Brainstorms

Melanin-based bioink for Light-Recording Materials

My individual final project is based on melanin and related compounds in an engineered living material (ELM) as a color-responsive bio-ink. Among many other factors, oxidation state, precursor availability / intermediate reaction pathways likely shape tone and long-term stability and may be modulated using a genetic system, be it a bacterium, a synthetic minimal cell, etc.

Melanin itself is a heterogeneous and hard-to-define analyte candidate, so my idea is to use its main defined intermediates, like L-DOPA, dopamine, and quinones, as analytes and use a high-resolution method like LC-MS for calibration/ground truth method aiming to understand and quantify melanin-related compounds that interfere in the darketing output of the ink/material. Than use protein design to build embedded sensing for spatial or real-time readouts inside the material aiming for building a fine-tuning system that can relate color tone of the material and the synthesis of the different melanin compounds as well as control mechanisms that can trigger it (different UV light wavelengths for instance).

Explore whether melanin-based optical outputs can be generated within different bio-materials such as bacterial cellulose (BC) and ELMs it for applications in fashion, design, and light-recording materials.

I want to establish a first melanin-producing genetic platform, and fine tune it’s pigmentation in a high resolution scale. The strongest version of the project, a bio-based material that gradually develops melanin-derived tonal variation in response to different input signals (i.e. different UV wavelenghts), behaving less like a dyed textile and more like an exposure-recording surface.

Since K. rhaeticus naturally produces cellulose, it also lets me focus on material-producing biology in a native chassis instead of forcing cellulose synthesis into a non-native organism. On top of that, I am interested in the possibility of later embedding synthetic minimal cells into the cellulose as localized, non-growing modules for sensing and pigment generation.

A major question for me is what the right analyte is. Since melanin is a heterogeneous polymer, I think it does not make sense to treat it as a single clean measurable output. Because of that, I am leaning toward focusing on using as analyte more tractable analytes such as the expressed enzyme itself, or melanin-related intermediates like L-tyrosine, L-DOPA, dopamine, quinones, DHI, or DHICA.

This is where LC-MS starts to feel really central to the project. I started thinking that maybe the application should be chosen based on what LC-MS is actually powerful enough to resolve. That led me to think about applications where fine control over color, stability, or chemical state is especially important:

• Bio-based inks or photography, where oxidation state could shape color and long-term stability.

The ink and photography direction is especially interesting to me because the final image might look stable, but what defines tone and durability may actually be determined much earlier by oxidation chemistry.

Two materials could look similar at first, but age very differently depending on how those intermediates evolved. In that case, LC-MS could help connect invisible intermediate chemistry to visible outcomes in the final material.

• Bioadhesives or coatings, where intermediate catechol chemistry may directly determine performance.

The bioadhesive or catechol-based coating direction also seems compelling. These systems often depend on catechol-containing molecules like dopamine or L-DOPA, which can oxidize into quinones and then participate in crosslinking. That balance between reduced catechol and oxidized quinone seems to shape adhesive behavior. So instead of only testing the final strength of an adhesive, LC-MS could potentially help track how the chemistry develops during formation and explain why some conditions produce better performance than others.

In these kinds of systems, LC-MS and fine tune control of synthesis of melanin-compounds does not feel like overkill to me. It feels like the right level of resolution for the chemistry that actually matters. So I am starting to think about the project less as “make a melanin material” in the broadest sense, and more as “choose a melanin-related material application where intermediate-state chemistry is central, measurable, and worth controlling.”

Project concept:

An engineered living material (ELM) based on bacterial cellulose (BC), using Komagataeibacter rhaeticus as the primary chassis, to produce melanin-based optical outputs in a cellulose material for fashion, design, and light-recording applications.

The current direction is not to maximize “smart material” complexity at once, but to first establish a robust melanin-producing BC platform, then evaluate whether additional functions such as keratin expression, self-repair, or embedded synthetic minimal cells are technically justified.

The strongest version of the project is a nude-toned or skin-adjacent material that gradually develops melanin-derived tonal variation in response to exposure conditions, producing a material that behaves less like a dyed textile and more like an exposure-recording surface.

Why bacterial cellulose?

BC is a strong candidate because it is:

• biogenic and directly fabricable as a sheet-like material
• compatible with engineered living material approaches
• mechanically robust relative to many other microbial matrices
• moldable as pellicles, spheroids, or printed structures
• already supported by the Komagataeibacter Tool Kit (KTK), a modular cloning toolkit for this genus

In carbon-rich media, Komagataeibacter polymerizes and secretes linear glucose chains that self-assemble into a dense interconnected cellulose mesh. This cellulose pellicle forms at the air-liquid interface and behaves like a biofilm-like material scaffold around the producing cells.

Which chassis?

Primary chassis: Komagataeibacter rhaeticus A high-yield bacterial cellulose producer and a strong chassis for BC-based ELMs.

Why Komagataeibacter rhaeticus?

• native bacterial cellulose production
• established relevance for BC-based material engineering
• allows the project to focus on more specific objectives for material-producing biology, rather than forcing cellulose synthesis into a non-native organism like E. coli

Secondary system: synthetic minimal cells embedded in BC

As a second aim, the project may incorporate synthetic minimal cells (SMCs) as embedded, non-replicating functional modules inside or on the cellulose material. As these SMCs would add localized, compartmentalized sensing and pigment-generation functions to the BC scaffold. Therefore, a useful synthetic minimal cell for this project would basically be a light-exposure logging vesicle embedded in or deposited onto bacterial cellulose.

The living BC producer: K. rhaeticus builds the material scaffold and the synthetic minimal cells allow vesicle-based modules provide controlled, non-growing sensing and melanin output. This separation may be useful if pigment production or sensing logic is easier to implement in a compartmentalized cell-free system than in the BC-producing chassis itself.

Main questions

1- Since melanin is a heterogeneous polymer, which analyte should I choose to analyse?

I might want to confirm the expressed enzyme/protein (for example tyrosinase, laccase, TyrP, or another melanin-related enzyme) or melanin intermediates: L-tyrosine, L-DOPA, dopaquinone-derived products, DHICA, DHI, etc since melanin is a heterogeneous polymer. so

These are often much more tractable by LC-MS than melanin itself.

Other questions

• Nutrient availability: If the final material remains living, nutrient supply becomes a major constraint.
• Biosafety: use of non-replicating synthetic minimal cells

Aims

AIM 1: Define and model a first light-responsive melanin-producing synthetic minimal cell for integration into bacterial cellulose

Develop a specific in silico design for a phospholipid vesicle-based synthetic minimal cell that uses EL222 to activate melA expression under blue light, with the goal of generating visible melanin production as a localized output that could later be embedded into bacterial cellulose made by K. rhaeticus. This aim focuses on specifying the exact first system, its required components, and whether its chemistry and logic are feasible before any experimental implementation.

AIM 1 Specific Objectives:

• define the exact genetic module to be tested first: EL222 + melA
• specify the full internal composition of the vesicle:
  • Tx/Tl source
  • ATP regeneration system
  • tyrosine
  • copper
  • salts/cofactors
• define the membrane composition for the first prototype, e.g. POPC + cholesterol
• map the input-output logic precisely:
  • input = blue light
  • regulator activation = EL222
  • output = tyrosinase expression
  • final material output = melanin accumulation / darkening
• determine which molecules must be pre-encapsulated and which, if any, must cross the membrane
• identify the minimum set of assumptions required for the system to function = specify the required materials, genes, lipids, cofactors, and readouts for the first prototype

AIM 2: Experimental planning and prototyping strategy for melanin integration into bacterial cellulose materials

Translate the selected design into a concrete experimental plan, prioritizing a staged workflow from simple proof of concept to material-level testing. This aim is not yet full implementation, but the preparation of a robust experimental roadmap that makes the project technically executable and testable.

Practical objectives:

• measures of success / failure:

  • define the first measurable success criteria: visible darkening? absorbance increase? spatially localized pigment formation?
  • identify the main failure points of this exact design, such as insufficient expression, low tyrosinase activity, substrate limitation, or poor melanin accumulation

• define the first build-test sequence, including which subsystem should be validated first:

• melanin pathway in a tractable chassis

• cell-free context

• BC production in K. rhaeticus

• integration of pigment module with BC

• plan how BC will be fabricated and presented for testing, e.g. pellicles, spheroids, molded sheets, or layered composites

• define how synthetic minimal cells would be embedded in, coated onto, or associated with BC

• determine the primary experimental readouts: visible pigmentation; image-based quantification of tone; spatial patterning under differential light exposure; material compatibility and stability

• define the controls needed to evaluate whether the system is functioning as intended identify the decision points that determine whether the project should proceed with:

  • direct microbial engineering only
  • synthetic minimal cells only or a
  • hybrid system

AIM 3: Evaluate secondary functional molecules only after establishing melanin as a robust first proof of concept

Keep melanin as the primary engineered output and assess other molecules only if they offer a clear, measurable improvement to the material. This aim is intended to prevent the project from becoming too diffuse too early and to ensure that any added complexity is justified by experimental value.

Practical objectives:

• define which secondary properties would be worth pursuing only after melanin is validated, such as:
  • increased abrasion resistance
  • reduced permeability
  • improved mechanical robustness
  • antimicrobial activity
• evaluate candidate molecules such as keratin or other structural/functional additives in terms of:
  • biological feasibility
  • compatibility with BC
  • expected measurable benefit
  • added engineering complexity
• establish criteria for whether a second molecule is worth integrating into the platform by prioritizing only additions that significantly improve the material’s performance or expand its application in a clear and testable way.

Previous ideas

Historical register of the brainstorm for the Individual Project:

Later, I added 3 slides with an updated version of those 3 ideas in the appropriate slide deck for Committed Listeners here.

However, the current project direction is a different idea: a bacterial cellulose-based material platform for melanin-derived tonal output, potentially extended with synthetic minimal cells for compartmentalized light-responsive pigment generation.

But I decided to devolop another idea not present in the inicial registers.

Individual Final Project: Melanin-based light-recording bioink/biomaterial

Important links:

• Commited Listener Slide Deck here.
• Benchling (TO BE ADDED)
• Asimov Kernel (TO BE ADDED)

Aim 1: Build a first melanin-producing cell-free DNA module based on melA tyrosinase + Define validation parameters

The melA gene is coding sequence of tyrosinase that catalizes the conversion of tyrosine to dopaquinone. Dopaquinone is intermediate product of melanin biosynthesis pathway that polymerizes in an enzyme-independent reaction to form melanin.

The pathway from L-tyrosine to Melanin with the use of the melA tyrosinase.

1. Design MelA expression constructs (Benchling and Twist)

Some of the construct variables to be considered because it will affect melA expression:

• Promoter
• RBS
• Terminator
• Codon usage
• Tag placement
• Vector context

Note: a construct that works in cells may not translate well to cell-free expression.

Previous expression constructs:

• iGEM 2004 - using the P(lac)IQ promoter (BBa_I14032), a work designed by Vikram Vijayan, Allen Hsu, Lawrence Fomundam Group of the iGEM04 (2004-08-04 Part:BBa_I14032).

• iGEM 2009 - using B0040 RBS (composite part: BBa_K193602) containing pLacIQ(BBa_I14032), RBS(B0030) and melA(BBa_K193600) on low copy vector(BBa_I52001 derived), a work designed by Kazuaki Amikura.

• iGEM 2017 - Erin Kelly Group (2017-10-27) have developed a different expression construct specifically for use in E. coli BL21(DE3) which takes advantage of the T7 RNA Polymerase expression construct in the DE3 cassette to provide tighter epxression control and help to prevent leaky expression of the tyrosinase. Additionally, a double terminator B0015 was added to increase control over the system. In order to maximize production of the tyrosinase and limit unnecessary energy expenditure by the cell, a transcriptional terminator ensure energy is not wasted on transcribing an overly-long mRNA transcript. They transformed the melA_pJET plasmid into E. coli BL21(DE3) and attempted to overexpress the MelA tyrosinase (~54kDa) and produce the pigment melanin. Four colonies from the transformation were picked and used to produce pre-cultures, which were then used to incoulate test expression cultures. During the test expression, cultures were also supplied with CuSO4 and extra tyrosine. Cultures were induced with IPTG at OD600 ~1 and a 1OD sample was taken (T0). Another 1OD sample was taken after the cultures were left to grow overnight (TON). The cultures were allowed to grow another three days (supplemented with tyrosine and ampicillin) to see if pigment would form, but we were unable to detect any melanin. The 1OD samples were run on a 12% SDS-PAGE to check for melA overexpression (Figure 3). The MelA tyrosinase is ~54 kDa in size. A faint band of approximately 54 kDa appears in the TON lane of culture 3. This indicated that we were successful in expressing the MelA tyrosinase from the pJET plasmid. Before the Jamboree, we will attempt another overexpression of MelA from the pSB1C3 plasmid.

My take: T7 can maximize protein yield but also overwhelm folding capacity, causing inactive protein accumulation (increase the likehood of tyrosinases misfolds, aggregation, or fail to incorporate copper correctly). I’d replace it by a moderated construct and compare the results in reference to the BBa_K2481108 (control).

Note: MelA expression is not the same as melanin production. Melanin polymerization is messy, dopaquinone polymerizes through non-enzymatic downstream chemistry. This means color output depends not only on MelA, but also on oxygen, pH, time, redox state, and local chemistry.

2. Prepare reagents and workflow (Ginko & Open AI, HW 11C)

Melanin production in E. coli or in a cell-free system is influenced by several parameters that actuate at the level of melA expression and enzyme activity / posterior reactions:

• L-tyrosine concentration (substrate, limited solubility)

• CuSO4 concentration: since this tyrosinase is a type 3 copper-containing enzyme, Cu2+ is a cofactor of the enzyme. Too much copper can also stress cells or inhibit cell-free reactions.

• Magnesium

• Energy mix

• Molecular oxigen avaliability for tyrosinase reactions

• pH: tyrosinase activity and melanin polymerization are pH-dependent. If the reaction acidifies over time, enzyme activity or pigment formation may decrease.

Note: Optimizing for sfGFP may not optimize for MelA.

3. Validation

The 2017 iGEM result is a useful warning: they may have produced a faint ~54 kDa MelA band, but still detected no pigment. For this reason, I am proposing a staged validation workflow that moves from simple expression and pigment checks to more refined mechanistic analyses, depending on the results obtained in each previous round.

Results interpretation framework:

Here’s a diagram of my proposed validation workflow

4. Model a light-activated expression circuit that could later support gradual tonal change in a material system (Asimov Kernel).

Post-Course: Towards the melanin-based light-recording bio-ink

5. Refine Asimov Kernel’s output for controlling melanin expression till the system is fine-tuned with my aesthetical needs (most controlled and previsible) or, if not possible develop a experimental design for embedded sensing (in this case map both the quantitative and qualitative workflows available).**

Decision point: Push for maximum molecular control first, even if the material context is still abstract? OR Move earlier into material-scale experiments?

6. Material engineering: Test different models of integration of the melanin cell-free module towards a intended function/product. Tests for integrating into material though ELM engineer Komagataeibacter rhaeticus + bacterial celulose (BC) or a hybrid system with BC scaffold / other biomaterial that can be embed with cell-free modules with synthetic minimal cells / K. rhaeticus.

7. Rethink the workflow for measurable readouts for the cell-free system. RGB image analysis in a controlled lighting box or spectral (semi-quantitative) with melanin absorbance ranging from 475 nm to 500 nm;

8. Optimization rounds looking into every step of the proposed workflow, including economic tests for modeling parameters (such as ink dilution)

9. Benchmarking partnerships + reach out plan (heavly based on the previous item conclusions (BioFabricate, Cultivarium)

Group Final Project

Bacteriophage Engineering

GROUP MEMBERS: Diogo Custodio; Flo Razoux; Katharine Kolin; Mariana Kanbe; Marisa Satsia.

PROJECT MAIN GOAL : Increase the stability of the L protein

GROUP PROPOSAL: We will use the same workflow than in previous HW (e.g. mutagenesis) but adapt it to specific aim(s) based on HW reading material of week 04 (e.g. shorten the L protein to make it not dependant on bacterial chaperone DnaJ anymore).

Please check our most recent updated Google Docs on this.

Here’s a summary of my main individual contributions to the plan for engineering the bacteriophage:

I ran the provided mutational scoring notebook to obtain per-substitution LLR scores for the MS2 L-protein and shortlisted substitutions with positive scores. The full scoring results are included in a table on my Homework 5 page.

I then cross-checked these shortlisted mutations against the provided experimental mutant dataset, L-Protein Mutants, which reports amino acid substitutions and their measured lysis phenotypes.

The overlap between the two data suggests that sequence-based LLR scores capture only part of the functional landscape of the MS2 L-protein. More broadly, positive LLR scores may reflect sequence plausibility or local biochemical compatibility, but they do not fully account for higher-order constraints such as host-factor dependence, membrane behavior, and oligomer formation.

Therefore, I decided to select five candidate mutations by combining positive LLR scores with biological reasoning about the protein’s distinct functional domains, treating LLR scores as a prioritization tool for experimental testing rather than as a direct predictor of lytic function.

The MS2 L-protein is organized into distinct functional domains:

1. Hydrophilic N-terminal region involved in DnaJ-mediated folding
2. Transmembrane/C-terminal region responsible for membrane insertion and pore formation

The two soluble-region mutants, S9Q and C29R, were chosen to probe effects on folding and possible DnaJ dependence, whereas the three transmembrane mutants, A45L, T52L, and N53L, were chosen to probe membrane insertion and oligomerization.

Mutant 1 - S9Q (soluble, LLR = 2.014)

Sequence: METRFPQQQQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFLAIFLSKFTNQLLLSLLEAVIRTVTTLQQLLT

Selection Rationale: High positive score in the soluble region (putative DnaJ-interaction domain). Ser→Gln increases hydrogen-bonding potential and may alter surface chemistry without strongly destabilizing the fold.

Mutant 2 - C29R (soluble, LLR = 2.395)

Sequence: METRFPQQSQQTPASTNRRRPFKHEDYPRRRQQRSSTLYVLIFLAIFLSKFTNQLLLSLLEAVIRTVTTLQQLLT

Selection Rationale: One of the strongest positive-scoring substitutions in the soluble region. Adds a positive charge that could reshape chaperone-recognition or interaction surfaces.

Mutant 3 - A45L (TM, LLR = 1.539)

Sequence: METRFPQQSQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFLLIFLSKFTNQLLLSLLEAVIRTVTTLQQLLT

Selection Rationale: Hydrophobic substitution in the transmembrane segment. Ala→Leu increases hydrophobicity and may stabilize membrane helix packing/insertion and oligomer stability.

Mutant 4 - T52L (TM, LLR = 1.814)

Sequence: METRFPQQSQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFLAIFLSKFLNQLLLSLLEAVIRTVTTLQQLLT

Selection Rationale: Polar→hydrophobic change in the TM region. Thr→Leu may increase membrane compatibility and reduce local insertion/misfolding penalties.

Mutant 5 - N53L (TM, LLR = 1.865)

Sequence: METRFPQQSQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFLAIFLSKFTLQLLLSLLEAVIRTVTTLQQLLT

Selection Rationale: Polar→hydrophobic change in the TM region with a strong positive score. Selected as an additional TM-stabilizing candidate.