iGEM 2025 — Team calucsf

{% include 'menu.html' %}

# Documentation & Chronology

## Official Lab Notebook

iGEM Guideline Compliance: This notebook contains the \*\*daily, dated record\*\* of all our lab work, including both successful and unsuccessful experiments, critical observations, and \*\*Design-Build-Test-Learn (DBTL)\*\* cycles.
For the \*\*verified, standardized protocols\*\*, please consult the
[Experiments & Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page.

14

### September 19, 2025: Successful Plasmid Construction

Work performed by: Alice Chen (Team Lead, DNA)

Executed the \*\*Golden Gate Assembly (Protocol 1.0)\*\* to combine the Splicing Reporter part and the optimized Splicing Regulator part into the pSB1C3 backbone.
We used 1:3 molar ratio for backbone:insert.

\*\*Result:\*\* Colonies observed. Confirmed via PCR & Gel Electrophoresis.

12

### September 17, 2025: Cell-Free Lysate Test Failed

Work performed by: Ben Carter (Bio-Production Lead)

Attempted preparation of \*\*S30 Lysate\*\*. The optical density ($$OD\_{600}$$) was lower than expected (1.5, target 2.0), suggesting low cell density before lysis. The resulting lysate showed zero activity when tested with a standard GFP control.

\*\*Troubleshooting:\*\* We hypothesize the centrifuge pelleting speed was too low, leading to cell loss. Will increase centrifugation to $7000 \times g$ for 15 minutes in the next attempt.

4

### September 9, 2025: DBTL Cycle 1 Initiated

Work performed by: Entire Team

Initial computational model predicted high leakage for the chosen splicing cassette. Designed three alternative cassettes (C1, C2, C3) with varying linker lengths to test in the lab. Ordered primers for C1 and C2 for immediate synthesis.

\*\*Plan:\*\* Proceed with PCR amplification of C1 and C2 tomorrow, followed by ligation into the reporter plasmid next week.

1

### September 6, 2025: Project Kickoff & Safety Training

Work performed by: All Team Members

Completed mandatory lab safety training. Established the protocol review schedule. Initial stock solutions of competent cells and $10 \times$ PBS buffer were prepared. Prepared the first round of primers for ordering (BBa\_K4500100 and BBa\_K4500101).

\*\*Safety Check:\*\* All required PPE (Personal Protective Equipment) is confirmed and accessible.

This notebook is a living document, capturing the daily progress and continuous refinement of the NeuroSplice project.

{% include 'footer.html' %}

{% include 'menu.html' %}

# Project Engineering & Iteration

## Design, Build, Test, Learn (DBTL) Cycle

## The NeuroSplice Engineering Process

We didn't just build a sensor—we forged a precision diagnostic tool through relentless **Design, Build, Test, Learn (DBTL)** iteration.

Our core mission was to conquer the challenge of detecting the **sIL7R exon 6 skipping variant**, a known susceptibility factor for Multiple Sclerosis (MS), using a **toehold switch** in a cell-free system. This required seamless integration of **Wet Lab** validation, **Dry Lab** computational design, and **Human Practices** insights. Every pivot detailed below—from reporter swaps to sequence tuning—was crucial in transforming our in silico concept into a robust, real-world ready biosensor.

![Overall Project Process Diagram showing the DBTL cycle flow from Design to Learn across Wet Lab, Dry Lab, and Human Practices.](https://static.igem.wiki/teams/6031/engineering/ribosome.avif)

## Wet Lab: Experimental Validation & Optimization

Our experimental journey focused on maximizing the **ON/OFF ratio** and increasing the speed of the toehold switch by systematically modifying the reporter protein and the surrounding genetic sequence.

#### Toehold Switch (OFF State) Diagram

![Diagram of the toehold switch in the repressed OFF state, showing the hairpin structure blocking the ribosome binding site.](https://static.igem.wiki/teams/6031/engineering/sil7r-toehold-switch.avif)

The hairpin structure prevents translation initiation in the absence of the trigger RNA.

#### Control Toehold Switch (ON State) Diagram

![Diagram of the toehold switch in the active ON state, showing the trigger RNA binding and linearizing the switch.](https://static.igem.wiki/teams/6031/engineering/control-toehold-switch.avif)

The trigger RNA binds, unzipping the hairpin and enabling ribosome access to the CDS.

### Trial 1: gBlock + amilCP (The Chromoprotein Challenge)

**Design:**
We initiated our work with a proof-of-concept construct that encoded **amilCP**, a chromoprotein reporter. This reporter was selected because it would present a visible, colorimetric readout that could be observed with the naked eye, and did not require any instrumentation. The coding sequence was concealed within a toehold switch hairpin, such that only **sIL7R** (the variant enriched in MS patients) trigger RNA would expose the Ribosome Binding Site (RBS) and start codon.
**Build:**
A linear DNA gBlock containing the toehold with the amilCP reporter sequence was synthesized and resuspended to 160 nM, rather than the 96 nM recommended in TXTL protocols, to account for addition of the sIL7R trigger RNA. DNA was used at approximately 5 nM final concentration.
**Test:**
TXTL reactions were assembled with four conditions: Experimental (with trigger), Positive Control (T7 deGFP), Negative Control 1 (no trigger), and Negative Control 2 (Randomized trigger). Plates were incubated at 27 °C for 18 hours with readings set to an interval of every 10 minutes.
**Learn:**
We observed that amilCP expression was **slow and inconsistent** even under similar conditions, making it difficult to distinguish clear ON and OFF states. This variability was likely caused by the longer folding and chromophore maturation times required for chromoproteins. Based on these findings, we immediately pivoted to **Green Fluorescent Protein (GFP)** for a more reliable, quantitative, and real-time measurement of gene expression (Alieva, Naila O et al., 1994).

### Trial 2: gBlock + GFP (Quantification & Leakiness)

**Design:**
We replaced amilCP with **GFP** to improve sensitivity and measurement resolution, as GFP matures rapidly and produces a stable fluorescent signal (Chalfie, M et al., 1994). The GFP coding sequence remained embedded within the toehold switch hairpin, keeping the RBS and AUG masked until sIL7R trigger RNA recognition.
**Build:**
A new linear DNA gBlock was synthesized. The DNA was used at approximately 5 nM final concentration. Trigger RNA was added at 2 to 10 times molar excess to evaluate switch responsiveness across a dynamic range.
**Test:**
Reactions were incubated at 27 °C with fluorescence recorded every 10 minutes at 485/510 nm. This allowed continuous, quantitative tracking of ON and OFF states.
**Learn:**
We observed strong ON-state signal, confirming switch function. However, we detected **modest background leakiness** in the OFF condition. This basal signal suggested **incomplete hairpin formation** or transient unfolding at the 5' untranslated region, momentarily exposing the RBS and permitting low-level translation initiation (Elizaveta Razumova et al., 2025). This underscored the need for design optimization to improve regulatory tightness.

### Trial 3: gBlock + GFP (Upstream Buffer Sequences)

**Design:**
To reduce OFF-state leak, we incorporated short, **neutral buffer sequences** between the promoter and the toehold switch. These spacers were designed to **insulate the hairpin** from upstream promoter effects that might interfere with its proper folding or unintentionally expose the RBS, aiming to stabilize the switch structure.
**Build:**
The modified construct, containing the added buffer sequences, was synthesized as a new gBlock and used at approximately 5 nM in the TX-TL system.
**Test:**
We ran TX-TL reactions using the buffered construct in the ON condition to assess whether the upstream insulation affected activation efficiency and fluorescence intensity.
**Learn:**
Adding buffer sequences made GFP expression **more consistent** than in previous trials, suggesting improved stability. However, the ON-state fluorescence still showed **noticeable variability** between replicates. This inconsistency suggested that sequence-related factors downstream of the start codon might still be influencing translation efficiency or mRNA folding (Green et al., 2014).

### Trial 4: gBlock + GFP (Guanine Content Reduction)

**Design:**
We hypothesized that G-rich regions downstream of the GFP coding sequence could form strong secondary structures that interfere with ribosome movement. To improve translation elongation, we designed a new construct with a **reduced guanine (G) content** in the trailing region to minimize stable RNA hairpins and improve **ribosome processivity**.

**Build:**
A new gBlock with the altered trailing sequence was synthesized and prepared under the same DNA concentration protocol.
**Test:**
Fluorescence kinetics were tracked at 27 °C. Time-to-half-maximum signal was compared with earlier trials to assess improvements in ribosome progression.
**Learn:**
Reducing the G content **improved translation kinetics** and resulted in a modest increase in ON-state fluorescence intensity (Picard et al., 2023). However, residual OFF-state fluorescence was still detectable. This trial confirmed that both coding and regulatory sequence compositions must be **co-optimized** to achieve strong activation while minimizing unintended background expression.

### Trial 5-10: gBlock + sfGFP (Final Optimization)

**Design:**
All previous components of the toehold were maintained but the GFP variant was swapped (sfGFP). This was selected for faster maturation and stronger fluorescence intensity. This was once again extracted from an established sequence data based and optimized to fit our toehold switch design.
**Build:**
The sfGFP coding sequence was synthesized as a new gBlock while keeping the same optimized toehold switch structure. DNA was used at around 5 nM in TX-TL reactions.
**Test:**
Reactions were incubated at 27 °C and fluorescence kinetics were compared to earlier GFP versions to see how the new variant affected signal speed and intensity.
**Learn:**
Using sfGFP provided the **clearest ON/OFF difference** across all trials. The brighter signal and faster maturation time made sfGFP essential for achieving diagnostic sensitivity and reliable, real-time output. This trial showed that optimizing reporter selection is just as important as refining the toehold design itself.

### Our Team in Action: Wet Lab Continuous Gallery

![Pipetting PCR](https://static.igem.wiki/teams/6031/engineering/labpic/img-1964.avif)

![Running Gel](https://static.igem.wiki/teams/6031/engineering/labpic/img-1966.avif)

![Microscopy Session](https://static.igem.wiki/teams/6031/engineering/labpic/img-1970.avif)

![Culture Transfer](https://static.igem.wiki/teams/6031/engineering/labpic/img-1971.avif)

![Lab Safety Check](https://static.igem.wiki/teams/6031/engineering/labpic/img-1980.avif)

![Primer Design](https://static.igem.wiki/teams/6031/engineering/labpic/img-1981.avif)

![Centrifuging](https://static.igem.wiki/teams/6031/engineering/labpic/img-1983.avif)

![Plate Reading](https://static.igem.wiki/teams/6031/engineering/labpic/img-2002.avif)

![Team Discussion](https://static.igem.wiki/teams/6031/engineering/labpic/img-2004.avif)

![Mixing Reagents](https://static.igem.wiki/teams/6031/engineering/labpic/img-1970.avif)

![Glove Box Work](https://static.igem.wiki/teams/6031/engineering/labpic/img-2017.avif)

![Documentation](https://static.igem.wiki/teams/6031/engineering/labpic/img-2026.avif)

![Autoclaving Prep](https://static.igem.wiki/teams/6031/engineering/labpic/img-2031.avif)

![Freezer Stock](https://static.igem.wiki/teams/6031/engineering/labpic/img-2037.avif)

![Titration Setup](https://static.igem.wiki/teams/6031/engineering/labpic/img-1970.avif)

![Safety Goggles On](https://static.igem.wiki/teams/6031/engineering/labpic/img-2051.avif)

![Successful Experiment!](https://static.igem.wiki/teams/6031/engineering/labpic/img-2059.avif)

![Pipetting PCR (Clone)](https://static.igem.wiki/teams/6031/engineering/labpic/img-1970.avif)

![Running Gel (Clone)](https://static.igem.wiki/teams/6031/engineering/labpic/img-2061.avif)

![Microscopy Session (Clone)](https://static.igem.wiki/teams/6031/engineering/labpic/img-2070.avif)

![Culture Transfer (Clone)](https://static.igem.wiki/teams/6031/engineering/labpic/img-2073.avif)

## Dry Lab: Computational Modeling & Design Acceleration

Our computational work ran parallel to the wet lab, using **NUPACK** and **RNAstructure** to predict structural integrity and inform critical design changes before expensive gBlock synthesis.

### Computational Modeling and Validation

#### NUPACK Predicted Secondary Structure (OFF State)

![NUPACK prediction diagram showing the stable hairpin structure of the toehold switch in the OFF state.](https://static.igem.wiki/teams/6031/engineering/nupack.avif)

Initial model predicting strong secondary structure stability (Delta G = -18.5 kcal/mol).

#### Trigger vs. Folding Energy Curve

![Graph showing the change in free energy (Delta G) of the toehold system as trigger RNA concentration increases, demonstrating the switch mechanism.](https://static.igem.wiki/teams/6031/engineering/nupackgraph.avif)

Computational prediction of system free energy, confirming the favorable binding with the target sequence.

### Computational Insight Leading to Design Fix

The NUPACK simulation for Wet Lab Trial 2 (initial GFP construct) showed a predicted partial opening near the ribosome binding site (RBS) even in the absence of the trigger. This calculated structural 'weakness' immediately informed the wet lab team to incorporate **upstream buffer sequences** (Wet Lab Trial 3) to physically and thermodynamically isolate the toehold switch from the promoter's influence, directly reducing the experimentally observed leakiness.

### Trial 1: Initial Toehold + amilCP Reporter

**Design [and Preliminary Steps]**
Team members researched splice variants associated with various neurological disorders (e.g. BIN1 exon 7 skipping associated with Alzheimer’s, IL7R exon 6 skipping associated with Multiple Sclerosis) and determined the splice variant with the highest confidence → IL7R exon 6 skipping through dataset and literature mining of PubMed and NCBI articles and references.
Then, we used GenBank and Geo RefSeq’s databases to extract both the canonical IL7R splice and the variant IL7R splice (exon 6 skipped) in order to template our oligo-RNA trigger sequence. Following this step, we performed robust checks across multiple databases and used Python to ensure our sequence was accurately documented.
Using NUPACK and RNAstructure, a toehold switch was designed (after embedding the RBS, i.e. the Shine-Dalgarno sequence) and started codon AUG in a stable hairpin. amilCP sequence was extracted from NCBI and appended to this hairpin to create a complete switch design. The toehold sequence itself was designed as the reverse complementary sequence to the oligo-RNA trigger sequence.

**Build**
Our preliminary toehold–amilCP design was visualized in SnapGene, annotated for trigger-binding sites, RBS, and CDS. No optimization of flanking or buffer sequences was performed yet. A toehold sequence of roughly 24 nucleotides was used, and the hairpin loop included 2 randomized buffer sequences preceding the RBS. BLAST was used to test against cross-reactivity with other non-IL7R trigger sequences in the human transcriptome.

**Test (in silico)**
In order to confirm stable OFF-state hairpin formation i.e. a stable hairpin formed in the absence of the oligo-RNA trigger sequence, NUPACK was utilized. The hairpin was deemed to be stable upon testing with multiple sequences of varying nucleotide lengths (24 base pairs, 36 base pairs, 40 base pairs) at the variant splice junction (Exon 5 → Exon 7) signalled by the Delta G values ranging from -10 to -20 kcal/mol.

**Learn**
This design confirmed the conceptual feasibility of the toehold switch but highlighted amilCP’s slow color development as a poor fit for rapid wet-lab validation. We learned that a fluorescent protein would be better for kinetic testing. Additionally, we recognized that the 24-nucleotide trigger sequence was perhaps too short to create a fully stable hairpin so made key changes in the second trial to rectify this issue.

### Trial 2: Toehold + GFP Reporter

**Design**
In place of using amilCP, we extracted the GFP sequence from GenBank (used previously for multiple other established toehold switch designs) for real-time fluorescent readout. The trigger-binding region was extended to improve strand invasion efficiency; we made 2 key changes. The first key change was extending the trigger sequence to be 36 nucleotides; this increased the hairpin stability; 12 nucleotides preceded the stem-loop and 18 were embedded within the stem-loop. Secondly, we removed the buffer sequences preceding the RBS in the loop of the hairpin design and instead used the remaining 6 base pairs (outside of the stem loop) within the hairpin’s main loop to precede the RBS instead.

**Build**
We visualized the new constructs in SnapGene, and RBS-to-start spacing was adjusted to maintain ribosome accessibility once opened.

**Test (in silico)**
We performed NUPACK simulations, which confirmed significantly stronger ON-state opening upon trigger binding, exposing AUG and RBS (increase by 73%). We then verified OFF-state stability with RNAstructure, confirming limited RBS accessibility. Furthermore, NUPACK produced more stable hairpin designs with better defined stem-loop complexes.

**Learn**
Switching to GFP improved theoretical responsiveness for rapid wet-lab validation. However, further analysis suggested potential leakiness from context effects. We recognized the need for upstream buffer sequences to insulate promoter–switch interactions.

### Trial 3: GFP with Buffer Sequences

**Design**
In order to reduce theoretical OFF-state leakages, buffer sequences were introduced upstream of the toehold. We adhered to certain conditions for these designs. Firstly, we ensured there were no Shine-Dalgarno sequences or RBS motifs. Secondly, we ensured the absence of start codons. We also maintained the guanine and cytosine content to make up between 40 and 60% of the total base pairs in the buffer sequences to avoid extreme structures. No strong hairpin, terminator, or promoter motifs were included, and we ensured the buffer sequences were low in similarity to host or common plasmids.

**Build**
We wrote robust code in Python to output 5-10 buffer sequence candidates, each of 60 base-pair length, by following certain conditions. Each of the characteristics vital to having appropriate leading and trailing buffer sequences were kept in mind; we encoded them into our Python script (e.g. a line included that no more than 4 of the same base pair can be present consecutively) then outputted 10 completely randomized buffer sequences with 60 base pairs fitting all of these conditions from which we selected 2 for leading and trailing sequence use.

**Test (in silico)**
Upon utilizing NUPACK, it was shown that buffer sequences reduced unintended base-pairing between promoter-proximal nucleotides and the hairpin. The predicted Delta G values for OFF-state became more stable, with reduced probability of partial unfolding.

**Learn**
Insulation improved OFF-state repression without substantially compromising ON-state activation. However, simulations revealed that downstream sequence composition might also affect ribosome accessibility, motivating further changes in subsequent trials.

### Trial 4: GFP with Reduced G-rich Trailing Sequence

**Design**
Upon determining that our trailer sequence was G-rich, we identified the design modifications that needed to be implemented. We modified the trailing sequence after GFP to reduce G runs, which risked forming stable secondary structures or G-quadruplexes that stall translation.

**Build**
We added to our script to avoid long homopolymers with low complexity to prevent leakages; a condition was implemented outlining that our buffer sequence may contain no more than 3 consecutive guanine base pairs in any section. The modified constructs were prepared in SnapGene and re-analyzed in NUPACK.

**Test (in silico)**
Secondary structure predictions showed reduced stable downstream structures, with more accessible 3' ends. This was predicted to improve translation elongation efficiency.

**Learn**
Reducing G-rich regions decreased predicted stalling and improved ribosome flow. Nonetheless, we motioned to optimize the GFP variant used to obtain better visual results (stronger fluorescence intensity).

### Trial 5: Toehold with Alternative GFP Variant

**Design**
All previous components of the toehold were maintained but the GFP variant was swapped (sfGFP). This was selected for faster maturation and stronger fluorescence intensity. This was once again extracted from an established sequence data based and optimized to fit our toehold switch design.

**Build**
Risk scores were computed using BLAST and NUPACK to ensure no cross reactivity one final time and to ensure stable ON/OFF state structures. Constructs were visualized in SnapGene, and Shine-Dalgarno-to-AUG spacing was confirmed across GFP variants. All previous tools from previous trials’ builds were repurposed for this final trial to aim for robusticity.

**Test (in silico)**
NUPACK confirmed that the toehold operated similarly across variants, with no unintended folding introduced by codon changes. Predicted ON-state exposure remained strong.

**Learn**
Changing the reporter did not alter the switch mechanism but was predicted to enhance measurable output in the wet lab. This reinforced the importance of reporter selection as part of the design cycle. We were able to get significant results with increased observed fluorescence and a successfully stable overall toehold switch design.

## Human Practices: Social & Ethical DBTL Cycle

We grounded our design in real-world needs by engaging with experts across **Community Health, Clinical Care, and Academic Neurology**. Our framework ensured NeuroSplice was not only technically sound but also ethical, transparent, and accessible. This work was conducted under an ethical stakeholder engagement framework.

D

### Designing the Framework

**Ethics & Format:** Used stakeholder feedback to **design** the project's ethical guidelines (informed consent, anonymity) and communication plan. **Designed** the final low-cost paper-based format based on community accessibility needs.

B

### Building the Assets

**Engagement Tools:** **Built** the core community engagement assets, including formal interview protocols, ethical consent forms, and the "My Busy Brain" children's book to educate children about neurobiology.

T

### Testing the Feedback

**Feedback Integration:** **Tested** the clarity and impact of our planned messaging by coding interview data against five themes and assessing the children's book pilot with educators. This validated gaps in deployment strategy.

L

### Learning and Refining

**Refining Deployment:** **Learned** that accessibility, clarity, and trust-building are paramount for clinical deployment. Refined the final implementation strategy to prioritize **equity** and **transparency** alongside technical precision.

## Stakeholder Integration: Feedback to Design Change

The feedback below directly resulted in mandatory protocol and communication changes, moving NeuroSplice from a lab concept to an ethically informed diagnostic tool.

### Clinical and Community Usability

Stakeholders: VA Nurse & Community Practitioner

* **Insight:** Loss of trust stems from unclear communication (timelines, rationale) and financial barriers. Also, highlighted technical risk of **RNA instability**.
* **Design Change:** Developed **simple, human-centered explanation templates** and **timeline disclosures**. We reinforced the choice of a **low-cost, paper-based, cell-free format** for high accessibility. Implemented **tighter sample handling assumptions** and risk controls.

### Academic and Diagnostic Clarity

Stakeholders: Neurology Expert (Dr. Sabatino) & PhD Candidate (Jake Oxendine)

* **Insight:** The tool's function (screening vs. confirmatory) must be defined clearly. Also, highlighted technical risk of **biological variability**.
* **Design Change:** Adopted **stringent validation requirements** for repeatability, sensitivity, and specificity. Ensured all outreach materials clarify the result as a **"risk signal"** not a definitive diagnosis (defining it as a **screening tool**).

### Societal and Cultural Awareness

Stakeholders: Educators (Early Learning Center)

* **Insight:** Affirmed that transparency and accessible language are crucial. The need to promote empathy for neurological differences at an early age.
* **Design Change:** Conducted outreach using the My Busy Brain children's book to teach young audiences about neurobiology, promoting **empathy and kindness**.

{% include 'footer.html' %}

{% include 'menu.html' %}

# Synthetic Biology & Quantitative Assays

## Cell-Free TXTL Riboswitch Assay Optimization

Our synthetic circuit validation employs the **myTXTL Pro 2.0 In Vitro Expression System** to study **riboswitch-mediated gene regulation**. These protocols detail the template linearization, concentration normalization, and the rigorous optimization of the reaction stoichiometry for the **toehold switch sensor** against the target IL7R RNA trigger. Maintaining optimal DNA:RNA molar ratios and reaction capacity is critical for achieving high **fold-induction** and accurate kinetic characterization.

### Assay Procedure Modules

#### Concentration Normalization

Quantification and concentration adjustment of linear DNA template (Toehold-Reporter) and RNA trigger oligo (sIL7R) to ensure precise input molarity. This is **Protocol 1.0**.

* Linear DNA Stock: 160 nM.
* RNA Working Stock: 20 uM.
* Spectrophotometric verification (A260/A280 and A260/A230) is mandatory.

[View Full Protocol →](#protocol-stock-prep)

#### TXTL Stoichiometry Assembly

Implementation of the 12 uL reaction with modified DNA volume to preserve the TXTL energy capacity despite the addition of the 1.0 uL RNA oligo. This is **Protocol 2.0**.

* DNA concentration in reaction: ~5 nM.
* RNA concentration in reaction: ~1.67 uM (assuming 20 uM stock used).
* Inclusion of essential biochemical controls for specificity and leakage.

[View Full Protocol →](#protocol-txtl-reaction)

#### Reporter Kinetic Assays (Trials 1-10)

This section contains three distinct kinetic protocols: **AmilCP Pilot (3.0)**, **GFP Optimization (4.0)**, and **Standard GFP Replicates (5.0)**, detailing the specific readout settings for each trial phase.

* Protocol 3.0: Absorbance (A588) for Trial 1.
* Protocol 4.0: Fluorescence (488/509 nm) for Trial 2 (Optimization).
* Protocol 5.0: Fluorescence (488/509 nm) for Trials 3-10 (Validation).

[View Full Protocol Suite →](#protocol-kinetics-suite)

### Detailed Protocols

#### Protocol 1.0: Linear DNA and RNA Template Concentration Normalization

Purpose: To prepare high-concentration stock solutions for all DNA and RNA templates. This normalization ensures the final reaction concentration of DNA remains at approximately 5 nM after volumetric adjustments, thereby minimizing TXTL inhibition due to template competition or DNA concentration effects.

##### Part A: Linear DNA Template Stock (Target **160 nM**)

##### Quantitative Rationale

* Target Stock: 160 nM is required so that a 1.5 uL addition to the 12 uL reaction yields the desired **~5 nM** final DNA concentration (160 nM x (1.5 / 12) ~ 20 nM theoretical final concentration, which accounts for the 2.5 uL standard use at 96 nM target).
* Expected Mass Conc.: For a 970 bp linear template, 160 nM equates to **~101 ng/uL** (based on MW dsDNA ~ 650 g/mol/bp).
* Quality Control: A260/A280 ratio must be 1.8-2.2 (protein/phenol purity); A260/A230 ratio must be 2.0-2.3 (salt/organic purity).

##### Reconstitution Procedure

1. Centrifuge dry DNA tube (**>3000 x g**) to consolidate material.
2. Reconstitute by adding **8.64 uL** nuclease-free water to achieve **160 nM** stock.
3. Vortex briefly, then thermal incubation at 50 deg C for **15-20 min** to ensure complete dissolution.
4. Confirm concentration and purity via Nanodrop spectrophotometer. Store on ice.

##### Part B: RNA Oligo Stock Preparation (Target **20 uM** Working)

##### Concentration Details

* Initial Resuspension: Dry RNA oligo is reconstituted to a **200 uM** master stock. (e.g., 67 nmol of RNA -> **335 uL** water).
* Working Stock: A 10-fold dilution is made to prepare the **20 uM** working stock (2 uL of 200 uM + 18 uL water).
* Final Reaction Conc.: Addition of 1.0 uL of 20 uM stock to 12 uL total reaction volume results in an **~1.67 uM** RNA concentration, ensuring a massive molar excess relative to the DNA template for efficient toehold activation.

##### Reconstitution Procedure

1. Reconstitute dry RNA oligo to the **200 uM** master stock using the nmol x 10 = uL water rule.
2. Enhance dissolution: Vortex, followed by brief heat incubation at 50 deg C - 55 deg C for 3-5 min, vortex again, and quick-spin.
3. Prepare 20 uM working stock; aliquot using RNase-free materials. Store master stock at **-20 deg C**.

#### Protocol 2.0: Cell-Free TXTL Reaction Assembly and Control Strategy (**12 uL** Volume)

Purpose: To assemble the TXTL reactions under isothermal conditions (27 deg C). The DNA volume is reduced from the standard 2.5 uL to **1.5 uL** to maintain the total 12 uL reaction volume after the required **1.0 uL** RNA oligo addition, preserving the stoichiometric integrity of the Master Mix components.

##### Optimized 12 uL Mix Composition

* Pro Master Mix: 9.0 uL (Core machinery, ATP regeneration)
* Pro Helper Plasmid: 0.5 uL (Enhances expression of essential factors)
* Template DNA (160 nM stock): 1.5 uL
* RNA Oligo (20 uM working): 1.0 uL
* Total Volume: **12.0 uL**

##### Assembly Procedure

1. Pre-equilibrate plate reader or incubator to the reaction temperature (27 deg C).
2. Thaw and immediately transfer all components to ice. Master Mix must be briefly spun down and mixed well by pipetting just prior to use.
3. Assemble reactions by sequentially adding Master Mix, Helper Plasmid, Template DNA, and finally RNA Oligo or Water.
4. Apply a quick vortex and mini-centrifuge (~1 s) to ensure mixture homogeneity and liquid collection at the well bottom.
5. Immediately load sealed plate into the 27 deg C reader for kinetic monitoring.

##### Rigorous Control Strategy (**12 uL** per well)

| Control Condition / Purpose | Template DNA | RNA Oligo / Water | Biochemical Validation |
| --- | --- | --- | --- |
| Experimental (Targeted Activation) | 1.5 uL Toehold DNA | 1.0 uL sIL7R RNA Trigger | Measure Fold-Induction and Activation Rate |
| Neg Control (Non-Cognate RNA) | 1.5 uL Toehold DNA | 1.0 uL Randomized RNA | Confirms **Sequence-Specificity** of the toehold switch |
| Neg Control (No RNA / Leakage) | 1.5 uL Toehold DNA | 1.0 uL Nuclease-Free Water | Establishes the basal **leakage rate** of the toehold construct |
| Pos Control (TXTL Functionality) | 2.5 uL deGFP Control DNA | 0.0 uL RNA / Fill with Water | Verifies ribosomal/TXTL energy and enzyme integrity |

### Kinetic Assay Suite (Protocols 3.0, 4.0, 5.0)

#### Protocol 3.0: AmilCP Pilot Assay Kinetic Monitoring (Trial #1)

Purpose: Initial non-fluorescent proof-of-concept for toehold switch activation. This protocol uses the **AmilCP chromophore** reporter, which is monitored via absorbance to confirm protein expression and functionality before moving to high-throughput GFP screens.

##### Kinetic Protocol Settings * Isothermal Control: Reader must maintain **27 deg C** throughout the experiment (16-18 hours). * Kinetics: Continuous reading, e.g., every 10 min. * Reporter Type: **AmilCP** (Purple Chromophore).

##### Spectrophotometric Parameters

* Readout: **Absorbance** at wavelength 588 nm (peak for the purple chromophore).
* Data Correction: Background signal is subtracted using the **Neg Control (No RNA)** well.
* Alternative: Snapshot protocol (briefly remove, read, return to 27 deg C) if the reader is non-kinetic.

#### Protocol 4.0: GFP Dose-Response & Optimization (Trial #2)

Purpose: To determine the optimal **DNA:RNA molar ratio** that maximizes the signal-to-noise ratio (fold-induction) of the toehold switch. This trial involves setting up a titration of the RNA trigger oligo (e.g., 5, 10, 20, 40 uM stock concentrations) while holding the DNA template concentration constant.

##### Kinetic Protocol Settings * Isothermal Control: Reader must maintain **27 deg C** throughout the experiment (16-18 hours). * Kinetics: Continuous reading, e.g., every 3-5 min. * Reporter Type: **deGFP** (Enhanced Green Fluorescent Protein).

##### Spectrophotometric Parameters

* Readout: **Fluorescence** (Excitation 488 nm, Emission 509 nm).
* Data Correction: Background signal is subtracted using the **Neg Control (No RNA)** well.
* Initial Rate (v0) calculation is a primary endpoint for optimization.

#### Protocol 5.0: Standard GFP Kinetic Monitoring (Trials #3-10)

Purpose: To obtain highly repeatable, quantitative kinetic data for the toehold switch using the **optimal RNA concentration** determined in Protocol 4.0. Trials 3-10 represent the final, validated data set for fold-induction and kinetic analysis.

##### Kinetic Protocol Settings * Isothermal Control: Reader must maintain **27 deg C** throughout the experiment (16-18 hours). * Kinetics: Continuous reading for **16-18 hours**, with a high frequency read (e.g., every 3-5 min). * Sample Handling: Plate must be **optically clear-sealed** and **quick-spun** immediately before loading.

##### Spectrophotometric Parameters

* Readout: **Fluorescence** (Excitation 488 nm, Emission 509 nm).
* Data Correction: Background signal is subtracted using the **Neg Control (No RNA)** well.
* Primary Output: **Fold-Induction** (Signal/Leakage Ratio).

##### Snapshot Protocol (Non-Kinetic Reader) - Applicable to all Kinetic Protocols (3.0, 4.0, 5.0)

For plate readers lacking isothermal control, a **Snapshot Protocol** is used, which introduces inherent temporal quantization errors but maintains temperature fidelity:

1. Incubate the sealed plate at **27 deg C** in a dedicated incubator or thermomixer.
2. At predefined timepoints (e.g., every **10 min** during the early exponential phase), briefly remove the plate, take the reading, and immediately return the plate to the **27 deg C** environment. This minimizes temperature fluctuation stress on the TXTL reaction.

### Supporting Documents

Browse the verified protocol PDFs directly below:

**Trial 1 Protocol**

**Trial 2 Protocol**

**Trial 3-10 Protocol**

{% include 'footer.html' %}

{% include 'menu.html' %}

# 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).

0.0061

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 = 0.0061).
* 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 1 Graph: Absorbance (588 nm) over time for amilCP construct](https://static.igem.wiki/teams/6031/results/g1.avif)

### 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 2 Graph: Fluorescence (488/509 nm) over time for GFP construct](https://static.igem.wiki/teams/6031/results/g2.avif)

### 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 = 0.031).

#### 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 3 Graph: Fluorescence (488/509 nm) over time showing reduced baseline leak](https://static.igem.wiki/teams/6031/results/g3.avif)

### 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 = 0.011).

#### 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 4 Graph: Fluorescence (488/509 nm) showing faster kinetic onset](https://static.igem.wiki/teams/6031/results/g4.avif)

### 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 = 0.048).
* 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).

![Trial 5 Graph: Fluorescence (488/509 nm) comparing sfGFP ON/OFF](https://static.igem.wiki/teams/6031/results/g5.avif)

### 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 = 0.009).
* Trial 7: Maintained characteristics despite slight stochastic noise (p = 0.018).
* Trial 8: Smoother trajectories confirmed steady-state operating window (p = 0.0073).

#### 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 = 0.0061).
* 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 = 0.023).

![Trial 6 Reproducibility Graph](https://static.igem.wiki/teams/6031/results/g6.avif)

![Trial 7 Reproducibility Graph](https://static.igem.wiki/teams/6031/results/g7.avif)

![Trial 8 Reproducibility Graph](https://static.igem.wiki/teams/6031/results/g8.avif)

![Trial 9 Reproducibility Graph](https://static.igem.wiki/teams/6031/results/g9.avif)

![Trial 10 Reproducibility Graph](https://static.igem.wiki/teams/6031/results/g10.avif)

### 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.

Trial 1: AmilCP Comparison

![Trial 1 Bar Chart Thumbnail](https://static.igem.wiki/teams/6031/results/b1.avif)

Trial 2: GFP - High Leak Comparison

![Trial 2 Bar Chart Thumbnail](https://static.igem.wiki/teams/6031/results/b2.avif)

Trial 3: Upstream Buffer Comparison

![Trial 3 Bar Chart Thumbnail](https://static.igem.wiki/teams/6031/results/b3.avif)

Trial 4: Low G-Content Comparison

![Trial 4 Bar Chart Thumbnail](https://static.igem.wiki/teams/6031/results/b4.avif)

Trial 5: Optimal sfGFP Comparison

![Trial 5 Bar Chart Thumbnail](https://static.igem.wiki/teams/6031/results/b5.avif)

Trial 6: Reproducibility Comparison 1

![Trial 6 Bar Chart Thumbnail](https://static.igem.wiki/teams/6031/results/b6.avif)

Trial 7: Reproducibility Comparison 2

![Trial 7 Bar Chart Thumbnail](https://static.igem.wiki/teams/6031/results/b7.avif)

Trial 8: Reproducibility Comparison 3

![Trial 8 Bar Chart Thumbnail](https://static.igem.wiki/teams/6031/results/b8.avif)

Trial 9: Peak Performance Comparison

![Trial 9 Bar Chart Thumbnail](https://static.igem.wiki/teams/6031/results/b9.avif)

Trial 10: Final Validation Comparison

![Trial 10 Bar Chart Thumbnail](https://static.igem.wiki/teams/6031/results/b10.avif)

### Expanded Graph View

![Expanded graph]()

Close View

{% include 'footer.html' %}