HTGAA 2026: Cell-Free Wearable Hydration Tracker SECTION 1: ABSTRACT Hi, I’m Alayah! And for those of you who don’t know me, this is how I spend most of my time.
Group Brainstorm on Bacteriophage Engineering Computational Engineering of the MS2 Lysis Protein (L) Background. The MS2 L protein is a 75-amino-acid polypeptide that lyses E. coli by an incompletely understood mechanism. Its C-terminal transmembrane (TM) domain inserts into the cytoplasmic membrane and oligomerizes, causing depolarization that triggers host autolytic enzymes to degrade the murein layer. Recessive, conservative missense mutations clustered around a conserved LS dipeptide strongly implies L engages an unidentified host protein target rather than simply disrupting the bilayer. The dispensable N-terminal domain binds chaperone DnaJ (with solved PDB structures), modulating lysis timing. Its removal causes lysis ~20 min earlier. No experimental structure of L exists. Goals. (1) Stabilize L for more robust membrane accumulation. (2) Accelerate lysis by bypassing DnaJ-dependent regulatory timing and improving delivery of functional L to the membrane. Because the downstream lytic target is unknown, we do not attempt to enhance per-molecule toxicity at the point of target engagement; we focus on removing regulatory brakes and increasing the supply of functional protein. Pipeline: Three Tools, Each Non-Redundant
Subsections of Projects
Individual Final Project
HTGAA 2026: Cell-Free Wearable Hydration Tracker
SECTION 1: ABSTRACT
Hi, I’m Alayah! And for those of you who don’t know me, this is how I spend most of my time.
I blow glass here at MIT's glass lab, and it is tons of fun. I make all sorts of glass animals and sculptural shapes (my favorites are turtles and elephants, see below)
As I was saying, blowing glass is tons of fun, but it is incredibly physically demanding; you spend hours in front of 2000°F ovens and need Kevlar sleeves just to protect you from the radiant heat of your piece, in short, you spend the whole time sweating buckets despite open windows and fans and ACs cranked up to max. Hydration is incredibly important in this kind of environment, and the consequences of dehydration can be catastrophic, yet it's hard to know when you can power through and when you should take a break.
Dehydration is a critical physiological risk in glass blowing, athletics, and any physically demanding occupations, yet continuous, real-time, non-invasive monitoring remains a significant challenge. In the glass lab, the gaffer, or project lead, will struggle through dehydration to finish their work to their own detriment, but having a noninvasive wearable sensor that measures dehydration and recommends that you take a hydration break can have a hugely positive impact on an individual's well-being.
This project aims to address this by developing a cell-free genetic circuit designed to detect lactate in sweat as a way of predicting hydration stress. The objective is to create a fast, safe, and fluorescent output without relying on living cells or slow protein translation and capturing this in a wearable paper-based system.
Specifically, the project works through a pathway where Lactate Oxidase (LOX) converts sweat lactate into hydrogen peroxide H2O2, which subsequently oxidizes the T7-driven transcription factor OxyR. Active OxyR will then trigger the PoxyS promoter to transcribe a specific RNA aptamer that binds to DFHBI dye, producing immediate fluorescence. By employing an *E. coli* lysate-based cell-free master mix. I will then test each step separately.
The central circuit is split into four phases:
Phase One - Input to Secondary Messenger: Lactate Oxidase breaks down Lactate into pyruvate and H2O2
- Test: Verify enzymes work in buffer conditions and H2O2 production
Phase Two - Secondary Messenger to Ac: H2O2 binds to OxyR enzyme produced by the first part of the genetic circuit
- Test: Verify OxyR is produced by looking for it in a gel
Phase Three: Activated OxyR binds to pOxyS promoter, starting the transcription of an RNA aptamer
- Tested with phase 4
Phase Four: The RNA aptamer binds to the DFHBI dye, causing it to fluoresce
- Visual examination of fluorescence
Here’s a handy diagram from ChatGPT showing a visual version of what my slightly convoluted system does.
SECTION 2: PROJECT AIMS
Aim 1: Experimental Aim (this project):
The first aim of my final project is to validate a cell-free genetic circuit that detects lactate and outputs fluorescence by utilizing purified Lactate Oxidase, and stock lactate, an OxyR transcription factor, and a PoxyS-driven RNA aptamer/DFHBI reporter system. I will accomplish this by ordering custom DNA constructs from Twist Bioscience, utilizing E. coli extract for my cell-free reactions, and measuring fluorescence.
Aim 2: Development Aim:
The next step following a successful Aim 1 would be to test the system with real sweat which contains a lot more noise and contaminants than a pure lactate input. For this aim I would also like to integrate my cell-free system into a dehydrated paper-based patch that would in turn be installed into a reusable wearable interface, either a watch or an arm band. This would involve engineering a semi-permeable membrane to continuously draw in sweat while preventing any leakage, and creating a miniature readout system that can fit in a wearable form factor.
Aim 3: Visionary Aim:
The visionary aim for this project is to create a new class of continuous, non-invasive biosensors that use biology in tandem with electronics. This technology could expand beyond hydration to monitor glucose, cortisol, or specific disease biomarkers continuously, allowing for easier access to personalized preventative healthcare and athletic performance tracking.
Note: Plan vs. Reality
This project started out with me wanting to detect dehydration. I wanted to detect sodium buildup in sweat but lactate proved to be easier. Lactate is not a direct indicator of dehydration, the same way sodium is, however it is an indicator of sweat which is an indicator of increased risk of dehydration. So, above a certain threshold of fluorescence would indicate increased risk of dehydration… but I built a sweat detector.
Or I would have built a sweat detector, but ordering delays had other plans. So instead, my project took a weird turn that was only slightly successful but extremely interesting. I’ll explain more later, but at the recommendation of an elementary school science experiment, I replaced most of my cell-free system with a potato.
SECTION 3: BACKGROUND
Background and Literature Context
Relevant Citations:
Citation 1 (https://pubmed.ncbi.nlm.nih.gov/26819044/): Gao W, Emaminejad S, Nyein HYY, Challa S, Chen K, Peck A, Fahad HM, Ota H, Shiraki H, Kiriya D, Lien DH, Brooks GA, Davis RW, Javey A. Fully integrated wearable sensor arrays for multiplexed in situ perspiration analysis. Nature. 2016 Jan 28;529(7587):509-514. doi: 10.1038/nature16521. PMID: 26819044; PMCID: PMC4996079.
Citation 2 (https://pmc.ncbi.nlm.nih.gov/articles/PMC6773238/): Baker LB. Physiology of sweat gland function: The roles of sweating and sweat composition in human health. Temperature (Austin). 2019 Jul 17;6(3):211-259. doi: 10.1080/23328940.2019.1632145. PMID: 31608304; PMCID: PMC6773238.
Summary of Literature & Knowledge Gap:
These citations establish both the physiological basis and the engineering precedent for using sweat lactate to monitor physical exertion and hydration. Baker (2019) outlines the complex physiology of eccrine sweat glands, demonstrating that sweat lactate is a primary metabolic byproduct of the gland’s intense activity; because sweat rate and volume directly reflect the body’s hydration status, measuring the onset and concentration of sweat lactate serves as a highly effective biological proxy for dehydration risk. Building on this physiological link, Gao et al. (2016) demonstrate that Lactate Oxidase (LOX) can successfully process sweat lactate in a wearable form factor to continuously monitor physical fatigue. However, their established approach relies entirely on rigid electrochemistry and traditional micro-controllers. My project addresses a distinct gap between the physiological realities of sweat and the limitations of electronic sensors by introducing a fully cell-free, synthetic biology alternative. By converting the LOX reaction into a visual output via a genetic circuit (or, in my revised prototype, a biological catalase reaction), this work pioneers a modular framework that requires no batteries or heavy hardware, moving diagnostics directly into the biological realm.
Novelty and Innovation:
This project represents a novel application of cell-free synthetic biology by using genetic circuits in a wearable device. Traditional synthetic biosensors rely on living cells that must undergo slow protein translation to produce a reporter protein (like GFP). This design was chosen because of its speed and ability to indicate right at the transcription phase. By using an RNA aptamer that immediately binds a fluorogen (DFHBI dye) upon transcription, the system drastically reduces response time. And the combination of an upstream enzymatic reaction (LOX) with a downstream genetic switch (OxyR) creates a safe framework that is highly modular.
Impact and Importance:
The problem this project addresses is the lack of accessible, real-time hydration and metabolic fatigue monitoring. Dehydration and metabolic exhaustion are critical barriers to safety in extreme environments, like glass shops or in sports and elderly care, where symptoms often appear too late for simple interventions. By developing a continuous, non-invasive sensor, this project could significantly improve personalized preventative healthcare. Beyond hydration, establishing a reliable, cell-free platform for wearable diagnostics provides a foundational template that can be adapted for different biomarkers simply by swapping the input enzyme or promoter.
Ethical Implications
The primary ethical principle guiding this project is non-maleficence (do no harm), along with a responsibility to user safety. While a continuous hydration sensor offers immense preventative benefits, relying on a diagnostic wearable introduces the severe risk of false readings. If the genetic circuit degrades and yields a false negative—failing to fluoresce when lactate levels are dangerously high—a user in a high-heat environment might push themselves past their physical limits, leading to severe heat exhaustion or heat stroke. Furthermore, there are important biosafety and human-interface considerations. Although utilizing a cell-free system avoids the biocontamination risks associated with living cells, the device still houses active biological lysates, DFHBI fluorogen dye, and actively generates localized hydrogen peroxide. If the wearable were to rupture or leak during intense physical activity it could cause skin irritation or allergic reactions.
To address these risks, Aim 1 must consist of enough testing to guage how often false positives and negatives occur, and Aim 2 must incorporate robust physical and structural barriers. I plan to use a selective, semi-permeable microfluidic membrane that relies on capillary action to pull sweat into the reaction chamber while restricting backflow onto the skin, it also helps that the system is designed to stay dehydrated until sweat rehydrates it. Despite these safety measures, a potential unintended consequence of this technology is physiological detachment; a user might become overly reliant on the device and begin to ignore their body’s natural thirst cues, stubbornly waiting for a visual signal to drink water. To counter this, the device should be framed as a secondary safety net rather than a primary hydration gauge. Alternatives to these proposed actions include implementing a secondary way of testing for dehydration risk to eliminate chances of errors.
SECTION 4: EXPERIMENTAL DESIGN, TECHNIQUES, TOOLS, AND TECHNOLOGY
Detailed Experimental Plan and Timeline
DNA Prep & Quantification (2 hours): Resuspend the dehydrated Twist Bioscience pTwist Chlor High Copy plasmids containing my DNA in nuclease-free water. Possible test: Quantify concentration using a NanoDrop/Qubit. Expected result: High-yield, pure DNA >50 ng/µL.
Exp 1: LOX Enzymatic Validation (2 hours): React purified Lactate Oxidase with 100 µM Lactate in PBS buffer. Use H2O2 test strips every 5 minutes to check H2O2 output. Expected result: Confirmation that H2O2 production stabilizes between the safe range of 10-100 µM and does not exceed the toxic 1 mM threshold.
Exp 2: OxyR Protein Synthesis Verification (6 hours): Incubate plasmid DNA in the cell-free system for 4 hours at 29°C. Denature and run a gel against a negative control. Expected result: A distinct band at ~34 kDa indicating successful T7-driven OxyR transcription/translation.
Exp 4 & 5: Circuit Bypass & Aptamer Validation (2 hours): Skip the LOX enzyme. Add 50 µM H2O2 directly into a cell-free reaction containing the DNA and DFHBI dye. Photograph for half an hour at 37°C. Expected result: The spiked tube fluoresces within 10 minutes, proving the OxyR to PoxyS to Aptamer circuit functions correctly.
Exp 6: Full System Integration (2 hours): Combine cell-free master mix, DNA, DFHBI dye, and LOX. Trigger with 100 µM Lactate. Track fluorescence over 1 hour. Expected result: A 30-60 minute dark “lag phase” while OxyR is produced, followed by a steady increase in green fluorescence as the full genetic circuit functiond functions.
Data Analysis: Test various amounts of lactate to figure out a quantitative brightness that represents dehydration risk.
Techniques Relevant to Project
Pipetting
Lab Safety
Bioethical Considerations (must check this box)
DNA Construct Design
Gel Electrophoresis (DNA)
Designing a Twist Order
Use of Asimov Kernel
Use of Benchling
Registry of Standard Biological Parts
Quality Control/Analysis (SDS-PAGE Protein Gel)
Cell Free Reactions
Freeze-Dried Cell Free Systems
Technique Expansion
Cell Free Reactions: I will utilize an E. coli extract-based cell-free system wiith with T7 RNA polymerase as the core master mix for my biosensor. Because my circuit relies on a T7 promoter to produce the OxyR switch, but requires native E. coli RNA polymerase to read the downstream PoxyS promoter, a hybrid lysate system is required rather than a pure recombinant system like PURExpress. This cell-free environment provides a bio-safe medium to execute my genetic circuit at human body temperature (37°C).
Designing a Twist Order: To physically acquire the genetic logic gates needed for this project, I designed a custom DNA sequence to be synthesized by Twist Bioscience. I did most of the design in Asimov Kernel using both parts that are readily available and ones found in the IGEM library. In the end, my order did not come in. I believe this is partially because of the complexity of my DNA, in particular, the RNA aptamer had a high number of repeats.
Here’s what my project outputs could have looked like, image credits to ChatGPT
Here’s how they actually ended up looking like, image credits to ChatGPT
Industry Council Companies Associated
Twist Biosciences: For synthesizing the custom DNA construct that produces OxyR and the RNA aptamer.
Asimov Kernel: For designing the DNA for the genetic circuit, it’s a really nice, intuitive platform.
SECTION 5: Results & Quantitative Expectations
Now that I went through and explained everything that my project was supposed to involve, I can explain how it evolved from a high-tech cell-free genetic circuit to a potato-based biosensor, and why I spent most of Monday night running up and down the stairs outside of the biolab.
As mentioned, my Twist DNA order was delayed indefinitely (likely because the RNA aptamer contained a high number of complex repeating sequences that are notoriously hard to synthesize). Without the genetic circuit to turn the H2O2 into fluorescence, I needed an alternative biosensor that I could dose with my Lactate Oxidase (LOX) reaction to prove Phase 1 of my circuit worked.
Ronan helped me scour the lab for any standard chemical alternative. We checked everywhere for potassium iodide, H2O2 test strips, or horseradish peroxidase assays but we found nothing. Ronan made a comment about the bubbling that occurs when H2O2 enters a cut, and I remembered a classic elementary school science experiment using potatoes.
Potatoes are packed with the enzyme catalase. Just like when you pour hydrogen peroxide on a scrape, and it fizzes, the catalase in a potato triggers a rapid redox reaction when exposed to H2O2, breaking it down into water and pure oxygen gas (2H_2O_2 to 2H_2O + O_2). So, I replaced my state-of-the-art, custom-synthesized, cell-free synthetic biology matrix… with a root vegetable.
Validation Selection:
Because the genetic reporter was unavailable, I chose to validate Phase 1 of my circuit: the successful enzymatic conversion of lactate into hydrogen peroxide H2O2 using Lactate Oxidase. By using the potato as my reporter, I could visually validate the presence of H2O2 through the generation of oxygen bubbles!
Detailed Protocol for Validation (The Potato Assay):
Preparation: Cut fresh wedges of a raw Golden Russet potato. To solve my first major challenge—the oxygen gas was escaping too quickly to see at low concentrations—I coated the potato surface with a thin layer of laboratory detergent (glorified expensive dish soap) to trap the escaping oxygen as visible foam.
Controls: Pipette 20 µL of 3% store-bought H2O2 onto one wedge (Positive Control) and 20 µL of 1X PBS onto another (Negative Control).
The Lab Lactate Reaction: Mixed 100 µM stock lactate with LOX in PBS buffer at 37°C. Pipetted 20 µL of this mixture onto a potato wedge after 15 minutes of incubation, and another 20 µL after 30 minutes of incubation.
Sidequest to Collect Sweat: I attempted to collect 10 µL of real human sweat. I tried running outside, but it was too cold. Then I went to the glass-blowing studio, but the ovens were off. Finally, I just ran up and down the stairs outside the biolab 10 times, but then realized that sweat evaporates really quickly. So I tried once more with a minicentrifuge tube in hand and finally got 10ul of sweat but after re-reading the protocol, realized I actually needed 20 µL, and had to run the stairs all over again.
The Sweat Reaction: Mixed my hard-earned 20 µL of sweat with LOX and buffer, incubated for 30 minutes at 37°C, and pipetted it onto a fresh detergent-coated potato wedge.
The Calibration Curve: To understand my results, I performed a serial dilution ofH2O2 (1%, 0.1%, 0.01%, and 0.001%) and dropped them on potatoes to visually map foam volume to concentration.
Synthetic Biology Techniques Utilized:
This validation relied on Enzymatic Reactions (using purified LOX), alternative Biological Assays (utilizing native plant catalase as a reporter), and Pipetting and Lab Safety. It also required extreme physical endurance for sweat collection.
Data Presentation and Analysis:
The Lab Lactate Test:
The positive control (3% H2O2) bubbled violently, creating a massive pile of foam, confirming the potato catalase was active. The negative control (PBS) did absolutely nothing. My lab lactate + LOX reaction worked! After 30 minutes of incubation, it produced a distinct, visible layer of bubbles. The 15-minute incubation produced fewer bubbles, proving that the enzymatic conversion of lactate to H2O2 was functioning correctly over time. I believe these H2O2 concentrations could be hazardous to the enzymes and cell machinery, so I’ll have to be careful about what concentration of lactate and LOX to use, denaturing can occur at as low as 0.005%.
The Calibration and Sweat Test:
My serial dilution test revealed that 1% and 0.1% H2O2 created highly visible foam, but 0.01% was very faint, and 0.001% was entirely invisible to the naked eye. However, out of sheer desperation, I held the 0.001% potato wedge up to my ear and realized I could hear it fizzing. (I did not expect to be potato whispering for this project)
When I tested my real sweat + LOX reaction, there were zero visible bubbles. I suspect the lactate concentration in my sweat simply generated an H2O2 concentration closer to the 0.001% range—too low to see the trapped oxygen, though I did hold the potato to my ear to listen for the reaction, it was inconclusive and demeaning. Upon further inspection, I noticed the sweat was heavily contaminated with hair oil, which could have also interfered with the enzyme kinetics.
Challenges and Problem Solving:
This project was a lesson on pivoting. Challenge one: My DNA didn’t arrive. Strategy: I pivoted to a biological catalase assay (a potato, I ran my tests on a potato). Challenge two: The biological H2O2 production was too low to create visible bubbles. Strategy: I added detergent to physically trap the oxygen as foam (I can’t take credit for this one, it’s often used for this experiment). Challenge three: Sweat collection was incredibly difficult, prone to evaporation, and full of contaminants (hair oil). Strategy: I exercised until I had enough volume to overcome evaporation, though the contaminants remained. Ultimately, I learned a vital engineering limitation: to make the sweat reaction visibly bubble on a potato, I would need to massively amp up the LOX concentration. However, in my actual wearable device design, raising the H2O2 concentration that high would bleach the DFHBI dye and denature the cell-free enzymes, destroying the genetic circuit. The potato proved that while my LOX enzyme works, biology requires highly sensitive genetic reporters (like my missing RNA aptamer) because observation with the naked eye just isn’t sensitive enough for micromolar concentrations. I believe this concentration issue can be addressed in the formulation of my cell-free mix. The fluorescence would be much more noticeable, especially with a digital readout from a photodiode. Also, less LOX means a slower rate of H2O2 production. I can also use a more aggressive buffer.
SECTION 6: ADDITIONAL INFORMATION
12. References
[1] Gao W, Emaminejad S, Nyein HYY, Challa S, Chen K, Peck A, Fahad HM, Ota H, Shiraki H, Kiriya D, Lien DH, Brooks GA, Davis RW, Javey A. Fully integrated wearable sensor arrays for multiplexed in situ perspiration analysis. Nature. 2016 Jan 28;529(7587):509-514. doi: 10.1038/nature16521. PMID: 26819044; PMCID: PMC4996079.
[2] Baker LB. Physiology of sweat gland function: The roles of sweating and sweat composition in human health. Temperature (Austin). 2019 Jul 17;6(3):211-259. doi: 10.1080/23328940.2019.1632145. PMID: 31608304; PMCID: PMC6773238.
Validation of a cell-free synthetic biology circuit designed to approximate hydration risk by detecting lactate. The system utilizes an E. coli lysate-based cell-free matrix supplemented with T7 RNA polymerase.
Dual-Temp Incubators / Heat Blocks (29°C and 37°C)
Electrophoresis Chamber & Power Supply
Blue Light Transilluminator (~470 nm) with amber filter
Digital Camera / Smartphone (mounted)
Image Analysis Software (e.g., ImageJ/Fiji)
Part 2: Experimental Design
Experiment 1: LOX to Hydrogen Peroxide Conversion
Objective: Confirm LOX enzyme activity and ensure hydrogen peroxide production remains between the safe activation threshold (10–100 µM) and the system-toxic threshold (>1 mM).
Prepare a buffered solution (PBS or Tris, pH ~7.4).
Add purified Lactate Oxidase (LOX).
Spike in 100 µM of Lactate.
Dip semi-quantitative hydrogen peroxide test strips into the solution at 5-minute intervals.
Compare the color change against the provided chart to estimate concentration over time.
Experiment 2: OxyR Protein Synthesis Verification
Objective: Verify that the T7 promoter is successfully driving the transcription and translation of the OxyR protein (~34 kDa) before relying on it as a genetic switch.
Prepare two 5 µL cell-free reactions:
Tube A (Negative Control): Cell-free extract + Water (No DNA).
Tube B (Test): Cell-free extract + OxyR plasmid DNA.
Incubate both tubes at 29°C for 3–4 hours.
Mix with Laemmli sample buffer and boil at 95°C for 5 minutes to denature.
Run samples on a 4-20% gradient SDS-PAGE gel alongside a protein ladder.
Stain with Coomassie Blue and look for a distinct, thick band at ~34 kDa in Tube B.
Experiment 3: Direct OxyR-Peroxide Binding
Note: Direct observation of binding requires SPR or similar techniques and is omitted in favor of downstream functional validation in Experiments 4 & 5.
Experiments 4 & 5: The Genetic Switch & Aptamer Validation
Objective: Isolate and validate the core genetic logic (Hydrogen Peroxide $\rightarrow$ OxyR $\rightarrow$ PoxyS $\rightarrow$ Aptamer $\rightarrow$ Dye) by bypassing the LOX/Lactate conversion step.
Prepare two 1.5 mL microcentrifuge tubes containing cell-free extract, plasmid DNA, and DFHBI dye.
Tube A (Baseline): No trigger added.
Tube B (Triggered): Spike in a final concentration of 50 µM hydrogen peroxide.
Incubate both tubes in a heat block at 37°C.
At intervals (T=0, 30m, 1h, 2h, 4h), place the tubes on a blue light transilluminator in a dark room.
Capture locked-exposure photos and return tubes to the heat block.
Use ImageJ to extract Mean Gray Value data from the photos to plot fluorescence over time. Tube B should exhibit significant fluorescence compared to Tube A.
Experiment 6: Full System Integration (The Wearable Simulation)
Objective: Verify that the entire cascade, from the initial biological proxy (lactate) to the final visual output, functions cohesively within a single matrix.
In a single 1.5 mL tube, combine cell-free extract, plasmid DNA, DFHBI dye, and purified LOX enzyme.
Add 100 µM of Lactate to trigger the system.
Incubate in a heat block at 37°C.
Capture locked-exposure photos on the blue light transilluminator at intervals (T=0, 30m, 1h, 2h, 3h, 4h).
Extract fluorescence data. Expect a “lag phase” during the first hour while LOX builds up hydrogen peroxide, followed by a steady increase in fluorescence.
Part 3: Experiment Protocols and Results
General Preparations Before Starting
DFHBI Dye Working Stock: Create a 200 µM working stock by diluting 1 µL of the commercial 10 mM stock into 49 µL of Nuclease-Free Water.
Hydrogen Peroxide Dilution: Perform serial dilutions of stock H2O2 in water immediately before use to create a 1 mM working stock. Keep wrapped in foil on ice.
Experiment 1: LOX to Hydrogen Peroxide Conversion
Detailed Protocol:
Tools/Reagents: 1.5 mL tubes, 37°C heat block, semi-quantitative peroxide test strips, 1X PBS buffer (pH 7.4), L-Lactate (100 mM stock), Lactate Oxidase (~1 U/µL).
Prepare the Matrix: In a 1.5 mL tube, add 980 µL of 1X PBS.
Trigger the Reaction: Add 10 µL of 100 mM Lactate (final concentration 1 mM). Mix gently.
Incubate & Measure: Place the tube in the 37°C heat block.
Time-points: At T=0, 5, 10, 15, 20, and 30 minutes, dip a fresh peroxide test strip into the solution.
Analysis: Compare the strip pad to the color chart to estimate concentration over time.
Experiment 1: LOX to Hydrogen Peroxide Conversion
Objective: Confirm that the Lactate Oxidase (LOX) enzyme successfully converts the Lithium Lactate substrate into Hydrogen Peroxide ($H_2O_2$) and calibrate the production rate to ensure it stays within the functional window (10–100 µM).
Phase Zero: Reagent Preparation
Before starting the experiment, you must reconstitute your dry reagents into liquid stocks.
Add 1 µL of cold 1X PBS for every 1 Unit of enzyme (e.g., add 100 µL PBS for a 100 U vial).
Do not vortex. Gently pipette up and down to dissolve.
Aliquot into 5–10 µL volumes and store at -20°C to avoid freeze-thaw cycles.
Detailed Protocol
Tools: 1.5 mL microcentrifuge tubes, P1000 & P20 micropipettes, 37°C heat block, semi-quantitative peroxide test strips.
Matrix: 1X PBS (pH 7.4).
Prepare the Reaction: In a clean 1.5 mL tube, combine:
980 µL 1X PBS buffer.
10 µL LOX Stock (1 U/µL).
The Trigger: Add 10 µL of 100 mM Lithium Lactate Stock (Final reaction concentration: 1 mM Lactate).
Incubation: Invert the tube 3 times to mix and place immediately in the 37°C heat block. Start a timer.
Data Collection: At T=0, 5, 10, 15, 20, and 30 minutes:
Dip a fresh peroxide test strip into the tube for 1 second.
Wait 15–30 seconds for color development (refer to strip manufacturer’s instructions).
Compare the strip to the colorimetric chart and record the concentration.
Results & Observations
(Record the color changes and corresponding $H_2O_2$ concentrations here. Note the time at which the reaction hits the 100 µM threshold.)
Time (min)
Color Observation
Estimated [$H_2O_2$] (µM)
0
5
10
15
20
30
Conclusion:(Summarize if the enzyme activity is sufficient and if the production rate allows for a safe 4-hour cell-free reaction window.)
Results & Observations:
(Describe how the procedure went, any unexpected issues, and visual observations here)
Conclusion:
(Summarize the findings and whether the target concentration was safely achieved)
Experiment 1: LOX Enzymatic Validation (The Potato Catalase Assay)
Objective: Qualitatively confirm that the Lactate Oxidase (LOX) enzyme is active and successfully converting the Lithium Lactate substrate into Hydrogen Peroxide ($H_2O_2$). This is achieved by utilizing the naturally occurring catalase enzyme found in raw potatoes to visually detect $H_2O_2$ via the generation of oxygen bubbles.
Procedure: Keep vial on ice. Add 1 µL of cold 1X PBS for every 1 Unit of enzyme listed on the vial. Gently pipette to dissolve (do not vortex). Aliquot into 5–10 µL volumes and store at -20°C.
3. The Sensor & Controls Preparation
The Sensor: Wash and cut a fresh, raw potato. (Do not cook it; heat denatures the catalase). Cut thick slices to expose fresh cellular surface area.
Positive Control: Standard store-bought 3% Hydrogen Peroxide.
Step 1: Establishing the Controls (Baseline Validation)
Positive Control: Using a pipette or dropper, place a single drop (~20 µL) of store-bought 3% $H_2O_2$ onto a fresh potato slice.
Expected Result: Immediate, vigorous white foaming and bubbling. This confirms the potato catalase is highly active.
Negative Control: Place a single drop (~20 µL) of 1X PBS buffer onto a different section of the potato.
Expected Result: No reaction. The liquid should sit flat. This confirms your buffer does not cause false positives.
Step 2: The LOX Reaction
In a clean 1.5 mL tube, combine:
80 µL of 1X PBS buffer.
10 µL of 100 mM Lithium Lactate Stock.
10 µL of LOX Stock (1 U/µL).
Invert the tube gently to mix and place it in the 37°C heat block.
Incubate for 30 minutes. (Because the potato assay is less sensitive than chemical test strips, we must give the LOX enzyme time to build up a larger concentration of $H_2O_2$).
Step 3: The Sensor Test
After 30 minutes, remove the reaction tube from the heat block.
Cut a completely fresh slice of potato to ensure the surface is moist and the cells are unoxidized.
Pipette 20 µL of your incubated LOX/Lactate mixture directly onto the fresh potato slice.
Observe closely for 1 to 3 minutes.
Results & Observations
(Record your visual observations below. Note that because the biological reaction produces micromolar concentrations of $H_2O_2$, the bubbling will be much finer and slower than the 3% commercial control).
Condition
Visual Observation (Foaming / Bubbling)
Conclusion
Positive Control (3% $H_2O_2$)
(e.g., Immediate violent foaming)
Catalase is active.
Negative Control (PBS)
(e.g., No reaction, flat liquid)
No background interference.
LOX Reaction (T=30 min)
(e.g., Slow, fine bubbles forming at the edges)
LOX is actively producing $H_2O_2$.
Conclusion:(Summarize the qualitative success of the enzyme. Did the reaction bubble as expected? How did it compare to the positive control?)
Expression: Incubate both tubes at 29°C for 4 hours.
Sample Prep: Remove 5 µL from each reaction. Add 5 µL of 4X Laemmli Buffer and 10 µL of Nuclease-Free Water to each.
Denaturation: Boil the samples at 95°C for 5 minutes.
Gel Loading & Run: Load 15 µL of Tube A, Tube B, and a Protein Ladder into the gel. Run at 150V for ~45 minutes.
Stain: Wash and stain with Coomassie Brilliant Blue. Look for a ~34 kDa band in Tube B.
Results & Observations:
(Describe the gel running process, staining clarity, and any observed banding)
Conclusion:
(Confirm if the OxyR band was successfully identified)
Experiment 3: Direct OxyR-Peroxide Binding
Results & Observations:(This step was bypassed in favor of functional testing in Experiments 4 and 5, as direct measurement requires Surface Plasmon Resonance (SPR) equipment.)
Experiments 4 & 5: The Bypass Test (Genetic Switch & Aptamer)
Detailed Protocol:
Tools/Reagents: 1.5 mL tubes, 37°C heat block, Blue light transilluminator, Camera, Cell-Free Master Mix, Plasmid DNA, DFHBI working stock (200 µM), H2O2 working stock (1 mM).
The Trigger: Add 0.4 µL of 100 mM Lactate (yielding ~3.3 mM final concentration).
Baseline Capture: Mix gently, snap T=0 photo on transilluminator.
Incubation: Place tube in 37°C heat block.
Time-points: Capture photos at T=30m, 1h, 2h, 3h, and 4h.
Analysis: Observe the lag phase followed by rising fluorescence. Extract data via ImageJ.
Results & Observations:
(Detail the observed lag phase, the ramp-up in fluorescence, and overall system performance)
Final Conclusion:
(Summarize the viability of the entire cell-free wearable circuit based on the experimental data)
Group Final Project
Group Brainstorm on Bacteriophage Engineering
Computational Engineering of the MS2 Lysis Protein (L)
Background. The MS2 L protein is a 75-amino-acid polypeptide that lyses E. coli by an incompletely understood mechanism. Its C-terminal transmembrane (TM) domain inserts into the cytoplasmic membrane and oligomerizes, causing depolarization that triggers host autolytic enzymes to degrade the murein layer. Recessive, conservative missense mutations clustered around a conserved LS dipeptide strongly implies L engages an unidentified host protein target rather than simply disrupting the bilayer. The dispensable N-terminal domain binds chaperone DnaJ (with solved PDB structures), modulating lysis timing. Its removal causes lysis ~20 min earlier. No experimental structure of L exists.
Goals. (1) Stabilize L for more robust membrane accumulation. (2) Accelerate lysis by bypassing DnaJ-dependent regulatory timing and improving delivery of functional L to the membrane. Because the downstream lytic target is unknown, we do not attempt to enhance per-molecule toxicity at the point of target engagement; we focus on removing regulatory brakes and increasing the supply of functional protein.
Pipeline: Three Tools, Each Non-Redundant
Clustal Omega (Conservation Map). Align L homologs across Leviviridae (MS2, f2, R17, GA, PP7, AP205, PRR1, M12, KU1, JP34). Conserved C-terminal residues, especially the LS motif, are presumed to mediate the unknown heterotypic interaction and are excluded from mutation. This map constrains all downstream design.
ESM2 + Deep Combinatorial Scanning (Fitness Oracle). Score every single-point mutation by log-likelihood change: increases at mutable positions indicate stabilizing substitutions (Goal 1). N-terminal scanning identifies mutations that disrupt DnaJ binding (Goal 2). A strict preservation rule applies near the LS motif: mutations are evaluated for maintenance of wild-type fitness, not improvement. The genetics show even conservative changes there cause recessive loss of function. Pairwise combinatorial scanning (about ~2M pairs) captures epistatic synergies at mutable positions. This could be potentially pushed further with enough compute.
AlphaFold 3 (Structural Filter + Complex Model). Predicts variant structures as a sanity check (does the TM helix survive?) and models the L–DnaJ complex to verify that N-terminal truncations/mutations disrupt the regulatory interface. Used as a filter, not a design engine. PAE matrix identifies confident interface contacts.
Ranking. Composite score: ESM2 log-likelihood gain (stability) + conservation preservation (all essential residues intact) + AF3-predicted DnaJ-binding disruption (for timing bypass). Top 10–20 variants advance to experimental validation.
Why Not More Tools? ProteinMPNN is excluded because it is trained on crystallized globular PDB proteins, not predicted structures of disordered membrane peptides. The compute is invested in combinatorial ESM2 depth.
Pitfalls
No experimental structure: All structural reasoning rests on AF3 predictions for a challenging target; mitigated by treating AF3 as a filter and cross-referencing against the conservation map.
Unknown lytic target: The central limitation. We cannot optimize target-binding affinity for an unidentified partner; engineering is restricted to upstream properties (stability, membrane delivery, DnaJ bypass).
Autolysin bottleneck: If lysis rate is limited by host autolytic enzyme activity rather than L accumulation, stabilization gains may show diminishing returns; the plaque assay will reveal this.
