Individual Final Project

Experimental Protocol: Cell-Free Hydration Wearable Sensor

Project Overview:

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.

Circuit Logic:

Lactate $\rightarrow$ Lactate Oxidase $\rightarrow$ Hydrogen Peroxide $\rightarrow$ OxyR (T7-driven) $\rightarrow$ PoxyS Promoter $\rightarrow$ RNA Aptamer $\rightarrow$ DFHBI Dye (Fluorescence)

Part 1: Bill of Materials & Equipment

1.1 Reagents & Biologicals

1.2 Consumables

1.3 Hardware

• Micropipettes
• Benchtop Centrifuge (10,000 x g)
• Spectrophotometer (NanoDrop or Qubit)
• 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).

1. Prepare a buffered solution (PBS or Tris, pH ~7.4).
2. Add purified Lactate Oxidase (LOX).
3. Spike in 100 µM of Lactate.
4. Dip semi-quantitative hydrogen peroxide test strips into the solution at 5-minute intervals.
5. 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.

1. 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.
2. Incubate both tubes at 29°C for 3–4 hours.
3. Mix with Laemmli sample buffer and boil at 95°C for 5 minutes to denature.
4. Run samples on a 4-20% gradient SDS-PAGE gel alongside a protein ladder.
5. 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.

1. 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.
2. Incubate both tubes in a heat block at 37°C.
3. At intervals (T=0, 30m, 1h, 2h, 4h), place the tubes on a blue light transilluminator in a dark room.
4. Capture locked-exposure photos and return tubes to the heat block.
5. 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.

1. In a single 1.5 mL tube, combine cell-free extract, plasmid DNA, DFHBI dye, and purified LOX enzyme.
2. Add 100 µM of Lactate to trigger the system.
3. Incubate in a heat block at 37°C.
4. Capture locked-exposure photos on the blue light transilluminator at intervals (T=0, 30m, 1h, 2h, 3h, 4h).
5. 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).

1. Prepare the Matrix: In a 1.5 mL tube, add 980 µL of 1X PBS.
2. Add Enzyme: Add 10 µL of LOX (final concentration ~0.01 U/µL).
3. Trigger the Reaction: Add 10 µL of 100 mM Lactate (final concentration 1 mM). Mix gently.
4. Incubate & Measure: Place the tube in the 37°C heat block.
5. Time-points: At T=0, 5, 10, 15, 20, and 30 minutes, dip a fresh peroxide test strip into the solution.
6. 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.

1. 100 mM Lithium Lactate Stock

• Product: Thermo Fisher L14500.06 (MW: 96.01 g/mol).
• Procedure:
  1. Weigh out 96 mg of Lithium Lactate powder.
  2. Dissolve in 10 mL of Nuclease-Free Water or 1X PBS.
  3. Vortex until clear.
  4. Store at 4°C.

2. 1 U/µL Lactate Oxidase (LOX) Stock

• Product: Sigma Aldrich L9795 (Lyophilized powder).
• Procedure:
  1. Keep the vial on ice.
  2. Refer to the vial label for the total Units (U).
  3. Add 1 µL of cold 1X PBS for every 1 Unit of enzyme (e.g., add 100 µL PBS for a 100 U vial).
  4. Do not vortex. Gently pipette up and down to dissolve.
  5. 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).

1. Prepare the Reaction: In a clean 1.5 mL tube, combine:
   • 980 µL 1X PBS buffer.
   • 10 µL LOX Stock (1 U/µL).
2. The Trigger: Add 10 µL of 100 mM Lithium Lactate Stock (Final reaction concentration: 1 mM Lactate).
3. Incubation: Invert the tube 3 times to mix and place immediately in the 37°C heat block. Start a timer.
4. 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.)

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.

Phase Zero: Reagent Preparation

1. 100 mM Lithium Lactate Stock

• Product: Thermo Fisher L14500.06 (MW: 96.01 g/mol).
• Procedure: Weigh out 96 mg of powder. Dissolve in 10 mL of 1X PBS or Nuclease-Free Water. Vortex until clear and store at 4°C.

2. 1 U/µL Lactate Oxidase (LOX) Stock

• Product: Sigma Aldrich L9795 (Lyophilized powder).
• 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.

Detailed Protocol

• Tools: 1.5 mL microcentrifuge tubes, P1000 & P20 micropipettes, 37°C heat block, scalpel/knife.
• Matrix: 1X PBS (pH 7.4).

Step 1: Establishing the Controls (Baseline Validation)

1. 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.
2. 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

1. 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).
2. Invert the tube gently to mix and place it in the 37°C heat block.
3. 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

1. After 30 minutes, remove the reaction tube from the heat block.
2. Cut a completely fresh slice of potato to ensure the surface is moist and the cells are unoxidized.
3. Pipette 20 µL of your incubated LOX/Lactate mixture directly onto the fresh potato slice.
4. 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).

Conclusion: (Summarize the qualitative success of the enzyme. Did the reaction bubble as expected? How did it compare to the positive control?)

Experiment 2: OxyR Protein Synthesis Verification

Detailed Protocol:

• Tools/Reagents: 1.5 mL tubes, 29°C & 95°C heat blocks, electrophoresis chamber, Cell-Free Master Mix, Plasmid DNA (100 ng/µL), Laemmli Buffer, 4-20% SDS-PAGE gel, Coomassie stain.

1. Set Up Reactions: Prepare two 12 µL reactions on ice:
   • Tube A (Negative Control): 9 µL Master Mix + 3 µL Nuclease-Free Water.
   • Tube B (Test): 9 µL Master Mix + 3 µL Plasmid DNA.
2. Expression: Incubate both tubes at 29°C for 4 hours.
3. Sample Prep: Remove 5 µL from each reaction. Add 5 µL of 4X Laemmli Buffer and 10 µL of Nuclease-Free Water to each.
4. Denaturation: Boil the samples at 95°C for 5 minutes.
5. Gel Loading & Run: Load 15 µL of Tube A, Tube B, and a Protein Ladder into the gel. Run at 150V for ~45 minutes.
6. 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).

1. Create Master Mix (for 2.5 reactions on ice): Combine 22.5 µL Master Mix, 5 µL Plasmid DNA, and 1.5 µL DFHBI stock.
2. Split Reactions: Aliquot 11.6 µL of this mix into Tube A and Tube B.
3. The Trigger:
   • To Tube A (Baseline), add 0.6 µL Nuclease-Free Water.
   • To Tube B (Triggered), add 0.6 µL of 1 mM H2O2 (final ~50 µM).
4. Baseline Capture: Place tubes on transilluminator, snap T=0 photo.
5. Incubation: Place tubes in 37°C heat block.
6. Time-points: Capture photos on the transilluminator at T=30m, 1h, 2h, and 4h.
7. Analysis: Extract Mean Gray Value data via ImageJ.

Results & Observations:

(Describe the visual fluorescence changes over time and the ImageJ data extraction process)

Conclusion:

(Discuss the effectiveness of the genetic switch and aptamer activation)

Experiment 6: Full System Integration (Wearable Simulation)

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, LOX enzyme, L-Lactate (100 mM).

1. Assembly: On ice, combine 9 µL Master Mix, 1.5 µL Plasmid DNA, 0.6 µL DFHBI stock, and 0.5 µL LOX enzyme.
2. The Trigger: Add 0.4 µL of 100 mM Lactate (yielding ~3.3 mM final concentration).
3. Baseline Capture: Mix gently, snap T=0 photo on transilluminator.
4. Incubation: Place tube in 37°C heat block.
5. Time-points: Capture photos at T=30m, 1h, 2h, 3h, and 4h.
6. 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)