Individual Final Project

Melanin-based light-recording bioink/biomaterial

Designing a MelC2-Based Cell-Free Module for Programmable Melanin Bioink

Reframing pigmentation from static dyeing to a programmable chemical state evolution, enabling materials that encode environmental history

Important links:

SECTION 1 - ABSTRACT

Melanin is a chemically heterogeneous dark biopolymer known for broadband UV-visible optical absorption, photoprotective behavior, photothermal conversion, redox activity, and long-term optical stability. These properties make melanin a compelling biological route to functional color: a pigment chemistry that can absorb and dissipate radiation, preserve optical traces, buffer oxidative stress, and interface with biological or electronic systems. This project proposes controlling melanin-forming chemistry in a synthetic biology system to develop a programmable bioink for engineered biomaterials. The broader vision is to create materials that combine biosensing and functional response: recording environmental inputs such as light, ionizing radiation, or oxidative stress through measurable optical change, while also enabling properties such as UV or radiation protection, photothermal conversion, antioxidant behavior, and bioelectronic interfacing. Depending on concentration, matrix composition, and material format, this melanin-based bioink could be explored for responsive textiles, UV-protective coatings, architectural and design surfaces, tattoo-like dermal pigments, space-oriented materials, bioelectronic interfaces, and localized radioprotective biomaterials. To move toward this goal, this project aims to design a first genetic module that generates measurable melanin-like optical changes in a controlled cell-free system, then use it as a foundation for future integration into engineered biomaterials such as bacterial cellulose. The central hypothesis is that a codon-optimized Streptomyces antibioticus MelC2 tyrosinase construct can provide a tractable route toward cell-free melanin-like pigment formation, with output shaped by tyrosinase activity, substrate availability, copper cofactor loading, oxygen, pH, redox state, and polymerization chemistry. During HTGAA 2026, I designed a MelC2 expression cassette for TX-TL / E. coli use and designed a validation workflow.

SECTION 2: PROJECT AIMS

Aim 1: Experimental Aim

Build and validate a first MelC2-based cell-free melanin module

The first aim of this project is to design a codon-optimized Streptomyces antibioticus MelC2 tyrosinase expression cassette for TX-TL / E. coli use and test whether it can generate measurable melanin-like optical changes in a controlled cell-free system. This aim uses DNA design, Benchling assembly, Twist synthesis, fluorescent protein controls, visible darkening, OD 400-500 nm absorbance, SDS-PAGE, and future LC-MS analysis to distinguish protein expression, enzymatic activity, pigment accumulation, and downstream oxidation chemistry.

Aim 2: Development Aim

Optimize the chemical and optical behavior of the melanin-forming system

After validating the first module, the next aim is to optimize the reaction conditions that shape pigment output, including L-tyrosine concentration, copper availability, pH buffering, oxygen exposure, magnesium, incubation time, and reporter choice. This aim will help determine whether the system can be tuned for stronger pigment formation, cleaner optical readouts, and more predictable color response before integration into a material matrix.

Aim 3: Visionary Aim

Develop programmable melanin bioinks for exposure-recording and functional biomaterials

The long-term aim is to integrate the optimized melanin-forming module into bacterial cellulose or other biomaterials to create bio-based surfaces that can both record environmental exposure and respond functionally. If successful, this could support responsive textiles, UV-protective coatings, design surfaces, tattoo-like dermal pigments, bioelectronic interfaces, space-oriented materials, and localized radioprotective biomaterials.

SECTION 3: BACKGROUND

3.1. Peer-reviewed research citations

Melanin is relevant to this project because its material properties extend beyond visible pigmentation. Menichetti et al. 2025 describe melanin photoprotection as a combination of broadband light extinction and antioxidant activity, supporting the idea that melanin-based materials could pair optical response with protection against light-induced damage. Dadachova and Casadevall 2009 further show that melanin changes how biological systems interact with ionizing radiation, with melanized fungi displaying radioprotective behavior and altered electronic properties under radiation exposure. Together, these studies support the central premise of this project: melanin can be treated not only as a pigment, but as a functional material chemistry for exposure-responsive systems.

This material potential has already been explored in several application directions relevant to the proposed bioink. In space-oriented materials, Cordero et al. 2025 showed that fungal melanin-polymer biocomposites exposed to low Earth orbit conditions had improved structural stability and radiation-shielding potential. In photothermal and bioelectronic materials, Yue and Zhao, 2021 review how melanin-like materials can convert absorbed optical energy into heat and support sensor or interface applications through redox activity and mixed ionic/electronic behavior. At the skin interface, Park et al. 2024 developed electroactive melanin tattoo inks using naturally derived melanin nanoparticles to reduce skin impedance, suggesting that melanin-based pigments may be useful for dermal bioelectronic interfaces as well as coloration.

The bioink and textile direction also has direct precedent. Walker et al. 2024 engineered cellulose-producing Komagataeibacter rhaeticus to express tyrosinase and grow self-pigmenting bacterial cellulose through melanin biosynthesis, showing that genetically encoded pigmentation can be integrated into a material-producing microbial platform. Ahn et al. 2021 produced melanin-like pigments microbially from caffeic acid and applied the pigment to cotton fabric dyeing, supporting the relevance of microbial melanin as a textile-compatible colorant.

These studies connect directly to this project’s direction, but also clarify its specific contribution: instead of starting with a finished textile, this work first builds a controlled MelC2-based cell-free module to make melanin-like optical output measurable, tunable, and chemically interpretable before later integration into bacterial cellulose or other biomaterial matrices.

3.2. Novelty and innovation

This project is innovative because it uses existing biological tools in a new material context: a MelC2 tyrosinase module is designed not only to produce pigment, but to generate a measurable and tunable optical output. The cell-free system makes this approach modular, allowing key variables such as copper loading, substrate availability, pH, oxygen, redox state, and polymerization conditions to be tested before introducing the system into more complex biomaterial matrices. This creates a controlled bridge between genetic design and material performance.

The project also challenges a common assumption in functional materials: that color, sensing, protection, and responsiveness must be added as separate components. Instead, it asks whether melanin-forming chemistry can be programmed as a single multifunctional layer that records exposure and produces useful material responses. In doing so, the project expands synthetic biology from making biological products toward engineering bio-based materials whose behavior can be designed, measured, and tuned.

3.3. Why the project matters and potential impact

The main ethical issue is not melanin itself, but the form in which the system is built and deployed. A melanin-based material can remain a controlled chemical module, become a non-replicating embedded system, or become part of a living material platform. Each design choice carries a different ethical burden, so the project should progress from the lowest-risk and most interpretable system toward more complex formats only after validation.

For this reason, the current project takes the cell-free route as an ethical and technical starting point. It validates the core chemistry - MelC2 expression, copper loading, substrate availability, pH, oxygen, and pigment formation - before adding living-system or material-scale complexity. This avoids treating a speculative material concept as a deployable product too early.

The practical ethical strategy is staged development: first validate pigment chemistry, then test material integration, then evaluate sensing or protective performance under relevant conditions. The main risks are overclaiming protection, treating color change as quantitative sensing too early, or moving into dermal / biomedical contexts before the material is characterized. The project could also be wrong if melanin pigmentation does not correlate reliably with exposure, if pigment chemistry is too variable to control, or if a simpler non-biological sensor performs better. Alternatives such as purified enzymes, synthetic melanin-like polymers, or conventional exposure sensors should remain available if they prove safer or more reliable.

SECTION 4: EXPERIMENTAL DESIGN, TECHNIQUES, TOOLS, AND TECHNOLOGY

4.1 Experimental plan and timeline in 15 steps

The experimental plan follows a build-test-learn structure. First, the MelC2 construct is selected, designed, ordered, and validated in silico. Next, the cell-free TX-TL system is tested using fluorescent protein controls. Then, pigment formation, protein expression, and pathway chemistry are validated separately. After the course, the project can move toward light-controlled expression modeling and material integration for a melanin-based light-recording bio-ink.

After the construct is designed, validation proceeds from simple functional checks to more specific analytical readouts. Each step is meant to isolate a different possible failure point: TX-TL expression capacity, MelC2 protein production, pigment accumulation, or pathway-level chemistry.

Previous iGEM tyrosinase projects (see references list at the end of this document) showed that tyrosinase expression can be detectable even when pigment formation is weak or absent. For this reason, protein production, enzyme activity, and optical output need to be validated separately rather than treated as a single result.

Post-course, the next conceptual step is to move this melanin-based light-recording bio-ink forward by modeling light-activated melanin expression in Asimov Kernel. This will help clarify whether the project should first push for tighter molecular control, or move earlier into material-scale experimentation. The longer-term goal is to connect the cell-free MelC2 module to a material system capable of controlled, spatially localized, and visually meaningful pigment formation.

The tables below summarize the validation logic for the cell-free MelC2 module. Each readout acts as a checkpoint, moving from general TX-TL expression capacity to visible pigment output, absorbance kinetics, protein expression, and finally chemical validation.

This staged logic keeps the first aim experimentally interpretable. Color change is treated as one readout among several, while protein expression and LC-MS provide the controls needed to distinguish enzyme production, catalytic activity, and downstream pigment chemistry.

An initial version of a visual workflow diagram for this validation logic generated using chatGPT can be found in my Brainstorms documentation.

4.2 Techniques relevant to this project

The checked techniques reflect the parts of the project that were used or directly planned, from MelC2 construct design and cell-free expression to validation readouts and future automated testing.

Pipetting

• Pipetting
• Lab Safety
• Bioethical Considerations

Pipetting, lab safety, and bioethical considerations are relevant because the project depends on careful preparation of small-volume cell-free reactions and controlled handling of reagents such as L-tyrosine, CuSO4, buffers, and DNA constructs. These techniques also support the project’s staged design: testing melanin-forming chemistry in a contained, non-deployable system before moving toward biomaterial applications (lab safety and bioethical consideration).

DNA Gel Art

• DNA Sequencing
• DNA Editing
• DNA Construct Design
• Restriction Enzyme Digestion
• Gel Electrophoresis
• DNA Purification From Gel
• Databases, e.g. GenBank, NCBI, Ensembl, and UCSC Genome Browser

DNA Gel Art / construct design was selected because the project required designing and verifying a MelC2 expression cassette. DNA construct design and databases are checked because Benchling, UniProt, BLASTP, and sequence databases were used to select, optimize, and verify the MelC2 construct. DNA sequencing, DNA editing, restriction digestion, gel electrophoresis, and gel purification are unchecked because they were not performed in this stage. However, they may become relevant after synthesis if the construct needs to be sequence-verified, edited, digested, visualized on a gel, or purified before downstream expression tests.

Bioproduction

• Bioproduction
• Chassis Selection, e.g. TX-TL / E. coli context
• Registry of Standard Biological Parts
• Plasmid Preparation
• Bacterial Culturing
• Quality Control / Analysis
• Bacterial Processing, e.g. centrifugation, lysis, DNA purification

Bioproduction was selected because the project aims to produce a functional biological output: MelC2-driven melanin-like pigmentation. Chassis selection is checked because the first expression context is TX-TL / E. coli, and the Registry of Standard Biological Parts informed the expression design. Quality control / analysis is checked because the workflow uses fluorescence, OD 400-500 nm, SDS-PAGE, and future LC-MS to validate expression, activity, and pigment output. Plasmid preparation, bacterial culturing, and bacterial processing are unchecked because this stage uses a synthesized construct and cell-free validation rather than live-cell propagation or processing.

Lab Automation

• Creating Code for Laboratory Automation
• Using Liquid Handling Robots, e.g. Opentrons
• Designing a Twist Order
• Creating a plan to use the Autonomous Lab at Ginkgo Bioworks

Lab automation was selected because the project includes automated experimental planning for the next validation stage. I checked code for laboratory automation, Twist order design, and Ginkgo Bioworks planning because I prepared the construct for synthesis and began designing a reaction matrix to test variables such as copper, tyrosine, buffering, magnesium, and reporter choice. I left liquid handling robots unchecked because I did not directly operate an Opentrons or similar robot in this stage.

Protein Design

• Protein Design
• Use of Boltz or PepMLM
• Use of Asimov Kernel
• Use of Benchling
• Models and Notebooks
• Databases

Protein design was selected because the project depends on choosing, analyzing, and eventually controlling a melanin-forming enzyme. I checked Benchling, databases, models, and notebooks because they were used to select MelC2, inspect sequence/function, support construct design, and analyze protein behavior. Asimov Kernel is checked because it was explored for future light-responsive control of MelC2 expression. Boltz and PepMLM are unchecked because they were not used in this stage.

Cell-Free Systems

• Cell-Free Reactions
• Freeze-Dried Cell Free Systems
• miniPCR Tools
• Protein Purification

Cell-free systems were selected because the first experimental goal is to test MelC2 expression and melanin-like pigment formation in a controlled, non-replicating format. Cell-free reactions and freeze-dried cell-free systems are checked because they are the planned platform for validating the module. miniPCR and protein purification are unchecked because this stage does not require PCR amplification or purified MelC2 protein.

Gibson Assembly

• Primer Design or Selection
• PCR Reactions
• Gibson Assembly
• Other Cloning Methods, e.g. Restriction Enzyme Digestion or Gateway Cloning

CRISPR

• CRISPR/Cas9
• Designing Prime Editing gRNA

Gibson Assembly and CRISPR were left unchecked because the current project does not involve cloning by PCR/Gibson methods or genome editing. The MelC2 module was designed digitally and prepared for synthesis through Twist, so primer design, PCR, Gibson Assembly, restriction-based cloning, CRISPR/Cas9, and prime-editing gRNA design were not part of this stage.

4.3 Two techniques expanded

The two most important techniques are DNA construct design, which creates the module, and cell-free reactions, which test whether the module produces an interpretable optical output.

DNA construct design: DNA construct design is central because Aim 1 depends on building a MelC2-based module that can be tested in TX-TL / E. coli conditions. I used database research to select MelC2, codon optimization to adapt the sequence for expression, and Benchling to assemble the cassette. The C-terminal 6xHis tag supports future protein-level validation. This matters because MelC2 expression must be distinguished from actual melanin-like pigment formation.
Cell-free reactions: Cell-free TX-TL is the cleanest first platform because the main uncertainty is chemical: whether MelC2 expression, copper loading, L-tyrosine availability, pH, oxygen, and downstream oxidation chemistry can generate optical change. Compared with living cells, the system is easier to control and interpret. It also avoids adding material-scaffold complexity too early. The first experiments use fluorescent protein controls, visible darkening, OD 400-500 nm absorbance, SDS-PAGE, and future LC-MS to validate the module step by step.

4.4 Industry Council companies relevant to the project

These companies are relevant because they map onto the main project needs: DNA synthesis, automation, modeling, chemical analysis, reagents, and future biomaterial translation.

A particularly relevant external benchmark is MelaTech, a startup focused on melanin-based materials for space applications.

SECTION 5: Results & Quantitative Expectations

5.1.1 Aspect of the final project validated

I validated the DNA design foundation of the project: the construction of a MelC2 tyrosinase expression cassette for TX-TL / E. coli use. This validation addresses the first build layer of the project, because a reliable genetic module is required before testing melanin-like pigment formation in a cell-free system.

The validated output is not melanin production itself, but a synthesis-ready construct designed to express a soluble, oxygen- and copper-dependent tyrosinase. This includes enzyme selection, codon optimization, expression cassette design, vector assembly in Benchling, Twist submission, and a planned validation workflow for future cell-free testing.

5.1.2 Validation protocol

The figure below, from my CL Final Project presentation, summarizes the project pipeline around the validated build layer: MelC2 selection, DNA construct design, Twist submission, and planned cell-free validation.

I followed this protocol:

5.1.2.1 Enzyme selection

I selected MelC2 from Streptomyces antibioticus as the target enzyme for the first construct. I chose MelC2 because it is a soluble, cytosolic, oxygen-, copper-dependent and reasonably small enzyme of about 273 amino acids, and has a reviewed Swiss-Prot annotation, which made it a strong first candidate for cell-free TX-TL expression.

Its dependence on copper, oxygen, substrate availability, pH, and downstream polymerization also gives the project a clear set of tunable variables for validating melanin-like pigment formation.

Useful references for this step included the UniProt P07524 entry and WT sequence in Benchling.

And here is the predicted structural model used as part of my design context:

5.1.2.2 Codon optimization

I codon-optimized the MelC2 sequence for E. coli K-12 expression in Benchling**

Codon-optimization of P07524 for E. coli K-12, to avoid BsaI/BsmBI/BbsI and add a C-terminal His-tag to quantify enzyme expression cleanly -> Results in Benchling here.

I’ve selected the region of the AA sequence I wish to back translate and right clicked on the highlighted region. From the the codon optimization tab:

• Host: E. coli K-12
• Method: Match codon usage
• GC content: Medium (0.33 to 0.66) cause the extremes may be inconvenient. High GC can create strong secondary structures and low GC can cause instability/repeats and can make synthesis harder.
• Uridine depletion: off (not relevant for bacterial expression)
• Hairpin parameters: Stem size: 8 and Window 50
• Restriction sites: avoid BsaI, BsmBI, BbsI (Type IIS restriction enzymes, the workhorses of Golden Gate assembly)
• Patterns to reduce: AAAAAA and ATATATATA

I clicked on “Preview Optimization” and got this result, which I’ve saved in the same Benchling folder here:

BLASTP verification of codon-optimized sequence:

I translated the codon-optimized DNA and ran BLASTP against nr/ClusteredNR. The top hits were MelC2 tyrosinases from Streptomyces spp., with 100% query coverage, E-value 0.0, 92% identity (251/273), 95% positives (261/273), and 0 gaps. Conserved domain analysis identified the Tyrosinase domain across the full sequence length. This confirms the optimized DNA still encodes a canonical tyrosinase.

melC2 tyrosinase (Streptomyces antibioticus, P07524, codon-optimized for E. coli K-12) DNA sequence Benckling link here.

5.1.2.3 Protein-detection design

I added a C-terminal 6xHis tag (CACCACCACCACCACCAC) before the stop codon to support future protein-level detection / quantification.

5.1.2.4 Expression cassette assembly

I assembled the TX-TL expression cassette using a T7 Promoter, RBS (Shine Delgarno) / AAATAT Spacer, codon-optimized melC2 CDS, C-terminal 6xHis tag, TAA stop codon, and T7 terminator BBa_B0015 Benchling link here.

To be considered: T7 can maximize protein yield but also overwhelm folding capacity, causing inactive protein accumulation (increase the likelihood 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).

So here’s my final melC2 tyrosinase CDS with annotations.

5.1.2.5 Vector assembly and construct inspection

I placed the full expression cassette into a pTwist Amp High Copy vector. Why: high-copy propagation in E. coli for easy plasmid prep; selection marker is standard.**

I inspected the final construct map in Benchling to confirm the organization of the insert, vector, promoter, terminator, and annotated CDS. Assemblings on Benchling here.

Final Construct: My melC2 construct submitted assembled into pTwist Amp High Copy on Benchling interface.

5.1.2.6 Twist submission

I submitted the final construct for synthesis through Twist. My Twist order for final Construct here

This completed the validated DNA-design layer of the project.

Planned next validation

After synthesis, the construct will be tested in a staged cell-free workflow: fluorescent protein controls for TX-TL capacity, visible darkening and OD 400-500 nm for pigment formation, SDS-PAGE / His-tag detection for MelC2 expression, and future LC-MS for tyrosine / L-DOPA-related intermediates.

I also began planning Ginkgo RAC-style cell-free reaction conditions to test key bottlenecks such as copper availability, tyrosine concentration, pH buffering, magnesium, incubation time, and reporter choice.

Prepare reagents and workflow (Ginkgo & Open AI)

I’ll first test my brainstormed HW 11C Cell-Free Master Mix experiment and then iterate aiming for a debugged and optimized workflow.

Here are some variables I had in mind when formulating this first 8 master mix compostion

Melanin production in E. coli or in a cell-free system is influenced by several parameters that act at the level of melC2 expression and enzyme activity / downstream 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.

My first 8 experiments at Ginkgo - aim is to successfully produce fluorescent protein and generate an initial dataset for analysis.

sfGFP → system calibration (TX-TL health) Melanin has a broad absorbance spectrum, but it absorbs much more strongly at shorter wavelengths (blue/green) than at longer wavelengths (red). Melanin interferes with optical readout since we will be trying to measure fluorescence in a reaction that is simultaneously getting darker, which creates optical interference broadening the wavelength spectrum of signal.

mScarlet-I → expression readout for melC2 tyrosinase specifically fluorescence is less sensitive to melanin, so it better tracks expression alone (sfGFP → Ex ~488 nm / Em ~510 nm → high overlap with melanin absorbance; mTurquoise2 → even worse (blue region); mScarlet-I → Ex ~569 nm / Em ~594 nm → less overlap).

For optimizing the Master Mix design for mScarlet-I in my melC2 tyrosinase cell-free system, I’d supplement CuSO4 since my analyte is a copper-dependent enzyme, HEPES-KOH pH 7.5 to have an additional buffer against acidification and magnesium glutamate to improve translation capacity.

At first I thought about adding glucose since it could extend energy regeneration, but then I wondered that it may also increase acidification. Since you’re worried about fluorescence readout in a pigment-producing system, I’d prioritize pH stability over extra glucose.

I’d actually supplement L-tyrosine that serves as a functional validation that my protein of interest MelC2 tyrosinase is being expressed and active.

Master Mix designs to be tested using mScarlet-I and sfGFP, the 8 reactions outlined are available here in Week 11 HW Documentation.

5.1.3 Synthetic biology techniques used

The main synthetic biology technique used was DNA construct design. I designed a codon-optimized MelC2 tyrosinase cassette for TX-TL / E. coli expression, added a C-terminal 6xHis tag for future protein detection, assembled the cassette in Benchling, and prepared it for synthesis through Twist.

I also used database-based sequence selection and verification. UniProt and Benchling were used to select and inspect the MelC2 sequence, while BLASTP and conserved-domain analysis were used to confirm that the codon-optimized DNA still encoded a canonical tyrosinase.

A third relevant technique was cell-free system planning. The construct was designed specifically for TX-TL / E. coli use, and the next validation workflow was planned around fluorescent protein controls, visible darkening, OD 400-500 nm absorbance, SDS-PAGE / His-tag detection, and future LC-MS analysis.

Finally, I used lab automation planning by preparing a Ginkgo RAC-style reaction matrix to test variables expected to affect MelC2 pigment formation, including copper availability, L-tyrosine concentration, pH buffering, magnesium, incubation time, and reporter choice.

5.1.4 Data and analysis

The validation data for this stage are design-level and sequence-level results generated during construct preparation. These data show that the MelC2 construct is synthesis-ready and still encodes the intended tyrosinase target.

These data validate the first build layer of the project: the DNA module is codon-optimized, annotated, compatible with the intended TX-TL / E. coli context, and submitted for synthesis. The results do not prove melanin production, but they confirm that the construct is coherent enough to justify downstream expression testing. The next quantitative expectation is that successful cell-free expression should produce detectable MelC2 by SDS-PAGE / His-tag detection and measurable pigment accumulation by OD 400-500 nm if the enzyme is active under the tested conditions.

5.2 Unexpected challenges, limitations, and alternatives

The main limitation is that a correct DNA construct does not automatically prove protein activity or melanin-like pigment formation. Tyrosinase expression can be detected while pigment remains absent if folding, copper incorporation, substrate availability, oxygen, pH, or downstream polymerization chemistry is limiting.

Another challenge is that melanin is a chemically heterogeneous output, so visible darkening alone is not enough to validate the system. To address this, the next validation stage separates TX-TL expression capacity, MelC2 protein production, pigment accumulation, and pathway-level chemistry using fluorescence controls, OD 400-500 nm, SDS-PAGE / His-tag detection, and future LC-MS. If the T7 design produces inactive or misfolded protein, an alternative strategy would be to test a moderated promoter, adjust copper and substrate concentrations, or compare purified enzyme / synthetic melanin-like polymer approaches before moving into more complex biomaterial systems.

SECTION 6: ADDITIONAL INFORMATION

6.1 References cited in this assignment

• Menichetti, L. et al. “Melanin as a photoprotective material,” 2025. https://www.mdpi.com/3235558
• Dadachova, E. and Casadevall, A. “Ionizing radiation: how fungi cope, adapt, and exploit with the help of melanin,” 2009. https://pmc.ncbi.nlm.nih.gov/articles/PMC2677413/
• Cordero, R. J. B. et al. “Fungal melanin-polymer biocomposites exposed to low Earth orbit conditions,” 2025. https://www.pnas.org/doi/10.1073/pnas.2427118122
• Yue, X. and Zhao, L. “Melanin-like materials for photothermal and bioelectronic applications,” 2021. https://www.mdpi.com/947490
• UniProt. Streptomyces antibioticus tyrosinase MelC2, P07524. https://www.uniprot.org/uniprotkb/P07524/entry
• iGEM Registry. BBa_I14032, P(lac)IQ promoter. https://parts.igem.org/Part:BBa_I14032
• iGEM Registry. BBa_K193600, melA tyrosinase. https://parts.igem.org/Part:BBa_K193600
• iGEM Registry. BBa_K193602, pLacIQ-RBS-melA composite construct. https://parts.igem.org/Part:BBa_K193602
• iGEM Registry. BBa_K2481108, MelA expression construct for E. coli BL21(DE3). https://parts.igem.org/Part:BBa_K2481108

6.2 Supply list and budget

This budget estimates the next practical stage of the project: validating the MelC2 construct in a cell-free TX-TL system before moving into material integration.

The cost ranges below were estimated with the assistance of ChatGPT and should be treated as approximate planning values. The estimation method was to break the project into major experimental cost categories - DNA synthesis, TX-TL reactions, controls, reagents, consumables, protein validation, chemical validation, and material integration - and assign conservative low/high ranges for each category based on typical small-scale synthetic biology workflows.

The lower end of each range assumes access to shared lab equipment, existing stocks of common reagents, and limited reaction numbers. The higher end assumes new reagent purchases, larger reaction matrices, external analytical services, or the need to purchase or arrange access to readout equipment. Exact costs would need to be confirmed through vendor quotes, institutional core facility pricing, or cloud-lab pricing.

Estimated total for first validation stage: approximately $850-3,600, assuming access to shared lab equipment.

This total excludes major equipment purchases and uncertain cloud-lab pricing. It includes the core experimental costs needed to move from a designed MelC2 construct to initial TX-TL expression, pigment-production screening, and basic validation.

Estimated total including purchased equipment or external analytical services: could exceed $5,000-10,000, depending on instrument access, number of samples, and whether LC-MS or optical readout must be outsourced or purchased.

This documentation was developed with the assistance of ChatGPT, which was used to support drafting, editing, organization, and figure generation. All scientific decisions, final content, and interpretations were reviewed and approved by the author.

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.

Note on project status

The Group Final Project became optional for Spring 2026, with collaborative work expected to resume later. Because of this, my documentation focuses on the individual contribution I made during the planning and design phase rather than on a completed experimental workflow.

My main contribution was to help define candidate MS2 L-protein mutations using a combination of protein language model scoring, experimental mutant data, and biological reasoning about L-protein functional regions. The goal was to identify a small set of interpretable mutations that could later be tested experimentally for effects on L-protein stability, DnaJ dependence, membrane insertion, and lysis function.

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.

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.

Validation workflow for MelC2 pigment-production analysis. Generated with ChatGPT.