Week 1 HW: Final Project Ideas
1. What you would like to biomanufacture and why?
I’m hoping to biomanufacture a bacterial cellulose pellicle (or SCOBY) that replicates heat sensation without any actual energy transfer, by leveraging a TRPV1 agonist embedded within the cellulose.
Given my general interest in bacterial cellulose and its fabrication possibilities (incidentally, my PhD topic), I would like to research more on this topic because bacteria are fun. While the TRPV1 topic is just one of many possibilities, the underlying living system I will be working with is bacterial cellulose. The idea of embedding heat-without-heat sensation came by accident as I was looking for ways to grow my own spicy chili peppers within university grounds and keep them alive during the cold winters of Kyoto. I myself am very sensitive to cold weather and often remain cold despite heat sources, which themselves don’t do much to fend off the winter cold. Plus this is a potential fire hazard if heat devices overheat or somehow malfunction, and heat packs like Kairo have dubious claims to sustainability. Since bacterial cellulose never wanders far from my mind, these lines of thought converged into this cold-spicy-cellulose axiom, and my proposition for HTBAA came about.
Ultimately my goal is to find ways to fabricate and functionalise bacterial cellulose making the most of its livingness qualities and potential to self design. As a sustainable material, it poses significant potential improvements in design areas beyond medical or purely technological applications that still may have considerable impact in industrial landscapes, such as textile materials.
Alternatively, if a TRPV1 agonist is impossible to biomanufacture in this way, I had been considering ways of engineering bacteria to turn on and off their cellulose-producing abilities based on certain materials they come in contact with. This relates to my investigations with textile structures integracted with cellulose, and the possibility of creating form, texture and patterns on the cellulose by designing the textile scaffold.
Prior Relevant Work:
2. What living system would you use?
· The starting point would be producing the cellulose sheet through bacterial fermentation of either Acetobacter Hansenii, a SCOBY with varying species, or another cellulose-producing species more compatible with the TRPV1 agonist.
Initially, I figured the TRPV1 agonist could be capsaicin itself, as seen with chili peppers. The TRPV1 agonist would be embedded through a co-culture, allowing the capsaicin (or alternative agonist) to be loaded onto the pellicle throughout growth, rather than a post-fermentation loading. Claude AI suggests Saccharomyces cerevisiae, since it tolerates BC’s pH levels comfortably (yeast+bacteria, as SCOBY grows in symbiosis). It further cites a joint initiative between MIT and Imperial validating this very coculture: https://www.nature.com/articles/s41563-020-00857-5
Claude further argues: “(my) version would swap their reporter/biosensor genes for the capsaicin-pathway genes instead: Pun1/AT3 (the acyltransferase sometimes called capsaicin synthase) and pAMT (the aminotransferase converting vanillin to vanillylamine), fed vanillin and a branched-chain fatty acid as precursor inputs — mirroring the same precursor logic the E. coli capsaicinoid patent attempt used.”
I need to verify this, and will do so throughout this weekend as I make my way through the reading list, and throughout the week after the homework review and classes. Corrections, amendments, and more are always welcome.
3. How would you use automation?
I imagine automation to be used in order to find an appropriate yeast-bacteria ratio, and to test the resulting co-cultured materials (as two separate processes). Research and AI sources make the following points the most critical:
· Yeast-bacteria production and screening: an Opentrons-style liquid handler to set up parallel co-culture vessels varying the yeast-to-Komagataeibacter inoculation ratio, feed timing, and vanillin-precursor concentration.
· Mechanism Verification: using a Franz diffusion cell, similar to the verification mechanism of the 8% capsaicin patch for the treatment of neuropathic pain, as stated in prior relevant work, paired with an automated diffusion-cell autosampler that pulls receptor-fluid samples at programmed intervals and routes them to HPLC. Claude AI further suggests: The TRPV1 calcium-imaging confirmatory assay as an already standard automated pharma infrastructure — a FLIPR-type fluorescence plate reader paired with an automated plate washer, running a 96-well plate’s worth of conditions simultaneously. Confirmation is needed on AI suggestions.
Furthermore, there is a potential for automation in engineering the TRPV1 agonist into the yeast through DNA or gene editing - although I need to research this part further.
4. How much would you need to make, and how long would it take? The aim of this project is to produce small material samples that can later be designed into garments and accessories. This way, the initial mechanism can be explored and tested without worrying about a larger artefact that needs to respond to a broader set of design demands, as well as biotechnological complexities explained thus far.
- How much
Scale is defined as proof-of-mechanism scale: enough material to run the verification pipeline (Franz-cell permeation + TRPV1 calcium-imaging confirmation) across a small test matrix, not enough to make wearable units. Concretely: 3 co-culture conditions (e.g., varying yeast: Komagataeibacter inoculation ratio or induction timing) × 4 replicates, plus untreated controls, gives roughly 16 BC test discs at ~1 cm² each — call it 20 cm² of BC pellicle total, rounding up for handling losses.
Converting to dry mass: static Komagataeibacter culture yields roughly 4.4–8 g/L dry cellulose over 5–10 days (K. rhaeticus in Hestrin-Schramm medium). Assuming a 1 cm medium depth, that’s ~10 L of medium per m² of culture surface, giving roughly 44–80 g dry BC per m² per fermentation cycle. Your 20 cm² (0.002 m²) requirement needs only about 0.09–0.16 g dry BC — one small culture dish, well under one growth cycle’s output.
Agonist quantity works backward from a target in-matrix concentration of ~0.05% w/w (OTC-equivalent) against that dry mass: roughly 45–80 µg of agonist needs to actually end up incorporated into the finished material. The harder number is upstream of that — how much the yeast partner needs to secrete into the medium to get that much captured by the forming cellulose fibers, since not everything secreted ends up trapped in the network rather than diffusing into the bulk broth. There’s no published retention-efficiency figure for this, so it has to be a stated assumption: at, say, 50% capture efficiency, the yeast needs to secrete on the order of 100–160 µg total across the fermentation. The number behind that — the yeast strain’s actual capsaicinoid titer — is the biggest open unknown in this whole calculation, since no titer has been published for an engineered yeast host expressing this pathway (only fatty-acid intermediates exist from the one E. coli attempt). Worth presenting explicitly as a placeholder assumption — e.g., “assuming a conservative early-stage heterologous titer on the order of low mg/L, by analogy to other engineered-yeast secondary-metabolite platforms in their first unoptimized iterations” — and flagging it as the assumption most likely to be wrong.
- How long
Production and incorporation happen simultaneously here, inside the same 5–10 day fermentation window Komagataeibacter needs to grow the pellicle in the first place — there’s no separate loading stage to add afterward.
The real timing constraint is synchronization: the yeast needs to be actively secreting agonist while the bacterium is actively laying down new cellulose fibers nearby, so the two get physically co-incorporated, the way Gilbert et al.’s Nature Materials work depends on. Secrete too early, before fiber deposition is underway, and the agonist likely diffuses into the bulk medium unclaimed; secrete too late, once the pellicle’s largely formed, and there’s no open network left to catch it in. If the yeast construct uses an inducible promoter (typical for heterologous pathways in S. cerevisiae), getting induction timing right relative to BC growth becomes its own sub-experiment — and you can’t tell whether it worked without harvesting and testing the finished pellicle, so a mistimed attempt costs a full 5–10 day cycle to find out.
That gives a per-round total of roughly 5–10 days (co-culture growth and incorporation) + 1–2 days (Franz cell) + ~1 day (TRPV1 assay, largely parallel) ≈ 7–13 days. Because this round is debugging three things at once — whether the yeast produces at all, whether timing actually captures product in the fibers, and whether concentration lands in range — budget more rounds than a simpler single-variable optimization would need: realistically 4–6 rounds rather than 2–3. That puts a defensible mechanism-proof timeline at roughly 6–10 weeks.