Individual Final Project

FINAL PROJECT DECK: https://www.figma.com/deck/G1Zvluf6m7BrTVp1HMR95F

SECTION 1: ABSTRACT

My final project explores whether ice cream can become a visible biological interface instead of a passive frozen dessert. The project began with a simple question: could an ice cream appear to “wake up” when it warms, by producing visible bioluminescence through a designed protein-substrate reaction? For this class, I narrowed the idea into a proof-of-concept experiment using NanoLuc luciferase and its substrate furimazine. NanoLuc is a small engineered luciferase derived from Oplophorus gracilirostris and designed to produce strong glow-type luminescence when paired with furimazine.

The broad objective is to test whether purified NanoLuc can produce naked-eye-visible light in or on ice cream when mixed with substrate under controlled conditions. My hypothesis is that if the protein and substrate are kept separated during frozen storage and then brought into contact during warming, the system can generate visible local light spots. During the course, I focused on designing a NanoLuc-GSGS-His6 construct, expressing or preparing NanoLuc through a cell-free or bacterial expression workflow, purifying the His-tagged protein with Ni-NTA resin, and testing protein-to-substrate ratios in a small-volume assay.

I also explored a food-material strategy where furimazine-containing substrate droplets could be physically separated from protein using cocoa butter as a temperature-responsive fat barrier. The current project is a lab demonstration only and should not be consumed, because Nano-Glo and furimazine are research reagents rather than approved food ingredients.

SECTION 2: PROJECT AIMS

Aim 1: Experimental Aim

The first aim of my final project is to produce and test visible NanoLuc-based bioluminescence in an ice cream setting by utilizing DNA construct design, cell-free or bacterial protein expression, Ni-NTA purification of NanoLuc-GSGS-His6, and controlled mixing of purified protein with Nano-Glo substrate.

Relevant methods and resources include:

  • NanoLuc-GSGS-His6 DNA design
  • pET-28a expression design
  • DH5alpha plasmid preparation
  • BL21(DE3) expression planning
  • Cell-free protein synthesis
  • Ni-NTA affinity purification
  • Promega Nano-Glo substrate assay
  • 96-well plate ratio testing
  • Dark-room imaging
  • Image-based brightness analysis

Aim 2: Development Aim

The next development aim is to build a more controlled frozen dessert prototype by encapsulating or physically separating the substrate inside small cocoa-butter-based particles, so the protein and substrate do not fully react during frozen storage but can contact each other when warmed locally or during simulated oral-temperature conditions.

This would move the project beyond simply dropping protein and substrate together. It would test whether a food-compatible fat matrix can act as a physical timing mechanism for a bioluminescent reaction. The main technical questions would be particle size, substrate loading, reaction brightness after storage, and whether the cocoa butter actually melts fast enough at the moment of contact.

Aim 3: Visionary Aim

The long-term vision is to develop a safe, food-grade version of a “living-feeling” ice cream that gives a visible biological response as it warms, melts, or is eaten.

A future version would need to replace the current research-use NanoLuc/furimazine system with food-safe or fully toxicology-tested components. It could also use a food-grade secretion host such as Pichia pastoris for protein production, and a thermally responsive split luciferase design, such as NanoBiT fused to a temperature-sensitive protein interaction pair. The goal would be a dessert that does not simply display color or decoration, but changes through a biological reaction that the eater can see.

SECTION 3: BACKGROUND

Background and Literature Context

Bioluminescence is widely used in biology because it can convert molecular activity into visible or measurable light. NanoLuc is especially useful for this project because it is small, bright, and stable compared with older luciferase reporters. Hall et al. engineered NanoLuc from a deep-sea shrimp luciferase system and paired it with a synthetic imidazopyrazinone substrate called furimazine. Their work reported glow-type luminescence and much higher specific activity than firefly or Renilla luciferase systems, which makes NanoLuc a good candidate for a visual proof-of-concept experiment rather than only a sensitive instrument-based assay.

A second relevant area is cell-free gene expression. Garenne et al. describe cell-free gene expression as a way to produce proteins outside living cells, and they point out that E. coli-based cell-free systems have become useful for synthetic biology, protein synthesis, and prototyping. This matters for my project because I do not need a living organism inside the ice cream. I only need the designed protein product. Cell-free expression gives a faster way to test whether the DNA design can produce functional NanoLuc before investing in a full bacterial expression and purification pipeline.

The safety context is also central to this project. Shipunova et al. studied furimazine toxicity in vitro and in vivo and reported hepatotoxic effects under prolonged intravenous administration in animals. That does not directly describe oral food exposure, but it clearly shows that furimazine cannot be treated casually as a food ingredient. Promega also labels the Nano-Glo Luciferase Assay System as “For Research Use Only. Not for Use in Diagnostic Procedures.” For this reason, the current project should be framed as a lab demonstration and a material-biology prototype, not as an edible product.

Novelty and Innovation

My project uses NanoLuc outside its usual role as a reporter in cells, tissues, or plate-reader assays. Instead of asking NanoLuc to report on gene expression, I am using it as the active component in a visible material experience. The novelty is in combining a designed protein, a substrate timing problem, and an ice cream material system into one prototype.

The project also treats frozen food as a reaction environment. Ice cream is cold, heterogeneous, fatty, and visually familiar, which makes it very different from a normal buffer or cell culture medium. The cocoa butter idea adds another layer: the fat is not just an ingredient, but a possible physical gate that controls when substrate contacts protein.

Why the Project Matters and Possible Impact

I care about this project because synthetic biology often stays invisible to people unless they are looking at a gel, a graph, or a screen. This project asks whether a biological reaction can become something direct and physical, something a person can see happening in a familiar object. Ice cream is a playful format, but the technical question is real: can we control the time, location, and visibility of a biological reaction inside a complex material?

The immediate impact is educational and experimental. A successful prototype could make enzyme-substrate reactions easier to understand because the reaction is visible without special imaging equipment. It could also show how biological tools can be combined with product design and material design, instead of staying only inside lab workflows.

The longer-term impact depends on safety and food compatibility. I do not think the current NanoLuc/furimazine system should be presented as edible. A future food-safe version could contribute to responsive foods, science communication tools, or controlled-release food materials. Even if the final product never becomes a commercial dessert, the project still tests a useful design pattern: separating biological components in a material until temperature, mixing, or physical contact triggers the reaction.

Ethical Implications

The main ethical issue is safety. The current version uses NanoLuc and furimazine through commercial research reagents, and these materials are not approved as food ingredients. Because of that, the project should follow non-maleficence: no one should eat the prototype, and no public demonstration should create the impression that the current sample is safe for consumption. The project also touches responsibility, because the language around “living ice cream” could be misleading. The prototype is not alive. It is a designed biochemical reaction placed in a food-like object.

To keep the project ethical, I would label all samples as non-edible research prototypes, perform tests only in appropriate lab or controlled demo settings, and dispose of materials as research waste. I would also avoid user testing that involves tasting unless every component is replaced with food-grade materials and reviewed under the appropriate food safety process. A possible unintended consequence is that the visual appeal of a glowing dessert could make people underestimate reagent risk. I could also be wrong about the stability of the reaction in ice cream, the effectiveness of cocoa butter encapsulation, or the safety pathway for future ingredients. A safer alternative for public display would be to keep the reaction sealed in a transparent food-shaped shell or use a non-food hydrogel model before moving back to real ice cream.

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

Detailed Experimental Plan

  1. Design the NanoLuc-GSGS-His6 DNA construct. The construct should include NanoLuc, a short flexible GSGS linker, and a C-terminal His6 tag for Ni-NTA purification.

  2. Place the construct into a pET-28a-style expression design so it can be expressed under a T7 promoter in BL21(DE3), while also being compatible with cell-free expression systems that use T7 RNA polymerase.

  3. Use Benchling or a similar DNA design tool to check the coding sequence, reading frame, His tag position, stop codon, and any required cloning or ordering details.

  4. Prepare the DNA template through plasmid preparation or ordered DNA, depending on the available timeline. Expected result: a verified DNA template that can express NanoLuc-GSGS-His6.

  5. Use a small-scale cell-free protein synthesis reaction to produce NanoLuc. Cell-free systems are appropriate here because they can synthesize protein within a few hours and can use plasmid or linear DNA templates.

  6. Incubate the cell-free reaction under the recommended temperature and time conditions. Expected result: functional NanoLuc protein is produced in the reaction mixture.

  7. Purify the His-tagged NanoLuc using loose Ni-NTA resin in a microcentrifuge tube. Ni-NTA resin is designed to purify recombinant proteins containing a polyhistidine tag.

  8. Equilibrate the Ni-NTA resin with binding buffer, bind the cell-free reaction or cleared lysate to the resin, wash away non-binding components, and elute NanoLuc using imidazole-containing elution buffer.

  9. Quantify the purified protein concentration. In the current experiment, the measured protein concentration was approximately 3.48 mg/L. Assuming NanoLuc is approximately 19.1 kDa, this corresponds to roughly 180 nM protein before dilution.

  10. Prepare a protein dilution series. My current planned set is: N1 as stock protein, N2 as 24 uL stock plus 6 uL PBS, N3 as 20 uL stock plus 10 uL PBS, and N4 as 15 uL stock plus 15 uL PBS.

  11. Prepare Nano-Glo reagent conditions. The main comparison should focus on 1x and 0.5x reagent because earlier trials suggested that excessive dilution makes the signal hard to see by eye.

  12. Set up a small plate assay using approximately 10 uL protein solution and 10 uL substrate reagent per well. Expected result: wells with higher protein and substrate concentration should show stronger visible luminescence.

  13. Record the reaction in a dark box or dark room using a phone camera or camera with fixed exposure. Capture images or video every few seconds for the first minute, then continue at longer intervals if the signal remains visible.

  14. Analyze brightness using image analysis. For each well, define a region of interest and extract average pixel brightness over time. Expected result: each condition produces a brightness-time curve rather than only a single visual observation.

  15. Transfer the best-performing condition to a real ice cream test. Because there was not enough time to make ice cream from scratch with protein and substrate built into the production process, the practical test is to make small holes in commercial ice cream, add the material locally, and image the response.

  16. For the first ice cream test, add purified protein and Nano-Glo reagent directly into small holes or surface pockets. Expected result: visible dots should appear if the reaction remains concentrated enough and the camera exposure is controlled.

  17. For the second ice cream test, test a cocoa-butter-based substrate particle or droplet. The substrate would be mixed or physically trapped with cocoa butter, cooled into small particles, and placed into the ice cream. Expected result: the fat barrier should slow premature contact between protein and substrate.

  18. Simulate warming by local heating or by adding warmed purified protein solution. Expected result: warming should soften the cocoa butter and increase protein-substrate contact.

  19. Compare three outcomes: direct mixing in buffer, direct mixing on ice cream, and cocoa-butter-mediated release on ice cream. The comparison will show whether the ice cream matrix and fat encapsulation reduce or preserve visible signal.

  20. Use the results to decide whether the next stage should focus on higher protein yield, better substrate encapsulation, or a different temperature-gated protein design.

Visual Workflow