial phases of the project.
 • He has worked with outer membrane vesicles of bacteria and helped us with how to approach the outer membrane vesicle isolation.
 • He also gave insights into vesiculating behavior of gram negative bacteria. He also suggested doing Bradford Assay and SDS-PAGE to characterize outer membrane vesicles.
 • He also suggested experiments to check fusion of OMVs secreted by E. coli with that of Xanthomonas.

10. Dr. Partho Sarothi Roy

Dr. Partho Sarothi Ray is an associate professor in the department of Biological Sciences at IISER Kolkata. He is interested in RNA-binding protein and molecular evolution.

Dr. Ray told us that designing CRISPR experiments is relatively straightforward, as researchers only need to design the guide RNA sequence for their target gene. In contrast, RNAi may require designing and validating multiple small interfering RNAs (siRNAs) to achieve effective gene silencing.

What did we learn from him?
 •He asked us to find a receptor ligand for Xanthomonas so that it can be incorporated into the membrane.  •He also suggested that we try out the Twin-Arginine Translocation system for loading proteins into OMVs, and track them with GFP if they are fusing with the desired cells  •He also suggested incorporating an effective oscillatory kill switch or an inducible one in our project.

11. Dr. Jayanta Kumar Tarafdar

During our conversation with Prof. Tarafdar, we gained valuable insights into the prevalence of bacterial blight, which appears to be a common and widespread issue in various regions, including Chakdah and Haringhata. Prof. Tarafdar also stressed the significance of the ooze test for diagnosing the problem, where cutting a leaf and immersing it in clear water can reveal the presence of ooze.
In response to our discussion about farmers' and pesticide dealers' suggestions regarding fungicides, Prof. Tarafdar recommended the use of Copper oxychloride, as it can effectively combat various bacterial-related diseases. Additionally, he pointed out the issue with urea, explaining that while it can address fungal problems in plants, it can exacerbate bacterial problems significantly. These insights were instrumental in shaping our project's direction and objectives.

What did we learn from him?
 •He raised a critical question about identifying the stage of plant life at which the infection occurs, whether during flowering or the development of food grains which would determine at what stage we use the detection kit.  •He advised us to develop a low-cost detection kit and highlighted the importance of selecting the right protein for this purpose

12. Dr. Dipshikha Chakravortty

Dr. Dipshikha Chakravortty is an Indian microbiologist, molecular pathologist and a professor at the department of Microbiology and Cell Biology at the Indian Institute of Science, Bangalore.

Dipshikha ma’am discussed with us the benefits of Cpf1 or CRISPR Cas12a systems over Cas9 systems. She told us about the smaller size of Cas12a with respect to Cas9, no requirement of tracrRNA for its maturation, ability of easily carry out multiplex genome editing

What did we learn from her?
 •She suggested using FnCas12a for our CRISPR system instead of Cas9, and enlightened us on the difference between the two.  •She also asked us to check the minimum concentration of DSF required for the activation of the detection circuit.

13. Dr. Partha Pratim Majumder

Distinguished Professor and Founding Director, National Institute of Biomedical Genomics

Dr. Majumdar during his interaction questioned whether we can use simple restriction endonucleases in place of CRISPR system for genome editing. To which we answered that we would lose the component of targeted specificity and secondly it is highly risky as it might cleave sequences in undesired cells.

What did we learn from him?
 •He gave us insights about the population modeling dynamics.  •And also asked us to carry out a consumer survey to check how many people are ready to accept rice produced by application of bacterial biocontrol agents.  •He also helped us to get numerous contacts of farmer associations and helped us in education part

14. Dr. Sharmila Mande

Dr. Sharmila Mande, Former Distinguished Chief Scientist, TCS Research

Prof. Mande being from both academia and industry backgrounds helped to analyze our project both scientifically and as a finished product in the market.

What did we learn from her?
 •She asked us to compare our solutions with already existing bio-control agents.  •She also asked us to apply for Patents once the Proof of Concept is successful.

15. Dr. Arabinda Chaudhuri

He is an expert in lipid nanoparticle bound delivery systems. His expertise in lipid synthesis and delivery systems guided us for many experimental designs like OMV purification post isolation. He had an active conversation with us to discuss the efficiencies of OMVs vs lipid nanoparticles as a delivery vector. He also suggested that in future we try fusion of both OMVs and Lipid Nanoparticles with one another using Polyethylene Glycol (PEG) use it for drug delivery research.

What did we learn from him?
 •He suggested various methods of CRISPR delivery systems and important factors to consider while creating such a system.  •He also introduced us to novel methods of CRISPR delivery and lipid target specificity

We are committed to improving our solutions to better cater to the needs of our local community. We engaged our stakeholders and academic advisors at every phase of our solutions. Starting from research and concept development, through the creation and evaluation of Xanthacinator, our design approach remained entirely focused on our stakeholders' perspectives and requirements.

16. Prasun Bhowmik

Distinguished Professor and Founding Director, National Institute of Biomedical Genomics

During our meeting with Prasun Bhowmik, the Agriculture Development Officer at the Block Development Office in Haringhata, we learned that local farmers frequently seek their assistance for a variety of agricultural concerns. The Agriculture Development Officer and their team actively work to engage with farmers by organizing awareness workshops, conducting survey campaigns, and occasionally inviting professors and scientists to provide guidance. A significant challenge they face is that farmers often present multiple issues without distinguishing between different problems, making it difficult to provide specific solutions. This is further complicated by the limited awareness among farmers. The Agriculture Development Officer is also actively involved with farmer associations and shared their contact information with us.

17. Mr. Amal

Farmer's Association Head

The Farmer's Association Head, Mr. Amal expressed similar concerns regarding the lack of awareness and knowledge among local farmers. They emphasized that farmers often struggle to distinguish between issues like bacterial blight and other problems that exhibit similar symptoms. Economic constraints were another challenge they highlighted, as many farmers found it difficult to afford advanced treatments or technologies.
We engaged our stakeholders and academic advisors at every phase of our solutions. Starting from research and concept development, through to the creation and evaluation of Xanthacinator, our design approach remained entirely focused on our stakeholders' perspectives and requirements.

Step 3: Implementation and Evaluation

18. Minshad Ansari

He is the CEO of World Bioprotection Forum and Bionema.

What did we learn from him?
 •We came to know about the importance of local agronomists, and extension programmes. He introduced us to 3 categories of biopesticide industry: biocontrol, biostimulant and biofertilizers (which comprise 90% of the industry market).  •He also educated us about different formulation and deployment methods, like talc based powder, microencapsulation method, and regular nozzles, whose size changes with frequency of usage.

19. Kanish Sarkar (online)

Microbac India, shyamnagar

He told us we need to approach the pesticide dealers and educate them as a first step to increase the acceptance of biocontrol agents among farmers.

What did we learn from him?
 •Biocontro agent is not a new thing in India, it is already established but farmers are not habituated with it.  •Educating pesticide dealers should be one of our priorities.

20. Amlan Datta (offline)

He is the CEO of startup BOM Life.

He told us that the primary goal of the biocontrol laboratory is to produce a large quantity of biological agents used to manage pests and diseases. These bio control agents are a crucial and essential component of Integrated Pest and Disease Management (IPDM).

What did we learn from him?
 •We learnt about the approved process of deployment of biocontrol agents in India.  •He talked about the challenges of non-genetically modified deployment, and the importance of formulations.  •He also talked about regulatory bodies and processes in the bio-control deployment process.

Consumer Survey Form

The data collected through the consumer survey form have laid a robust foundation for guiding our research and assessing societal approval. Dr. Malancha Ta assisted us in reviewing our survey and interview questions to ensure their logical and meaningful structure. She is a member of the Institutional Ethics Committee that granted approval for our survey and interview inquiries.
Our engagement with the farming communities in Birohi and Madandanga, nestled within the Kalyani Subdivision of Nadia District, and Hili in Balurghat Sadar Subdivision of Dakshin Dinajpur District, extended beyond awareness campaigns. To develop our project and gain a comprehensive understanding of the challenges faced by farmers in crop identification and management, we undertook a series of surveys. Total 172 people participated in the consumer survey. Some of the responses to the key questions are:

Safety

21. Dr. G. Lekha

Dr. Lekha Scientific Officer at IISER Kolkata.

She provided valuable insights on waste disposal protocols and played a pivotal role in guiding us through all safety considerations throughout the project. Her expertise was instrumental in the formulation of our project's safety procedures and documentation. As the biosafety officer for our institute, she conducted a comprehensive workshop on safety and security, ensuring that we received adequate training to prioritize the safety aspects of our project.

Conclusion

The surveys and interactions were meticulously designed to capture firsthand insights from both the farmers and the pesticide dealers. By engaging with these key stakeholders, we sought to unearth the core issues plaguing agricultural practices in these regions. The information gathered through these surveys played a pivotal role in shaping our project and ensuring that our solutions were aligned with the real needs of the farming communities. Thus, we have effectively established feedback loops connecting our synthetic biology project to its real-world context, encompassing Reflection, Responsibility, and Responsiveness.