Project Description
EPIC: Laying the Software Layer of Bacterial Synthetic Biology for a Biomanufacturing Village
We Domesticate Epigenetics. Biology works on multiple levels. The genetic code (DNA) is the hardware: stable, information-rich, and widely edited by genetic engineering. However, there is another essential layer - epigenetics - that does not alter the DNA sequence but controls when, where, and how genes are expressed. Epigenetics is the cell's software: it adds memory and context to the hardware.
The Foundational Gap in Bacterial Synthetic Biology
In eukaryotes (plants, animals, humans), targeted epigenetic tools already let researchers program long-term memory, reversible switches, and context-dependent behavior. In bacteria, however, epigenetic control remains poorly accessible: the main bacterial methyltransferase (Dam) acts broadly across the genome, making it hard to study or use methylation at single loci.
This is not a quibble about marginal control of expression. It is a foundational gap. Without a programmable bacterial "software" layer, synthetic biology in microbes remains largely limited to hardware-style fixes (promoters, RBS libraries, plasmid copy-number tweaks). Those are powerful, but they do not enable engineered cells to remember states, adapt across generations without constant input, or stably encode low-overhead regulatory programs that reduce long-term instability in industrial settings. EPIC's mission is to build that foundational layer for bacteria.
Why This Matters for a "Biomanufacturing Village"
Industrial biomanufacturing is evolving from using single strains for performing single tasks to using ecosystems of engineered microbes collaborating across time and processes. For that village vision to work, reliable, low-cost, long-duration production with minimal manual intervention requires cells to be able to store, switch, and stably maintain regulatory states without incurring large metabolic overheads.
Epigenetic programming is uniquely suited for this: it can encode memory, reduce the need for continuous induction, and enable dynamic, low-cost adaptation of production strains during long fermentations. Building a reliable, programmable epigenetic layer in bacteria helps create infrastructure for future, resilient biomanufacturing.
Inspiration and Scope
Discussions with biomanufacturing teams highlighted real industrial problems, for example, amplification strategies in mammalian systems (like DHFR amplification) create metabolic burdens and long-term silencing that reduce production consistency. While such specific fixes often have simpler short-term remedies, they inspired a broader question: can we create a general, programmable epigenetic toolkit that enables new classes of solutions across microbial and mammalian platforms?
EPIC focuses on the foundational work: domesticating bacterial epigenetics so that future teams can build robust, lower-cost production schemes on top of it. This is not a one-off "fix" for a particular marker, it is the road on which future "cities" of innovation can be built.
Local Impact
This technical challenge is acutely felt in India. High production costs, driven partly by inefficiencies like persistent metabolic burdens, make many essential biologics unaffordable for the mass population. EPIC can significantly reduce production costs, thereby improving the biomanufacturing sector in India and aiding the national mission of Atmanirbhar Bharat (Self-Reliant India).
What EPIC Is
EPIC (Epigenetic Programmable Intervention & Control) is a CRISPR-based toolkit that converts bacterial DNA methylation from a global, untargeted process into a programmable, site-specific regulatory tool.
dCas9
A catalytically dead Cas9 that binds DNA at sequences specified by short, easy-to-design guide RNAs (gRNAs) without cutting it.
Dam Methyltransferase
The bacterial enzyme that methylates adenine within GATC sequences, naturally involved in DNA replication timing and gene regulation.
Flexible Linker
A 15-amino-acid glycine-serine linker connecting dCas9 to Dam to preserve folding and activity of both domains.
Inducible Control
A doxycycline-responsive Tet-On promoter to titrate dCas9-Dam expression for temporal and quantitative control.
gRNA Library
Sequence-based targeting to direct methylation to any locus containing (or adjacent to) GATC sites.
EPIC is built by fusing Dam to dCas9 via infusion cloning, inserting gRNA expression cassettes using restriction cloning, and controlling fusion expression with an inducible promoter. This design makes targeting as simple as designing a 20-nt gRNA, no protein redesign or months of optimization.
Proof of Concept
Target Choice
The dnaAP2 promoter, a methylation-sensitive region with multiple GATC sites. This promoter is well-characterized for methylation sensitivity and provides a clear readout of methylation-dependent regulation.
Reporter
GFP is used under the control of the methylation-responsive promoter. The dnaA1 region and known repressor binding sites were removed/mutated to isolate methylation effects.
Constructs and Controls
- dCas9-linker-Dam under Tet-On control (inducible with doxycycline)
- gRNA(s) targeting the dnaA2p region
- Controls: dCas9 alone (no Dam), Dam alone (no dCas9 targeting), and catalytically inactive Dam mutant as negative control
Assays
Functional readout: GFP fluorescence measured by plate reader and flow cytometry to quantify expression changes.
Methylation mapping: DpnI/DpnII restriction-digest assays and qPCR to detect methylated versus unmethylated GATC sites; for genome-wide off-target assessment we use long-read methylation-sensitive sequencing (e.g., SMRT/PacBio methylome profiling).
Physiological metrics: Growth curves, viability, and protein yield to assess metabolic burden and any unintended fitness costs.
Expected behavior: Targeted dCas9-Dam recruitment increases local GATC methylation at dnaAP2. Depending on the promoter architecture, methylation produces predictable shifts in promoter activity measurable by GFP. The inducible Tet-On system allows us to tune methylation intensity and observe reversibility or persistence across generations.
This bottom-up experiment demonstrates controllable, locus-specific methylation in bacteria and provides quantitative assays for both on-target function and genome-wide safety.
Why This Is Not "Just Another Expression Tool"
Gene expression control in bacteria is already well-established, and EPIC isn't a superior method to tune expression when simpler approaches exist. Rather, EPIC creates a new engineering dimension:
Memory
Epigenetic marks can persist across cell divisions, enabling programmable inheritance of a production state without continuous inducer.
Low-overhead Regulation
Epigenetic switches can reduce the need for continuous, metabolically costly induction, lowering the resource load on production strains.
Novel Logic & Architectures
Combining epigenetic marks with synthetic logic gates enables circuits that change behavior in response to environmental triggers and then maintain that state.
Domestication
EPIC enables us to study how bacterial methylation affects physiology in a tractable and targetable way, the first step toward safely harnessing epigenetics at scale.
Immediate Outcomes and Longer-Term Impact
Short Term
- Demonstrate robust, guide RNA–directed methylation at dnaAP2 with clear GFP readout
- Show inducible control, titratability, and measurable persistence or reversibility of methylation marks
- Provide open protocols, plasmids, and gRNA design rules for the community
Long Term
- Stable production strains that maintain production states without continuous induction
- Epigenetic logic devices to coordinate cell consortia and task allocation in fermentation
- Lower-cost, more resilient biomanufacturing platforms (supporting Atmanirbhar Bharat ambitions) by enabling designs that reduce metabolic waste and production inconsistency over long runs
Limitations, Safety, and Responsible Deployment
EPIC introduces methylation in a targeted fashion, but Dam can have genomic off-targets if overexpressed. We mitigate this by using inducible, titratable expression, comprehensive off-target methylome profiling, and negative controls. Any industrial deployment will require rigorous fitness, stability, and biosafety testing to ensure no unintended behaviors in production or release scenarios.