Integrated Human Practices
Our comprehensive approach to understanding and addressing the social, ethical, and environmental implications of our project through meaningful stakeholder engagement.
Academia
Academic perspectives form the backbone of any synthetic biology project that aims to bridge innovation with real-world application. Engaging with researchers and professors allowed us to look beyond the immediate technical details of our system and understand how our work fits within the broader scientific and ethical landscape. Their insights helped us refine our problem statement, align our approach with current research, and recognize the limitations and possibilities of using synthetic biology as a tool to address complex biological problems. These interactions shaped our project not just as an experiment, but as a scientifically grounded exploration that builds upon established knowledge while contributing to the growing dialogue between academic research and applied synthetic biology.
Dr. Bianca Sclavi
Who We Contacted
Prof. Bianca Sclavi - Director of Research at CNRS, France
Why
Her team's work on oscillations in dnaA expression during the bacterial cell cycle has been foundational to our project. Prof. Scalvi's research group used the dnaA operon as a reporter to study the activity of DnaA-ATP. The paper explained the dual role of dnaA as both an activator and repressor for the dnaA operon; and the interplay with SeqA-dependent repression which in turn is affected by GATC methylation. This provided us with critical context for designing our own GATC methylation-sensitive promoter based on the dnaA operon.
What We Learnt
Promoter engineering: Adding GATC sites at chosen promoters could help regulate gene expression through methylation, though SeqA binding effects need careful consideration.
Epigenetic Memory System: Bacterial "memory" systems differ from eukaryotic ones but could be powerful in biosensing. We need to think critically about mechanisms to turn off such systems and control background expression levels.
Alternative strategies: We need to reflect on the necessity of methylation marks for memory systems. Prof. Sclavi reminded us that even inducible promoters with positive feedback can provide heritable memory.
Applications: She advised us to explore applications of targeted methylation such as stress response systems, DNA repair, and synthetic biology solutions for microbiome engineering and plastic degradation.
Impact on our project
These interactions provided valuable technical validation for our project and highlighted several promising directions for future work. The discussion also addressed practical lab challenges, including transformation efficiency and plasmid compatibility, giving us effective strategies to enhance our workflows and troubleshooting. Overall, this guidance not only strengthened our current project design but also sparked ideas for expanding the scope of our synthetic epigenetics research.
Dr. Shamlan Reshamwala
Why We Met
Since our iGEM project is rooted in applications for biomanufacturing, we were keen to interact with experts who are working at the intersection of synthetic biology and industrial biotechnology. Dr. Shamlan Reshamwala is an Assistant Professor at the Institute of Chemical Technology (ICT), Mumbai, and serves as a faculty lead and primary investigator for the ICT Mumbai iGEM team. His work focuses on advancing biomanufacturing using synthetic biology frameworks.
We first came across Dr. Reshamwala at this year's All India iGEM Meet (AIIM), where he shared perspectives on biomanufacturing and its potential for scaling sustainable solutions. We really enjoyed our initial discussion with him at AIIM, which made us curious to learn more about his research and explore possible directions where his insights could connect with our project. This led us to schedule a dedicated meeting with him, building on that earlier interaction.
What we learnt
During our meeting with Dr. Shamlan Reshamwala, we explored ideas for future projects and gained valuable advice on the iGEM jamboree experience. He emphasized the importance of attending the jamboree not just with the aim of winning, but to learn from others and return inspired to further our scientific journey.
For our dry lab work, Dr. Reshamwala recommended studying research by Dhananjaya Sarnath on the epigenetics of oral cancer, as well as interesting findings on age-related methylation patterns in sperm cells that contribute to infertility.
We also gained critical insight into the challenges faced by genetically modified organisms (GMOs) in India. Dr. Reshamwala highlighted that regulatory hurdles and public perception significantly limit the mainstream use of GMOs, causing many successful experiments to remain confined to the lab stage despite their potential applications.
Impact on our project
The meeting inspired new ideas for future work and shaped our dry lab focus, grounding our project in both innovative epigenetic insights and practical challenges in biomanufacturing.
Prof. Subhojit Sen
Why we spoke to him
Professor Subhojit Sen's work on Chlamydomonas-based epigenetics assays for drug discovery is truly inspiring. His innovative paper, "A potential screening method for epigenetic drugs," presents a three-step epigenetic assay that tracks the variegation of randomly integrated transgenes to identify compounds with epigenetic activity, using a cost-effective spot-dilution analysis system that makes complex epigenetic measurements achievable in basic undergraduate laboratory settings. The screening platform successfully identified plant-derived compounds like cinnamic acid that show similar phenotypic responses to known histone deacetylase inhibitors, opening new avenues for discovering epigenetic drugs from traditional medicinal sources.
We networked and connected with Professor Sen during this year's All India iGEM Meet (AIIM), where he presented his work on "Synthetic Models for Epigenetic Drug Screens".
Professor Sen is a leading researcher in cancer epigenomics and environmental epigenetics at UM-DAE Centre for Excellence in Basic Sciences, with research focusing on understanding how environmental factors induce heritable changes in gene expression through epigenetic modifications. His research specifically examines stress-induced epigenetic memories that lead to gene silencing and transgenerational inheritance patterns, which directly relates to synthetic biology applications where environmental triggers are used to control epigenetic switches.
Alternative applications were suggested for the dCas9-Dam system, including applications in algae for upregulating genes involved in green hydrogen production. The meeting also explored replacing GFP with kill switch genes to cause controlled cell culture death when the cell culture encounters an ON signal or stimulus indicative of uncontrolled growth. In the proposed memory system, a methylation-sensitive gene repressor is a must, and the current part, the zinc fingers, are very hard to design and non-modular in nature. Therefore, there is a need for a more reliable and programmable alternative. The discussion on this matter led to TALENs being recommended as a superior option. It was suggested that we must verify the expression and translation of the dCas9-Dam fusion protein, using validation methods such as SDS-PAGE or tagging the fusion construct with an antibody. The transformation efficiency in our dam(-) cells was greatly reduced, and we obtained very few colonies. To address this issue, sequential transformation rather than co-transformation was recommended. When we discussed the issue of the dam(-) strain sharing the same antibiotic resistance as the dCas9-Dam plasmid, thereby nullifying the selection pressure needed for plasmid uptake, potential resource acquisition strategies were considered. It was suggested that we contact research laboratories to obtain small samples of specialized bacterial strains instead of relying on costly commercial stocks. The meeting provided comprehensive technical guidance spanning validation methods and alternative approaches for advancing the dCas9-Dam epigenetics project.
What We learnt
These interactions provided valuable technical validation for our project and highlighted several promising directions for future work. The discussion also addressed practical lab challenges, including transformation efficiency and plasmid compatibility, giving us effective strategies to enhance our workflows and troubleshooting. Overall, this guidance not only strengthened our current project design but also sparked ideas for expanding the scope of our synthetic epigenetics research.
DNA-Binding Alternatives: TALENs were recommended as a superior alternative to zinc finger proteins for methylation-sensitive gene repression/DNA binding. The Tet-On system was also suggested as another viable option for the team's epigenetic regulation purposes.
Validation methods: Methods for verifying dcas9 Dam fusion protein production inside the cells were discussed, including running the proteins on a gel and performing a western blot analysis utilising a dCas9 antibody.
Transformation Strategy Optimization: Sequential transformation rather than co-transformation was recommended when working with dam-negative bacterial strains. This approach could address the reduced colony numbers observed in dam-negative experiments.
Resource Acquisition Strategies: Cost-effective strain sourcing was discussed, with suggestions to contact research laboratories for small samples of specialized bacterial strains rather than purchasing expensive commercial stocks.
Co-transformation Optimization: An alternative strategy using high ratios of dCas9 plasmid was proposed, based on the principle that cells taking up one plasmid are likely to take up additional plasmids.
Impact on our project
These interactions provided valuable technical validation for our project and highlighted several promising directions for future work. The discussion also addressed practical lab challenges, including transformation efficiency and plasmid compatibility, giving us effective strategies to enhance our workflows and troubleshooting. Overall, this guidance not only strengthened our current project design but also sparked ideas for expanding the scope of our synthetic epigenetics research.
Prof. Anubama Rajan
Why we approached her
We interacted with Prof. Anubama, Department of Medical Sciences and Technology, IITM, who specializes in patient-derived organoids, particularly lung and pancreatic models, to understand how the EPIC system could eventually be used as a therapeutic tool for diabetes. Since our project focuses on using targeted epigenetic modulation to influence gene expression, we wanted her perspective on how our approach might fit into real-world disease models.
What insights we obtained
Prof. Anubama found our idea complex yet interesting, acknowledging its potential for long-term therapeutic applications. She pointed out that while our system could have promising implications for diabetes, implementing it in mammalian or pancreatic cells would require strict ethical and biosafety clearance. Specifically, she advised us to obtain approval from the Institutional Biosafety Committee (IBSC) at IIT Madras before starting any work involving mammalian or even E. coli systems under this project.
She also encouraged us to think carefully about the translational process and how a platform like ours could move from basic proof-of-concept to potentially act as a therapeutic platform. She generously offered lab support for mammalian cell work, which would help us begin testing the system in more physiologically relevant conditions once the necessary approvals were in place.
How we implemented these insights in our project
Based on her advice, we initiated the process of obtaining IBSC clearance for our project to ensure compliance with biosafety and ethical guidelines. We also started planning how to adapt our EPIC system for potential testing in mammalian cells. Her feedback helped us ground our long-term goals in both ethical responsibility and real-world feasibility, guiding us to think more carefully about how our epigenetic system could one day evolve into a practical therapeutic approach.
Prof. Víctor de Lorenzo
Why we met him
As part of our Integrated Human Practices journey, we reached out to Prof. Víctor de Lorenzo, a pioneer in metabolic engineering and synthetic biology, to gain perspective on how our project — the EPIC (Epigenetic Programmable Intervention and Control) system — fits into the broader context of synthetic biology. His deep experience in microbial engineering and genetic circuit design made him the ideal person to guide us on translating our ideas on epigenetic control into a framework with lasting impact.
What insights we obtained
Prof. de Lorenzo described epigenetics as the "software upgrade" to synthetic biology — if traditional synthetic biology forms the hardware, epigenetics adds the layer of adaptability, tunability, and memory. Through our conversation, several key insights stood out:
- Epigenetics as an enabling technology: Our work represents a foundational tool rather than an immediate industrial solution — something that could redefine how we understand and manipulate biological information flow.
- Domestication of epigenetic marks: Instead of focusing on demonstrating simple gene expression control, we should emphasize how EPIC helps in understanding and "domesticating" epigenetic processes, turning them into reliable, engineerable systems.
- Integrating regulatory layers: Gene expression outcomes depend not only on methylation but also on promoter strength and translational efficiency, highlighting the need for holistic system design.
- Future-facing framework: He encouraged us to view EPIC as infrastructure for future innovations, "cities built upon our road", rather than a standalone construct.
- Programmable memory and logic potential: Our dCas9-based memory and logic gate designs have immense potential for exploring adaptive and heritable control in synthetic systems.
How we implemented these insights
Prof. de Lorenzo's feedback helped us look at our project with more clarity and realism. Instead of trying to show that our toolkit can directly improve gene expression control, we began positioning EPIC as a system that helps us study and understand how epigenetic mechanisms themselves can be engineered. This shift made our goals more exploratory and research-oriented rather than purely application-driven.
We also started paying more attention to other layers influencing expression, such as promoter strength and translation efficiency, to design experiments that reflect the complexity he mentioned. His comments encouraged us to think of EPIC as an early step toward building tools that could eventually support applications like gene therapy or organoid-based studies.
Overall, his perspective grounded our project. It reminded us that EPIC's strength lies in its potential to uncover how epigenetic control can be rationally tuned, rather than in trying to immediately demonstrate industrial-level outcomes.
Prof. Greeshma Thrivikraman
Why
As part of our efforts to validate and refine our project, we met with Prof. Greeshma Thrivikraman from the Department of Biotechnology, IITM, whose work lies at the intersection of biomaterials, cell–matrix interactions, and tissue engineering. We wanted to understand the translational potential and feasibility of our proposed system from a cell biology and engineering standpoint.
What insights we obtained
1. Feasibility and Scope of the Project: Prof. Greeshma found our idea both novel and impactful but noted that it was quite ambitious, something that could easily grow into a PhD-level project. She encouraged us to stay realistic about what we could achieve within the iGEM timeframe, especially since mammalian cell systems are far more time and resource-intensive than bacterial ones and require careful optimization at every step.
2. Strategic Phasing of the Project: She endorsed our phase-wise approach: developing and validating the CRISPR-dCas9-based system first, followed by its application in mammalian cells. However, she emphasized that for iGEM, we should prioritize mammalian cell validation to make the proof-of-concept more impactful, encouraging us to begin experiments as early as possible.
3. Collaborations and Technical Guidance: Prof. Greeshma recommended that we consult Prof. Nathia for technical inputs on mammalian cell work and CRISPR methodologies. She also informed us that CHO cells could be obtained from Prof. Meiyappan, provided we arrange a proper workspace for long-term experiments.
4. Application Direction and Challenges: While she found the therapeutic application in tissue engineering and stem cells to be interesting, she cautioned that these systems involve multiple interconnected regulatory layers. She suggested we first demonstrate our system's tunability and robustness before extending it to complex differentiation models.
How we implemented these insights in our project
Taking her advice, we refined our experimental plan into clear stages:
- Phase 1: Establish proof-of-concept for our epigenetic regulation system in E. coli, focusing on dynamic methylation control and reporter-based validation.
- Phase 2: Extend the system to CHO cells for controlled gene expression, leveraging the resources she pointed us to.
Her inputs helped us ground our project in practical feasibility while maintaining its translational vision, optimistically paving a path from a competition-scale proof of concept toward future applications in epigenetic control and regenerative medicine.
Industry
Engagement with Industry experts helped us understand how our work could eventually translate from a conceptual framework to a usable technology. Conversations with professionals working in applied biotechnology and production systems gave us perspective on scalability, reproducibility, and regulatory considerations — aspects that often remain distant from academic settings. Their feedback pushed us to think about how our toolkit could be adapted to real operational environments, where control, efficiency, and reliability are key. These discussions reminded us that innovation in synthetic biology gains true value when it can integrate seamlessly into existing workflows, helping us refine our project's goals toward creating systems that are both scientifically sound and practically adaptable.
Ms. Sailaja Nori (Sea6 Energy)
Introduction & Context
We met with Ms. Sailaja Nori, co-founder and R&D lead at Sea6 Energy, to gain perspective on biomanufacturing at scale and to integrate industry insights into our project. As part of the Biomanufacturing Village at iGEM 2025, this conversation was a crucial element of our Integrated Human Practices, helping us align design choices with practical and sustainable industrial needs.
What We Learned
Our project uses a CRISPR-dCas9–DNA adenine methyltransferase (Dam) fusion to achieve targeted, reversible gene silencing. We aim to apply this in biomanufacturing, where regulating pathway enzymes can improve yields, reduce toxic buildup, and adapt to stress conditions.
Validation across hosts: Ms. Nori emphasized that demonstrating our tool in both E. coli and eukaryotic systems, like yeast or Aspergillus, is essential. While E. coli demonstrates feasibility, showing robustness in yeast signals, it has greater industrial relevance.
Tolerance thresholds: She highlighted how microbes collapse once product concentrations exceed limits—for example, yeast struggling beyond ~6% ethanol. To overcome this, strategies such as promoter tuning, using more tolerant strains, or engineering enzymes to degrade toxic intermediates should be integrated from the start rather than treated as afterthoughts.
Promoter control and sensing: We also discussed the role of titratable promoters, which she described as effective "safety valves." These dynamic sensors allow cells to balance growth and production, preventing collapse under metabolic stress. Linking our dCas9–Dam system to such promoters could improve stability and industrial adoption.
Together, these insights refined our vision: demonstrate cross-system robustness, integrate tunable promoter control, and explicitly design with toxicity thresholds in mind by prioritizing resistant strains or detox pathways.
Impact on Our Design Strategy
This meeting directly influenced our next steps:
- Dual-host validation (E. coli and yeast) to strengthen translational credibility.
- Incorporation of tunable promoters for graded silencing control.
- Explicit modeling of metabolite stress thresholds to guide system design.
- Selection or engineering of stress-tolerant strains early in development.
In summary: This meeting reinforced the industrial relevance of our approach and guided us toward concrete improvements. By combining epigenetic gene silencing with tunable promoter systems and validating across hosts, we are aligning our project with real-world biomanufacturing needs while staying grounded in sustainable design.
Dr. Avaneesh Thautam (Trinity Life Sciences)
Background
We had a chat with Avaneesh Thautam, who was part of the IIT Madras iGEM 2010–11 team that won the Best New Biobrick award for creating a tunable pH-sensitive promoter. Now working at Trinity Life Sciences, Avaneesh shared his experiences from his own iGEM journey and gave us a fresh perspective on how to tell the story of our project.
Key Insights
He didn't dive deep into technical details, but he helped us see the value of a clear narrative - something that connects science to its purpose. He suggested we share the judging criteria with him so he could point us toward the prize categories that might suit our work best. On top of that, he connected us with a few of his teammates from that year who could give more hands-on technical feedback.
Talking to someone who's been in our shoes reminded us that iGEM isn't just about experiments - it's also about how you present your idea. It gave us a bit more clarity on how to frame EPIC, not just as a project, but as a story worth telling.
Dr. Reshma Shetty (Ginkgo Bioworks)
Overview of Our Ginkgo Bioworks Session
We had the unique opportunity to engage in a one-on-one discussion with the CEO of Ginkgo Bioworks in Boston. This was carried out by our Team Leader R Karthik while he was in Boston during the summer. The goal was to learn how vision-driven projects like ours can actually become successful biotech startups with the right mix of guidance, resources, and strategic thinking. For us, this session mapped out how research, innovation, and entrepreneurship merge in biotech, a question central for iGEM teams who hope to go beyond the competition and make a real-world impact.
Insights from Ginkgo Bioworks
Ginkgo Bioworks started in 2008 from an MIT research lab. Today, it's recognized as a global leader in synthetic biology and engineering microbes for all sorts of applications, like medicine, agriculture, food, and materials. Ginkgo's main edge is its "foundry" model – a system that lets startups quickly prototype, test, and scale their bio-projects using shared lab space, automation, and advanced AI tools. This makes it way less expensive and much faster for new founders to work on real products rather than spending years building their own lab.
During our conversation, the CEO explained the nuts and bolts of turning a lab project into a biotech startup:
- Product-market fit: First you need to prove your science solves a real problem that people care about. Because biology experiments take a long time, technical validation and working demos matter a lot.
- Building a team and network: Success comes from connecting with incubators, accelerators, and mentors (not just classmates or faculty). Programs like Y Combinator and Petri have backed startups all the way from graduation to billion-dollar valuations.
- Ownership and milestones: To attract investors, it's important to set up clear milestones and think strategically about patents/IP. Showing progress and protecting your ideas helps secure funding and keep your project moving forward.
Ginkgo went from just five MIT grads with an idea to one of the leading companies in biotech, making their journey a reference for anyone trying to launch a startup from scratch.
How to Turn an iGEM Project Into a Startup
The meeting with her also gave insights into how we can hope to turn an iGEM project into a Startup. Some of the key takeaways from the conversation include:
- Think like a founder: Be clear about the problem your project solves, stay flexible, and don't be afraid to fail and try again. Surround the team with curious, smart people and learn from anyone willing to give real feedback.
- Find the right incubator: Look for programs and incubators (Y Combinator, IndieBio, iGEM Venture Foundry, Petri) that support student entrepreneurs—these can offer lab space, funding, and mentorship to speed things up.
- Get funding and advice early: Pitch your idea even if it's not perfect, get business advice from mentors, and start building connections with partners in industry, academia, and other startups.
- Work towards good demos and proof-of-concept: Investors and sponsors want to see working results, not just theory.
- Understand commercialisation: Learn about IP, patents, regulations, and manufacturing as soon as possible. Getting expert help early makes scaling up smoother.
- Stay motivated and resilient: A lot of startup work is about persistence - keep going, iterate, and use setbacks to get smarter.
Conclusion
The discussion with Dr. Reshma Shetty gave us an honest, inside look at what it really takes to move from a student project to a working biotech company. By building the right mindset, networking with the smartest people, and working towards clear milestones and demos, iGEM teams can make the jump from competition entries to companies that solve real problems in biomanufacturing and synthetic biology.
Engagement
These are meetings with people in different fields not directly related to our project but whose perspectives helped us understand the broader implications and potential applications of our work.
All India iGEM Meet 2025
Overview
We had the privilege of presenting our project at the All India iGEM Meet (AIIM) 2025, held at the Institute of Chemical Technology, Mumbai, from July 25–27. AIIM is an annual initiative where all Indian iGEM teams gather before the Grand Jamboree to share their work, receive constructive feedback, and refine their ideas through a mock jamboree format. It provides an invaluable platform for dialogue and collaboration, ensuring that each team's project moves further along the Design–Build–Test–Learn (DBTL) cycle with deeper insights and stronger foundations.
Presentation and Feedback
Presenting in front of other iGEM teams and a group of judges allowed us to share our ideas and get helpful feedback. The judges really liked how clearly we explained our project — highlighting not just our goals, but also the choices we made, our plan for experiments, and the evidence we had collected so far. Our wet lab work, particularly using the In-Fusion cloning method to create the CRISPR dCas9-Dam fusion protein, was noted as a strong point that showed our creativity and technical skill.
The dry lab component of our project also attracted keen interest. We presented our AI-driven systems biology framework, where machine learning models are used to translate DNA methylation patterns into predicted gene expression values for cancer-related genes, and these inferred values are integrated into a genome-scale metabolic model (Human-GEM). By using context-specific constraint methods such as eFlux, GIMME, and iMAT, we can dynamically adjust flux bounds and simulate how epigenetic changes reshape tumour metabolism. The judges noted that this integration of AI pipelines with metabolic modelling gave our project a strong systems-level perspective, allowing us to explore not only mechanisms but also potential interventions.
Areas for Improvement
Along with this appreciation, we also received feedback on areas where we could make our presentation more effective. The judges encouraged us to develop a clearer visual illustration of our gene circuit design, showing the dCas9-Dam fusion, gRNA, and reporter plasmid, in order to make the mechanism accessible even to non-specialist audiences. They also advised us to highlight aspects of our work that could align with special prizes, ensuring that our contributions are recognised not only for scientific rigour but also for their broader innovation and impact.
Impact on Our Journey
Participating in AIIM was thus a turning point in our Human Practices journey. It provided us with affirmation of the strengths of our project while also giving us tangible directions for improvement. The feedback we received continues to shape how we communicate, design, and refine our work, preparing us more effectively for the iGEM Grand Jamboree.
Prof. Mriganka Sur
Why we approached him
Understanding epigenetics' role in neurodegenerative disease treatment led us to interact with Prof. Mriganka Sur, a leading neuroscientist at MIT. Our team is exploring the use of CRISPR-dCas systems fused with DNA adenine methyltransferase (Dam) and guided by specific gRNAs to achieve targeted DNA methylation. This approach allows precise control over gene expression by epigenetically modifying specific neuronal genes without altering the DNA sequence.
What we learnt
Speaking with Prof Sur we understood that such targeted methylation tools are highly relevant to his ongoing research in brain plasticity and gene regulation in neurodegenerative diseases. By selectively methylating regulatory regions of genes involved in neural circuit function, this technology can potentially reverse or modulate pathological gene expression patterns linked to disorders like Alzheimer's or Rett syndrome. Prof Mirganka Sur mentioned that this system has its potential application to fine-tune gene networks in brain cells, offering new therapeutic avenues in epigenetic editing and neurodegenerative disease research.
Prof. David Low
Why we spoke to him
We reached out to Prof. David Low whose work in prokaryotic epigenetics has been greatly inspiring for us. Prof. Low's expertise in how bacteria natively utilize methylation for gene regulation is directly relevant to our project's goal of engineering programmable, synthetic epigenetic memory E. coli.
Prof. Low is a Professor in the Department of Molecular, Cellular, and Developmental Biology at UC Santa Barbara. His lab has made important contributions to understanding how site-specific DNA methylation, beyond the dam methylase, controls virulence, host adaptation, and antibiotic resistance in bacterial pathogens.
What we learnt
Plasmid Copy Number is Critical: He emphasized the importance of using low-copy-number reporter plasmids to study methylation-based regulation. High-copy plasmids can interfere with the precise regulatory effects we aim to measure due to their high expression rates. To ensure accurate and measurable regulation, he strongly recommended using low-copy reporter plasmids, which better reflect the true dynamics of methylation-dependent gene control.
Controlling the Methylation Source: Prof. Low also addressed a major experimental problem of poor transformation efficiency in our Dam(-) strain. He proposed a very clever solution. He recommended transforming the Dam(-) cells with a temperature-sensitive or regulatable plasmid carrying the Dam gene. This way, Dam methylase remains active during transformation to improve efficiency and can later be deactivated or removed by shifting the temperature or changing growth conditions, ensuring a Dam(-) genetic background for our methylation assay.
Refining the Memory System: He provided mechanistic insights into how our methylation based memory system works. He explained that the ON/OFF switching is due to competition between the Zinc finger proteins and dCas9-DAM. According to him, maintaining the correct balance between these factors is essential for stable memory formation. He advised us to ensure that the designed zinc finger proteins selectively recognize hemimethylated DNA, as this intermediate state is key to sustaining epigenetic "memory" through successive cell generations.
Impact on our project
Based on Prof. Low's feedback, we gained a clearer understanding of how critical experimental design factors, such as plasmid copy number, methylation control, and protein–DNA interactions, can affect the reliability of a methylation-based memory system. His suggestions on using low-copy plasmids, controlling Dam expression during transformation, and carefully designing zinc finger proteins that interact with hemimethylated DNA gave us valuable direction for improving our future experimental plans. His insights helped us identify key parameters we need to consider before moving forward with the bacterial implementation of our EPIC system.
