Validation & Data
Experimental Results
This section presents the quantified data from our cell-free optimization experiments. We used the Design-Build-Test-Learn (DBTL) cycle across 10 trials to optimize the final sfGFP construct, focusing on maximizing the signal-to-noise ratio.
1.0 Key Performance Metrics (Trials 1-10 Summary)
Peak System Fold-Activation
Ratio of Peak sfGFP Output (~70,000 a.u.) / Stable Baseline (~35,000 a.u.).
~2.0x
Signal Induction Ratio
Best Specificity (p-value)
Lowest probability of ON/OFF results being random (Achieved in Trial 9).
1.43 x 10^-111
Statistical Confidence
Optimal Reporter
Best choice for fast folding and high-intensity signal (Established in Trial 5).
sfGFP
Superfolder GFP
2.0 Individual Trial Optimization (DBTL Cycle)
Trial 1: Proof-of-Concept (amilCP Chromoprotein)
Goal and Design
Used the gBlock–amilCP construct to test specific activation by sIL7R RNA. AmilCP was chosen for its simple, colorimetric readout.
- Test: Toehold switch -> AmilCP reporter.
- Protocol: Adjusted DNA prep by resuspending the gBlock at 160 nM to maintain consistent TXTL composition.
Activation Results
- Activation Confirmed: Statistically significant increase in absorbance compared to controls (p = 1.43 x 10^-111).
- High Specificity: Wells with no RNA or non-cognate RNA remained flat, verifying minimal leaky expression.
DBTL Learning
Limitation: AmilCP's slow maturation and non-fluorescent nature hindered precise quantitative and kinetic analysis.
- Decision: Chromoproteins are unsuitable for early-cycle characterization. Must switch to deGFP or sfGFP for faster, quantitative, real-time feedback.

Trial 2: Replacing Chromoprotein with GFP for Kinetic Insight
Design Change & Goal
Replaced AmilCP with GFP to improve sensitivity and temporal resolution. Retained the original toehold hairpin structure.
- Test: Toehold switch -> GFP reporter.
- Goal: Confirm specificity while enabling continuous fluorescence tracking (488/509 nm).
Fluorescence Results
- Signal Output: Experimental well averaged around 400,000 a.u. (ON state).
- High Significance: Activation was highly significant (p = 5.00 x 10^-39).
- Validation: GFP is superior for quantitative, real-time measurement.
DBTL Learning: Leakiness
Observed significant leakiness in the OFF-state wells (~200,000–250,000 a.u.), suggesting low-level GFP expression without trigger RNA.
- Hypothesis: Residual partial unfolding of the hairpin or unanticipated secondary structures allowing ribosome access.
- Decision: Next step must focus on minimizing spontaneous activation by introducing additional upstream stabilizing stems.

Trial 3: Minimizing Leak with Upstream Buffer Sequences
Strategy & Execution: Upstream Insulation
Introduced short, neutral buffer sequences between the promoter and toehold to insulate the hairpin and stabilize the OFF configuration.
- Test: Buffered Toehold -> GFP.
- Goal: Reduce the high OFF-state leakiness observed in Trial 2.
Repression Improvement (Success)
- Leak Reduction: Negative controls reduced to low, stable signals (~25,000–30,000 a.u.). Success in Repression.
- Specificity: Activation remained highly significant (p = 3.06 x 10^-31).
DBTL Learning: ON-State Trade-off
Improved repression but observed lower and inconsistent ON-state intensity (~30,000–35,000 a.u.).
- Hypothesis: Buffer sequence impedes translation efficiency after switch opening.
- Decision: Focus next on optimizing the downstream sequence to enhance translational throughput.

Trial 4: Enhancing Translation by Reducing Downstream G-Content
Kinetic Improvement (Primary Result)
- Faster Onset: Fluorescence curve rose earlier, consistent with smoother ribosomal elongation.
- Modest Yield: Slightly higher mean absorbance (~30,000–35,000 a.u.).
- High Specificity: Maintained (p = 6.55 x 10^-38).
Strategy & Execution
Modified the trailing sequence to lower Guanine (G) content to minimize stable G-quadruplexes that stall translation elongation.
- Test: Buffered Toehold -> Low G-content GFP.
- Conditions: Elevated incubation temperature (31–33°C) used to accelerate kinetics.
DBTL Learning: Structural Limits
Residual OFF-state leak persists (~25,000–30,000 a.u.), suggesting the core toehold stem itself needs better thermodynamic stability.
- Next Step: Co-optimize the stem/hairpin structure and consider a faster reporter protein to achieve sharper ON/OFF contrast.

Trial 5: Maximizing Signal with Superfolder GFP (sfGFP)
Strategy & Execution
Replaced standard GFP with Superfolder GFP (sfGFP), which folds efficiently and matures quickly for stronger output.
- Test: Optimized Toehold -> sfGFP reporter.
- Goal: Improve signal clarity and kinetic responsiveness.
Performance Boost (Clearer Discrimination)
- High Peak Intensity: ON-state reached ~45,000–50,000 a.u.
- Sharper Contrast: Clearer ON/OFF separation than previous trials (p = 7.87 x 10^-25).
- Validation: sfGFP allows more accurate kinetic tracking and sharper discrimination.
DBTL Learning: Finalized Reporter
The sfGFP reporter provides the best balance of speed, brightness, and reliability (OFF-state near ~25,000–30,000 a.u.).
- Decision: Proceed with the sfGFP construct for final validation and reproducibility testing (Trials 6-10).

Trials 6–10: Validation, Reproducibility, and Final Optimization (sfGFP)
Reproducibility Validation (Trials 6-8)
Focused on ensuring the sfGFP construct was stable and reproducible across biological repeats (~45,000–50,000 a.u. ON vs ~20,000–30,000 a.u. OFF).
- Trial 6: Confirmed reliability with near-identical results (p = 5.76 x 10^-44).
- Trial 7: Maintained characteristics despite slight stochastic noise (p = 5.74 x 10^-37).
- Trial 8: Smoother trajectories confirmed steady-state operating window (p = 8.60 x 10^-52).
Peak Performance (Trials 9-10)
Extended testing to confirm long-term stability and quantitative reliability.
- Trial 9 (Best): Achieved the highest statistical significance of all trials (p = 1.98 x 10^-78).
- Trial 10: Robust validation, showing peak signal output (~60,000–70,000 a.u.) and stable baseline (~30,000–35,000 a.u.).
- Trial 10 Specificity: Confirmed high fidelity (p = 7.42 x 10^-34).





3.0 Graph Data Visualization (Comparison Bar Charts)
Experimental and positive control absorbance data were normalized to negative control absorbance values to assess fluorescence fold increase
Click on any graph thumbnail below to view the full-size comparison bar chart.









