iGEM 2025 — Team warsaw

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# Notebook

![ikona 1](https://static.igem.wiki/teams/5865/icons/nb1.webp)

Documents our daily lab workflow step by step, including observations, deviations, and decision points.

![ikona 2](https://static.igem.wiki/teams/5865/icons/nb2.webp)

Chronicles the detailed progress of experiments, trials, and adjustments, in a transparent format.

![ikona 3](https://static.igem.wiki/teams/5865/icons/nb3.webp)

Serves as a living record for other teams and researchers to track, evaluate, and extend our work.

* [OBTAINING PLASMIDS FROM THE DISTRIBUTION KIT](#ch1)
* [OPTIMIZATION OF CELL CULTURE](#ch2)
* [ASSESSING ENDOGENOUS AFP MRNA LEVELS IN CELL LINES](#ch3)
* [ASSEMBLY OF DUAL-REPORTER CONSTRUCT](#ch4)
* [TOEHOLD SWITCH ASSEMBLY](#ch5)
* [FIRST VERIFICATION OF TOEHOLD SWITCHES](#ch6)

## Obtaining plasmids from the Distribution Kit

#### Optimization of Cell Culture

#### Assessing endogenous AFP mRNA levels in cell lines

#### Assembly of dual-reporter construct

#### Toehold switch assembly

#### First verification of Toehold Switches

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![Engineering graphic](https://static.igem.wiki/teams/5865/banners/h-engineering.avif)

# Engineering

INTRODUCTION

CLONING

CHOOSING OUR BESTS

#### Chapter I - Introduction

Our primary goal was to engineer novel toehold switches capable of detecting α-fetoprotein (AFP) mRNA within mammalian cells with expression of our protein of interest.
We achieved this through a single, comprehensive Design-Build-Test-Learn (DBTL) cycle that took us from initial research to a final, curated list of candidates ready for synthesis.

![DBTL inner illustration](https://static.igem.wiki/teams/5865/animations/engineering-wheel-inside1.webp)

DESIGN
BUILD
TEST
LEARN

#### Design: laying the foundation

The **design** phase of our cycle began with foundational learning. A thorough review of existing literature taught us
the critical design principles of toehold switches, especially the nuances of adapting them for eukaryotic systems.
We started our journey on classical paper by Alexander Green from 2014, and analyzed number of papers up to the current year.

This knowledge informed our core design strategy:

* **Target:** α-fetoprotein (AFP) mRNA.
* **Chassis:** mammalian HEK cells.
* **Architecture:** we adopted an innovative structure, inspired by the 2019 iGEM Tel-Aviv team,
  placing the Kozak sequence after the toehold stem. This design promised a more stable off-state and simplified construction
  by removing the need for an N-terminal peptide linker.
* **Tool:** we chose the Triggate web server, as it was specifically developed to support this novel architecture.

#### Build: executing the design

The **build** phase transitioned our design into practice. Initially, this involved using the Triggate server to generate toehold structures.
Our first "test" of this process yielded promising results for simple queries, validating our initial approach.

However, as we moved to more complex and optimized queries, our process failed its test. The server consistently timed out, unable to complete the calculations.
This was a critical roadblock. We were faced with a choice: abandon our innovative design or engineer a solution.

This challenge triggered a rapid, internal "learn-and-rebuild" loop within our main cycle. We **learned** by contacting Triggate's creator,
Yehuda Almog, who confirmed the issue was a server-side timeout, not a fundamental flaw in the algorithm. This insight was pivotal.
We then **built** a new, robust computational pipeline to overcome this limitation:

* **A "divide and conquer" script:** I developed a Python script using **Selenium** to programmatically split our large AFP target
  into smaller, overlapping fragments and submit them as individual jobs.
* **An automated data pipeline:** I created a Google Apps Script to automatically capture the hundreds of results and pipe them into Google Drive,
  followed by a Colaboratory script to parse and organize the data into a master Google Sheet.

This custom-built pipeline effectively solved the server-side timeout issue and allowed our team to generate the comprehensive dataset we needed.

#### Test: validating the pipeline

The **build** phase transitioned our design into practice. Initially, this involved using the Triggate server to generate toehold structures.
Our first "test" of this process yielded promising results for simple queries, validating our initial approach.

However, as we moved to more complex and optimized queries, our process failed its test. The server consistently timed out, unable to complete the calculations.
This was a critical roadblock. We were faced with a choice: abandon our innovative design or engineer a solution.

This challenge triggered a rapid, internal "learn-and-rebuild" loop within our main cycle. We **learned** by contacting Triggate's creator,
Yehuda Almog, who confirmed the issue was a server-side timeout, not a fundamental flaw in the algorithm. This insight was pivotal.
We then **built** a new, robust computational pipeline to overcome this limitation:

* **A "divide and conquer" script:** Python + **Selenium** to split AFP target and submit jobs.
* **Automated pipeline:** Google Apps Script → Drive → Colab → master Sheet.

This custom-built pipeline effectively solved the server-side timeout issue and allowed our team to generate the comprehensive dataset we needed.

#### Learn: analysis and selection

With a comprehensive dataset in hand, we entered the final, crucial Learn phase of our cycle. The goal was to derive actionable knowledge from
the hundreds of candidates our pipeline had generated.

First, we learned from experts, consulting with several toehold switch researchers to establish a rigorous set of selection criteria.
We consulted with several toehold switch researchers to refine our selection criteria.

* **Allen Liu** – to consult the architecture of our toehold we contacted Dr. Liu. He kindly explained our doubts about integration of 5’UTR into our toehold and clarified that as the toehold is fully 5’UTR, additional stimulation is not necessary.
* **Antonis Guakonutis (iGEM Thessaly)** – emphasized the importance of secondary structures of target mRNA and how to counteract them, prompting deeper analysis of predicted toehold structures.
* **Francois Rouleau** – helped us understand how to travel the space of various toehold parameters and compare candidates across them.

Armed with this expert advice, we analyzed our data through a multi-step filtering process:

1. **Functional consistency:** robust predicted structures for both our reporter (GFP) and therapeutic (Gasdermin D) genes.
2. **Specificity:** BLAST against the human transcriptome to eliminate any candidates with potential off-target effects.
3. **Biophysical ranking:** prioritize a **low stem Gibbs free energy (ΔG)** to ensure a stable "off" state and minimize leakiness.

#### Chapter II - Cloning

We engineered two transcription units — **TU1** (toehold–eGFP) for sensing and **TU2** (RFP) as a transfection/control readout — and iterated through short **DBTL** cycles to de-risk cloning and screening. Below is a structured recap of what we planned — built — tested — learned across three iterations.

![DBTL inner illustration](https://static.igem.wiki/teams/5865/animations/engineering-wheel-inside1.webp)

DESIGN
BUILD
TEST
LEARN

#### Iteration I Design: plan with Asimov set

We initially planned to use the **iGEM Parts Registry**, basing our work on the
**Asimov Mammalian Parts Collection** from the **2024** and **2025 Distribution Kits**,
generously shared by the **iGEM JU 2024** team and **Dr. Mateusz Wawro**.
Our goal was to assemble a transcription unit (**TU1**) for each **toehold switch** using
**Modular Cloning (MoClo)**: **CMV promoter (BBa\_J433000)**, the toehold switch sequence as the 5′UTR,
an **eGFP** protein coding sequence lacking a start codon and Kozak sequence (included in the toehold switch),
a **synthetic 3′ UTR (BBa\_J433018)**, and the **rabbit β-globin poly(A) signal (BBa\_J433023)**.

As a control for transfection efficiency and plasmid uptake, we also designed **TU2**,
composed of the **CMV promoter**, strong **5′ UTR (BBa\_J433003)**, a Kozak sequence with **RFP** CDS,
the **3′UTR**, and the **rabbit β-globin poly(A) signal**. Both TUs would later be merged
into a **Level-2 multigene construct** for screening.

#### Iteration I Build: transformations from kits

Dried DNA from the 2024 and 2025 kits was rehydrated in 10 µL of mQ water and 1–2 µL was used
to transform 50 µL of *E. coli* **DH5α** and **DH10β**. Despite correct positive (**pUC control**)
and negative (**mQ**) controls, only a few colonies appeared even after three attempts.

#### Iteration I Test: sequence verification

Mini-prepped colonies were sequenced with primers flanking the inserts. Sequencing revealed that several
plasmids did not match their reported identities, and even clones from the same transformation showed
inconsistent results — including one that showed **bGH (BBa\_J433021)**, a part we had never attempted to extract —
ruling out cross-contamination.

#### Iteration I Learn: confirm & pivot

After consulting **iGEM’s** representative **Cristiane Toledo** and **Asimov’s** representative
**Traci Haddock**, we confirmed our protocol was correct. Additionally, we learned that other teams
(e.g., **Barnacure WCIS iGEM 2025**) had similar issues. Due to time constraints, we abandoned the
**Distribution Kit** strategy and opted to **synthesise redesigned parts** directly, which also
simplified our cloning workflow.

#### Iteration II Design: GG-ready, no scars

We ordered sequences designed identical to the **Asimov collection**, but already assembled, with a
**Golden Gate Assembly** cloning site allowing insertion of 5′UTR and CDS. Because the **toehold switch**
and protein coding sequences cannot tolerate Golden Gate scars (a 4-nt scar would cause a frameshift),
we ordered them as continuous fragments **without scar**. We also ordered an already assembled
**transcription unit 2** with overhangs necessary for assembly; however, the vendor could not synthesise the full
transcription unit due to heavy secondary structure, so we split it into three fragments with 20-nt overlaps
for **Gibson Assembly**.

#### Iteration II Build: assembly attempts

Ordered constructs were transformed and plasmid stocks prepared. We attempted to first assemble **TU2**
as a linear construct and then use it in **Golden Gate Assembly** with **TU1** and a **Level 2 backbone**,
although it was unsuccessful. Then we prepared a linear vector to first clone **TU2** into a plasmid and
then proceed with Golden Gate Assembly, but it also failed.

#### Iteration II Test: no correct clones

Unfortunately, we were unable to obtain any positive colonies, even after several repetitions and
modifying the protocol.

#### Iteration II Learn: narrow scope

With deadlines approaching, we chose to proceed using only **TU1** and interpret results conservatively.
Winning an **Ansa award** for iGEM teams later enabled us to order highly structured sequences like **TU2**
for future work.

#### Iteration III Design: *lacZ* for blue–white

To simplify screening, we inserted a *lacZ* cassette into the replaceable region of the **TU1** plasmid,
allowing **blue–white screening** during insertion of toehold switches. We did not do it initially,
because it would make blue–white screening of colonies during combining **TU1** and **TU2** impossible.

#### Iteration III Build: Golden Gate insertions

We cloned the *lacZ* insert into **TU1** and used **BsaI Golden Gate Assembly** to introduce different
toehold switches with **eGFP**.

#### Iteration III Test: PCR & prep

Correct insertions were confirmed by **colony PCR**, followed by **midipreps** on 100 mL of overnight culture
to obtain **endotoxin-free plasmids** for human cell transfection.

#### Iteration III Learn: ready for transfection

This approach proved efficient and fast. **Sanger sequencing** verified the constructs, and they were
ready for experiments to assess **toehold switch** activity.

#### Chapter III - Choosing our bests

To design novel toehold switches that could serve as targeted therapy for hepatocellular carcinoma, we refined our validation and selection strategy.
After several DBTL loops we converged on a procedure that fits our scope, resources, and timelines.

![DBTL inner illustration](https://static.igem.wiki/teams/5865/animations/engineering-wheel-inside1.webp)

DESIGN
BUILD
TEST
LEARN

#### Iteration I Design: evolution of complexity

The **Design** phase of our cycle began with the simple idea of checking our
**toehold switches** designs in two steps. First, we wanted to do the primary screening process
on a simple and cheap organism, then we wanted to test only those promising ones on
a second, more complex object (just like it's usually done during drug development
research — first tests on mice and the most promising on people).

#### Iteration I Build: choice of screening hosts

It was obvious that the second step would be tested on the target cells — human
hepatocellular cancer (cell line: **HepG2**).

We spent more time on choosing our first screening host. A brief idea was tests on
commonly used bacteria, but due to the different structure of prokaryotic translation
initiation sites we quickly abandoned this solution. Then we turned to the most
simple and commonly used eukaryotic organisms — yeasts. It was supposed to be a
time- and budget-friendly preselection, and only five most promising **toehold** designs
would be checked on the more expensive in culture mammalian cells.

#### Iteration I Test: talk with expert

We consulted our idea with **Magda Konarska**, who worked on the yeast genome. We
learned from her that expression between yeast and mammalian cells can differ due
to other components and complexes involved in the mRNA-binding process in the cytosol.
That could possibly lead to the choice of wrong **toehold** designs.

#### Iteration I Learn: overcomplicated?!

After learning that we could have selected wrong **toehold** designs by mistake while
performing prescreening on yeast, we discussed with **Magda Konarska** different screening
methods. The conclusion of our talks was quite surprising: our selection process
was **overcomplicated** for the scale of our project. An easier, less time-consuming, and
affordable solution was to perform all selection on the **target cells**.

#### Iteration II Design: what about controls?

When we decided on tests on the **HepG2** cell line, we considered different cell lines for
negative control. Our **toehold** designs are capable of detecting *AFP mRNA* within
mammalian cells; after detection the toehold becomes active, so we needed a cell line
where the toehold would stay in the inactive form. One of the most popularly used
human cell lines without α-fetoprotein (**AFP**) expression were **HEK293T**.

We felt conflicted about comparing tests on different cell lines and came up with the
idea of developing a **HEK293** cell line with expression of α-fetoprotein (**AFP**) and
using it as a positive control instead of **HepG2**.

#### Iteration II Build: talk with experts

We discussed the development of the new **HEK293** cell line with **Pawł Sikorski**.
He told us that while it is possible to make cells express α-fetoprotein, establishing a
**stable cell line** with α-fetoprotein expression will be extremely time-consuming.

We also consulted with **Maciej Cieśla** on the idea of using **lentiviruses** or
**plasmids** to make new **HEK293** cells with α-fetoprotein expression. He revealed that
unfortunately this method would be quite resources-consuming. Conclusions from both
experts were that it would be better to use already well-established cell lines. They
also reminded us that the human body is built out of different cell types and
suggested that it might be acceptable to test our **toehold switches** on different cell lines.

#### Iteration II Test: literature search

We decided to check in literature whether people test subjects of their research on
different cell lines and compare the results. We found that it is a **frequent methodology**
used by scientists.

#### Iteration II Learn: back to original

We learned that our original idea — to test our **toehold** designs on different cell lines and
collect results on **flow cytometry** — was well thought-out. We also considered more tests on
other cell lines in the future.

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![Experiments graphic](https://static.igem.wiki/teams/5865/banners/h-experiments.avif)

# Experiments

![ikona 1](https://static.igem.wiki/teams/5865/icons/exp1.webp)

Contains all experimental protocols applied throughout the project.

![ikona 2](https://static.igem.wiki/teams/5865/icons/exp2.webp)

Provides clear and accessible records of every conducted experiment.

![ikona 3](https://static.igem.wiki/teams/5865/icons/exp3.webp)

Offers a comprehensive resource for other researchers to reference and build upon.

* [INTRODUCTION](#ch1)
* [PROTOCOLS](#ch2)
  + [BACTERIAL PROTOCOLS](#ch2-1)
  + [CELL CULTURE PROTOCOLS](#ch2-2)
  + [MOLECULAR BIOLOGY PROTOCOLS](#ch2-3)
  + [NUCLEIC ACIDS PURIFICATION AND QUANTIFICATION PROTOCOLS](#ch2-4)
* [MATERIALS & METHODS](#ch3)
* [SEQUENCES](#ch4)
* [REFERENCES](#ch5)

#### Introduction

The "Experiments" section of our iGEM 2025 project provides a comprehensive overview of the experimental methodologies employed throughout our research. This includes detailed protocols, materials and methods, and experimental designs that formed the foundation of our project. Each protocol is meticulously documented to ensure reproducibility and transparency, featuring complete reagent lists, parameters, and control setups. Wherever applicable, we have included notes on adaptations or optimizations made to standard methods to enhance their effectiveness or suitability for our specific objectives. This section serves as a valuable resource for other researchers aiming to replicate our work or build upon our findings.

#### Protocols

##### Bacterial protocols

14 Minute Transformation Protocol
+

The 14 Minute Transformation Protocol for NEB 5-alpha and 10-beta competent E. coli offers a balance between speed and efficiency,
making it suitable for routine cloning with higher transformation efficiency than ultra-fast protocols. The protocol includes critical steps such
as ice incubation, heat shock, and outgrowth to maximize colony formation. The following protocol results in only 25% efficiency compared
to the High Efficiency Transformation Protocol. For C2987H, perform steps 1-6 in the tube provided.

1. Prepare all required reagents and consumables:
   * NEB 5-alpha or 10-beta competent cells (thawed on ice)
   * Plasmid DNA (1 pg–100 ng in 1–5 μl)
   * SOC or NEB 10-beta/Stable Outgrowth Medium (950 μl, room temperature)
   * Sterile 1.5 ml microtubes
   * Ice and ice bucket
   * Water bath at 42°C
   * 37°C incubator
   * Sterile pipettes and tips
   * LB agar plates with appropriate antibiotic

- Procedure (C2987H/C2987I)
  * For C2987H: Remove cells from -80°C freezer and thaw in your hand. For C2987I: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix
    gently and carefully pipette 50 μl of cells into a transformation tube on ice.
  * Add 1-5 μl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells
    and DNA. **Do not vortex.**
  * Place the mixture on ice for 10 minutes. Do not mix.
  * Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
  * Place on ice for 3 minutes. Do not mix.
  * Pipette 200 μl of room temperature SOC into the mixture. Immediately spread 50-100 μl onto a selection plate and
    incubate overnight at 37-42°C. NOTE: Selection using antibiotics other than ampicillin may require some outgrowth
    before plating on selective media. Colonies develop faster at temperatures above 37°C, however some constructs
    may be unstable at elevated temperatures.

High Efficiency Transformation Protocol
+

The High Efficiency Transformation Protocol by NEB optimizes the uptake of plasmid DNA into E. coli strains 5-alpha and 10-beta,
achieving efficiencies of 1–3 × 109 cfu/μg. It involves heat shock (42°C, 30 seconds) and recovery in specialized media, ideal for cloning
large plasmids or BACs (10-beta).

1. Prepare all required reagents and consumables:
   * NEB 5-alpha or 10-beta competent cells (thawed on ice)
   * Plasmid DNA (1 pg–100 ng in 1–5 μl)
   * SOC or NEB 10-beta/Stable Outgrowth Medium (950 μl, room temperature)
   * Sterile 1.5 ml microtubes
   * Ice and ice bucket
   * Water bath at 42°C
   * 37°C incubator
   * Sterile pipettes and tips
   * LB agar plates with appropriate antibiotic

- Procedure (C2987H/C2987I, for C2987H, perform steps 1-7 in the tube provided)
  * For C2987H: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes. For C2987I: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix
    gently and carefully pipette 50 μl of cells into a transformation tube on ice.
  * Add 1-5 μl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells
    and DNA. **Do not vortex.**
  * Place the mixture on ice for 30 minutes. Do not mix.
  * Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
  * Place on ice for 5 minutes. Do not mix.
  * Pipette 950 μl of room temperature SOC into the mixture.
  * Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
  * Warm selection plates to 37°C.
  * Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
  * Spread 50-100 μl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at
    30°C for 24-36 hours or 25°C for 48 hours.

Colony PCR
+

Colony PCR is a rapid screening method used to verify whether bacterial colonies contain the expected plasmid
insert. Instead of isolating plasmid DNA, bacterial cells from individual colonies are lysed directly and used as
template in a PCR reaction. This approach saves time and allows quick identification of positive clones. It is widely
used after transformations, though positive results should always be confirmed by sequencing.

1. Prepare all required reagents and consumables:
   * Agar plates with selective antibiotic (colonies to be tested)
   * Sterile pipette tips and pipettes
   * Microcentrifuge tubes (1.5mL) and PCR tubes
   * Sterile nuclease-free water
   * PCR Master Mix (commercial 2× mix or individual components: polymerase, buffer, dNTPs)
   * DNA polymerase (RUN, A&A)
   * Buffer for PCR (RUN, A&A)
   * dNTPs
   * Forward and reverse primers specific to the insert/construct
   * Thermocycler
   * Agarose gel electrophoresis setup (gel, DNA ladder, staining solution, power supply, UV or
     blue-light imager)
   * Laboratory safety equipment: gloves, lab coat, goggles
2. Prepare primers and reagents
   * Design primers to amplify the region of interest (insert-specific or vector flanking primers).
   * Prepare the PCR Master Mix according to manufacturer’s instructions.
3. Pick bacterial colonies
   * Touch a single colony with a sterile pipette tip.
   * Resuspend the cells in a small volume of sterile water (100μL) or buffer in a microcentrifuge tube.
4. Lyse the cells
   * Perform cell lysis by heat treatment: 10 min in 95°C
   * Use the resulting lysate as template DNA
5. Set up PCR reactions
   * Aliquot all of the ingredients of PCR into PCR tubes. Use the table below to calculate the volumes.

   | Reagent | Volume |
   | --- | --- |
   | DNA template | 2 μL |
   | Buffer | 2 μL |
   | dNTPs | 0.4 μL |
   | Primer Forward | 1 μL |
   | Primer Reverse | 1 μL |
   | DNA polymerase | 0.2 μL |
   | Distilled water | 13.4 μL |

   * Include a negative control (no template) and a positive control (known plasmid).
6. Run PCR
   * Place the reactions in a thermocycler. Set the program with settings listed below:

   | Step | Temparature | Time | Cycles |
   | --- | --- | --- | --- |
   | Initial denaturation | 95°C | 3 min | 1 |
   | Denaturation | 95°C | 30 s | 35 |
   | Annealing | 55°C | 30 s | 35 |
   | Elongation | 72°C | 60 s | 35 |
   | Final extension | 72°C | 5 min | 1 |
   | Hold | 4°C | ∞ | 1 |

   * Run the cycling program optimized for your polymerase and primers (denaturation, annealing, extension).
7. Analyze products
   * Load PCR products on an agarose gel with a DNA ladder.
   * Compare observed band sizes with the expected amplicon.
   * Positive clones can be confirmed by sequencing.

Competent cell preparation
+

This protocol describes how to prepare chemically competent E. coli cells capable of efficiently taking up
plasmid DNA through heat shock transformation. Using calcium chloride (CaCl2) treatment, the cells become
permeable to DNA, achieving transformation efficiencies typically ranging from 106 to 108 colony-forming
units (cfu) per microgram of DNA, depending on the strain and conditions.

1. Prepare all required reagents and consumables:
   * E. coli strain (e.g., DH5α, BL21)
   * LB or SOB broth (sterile)
   * Ice-cold 0.1M Calcium chloride (CaCl2) solution (sterile)
   * Ice-cold 15% glycerol solution (sterile)
   * Shaking incubator at 37°C
   * Refrigerated centrifuge (4°C)
   * Ice bucket
   * Sterile 50mL and 1.5mL centrifuge tubes
   * Sterile pipettes and tips
2. **Starter Culture**: Inoculate a single colony of E. coli into 2–5mL LB broth. Incubate overnight at 37°C
   with shaking (200–250 rpm).
3. **Main Culture**: Dilute 1mL of the overnight culture into 100mL fresh LB or SOB medium. Grow at
   37°C with shaking until the culture reaches an OD600 of approximately 0.4–0.6 (about 2–3 hours).
4. **Cooling**: Place the culture on ice for 15–30 minutes to chill the cells.
5. **Centrifugation**: Centrifuge the culture at 2500–4000 × g for 10 minutes at 4°C. Carefully discard
   the supernatant.
6. **Calcium Chloride Wash**: Gently resuspend the cell pellet in 25–50mL of ice-cold 0.1M CaCl2
   solution. Incubate on ice for 30 minutes. Centrifuge again under the same conditions and discard
   the supernatant.
7. **Final Resuspension**: Resuspend the pellet in 2–5mL of ice-cold 0.1M CaCl2 containing 15% glycerol.
   Mix gently to avoid shearing the cells.
8. **Aliquoting and Storage**: Dispense the competent cells into sterile microcentrifuge tubes (50–
   200μL per tube). Immediately flash-freeze the aliquots in a -80°C freezer.

##### Cell culture protocols

Cell culture protocol
+

**Introduction**

This protocol provides an optimized and standardized approach for the maintenance of
HEK293T, WI-38, HepG2, and A549 cell lines, ensuring optimal viability and reproducibility.
It covers cell thawing, passaging, transfection and cryopreservation, with tailored guidelines
that incorporate specific modifications to accommodate the unique growth characteristics
and requirements of each cell type.

**Safety note**

This protocol provides an optimized and standardized approach for the maintenance of
HEK293T, WI-38, HepG2, and A549 cell lines, ensuring optimal viability and reproducibility.
It covers cell thawing, passaging, transfection and cryopreservation, with tailored guidelines
that incorporate specific modifications to accommodate the unique growth characteristics
and requirements of each cell type.

**Characteristics of cell lines**

HepG2

HepG2 is a human hepatocellular carcinoma cell line derived from the liver tissue of a
15-year-old male with hepatoblastoma. These cells are widely used as an in vitro model of
human hepatocytes due to their ability to retain many liver-specific metabolic and synthetic functions, including the expression of plasma proteins and certain cytochrome P450
enzymes. Being a tumor-derived cell line, HepG2 cells are immortal and proliferate
indefinitely under standard culture conditions.

HEK293T

HEK293T cells are a derivative of the human embryonic kidney 293 (HEK293T) cell line,
originally established from embryonic kidney tissue transformed with adenovirus 5 DNA. The
“T” variant is stably transfected with the SV40 large T antigen, enabling episomal replication
of plasmids containing the SV40 origin of replication. HEK293T cells are immortalized and
tumorigenic, and they are extensively employed in molecular biology and biotechnology,
particularly for high-efficiency transient transfection and recombinant protein or viral particle
production.

A549

A549 is a human lung carcinoma cell line derived from the alveolar basal epithelial cells of a
58-year-old Caucasian male with adenocarcinoma. These cells exhibit epithelial morphology
and retain many features of alveolar type II pneumocytes, including the ability to synthesize
and secrete pulmonary surfactant components. A549 cells are cancer-derived and therefore
immortal, and they are frequently used as a model for respiratory epithelium, lung cancer
biology, and drug testing.

WI-38

WI-38 is a normal human diploid fibroblast cell line derived from lung tissue of a 3-month-old
female fetus. Unlike the previously mentioned tumor-derived lines, WI-38 cells are
non-tumorigenic and exhibit a finite lifespan in culture, undergoing replicative senescence
after approximately 50 population doublings (the Hayflick limit). WI-38 has been widely used
in research on cellular aging, vaccine production, and normal human cell biology, serving as
a reference for comparison with immortalized or transformed cell lines.

**Thawing cells**

Goal of the experiment

The goal of this experiment is to efficiently and aseptically thaw cryopreserved cells,
restoring their viability and functionality for subsequent culture. This procedure ensures
minimal cellular stress and maximizes post-thaw recovery by rapidly transitioning cells from
cryogenic storage to optimal growth conditions.

1. Prepare all required reagents and consumables:
   * Complete culture medium - DMEM + 10% FBS + 1× P/S + 1× GlutaMAX
   * Cryovial containing frozen cells of appropriate cell line
   * Tissue culture dish Ø 60 mm
   * Pipet controller and sterile serological pipettes (5 mL and 10 mL)
   * Pipet and sterile 1 mL tips
   * 15 mL centrifuge tube
   * Centrifuge compatible with 15 mL tubes
   * Brightfield (inverted) microscope
   * 37°C water bath
   * CO2 incubator set to 37°C and 5% CO2
2. Procedure

   Important: During thawing cells are in a vulnerable state. Before you start prepare all the
   materials, set the pipet for 1 mL, label tissue culture dish, prepare 5 mL of complete culture
   medium in a centrifuge tube and keep the frozen cryovial on dry ice.

   * Preheat complete culture medium in warm water bath to 37°C
   * Put the cryovial containing cells in a warm water bath at 37°C, making sure that the
     lids stay out of the water. It should take no more than 2 min.
   * When only a small piece of frozen cell suspension remains in the cryovial,
     immediately transfer it under sterile conditions to the biosafety cabinet and gently
     pipette the contents into a pre-warmed 15 mL centrifuge tube containing preheated 5
     mL of complete culture medium.
   * Immediately spin the cells down in the centrifuge at appropriate **settings.**

   | Cell line | centrifuge settings |
   | --- | --- |
   | HepG2 | 100 × g, 8 min, RT |
   | HEK293T | 500 × g, 5 min, RT |
   | A549 | 500 × g, 5 min, RT |
   | WI-38 | 100 × g, 8 min, RT |

   * Carefully discard the medium. Resuspend the cells in 4 mL of medium.
   * Transfer the suspended cells onto Ø 60 mm tissue culture dish.
   * Incubate cells in standard conditions: 37°C, 5% CO2.
   * Cells should be passed when they reach approximately 80% confluency (about 1
     day) onto a Ø 100 mm culture dish. For experimental use, cells are recommended to
     be utilized after minimum two passages to ensure phenotypic stability and optimal
     viability.

**Passing cells**

Goal of the experiment

The aim of this experiment is to subculture adherent cell lines under optimal and aseptic
conditions to maintain their viability, phenotypic stability, and experimental reproducibility.
Proper passaging prevents overconfluence, senescence, and phenotypic drift, ensuring
consistent cellular behavior across experiments.

1. Prepare all required reagents and consumables:
   * Complete culture medium - DMEM + 10% FBS + 1× P/S + 1× GlutaMAX
   * Trypsin-EDTA
   * PBS
   * Cell culture with confluence of about 80%
   * Tissue culture dish Ø 100 mm
   * Pipet controller and sterile serological pipettes (5 mL and 10 mL)
   * Pipet and sterile 1 mL tips
   * Brightfield (inverted) microscope
   * 37°C water bath
   * CO2 incubator set to 37°C and 5% CO2
2. Procedure

   * Preheat complete culture medium in warm water bath to 37°C
   * Check confluency of cells using the brightfield microscope. Cells should cover about
     80% of the tissue culture dish.
   * Carefully pipette out the old growth medium from the corner of the dish.
   * Gently wash the cells with an appropriate amount of PBS by pouring it onto the walls.
     Swirl it around and pipette it out from the corner of the dish.
   * Add the appropriate amount of Trypsin-EDTA, it should cover the surface. Incubate
     for a maximum of 10 min in standard conditions. Check dissociation every 2 mins
     under the brightfield microscope.
   * After all cells dissociate, add a complete culture medium to neutralize the
     Trypsin-EDTA. Suspend the cells.
   * Optional: Prior to plating for the experiment, cells should be centrifuged to eliminate
     residual trypsin-containing medium.
   * Determine the amount or ratio of cells that should be added to the new dish. For
     standard passing every 2 or 3 days ratio is listed below.

   | Cell line | centrifuge settings | ratio |
   | --- | --- | --- |
   | HepG2 | 100 × g, 8 min, RT | 1:3 – 1:4 |
   | HEK293T | 500 × g, 5 min, RT | 1:4 – 1:6 |
   | A549 | 500 × g, 5 min, RT | 1:4 – 1:5 |
   | WI-38 | 100 × g, 8 min, RT | 1:2 – 1:3 |

   | Reagent | 100 mm | 60 mm | 6-well | 24-well | 96-well |
   | --- | --- | --- | --- | --- | --- |
   | Volume of medium in which the culture is carried out [mL] | 10 | 4 | 2 | 0.4 | 0.1 |
   | Volume of PBS used to wash the cells [mL] | 11 | 5 | 3 | 0.5 | 0.15 |
   | The volume of Trypsin-EDTA used to detach the cells [mL] | 1.5 | 0.5 | 0.25 | 100 | 0.05 |

**Cryopreservation**

Goal of the experiment

The goal of this experiment is to cryopreserve viable and functionally stable cell stocks of
HEK293T, THLE-2, HepatoXcell, and HepG2 lines for long-term storage. By freezing cells at
optimal confluency in appropriate cryoprotective media under controlled-rate cooling
conditions, this protocol ensures minimal cellular damage and maximizes post-thaw recovery
and reproducibility in future experiments.

1. Prepare all required reagents and consumables:
   * Complete culture medium - DMEM + 10% FBS + 1× P/S + 1× GlutaMAX
   * Freezing medium - 90% complete DMEM + 10% DMSO
   * Trypsin-EDTA
   * PBS
   * Cryovials
   * Freezing container
   * Cell culture with confluence of about 80%
   * Pipet controller and sterile serological pipettes (5 mL and 10 mL)
   * Pipet and sterile 1 mL tips
   * 15 mL centrifuge tube
   * Brightfield (inverted) microscope
   * 37°C water bath
   * CO2 incubator set to 37°C and 5% CO2
2. Procedure

   Important: During cryopreservation, after suspending cells in freezing medium, cells are in a
   vulnerable state. Before you start prepare all the materials, set the pipet for 1 mL, label
   cryovials, prepare freezing medium and freezing container. Work fast and immediately after
   loading the freezing container transfer it into the -80°C freezer.

   * Preheat complete culture medium in a warm water bath to 37°C, prepare appropriate
     amount and type of freezing medium (90% complete DMEM + 10% DMSO).
   * Check confluency of cells using the brightfield microscope. Cells should cover about
     80% of the tissue culture dish.
   * Carefully pipette out the old growth medium from the corner of the dish.
   * Gently wash the cells with an appropriate amount of PBS by pouring it onto the walls.
     Swirl it around and pipette it out from the corner of the dish.
   * Add the appropriate amount of Trypsin-EDTA, it should cover the surface. Incubate
     for a maximum of 10 min in standard conditions. Check dissociation every 2 mins
     under the brightfield microscope.
   * After all cells dissociate, add a complete culture medium to neutralize the
     Trypsin-EDTA. Suspend the cells.
   * Transfer cells to the centrifuge tube and spin the cells down in the centrifuge at
     appropriate settings. Discard the supernatant.
   * Resuspend the cells in freezing medium and immediately distribute 1 mL of the
     suspension into each cryovial.

   | Cell line | centrifuge settings |
   | --- | --- |
   | HepG2 | 100 × g, 8 min, RT |
   | HEK293T | 500 × g, 5 min, RT |
   | A549 | 500 × g, 5 min, RT |
   | WI-38 | 100 × g, 8 min, RT |

**Transfection**

Introduction

LipofectamineTM 3000 (Thermo Fisher) is a lipid-based transfection reagent optimized for
efficient delivery of plasmid DNA into mammalian cells, including hard-to-transfect lines.
Together with the P3000TM enhancer, it promotes complex formation between DNA and
cationic lipids, resulting in high transfection efficiency and low cytotoxicity. Complexes are
prepared in serum-free medium (Opti-MEMTM) and directly added to adherent cells in culture
without the need to replace the medium after transfection.
This protocol describes how to prepare DNA–lipid complexes and transfect adherent cells in
24-well plates. Conditions can be scaled up or down depending on the culture vessel.

1. Prepare all required reagents and consumables:
   * Cells and culture
     + Mammalian cells for transfection
     + Complete growth medium appropriate for the chosen cell line
     + 24-well tissue culture plates (or other format)
     + PBS (without Ca2+/Mg2+)
     + Trypsin or dissociation reagent
   * Transfection reagents
     + LipofectamineTM 3000 (Thermo Fisher)
     + P3000TM reagent (Thermo Fisher)
     + Opti-MEMTM Reduced Serum Medium
     + Plasmid DNA to be transfected (purified, endotoxin-free)
     + Plasmid DNA to be transfected (purified, endotoxin-free)
   * + Sterile 1.5 mL microcentrifuge tubes
     + Filtered sterile pipette tips (P10, P200, P1000)
     + Micropipettes (P10, P200, P1000)
     + Biological safety cabinet (laminar flow hood)
     + CO2 incubator (37°C, humidified atmosphere)
     + Vortex mixer and benchtop centrifuge
     + Inverted microscope for cell morphology check
     + Personal protective equipment (lab coat, gloves, goggles)
2. Procedure

   Note: Seed cells one day prior to transfection so they reach ~70–90 % confluency on
   the day of transfection.

   * Prepare DNA solution and Lipofectamine 3000 solution
     + Use the amounts listed in the table below to prepare both solutions.
     + Mix gently by pipetting.

   |  | Solution 1 | | | Solution 2 | |
   | --- | --- | --- | --- | --- | --- |
   | Reagent | Opti-MEM | P3000TM | DNA | Opti-MEM | LipofectamineTM 3000 |
   | Volume per 1 well (24-well plate) | 25 uL | 1 uL | 500 ng | 25 uL | 1,5 uL |

   Form DNA-lipid complexes
   + Combine the DNA/P3000TM solution with the LipofectamineTM 3000 solution.
   + Mix gently by pipetting up and down.
   + Incubate at room temperature for 10–15 min to allow complex formation.
   Add complexes to cells
   + Add the entire portion of complexes dropwise to each well containing cells in
     complete growth medium.
   + Gently rock the plate to evenly distribute the complexes.
   Incubation
   + Return the cells to the CO2 incubator (37°C, humidified, 5 % CO2).
   + Do not change the medium; complexes remain with the cells throughout the
     incubation.
   Post-transfection analysis
   + After 24–48 h, assess transfection efficiency (e.g. via reporter gene
     expression or downstream assay).
   + Monitor cell morphology and viability using a microscope or flow cytometry.

Flow cytometry cells preparation
+

This protocol describes the preparation of adherent HepG2 (hepatocellular carcinoma) and HEK (human embryonic kidney) cells cultured
in 24-well plates for flow cytometry analysis.

1. Prepare all required reagents and consumables:
   * Adherent HepG2 or HEK cell cultures in 24-well plates (70-90% confluent)
   * Phosphate-buffered saline (PBS), calcium and magnesium-free, sterile
   * 0.25% trypsin-EDTA solution (pre-warmed to 37°C)
   * Complete growth medium containing serum (pre-warmed to 37°C)
   * PBS buffer (cold, 4°C)
   * PBS buffer (room temperature)
   * 15 mL conical centrifuge tubes
   * Hemocytometer or automated cell counter

- Procedure (C2987H/C2987I)
  * Pre-Protocol preparation
    + Pre-warm 0.25% trypsin-EDTA solution and complete growth medium to 37°C
    + Keep PBS buffer cold at 4°C
    + Label conical centrifuge tubes for each sample
    + Ensure cells are 70-90% confluent before harvesting
  * Cell washing
    + Remove culture medium from each well by gentle aspiration
    + Rinse wells with 500 μL sterile, room temperature PBS to remove residual serum
  * Enzymatic Detachment
    + Add 200 μL of pre-warmed 0.25% trypsin-EDTA solution per well
    + Incubate plates at 37°C for 5 - 8 minutes
      - Monitor cell detachment under inverted microscope
      - Gently tap or rock the plate if needed to facilitate detachment
  * Trypsin Neutralization and Cell Collection
    + Add 500-800 μL complete growth medium containing serum to each well to neutralize trypsin
    + Pipette cell suspension up and down 3-5 times to ensure single-cell suspension
    + Transfer cell suspension from each well into labeled 15 mL conical centrifuge tubes
  * Cell Washing and Preparation
    + Centrifuge tubes at 500 × g for 5 minutes at room temperature
    + Carefully discard supernatant without disturbing cell pellet
    + Resuspend pellet in 2 mL cold PBS buffer
    + Centrifuge again under the same conditions
    + Discard supernatant and resuspend pellet in 1 mL cold PBS buffer
  * Cell Count
    + Perform cell count using hemocytometer with trypan blue
    + Assess cell viability (should be >90% for optimal results)
  * Final Preparation for Flow Cytometry
    + Keep cells on ice (4°C) until analysis
    + Use cells for flow cytometry within 2-4 hours of preparation for best results

##### Molecular biology protocols

Golden Gate Assembly - BsaI
+

Based on Golden Gate Assembly Protocol for BsaI-HF v2 (NEB R3733)

1. Prepare all required reagents and consumables:
   * T4 DNA Ligase (NEB #M0202)
   * BsaI-HFv2 (NEB #R3733)
   * plasmid with backbone
   * NEB 10-beta competent E. coli

- Procedure
  * Set up 25μL assembly reactions as follows:

  | Reagent | Assembly reagents |
  | --- | --- |
  | backbone vector | 75 ng |
  | 1-3 inserts | 2:1 molar ratio to backbone |
  | T4 DNA Ligase Buffer (10X) | 2.5 μl |
  | T4 DNA Ligase (NEB #M0202), 2000 U/μl | 0.5 μl (1000 units) |
  | T4 DNA Ligase (NEB #M0202), 2000 U/μl | 1.5 μl (30 units) |
  | Nuclease-free H2O | to 25 μl |

  * Mix gently by pipetting up and down 4 times.
  * Briefly microcentrifuge (1 sec.) to bring material to the
    bottom of tube.
  * Transfer to thermocycler and program as follows: (5 min
    37°C → 5 min 16°C) x 30 cycles followed by 5 min 60°C. If
    reactions are done overnight, add a 4°C terminal hold to
    the protocol, but repeat the final 5 min 60°C step the next
    day before the transformations.

Golden Gate Assembly - SapI
+

Based on Golden Gate Assembly Protocol for SapI (NEB #R0569)

1. Prepare all required reagents and consumables:
   * T4 DNA Ligase (NEB #M0202)
   * SapI (NEB #R0569)
   * Destination Plasmid
   * NEB 10-beta Competent E. coli

- Procedure
  * Set up 20μL assembly reaction as follows:

  | Reagent | Assembly reagents |
  | --- | --- |
  | Destination vector | 3 nM (final concentration) |
  | Amplicon inserts | 3 nM each amplicon (final concentration) |
  | SapI (NEB #R0569), 10 U/μl | 1.5 μl (15 units) |
  | T4 DNA Ligase (NEB #M0202), 2000 U/μl | 0.25 μl (500 units) |
  | T4 DNA Ligase Buffer (NEB #B0202) (10X) | 2 μl |
  | Nuclease-free H2O (NEB #B1500) | to 20 μl |

  * (37°C, 5 min → 16°C, 5 min) x 30 → 60°C, 5 min → 4°C.
  * Cool reaction to 4°C prior to transformation, or store
    completed assembly reactions at -20°C.

T4 DNA Ligase Protocol
+

T4 DNA ligase is a crucial ATP-dependent enzyme utilized in molecular biology for joining DNA fragments. This
enzyme was isolated from bacteriophage T4 and catalyzes the formation of phosphodiester bonds between adjacent
3'-OH and 5'-phosphate termini in double-stranded DNA.

1. Prepare all required reagents and consumables:
   * NEB T4 DNA Ligase
     + T4 DNA Ligase
     + 10X T4 DNA Ligase Reaction Buffer
   * Linearized DNA vector
   * DNA insert
   * RNase free water

- Procedure
  * Set up the following reaction in a microcentrifuge tube on ice. Note: T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3
    vector to insert for the indicated DNA sizes.

  | Reagent | Assembly reagents | Comments |
  | --- | --- | --- |
  | Component | 20 μl reaction |  |
  | T4 DNA Ligase Buffer (10X)\* | 2 μl | \* The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature. |
  | Vector DNA (4 kb) | 50 ng (0.020 pmol) |  |
  | Insert DNA (1 kb) | 37.5 ng (0.060 pmol) |  |
  | Nuclease-free water | to 20 μl |  |
  | T4 DNA Ligase | 1 μl |  |

  * Gently mix the reaction by pipetting up and down and microfuge briefly.
  * For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
  * For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours
    (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation)
  * Heat inactivate at 65°C for 10 minutes.
  * Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.

HiFi assembly
+

HiFi assembly protocol is designed for multiple fragments seamless
assembly. Based on:
[NEB HiFi DNA Assembly reaction protocol](https://www.neb.com/en/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol).

1. Prepare all required reagents and consumables:
   * Proper vector
   * Proper inserts
   * NEBuilder HiFi DNA Assembly Cloning Kit

- Procedure
  * Set up reaction on ice:

  | Reagent | Assembly reagents | Comments |
  | --- | --- | --- |
  | Recommended DNA Molar Ratio | vector:insert = 1:2 |  |
  | Total Amount of Fragments | X μl (0.03–0.2 pmols) | Optimized cloning efficiency is 50–100 ng of vector with 2-fold excess of each insert. Use 5-fold molar excess of any insert(s) less than 200 bp. Total volume of unpurified PCR fragments in the assembly reaction should not exceed 20%. To achieve optimal assembly efficiency, design 15-20 bp overlap regions between each fragment. |
  | NEBuilder HiFi DNA Assembly Master Mix | 10 μl |  |
  | Nuclease-free Water | 10-X μl |  |
  | Total Volume | 20 μl |  |

  * Incubate samples in a thermocycler at 50°C for 15 minutes.
  * Following incubation, store samples on ice or at –20°C for
    subsequent transformation.
  * Transform to proper bacterial strains (e.g. NEB 10-beta
    competent E. coli)

Restriction digestion - BsaI
+

Digestion for insert verification

1. Prepare all required reagents and consumables:
   * Plasmid DNA (500ng–1μg per reaction)
   * BsaI-HF®v2 (NEB R3733)
   * 10X rCutSmartTM Buffer
   * mQ water

- Procedure
  * Set up reaction on ice:

  | Components | 50 μl reaction |
  | --- | --- |
  | DNA | 1 μg |
  | 10X rCutSmartTM Buffer | 5 μl |
  | BsaI-HF®v2 (NEB R3733) | 1 μl |  |
  | Nuclease-free Water | to 50 μl |  |

  * Gently mix the reaction by pipetting up and down and
    microfuge briefly.
  * Incubate at 37°C for 1 hour.
  * Denaturate for 20 minutes at 65°C

Restriction digestion - SapI
+

Digestion for insert verification

1. Prepare all required reagents and consumables:
   * Plasmid DNA (500ng–1μg per reaction)
   * BsaI-HF®v2 (NEB R3733)
   * 10X rCutSmartTM Buffer
   * mQ water

- Procedure
  * Set up reaction on ice:

  | Components | 50 μl reaction |
  | --- | --- |
  | DNA | 1 μg |
  | 10X rCutSmartTM Buffer | 5 μl |
  | SapI | 1 μl |  |
  | Nuclease-free Water | to 50 μl |  |

  * Gently mix the reaction by pipetting up and down and
    microfuge briefly.
  * Incubate at 37°C for 1 hour.
  * Denaturate for 20 minutes at 65°C

IVT HighScribe
+

The HiScribe T7 High Yield RNA Synthesis Kit (NEB) is designed for efficient in vitro transcription (IVT) of RNA from
a DNA template containing a T7 promoter. This kit enables the synthesis of high yields of RNA in a short reaction
time, and is commonly used in applications such as mRNA production, probe synthesis, or synthetic biology
constructs. The kit typically includes T7 RNA polymerase, NTPs, buffer, and optional DNase I for template removal.
The RNA product can be used directly (after cleanup) for downstream applications (e.g. qRT-PCR, in vitro translation,
transfections). In this protocol, we outline the steps using the HiScribe kit: template preparation, transcription reaction
setup, DNase treatment (if applicable), RNA purification (if desired), and quality control checks.

1. Prepare all required reagents and consumables:
   * HiScribe T7 High Yield RNA Synthesis Kit (NEB)
   * Linearized DNA template containing a T7 promoter (purified, appropriate concentration)
   * Nuclease-free water
   * DNase I
   * Reaction tubes (RNase-free)
   * Pipettes and RNase-free filter tips
   * RNase inhibitor
   * RNA cleanup kit or reagents (e.g. spin columns, LiCl precipitation, phenol:chloroform,
     ethanol precipitation)
   * RNAse-free microcentrifuge
   * RNase-free tubes for storage
   * Quantification equipment: NanoDrop
   * Agarose gel electrophoresis
   * Ice bucket, gloves, RNase-free gloves, RNase-away reagent, etc.
2. Template preparation
   * Ensure DNA template is linear (avoid circular plasmid for run-off transcription).
   * Purify the DNA (remove proteins, residual salts). Determine concentration and check purity (A260/A280).
3. Set up transcription reaction
   * Thaw the necessary components at room temperature. Keep the T7 RNA Polymerase Mix on ice.
   * Mix and pulse-spin in a microfuge to collect the solutions to the bottom of the tubes.
   * Set up the reaction at room temperature in the order listed in the table below:

   | Components | 20 μl reaction | Final amount |
   | --- | --- | --- |
   | Nuclease-free water | X μL |  |
   | 10X T7 Reaction Buffer | 2 μL | 1x |
   | 100 mM ATP | 2 μL | 10 mM |
   | 100 mM GTP | 2 μL | 10 mM |
   | 100 mM UTP | 2 μL | 10 mM |
   | 100 mM CTP | 2 μL | 10 mM |
   | Linear Template DNA | X μL | 1 μg |
   | DTT (0.1M) | 1 μL | 5 mM |
   | T7 RNA Polymerase Mix | 2 μL |  |
4. Incubation / transcription
   * Incubate at 37°C for the duration 2 hours
5. DNase treatment
   * Add DNase I to degrade the DNA template
   * Incubate for the recommended time/temperature to ensure DNA removal.
   * RNA purification (optional but recommended)
6. Quality control and quantification
   * Measure RNA concentration (e.g. NanoDrop).
   * Assess purity (A260/A280, A260/A230).
   * Check integrity and size (agarose gel).
7. Storage
   * Aliquot RNA and store at −80°C (avoid repeated freeze-thaw).
   * Optionally, add RNase inhibitor or buffer stabilizer if needed.

PCR - Q5® High-Fidelity
+

This protocol describes methods for PCR using Q5 High-Fidelity
DNA Polymerase, which offers high fidelity (~280X higher than Taq),
resulting in ultra-low error rates. Please note that protocols with Q5
High-Fidelity DNA Polymerase may differ from protocols with other
polymerases. The conditions recommended below should be used
for optimal performance.

1. Prepare all required reagents and consumables:
   * 5X Q5 Reaction Buffer
   * 10mM dNTPs
   * 10μM Forward Primer
   * 10μM Reverse Primer
   * Q5 High-Fidelity DNA Polymerase
   * 5X Q5 High GC Enhancer (optional)
   * Nuclease-Free Water

- Procedure
  * Assemble all reaction components on ice. Each
    component should be gently mixed before adding to the reaction in a sterile thin-walled PCR tube. The Q5 High-
    Fidelity DNA Polymerase may be diluted in 1X Q5 Reaction Buffer just prior to use to reduce pipetting errors. The
    entire reaction should be mixed again to ensure
    homogeneous, consistent mixture. Collect all liquid to the
    bottom of the tube with a quick centrifuge spin if
    necessary. Overlay the sample with mineral oil if using a
    PCR machine without a heated lid.
  * Quickly transfer the reactions to a thermocycler preheated
    to the denaturation temperature (98°C) and begin
    thermocycling. According to the table 1 below.

  | Component | 25 μl reaction | 50 μl reaction |
  | --- | --- | --- |
  | 5X Q5 Reaction Buffer | 5 μl | 10 μl |
  | 10 mM dNTPs | 0.5 μl | 1 μl |
  | 10 μM Forward Primer | 1.25 μl | 2.5 μl |
  | 10 μM Reverse Primer | 1.25 μl | 2.5 μl |
  | Template DNA | variable | variable |
  | Q5 High-Fidelity DNA Polymerase | 0.25 μl | 0.5 μl |
  | 5X Q5 High GC Enhancer (optional) | (5 μl) | (10 μl) |
  | Nuclease-Free Water | to 25 μl | to 50 μl |

  * Thermocycling Conditions for a Routine PCR:

  | Step | Temp |  |
  | --- | --- | --- |
  | Initial denaturation | 98°C | 10 μl |
  | 25–35 Cycles | 98°C, 50–72°C, 72°C | 5–10 seconds, 10–30 seconds, 20–30 seconds/kb |
  | Final Extension | 72°C | 2 minutes |
  | Hold | 4–10°C | ∞ |

RT-qPCR
+

Quantitative Reverse Transcription PCR (RT-qPCR) is a sensitive and specific technique used to detect and
quantify RNA levels by first converting RNA into complementary DNA (cDNA) using reverse transcriptase,
followed by amplification and real-time quantification of specific DNA targets during PCR cycles. RT-qPCR is
widely applied in gene expression analysis, pathogen detection, and validation of RNA interference studies.
Fluorescent chemistries such as DNA-binding dyes (e.g., SYBR Green) or hydrolysis probes (e.g., TaqMan
probes) enable real-time monitoring of amplification, allowing quantification based on fluorescence intensity
correlated to the amount of target nucleic acid.

1. Prepare all required reagents and consumables:
   * Luna Universal One-Step Reaction Mix (2X)
   * Luna WarmStart RT Enzyme Mix (20X)
   * Forward primer (10μM) specific to target gene
   * Reverse primer (10μM) specific to target gene
   * RNA samples (purified total RNA, ≤1μg per reaction)
   * Nuclease-free water
   * qPCR tubes or optical 96/384-well plates with sealing film or caps
   * Real-time PCR instrument with SYBR or FAM detection capability
   * Pipettes and sterile filtered tips
2. RNA Preparation
   * Extract and purify RNA from samples using appropriate methods.
   * Quantify RNA concentration by measuring OD260.
   * Prepare RNA dilutions fresh before use, diluting in nuclease-free water or TE buffer if needed.
3. Reaction Setup (20μL total volume)
   * Thaw Luna Universal One-Step Reaction Mix and Luna WarmStart RT Enzyme Mix at room
     temperature, then place on ice. Mix gently.
   * Prepare a master mix for all reactions (including 10% extra volume) containing:
     + 10μL Luna Universal One-Step Reaction Mix (2X)
     + 1μL Luna WarmStart RT Enzyme Mix (20X)
     + 0.8μL Forward primer (10μM) (final 0.4μM)
     + 0.8μL Reverse primer (10μM) (final 0.4μM)
     + Nuclease-free water to adjust volume before adding RNA template
   * Aliquot 18–19μL of the master mix into each qPCR tube or well.
   * Add 1–2μL of RNA template (≤1μg total RNA) to each tube/well.
   * Seal tubes or plate with optical caps or film.
   * Briefly centrifuge to remove bubbles and collect liquid at the bottom (1 min at 2,500–3,000 rpm)
4. Thermal Cycling Conditions
   * Program the real-time PCR instrument as follows:

   | Step | Temperature | Time | Cycles |
   | --- | --- | --- | --- |
   | Reverse transcription | 55°C | 10 minutes | 1 |
   | Initial denaturation | 95°C | 1 minute | 1 |
   | Denaturation | 95°C | 10 seconds | 40-45 |
   | Annealing/Extenstion | 60°C | 30 seconds\* (+ plate read) | 40-45 |
   | Melt Curve (optional) | 60-95°C | Instrument-specific | 1 |

   \*For Applied Biosystems instruments, use a 60-second extension step.
5. Data Collection and analysis
   * Use SYBR or SYBR/FAM detection mode on the instrument.
   * Collect fluorescence data at the end of each annealing/extension step.
   * Analyze Cq values and perform relative or absolute quantification as needed.
   * Analyze Cq values and perform relative or absolute quantification as needed.
6. Notes
   * Avoid reverse transcription temperatures below 50°C to ensure full WarmStart RT activation and
     optimal performance.
   * Primer concentrations and annealing temperatures can be optimized for specific targets.
   * The one-step format reduces contamination risk and simplifies workflow compared to separate RT and qPCR steps.
   * For multiplex assays or probe-based detection, use compatible Luna probe kits and adjust reaction
     components accordingly.
   * This protocol provides an efficient and reliable method for RNA quantification using the Luna
     Universal One-Step RT-qPCR Kit, suitable for a wide range of applications including gene
     expression analysis and pathogen detection

Primers treatment
+

This protocol describes how to properly resuspend lyophilized DNA oligonucleotide primers supplied by Genomed to a final concentration of
100 μM using 10 mM Tris buffer, pH 7.5. The protocol includes preparation of the resuspension buffer and step-by-step instructions for
dissolving the primers.

1. Prepare all required reagents and consumables:
   * Lyophilized DNA oligonucleotide primers (ordered from Genomed)
   * Tris base (molecular biology grade)
   * Concentrated HCl (for pH adjustment)
   * Nuclease-free water
   * pH meter or pH indicator strips
   * 0.22 μm sterile filter (for buffer sterilization)
   * Sterile microcentrifuge tubes
   * Pipettes and sterile tips
2. Prepare 1 M Tris-HCl stock solution:
   * Dissolve 12.1 g Tris base in ~80 mL nuclease-free water.
   * Slowly add concentrated HCl while monitoring pH, until pH reaches 7.5.
   * Allow the solution to cool to room temperature, then adjust the pH again if needed.
   * Bring the final volume to 100 mL with nuclease-free water.
   * Sterilize the solution by passing it through a 0.22 μm filter.
3. Prepare 10 mM Tris buffer
   * Dilute the 1 M Tris-HCl stock 1:100 with nuclease-free water (e.g., 1 mL 1 M Tris-HCl + 99 mL water).
4. Resuspension of Lyophilized Primers
   * Determine the required volume. Refer to the information provided by Genomed specifying the volume needed to achieve 100 μM concentration for
     your primer batch.
   * Add buffer. Add the calculated volume of 10 mM Tris, pH 7.5, directly to the lyophilized primer in its tube.
   * Mix thoroughly. Vortex gently or pipette up and down to ensure complete dissolution.
     If the primer does not fully dissolve, briefly centrifuge and incubate at room temperature for 10–15 minutes, then
     mix again.
   * Label and store. Clearly label the tube with primer name, concentration, and date.
     Store the resuspended primer at –20°C for long-term storage.

##### Nucleic acids purification and quantification protocols

Clean-up Concentrator
+

The procedure for purifying DNA products from an enzymatic reaction involves adding a DNA-binding buffer to the sample, then applying
the mixture to a silica column, where the DNA selectively adsorbs. Subsequent steps include multiple wash cycles to remove residual
reagents, followed by elution of the purified DNA using elution buffer or DEPC-treated water. The resulting DNA is inhibitor-free and ready
for downstream applications such as ligation, PCR, sequencing, or cloning.

1. Prepare all required reagents and consumables:
   * Clean-Up Concentrator Kit from A&A Biotechnology
   + Microcolumns
   + 1.5 mL elution tubes
   + GI binding solution
   + A1 wash solution
   + Sodium acetate (3 M, ph 5.5)
   + Tris buffer (10 mM, pH 8.5)* Additional equipment and reagents
   + 1.5 mL sterile Eppendorf tubes
   + Vortex
   + Microcentrifuge
2. Mix DNA samples (up to 100 μL) with 5 volumes of GI binding solution. Mix the samples by inverting the tubes or vortexing.
   GI binding solution contains the color pH indicator. Upon mixing the DNA sample with GI binding solution, yellow color of the mixture indicates an optimal pH for DNA binding. If the mixture color turns pink the pH of the solution is too high. In such conditions DNA binds ineffectively to the silica membranes and may be lost.
   Too high pH can be corrected by adding 1-10 µL of 3 M sodium acetate solution (pH 5.5) (included) and mix. Purification can be continued after reaching a yellow color.
   optimal condition pH ≤ 7.2 too high pH.
3. Briefly centrifuge the samples to remove the leftovers of solution from the tube walls and caps.
4. Apply samples onto the microcolumns. Close the tubes with the caps.
5. Centrifuge for 30-60 s at 10 000-15 000 RPM.
6. Remove the microcolumns, discard the filtrates. Place the microcolumns to the same tubes.
7. Add 300 µL of A1 wash solution. Close the tubes with the caps.
8. Centrifuge for 30-60 s at 10 000-15 000 RPM.
9. Add 200 µL of A1 wash solution. Close the tubes with the caps.
10. Centrifuge for 2 min at 10 000-15 000 RPM.
11. Transfer the microcolumns to new 1.5 mL tubes (not included).
12. Add 15-30 µL of Tris buffer directly onto the microcolumn resin. Close the tubes with the caps. Applying Tris buffer onto the column be sure that liquid is applied directly onto the resin. If some of the liquid stays on the column wall the elution will be less effective.
    Elution in a smaller volume is less efficient, but the extracted DNA has a higher concentration. Elution in 30 µL is recommended for fragments over 2000 bp.
13. Incubate for 3 min at room temp.
14. Centrifuge for 2 min at 10 000-15 000 RPM.
15. Remove the microcolumns, close the tubes. Store the tubes with purified DNA at 4-8°C until later use.
    Elution tube has a long elastic cap connector. It's important to start closing the tube by carefully pressing the cap on the connector side. A "click" - sound confirms proper closure. Different ways of closing may cause opening of the tube during storage.

DNA concentration measurement
+

The NanoDrop is a UV–Vis spectrophotometer that employs surface tension technology to rapidly and accurately assess nucleic acid
concentration and purity without requiring cuvettes.

1. Prepare all required reagents and consumables:
   * Autoclaved dH2O
   * Fiber-free tissues
   * NanoDrop spectrophotometer
   * Pipettes and tips
   * Samples
2. Start the NanoDrop spectrophotometer.
3. Select the DNA measurement ‘Nucleic Acid’ in the NanoDrop menu for examining DNA sample.
4. Clean the surface of the NanoDrop with dH2O and a fiber free-tissue.
5. Perform a calibration and blank measurement by entering one drop of 1 μL autoclaved dH2O.
6. Clean the surface again and place 1 μL per sample on the NanoDrop and measure the concentration.

DNA electrophoresis
+

DNA electrophoresis is a technique used to separate fragments of deoxyribonucleic acid (DNA) in an electric field based on their size. Due
to the negative charge of phosphate groups in the DNA backbone, the molecules migrate towards the anode. Smaller fragments move more
rapidly through the polymer network of the gel than larger ones, which allows for the assessment of fragment length, verification of PCR or
cloning quality, and the isolation of specific fragments for further analyses.

1. Prepare all required reagents and consumables:- Reagents
     * Agarose
     * 1X TBE Buffer
     * GelRed nucleic acid stain
     * 6X Loading dye
     * DNA Ladder (e.g. 1 kb Plus Ladder)
     * Samples (PCR product or purified DNA)
   - Equipment
     * Electrophoresis chamber with power supply
     * Gel casting trays with combs
     * Casting stands
     * Microwave oven
     * UV lamp or blue-light transilluminator for gel visualization
2. Gel preparation
   * Calculate the gel volume and percentage based on the number and characteristics of the samples.
   * Use an analytical balance to weigh the appropriate amount of agarose (e.g., 1 g of agarose per 100 mL of 1X TBE
     for a 1 % gel).
   * Pour 1X TBE buffer into a flask in a volume equal to the final gel volume, then add the agarose.
   * Heat the mixture in a microwave until the agarose is completely dissolved. Remove every 30 seconds to swirl
     gently and verify that a clear solution has formed.
   * Cool the gel solution under running tap water until it reaches approximately 55°C.
   * Add the recommended amount of GelRed according to the manufacturer’s instructions, and mix gently.
   * Attach casting stands to the gel tray, insert the comb, pour in the gel solution, and allow it to solidify at room
     temperature for 20–30 minutes.
   * Once polymerized, remove the casting stands and carefully extract the comb to create the sample wells.
3. Sample Treatment
   * Prepare DNA samples and ladders in Eppendorf tubes.
   * Add loading dye to each sample at a ratio of 1 part loading dye to 5 parts sample (e.g., 5 μL sample + 1 μL loading
     dye).
   * Gently mix by pipetting, avoiding the introduction of air bubbles.
   * Briefly centrifuge the samples (1–2 s) to collect all contents at the bottom of the tube.
4. Electrophoresis & Analysis
   * Place the concentrated gel in the electrophoresis chamber and add 1X TBE buffer until it covers the gel
     (approximately 3–5 mm above the surface).
   * Carefully load the ladder and samples into the wells, loading the ladder first, followed by the samples.
   * Connect the electrodes and set the voltage to 5–10 V/cm (for example, for a 10 cm gel, set to 100 V).
   * Start the power supply and run the electrophoresis for the appropriate duration (usually 30–60 minutes) until the
     tracking dyes have migrated to the desired distance from the origin.
   * After completion, turn off the power supply, remove the gel, and place it on a transilluminator to visualize the DNA
     fragments.

DNA Gel Extraction
+

The DNA extraction process from agarose gel involves excising the target band from the gel, dissolving the matrix in a dissolving buffer, and
then selectively binding the DNA to a silica column. Subsequent steps include the removal of contaminants through multiple wash steps,
followed by elution of the DNA in a small volume of buffer or water. The purified DNA obtained is ready for downstream applications such as
ligation, sequencing, cloning, or PCR.

1. Prepare all required reagents and consumables:- Gel-Out kit A&A Biotechnology
     * Minicolumns
     * R7SI agarose solution
     * A1 wash solution
     * Sodium acetate (3 M, pH 5.5)
     * Isopropanol
     * TE buffer
   - Additional Required Reagents/Equipment
     * 1.5 mL sterile Eppendorf tubes
     * Incubator or thermoblock set to 50°C
     * Vortex
     * Microcentrifuge
2. Cut out the agarose slices (up to 200 mg) containing DNA. Transfer agarose slices to Eppendorf tubes (not
   included). Agarose gel electrophoresis can be performed in the presence of either TAE or TBE buffer.
3. Add an appropriate volume of R7SI agarose melting solution:
   * < 2% agarose gel – 400 μL
   * ≥ 2% agarose gel – 500 μLIncubate samples at 50°C until complete dissolution of agarose slices. Mix the samples by inverting the tubes or
   vortexing a few times. Agarose melting solution R7SI contains the color pH indicator. Upon mixing the DNA sample with R7SI agarose
   melting solution, yellow color of the mixture indicates an optimal pH for DNA binding. If the mixture color turns
   pink the pH of the solution is too high. In such conditions DNA binds ineffectively to the silica membranes and
   may be lost. Too high pH can be corrected by adding 1–10 μL of 3 M sodium acetate solution (pH 5.5) (included)
   and mix. Purification can be continued after reaching a yellow color.
   optimal condition pH ≤ 7.2 too high pH
4. Add an appropriate volume of isopropanol:
   * < 2% agarose gel – 200 μL
   * ≥ 2% agarose gel – 250 μLMix by inverting the tubes.
5. Briefly centrifuge the samples to remove the leftovers of solution from the tube walls and caps.
6. Apply samples onto the minicolumns.
7. Centrifuge for 30 s at 10 000–15 000 RPM.
8. Remove the minicolumns, discard the filtrate. Place the minicolumns to the same tubes.
9. Add 600 μL of A1 wash solution.
10. Centrifuge for 30 s at 10 000–15 000 RPM.
11. Remove the minicolumns, discard the filtrate. Place the minicolumns to the same tubes.
12. Add 300 μL of A1 wash solution.
13. Centrifuge for 1 min at 10 000–15 000 RPM.
14. Remove the minicolumns, discard the filtrate. Place the minicolumns to the same tubes.
15. Centrifuge for 1 min at 10 000–15 000 RPM.
16. Transfer the minicolumns to new 1.5 mL tubes.
17. Add 50 μL of TE buffer or sterile water (not included) directly onto the minicolumn resin. Applying elution liquid (TE buffer or sterile water) onto the minicolumn be sure that liquid is applied directly onto
    the resin. If some of the liquid stays on the column wall the elution will be less effective. Elution in a smaller
    volume is less efficient, but the extracted DNA has a higher concentration. Elution in 50 μL volume is more
    efficient, but DNA has a lower concentration.
18. Incubate for 3 min at room temp.
19. Centrifuge for 1 min at 10 000–15 000 RPM.
20. Remove the minicolumns and store the tubes with purified DNA at 4–8°C until later use.

Plasmid Isolation - Plasmid Midi AX Endotoxin-Free
+

Isolation of plasmids using the Midi‐prep method is a process for purifying plasmid DNA from a large volume of bacterial culture via kits
equipped with ion‐exchange columns. This technique relies on alkaline lysis of the cells, followed by selective binding of plasmid DNA to the
column matrix, washing away contaminants, and eluting the pure DNA, which is then suitable for downstream applications such as
transfection, PCR, or sequencing.

1. Prepare all required reagents and consumables:
   * Kit Components (per 1 isolation)
   + Plasmid 200 column (1x)
   + Filtration column (1x)
   + 50 mL tubes (2x)
   + L1 cell suspension solution (5 mL)
   + L2 lysis solution (5 mL)
   + L3 neutralizing solution (5 mL)
   + LPS-out endotoxin removal solution (15mL)
   + K2P wash solution (20 mL)
   + K3 elution solution (6 mL)
   + Precipitation enhancer (25 μL)
   + Isopropanol (5 mL)
   + TE buffer (0.2–1 mL for resuspension)* Additional Required Reagents/Equipment
   + 70% Ethanol (2 mL)
   + Sterile 15 mL and 50 mL Falcon tubes
   + Microcentrifuge capable of 11,000 × g
   + Sterile water (optional)
   + Pipettes and tips
   + Sterile cotton buds (for drying)
   + Personal protective equipment (PPE)* Safety notes
   + L2 lysis solution and K3 elution solution contain hazardous chemicals. Refer to the kit’s safety data for handling instructions.
   + Isopropanol is flammable and an irritant-work in a ventilated area and away from ignition sources.
   + Wear gloves, lab coat, and eye protection throughout the procedure.
2. Cell harvesting
   * Centrifuge up to 100 mL of overnight E. coli culture at 4,000–6,000 × g for 10 min.
   * Discard the supernatant.
   * Resuspend the bacterial pellet in 5 mL of L1 cell suspension solution. Note: Solution should change from deep pink to opaque light pink; ensure no pellet remains at the bottom.
3. Cell lysis
   * Add 5 mL of L2 lysis solution.
   * Gently invert the tube several times (do not vortex).
   * Incubate at room temperature for 5 min. Note: Lysate should become clear and raspberry-colored. If not, mix gently and incubate for an additional 3 min.
4. Neutralization
   * Add 5 mL of L3 neutralizing solution. Note: Solution should turn yellowish, indicating complete neutralization.
5. Filtration
   * Transfer the lysate to the filtration column.
   * Centrifuge at 1,500 × g for 5 min.
6. Endotoxin removal
   * Add 1.2 mL LPS-out solution to the filtered lysate. Mix and keep on ice for 30 min.
7. DNA binding
   * Place a Plasmid 200 column into a clean 50 mL tube.
   * Apply the clear filtrate onto the column and allow it to flow through completely.
8. Column Wash
   * Add 20 mL of K2P wash solution to the column.
   * Allow the solution to flow through.
9. Elution
   * Transfer the Plasmid 200 column to a new 50 mL tube.
   * Add 6 mL of K3 elution solution.
   * Collect the eluate.
10. DNA Precipitation
    * Transfer the eluate to a new 15 mL tube.
    * Add 25 μL precipitation enhancer and 5 mL isopropanol. Note: For sensitive applications, omit the enhancer if desired.
    * Mix by gentle inversion.
    * Centrifuge at 11,000 × g for 10 min. Note: A light-blue DNA pellet should be visible.
11. Wash DNA Pellet
    * Carefully discard the supernatant (do not lose the pellet).
    * Add 2 mL of 70% ethanol.
    * Mix and centrifuge at 11,000 × g for 3 min.
12. Dry DNA Pellet
    * Carefully discard the supernatant.
    * Air dry the pellet at room temperature (10 min, tube inverted).
    * Remove any residual ethanol with a sterile cotton bud if necessary.
13. DNA Resuspension
    * Dissolve the DNA pellet in 0.2–1 mL of TE buffer or sterile nuclease-free water. Note: The blue color aids in visual confirmation of dissolution.
14. Storage
    * Store the purified plasmid DNA at 4–8°C.
15. Color Indicators (LySee System)
    * **L1 addition:** Deep pink → opaque light pink (complete resuspension)
    * **L2 addition:** Opaque light pink → clear raspberry (complete lysis)
    * **L3 addition:** Raspberry → yellowish (complete neutralization)
16. Troubleshooting
    * **L2 solution cloudy:** Warm at 40°C until clear before use.
    * **Pellet loss:** Always pour supernatant into a separate tube to recover pellet if accidentally lost.
    * **Incomplete resuspension/lysis/neutralization:** Confirm color changes at each step.
17. Waste Disposal
    * Dispose of all hazardous liquids and biological waste according to institutional guidelines.

Plasmid Isolation - Plasmid Mini
+

Isolation of plasmids using the Mini-prep method is a process for purifying plasmid DNA from a small volume of bacterial culture. This
technique relies on alkaline lysis of bacterial cells, which causes denaturation of genomic DNA and cellular proteins, while small, circular
plasmid DNA retains its structure. Following lysis, contaminant precipitation occurs and selective recovery of plasmid DNA is achieved
through binding to the column matrix, washing, and elution of pure DNA in small volumes. Plasmids obtained through such isolation can be
applied in processes such as bacterial transformations, PCR reactions, or sequencing.

1. Prepare all required reagents and consumables:
   * Centrifuge
   * Sterile 1.5 mL Eppendorf-type tubes
   * Sterile nuclease-free water
   * Plasmid Mini Kit A&A Biotechnology
   + Microcolumns
   + L1 Cell Resuspension Solution
   + L2 Lysis Solution
   + GL3 Neutralization Solution
   + W First Wash Solution
   + A1 Second Wash Solution
   + TE Buffer
2. Centrifuge 1.5–3 mL of overnight bacterial culture in 12 500 RCF for 10 minutes.
3. Remove the supernatant and thoroughly resuspend the pellet in 200 μL of L1 Cell Resuspension Solution. Note: During resuspension of the bacterial pellet, the solution will change in appearance from completely
   transparent with a dark pink tint to opaque with a light pink tint. Resuspension can be considered complete once
   the pellet at the bottom of the tube has completely disappeared.
4. Add 200 μL of L2 Lysis Solution and gently mix until complete lysis occurs. Note: After adding L2 Lysis Solution, gently mix the tube contents to avoid chromosomal DNA fragmentation.
   Several gentle tube inversions are typically sufficient. The mixture should change in appearance and coloration.
5. Incubate for 3 minutes at room temperature. Note: After 3 minutes of incubation, the lysate should be completely clear and uniformly pink. If not, mix the lysate
   several times and extend incubation for an additional 3 minutes.
6. Add 400 μL of GL3 Neutralization Solution and gently mix until the pink coloration of the lysate disappears. Note: Upon addition of GL3 Neutralization Solution, rapid precipitation of potassium SDS salts, chromosomal
   DNA, and some proteins occurs. After mixing, the tube contents should change to a light yellow color. The
   absence of pink coloration indicates complete neutralization and successful completion of alkaline lysis.
7. Centrifuge the lysate for 10 minutes at 12,500 RCF.
8. Carefully apply the lysate (supernatant) to the minicolumn.
9. Centrifuge for 1 minute at 12,500 RCF.
10. Remove minicolumns from tubes, discard the flow-through, and replace minicolumns in the same tubes.
11. Add 500 μL of first wash solution W.
12. Centrifuge for 1 minute at 12,500 RCF.
13. Remove minicolumns from tubes, discard the flow-through, and replace minicolumns in the same tubes.
14. Add 600 μL of second wash solution A1.
15. Centrifuge for 2 minutes at 12,500 RCF.
16. Transfer minicolumns to new 1.5 mL tubes.
17. Apply 60 μL of TE buffer or sterile water to the column matrix.
18. Incubate samples for 3 minutes at room temperature.
19. Centrifuge for 1 minute at 12,500 RCF.
20. Remove minicolumns and store plasmid DNA at 4-8°C until further analysis.

RNA concentration measurement
+

The NanoDrop is a UV–Vis spectrophotometer that employs surface tension technology to rapidly and accurately assess nucleic acid
concentration and purity without requiring cuvettes.

1. Prepare all required reagents and consumables:
   * Autoclaved dH2O
   * Fiber-free tissues
   * NanoDrop spectrophotometer
   * Pipettes and tips
   * Samples
2. Start the NanoDrop spectrophotometer.
3. Select the RNA measurement ‘Nucleic Acid’ in the NanoDrop menu for examining RNA sample.
4. Clean the surface of the NanoDrop with dH2O and a fiber free-tissue.
5. Perform a calibration and blank measurement by entering one drop of 1 μL autoclaved dH2O.
6. Clean the surface again and place 1 μL per sample on the NanoDrop and measure the concentration.

RNA Isolation - Total RNA Mini
+

The total RNA was isolated using a protocol involving cell lysis in a phenol–guanidine buffer, followed by specific binding of RNA to a silica
column. The purified RNA can be employed, among other applications, for cDNA synthesis and RT-qPCR analysis.

1. Prepare all required reagents and consumables:
   * Total RNA Mini Kit
   + Minicolumns – 100 pcs
   + 2 mL Tubes – 100 pcs
   + A1 Washing Solution – 200 mL
   + Phenol – 100 mL
   + Isopropanol – 30 mL
   + Ultrapure Water – 15 mL* Sterile 1.5 mL Eppendorf-type tubes
   * Chloroform
   * Microcentrifuge
   * Incubator at 50°C
2. Preparation of Cell Culture Material
   * Centrifuge cells (1×106) and remove the supernatant.
3. RNA Isolation
   * Add 800 μL of Fenozol (phenol-based solution) to the prepared sample. Mix by pipetting until complete cell lysis. Notes: Fenozol inactivates endogenous RNases. Samples in Fenozol can be stored:
     + Up to 1 year at -80°C
     + Up to 1 week at 2–8°C
     + Up to 24 hours at room temperature
     + ⚠️ Fenozol contains phenol. Avoid skin contact; wear protective gloves.
   * Incubate the sample for 5 minutes at 50°C.
   * Add 200 μL chloroform (not included in the kit) to the lysate. Gently mix by inverting the tube several times.
   * Let the sample sit for 3 minutes at room temperature (20–25°C).
   * Centrifuge for 10 minutes at 10,000–12,000 RPM.
   * Transfer the upper aqueous phase (~450 μL supernatant) to a new 1.5 mL tube.
   * Add 250 μL isopropanol and mix thoroughly.
   * Apply the mixture to a minicolumn.
   * Centrifuge for 1 minute at 10,000–12,000 RPM.
   * Transfer the minicolumn to a new 2 mL tube.
   * Add 700 μL Wash Solution A1.
   * Centrifuge for 1 minute at 10,000–12,000 RPM.
   * Discard the flow-through and reinsert the minicolumn into the same tube.
   * Add 700 μL Wash Solution A1 and centrifuge again for 1 minute at 10,000–12,000 RPM.
   * Discard the flow-through and reinsert the minicolumn.
   * Add 200 μL Wash Solution A1 and centrifuge for 2 minutes at 10,000–12,000 RPM.
   * Transfer the minicolumn to a new 1.5 mL tube.
   * Add 100 μL ultrapure water directly to the center of the minicolumn membrane.
   * Let the column sit for 3 minutes at room temperature.
   * Centrifuge for 1 minute at 10,000–12,000 RPM.
   * Discard the minicolumn. Store the purified RNA in the tube at -20°C or -80°C until further analysis.

#### Materials & Methods

Toehold Switch Assembly
+

##### Introduction

Toehold switches are advanced tools in synthetic biology that enable precise regulation of gene expression at the translational level. They are artificially engineered single-stranded RNAs that function as sensors within the 5′ untranslated region (5′ UTR) of a transcript. In their inactive state, a toehold switch adopts a complex secondary structure that occludes the ribosome binding site and the start codon, thereby preventing the initiation of translation. Only in the presence of a specific RNA sequence (the so-called trigger) does the toehold switch undergo a conformational change, allowing the ribosome to initiate protein synthesis.

The objective of this experiment is to obtain 21 constructs, each consisting of Transcription Unit 1 (TU1) (BBa\_25SH329F) and a genetic element comprising one of the twenty-one toehold switches placed upstream of the eGFP coding sequence (parts ID). The prepared constructs will provide an effective platform for testing all of the designed toehold switches.

##### Materials & Methods

Twenty-one designed toehold switches (Toehold switches collection, Parts) as well as the TU1 construct (BBa\_25SH329F) were ordered for synthesis from Twist Bioscience. The lyophilized constructs were dissolved in Milli-Q water to a final concentration of 100 mM. One microliter of each stock solution was used for bacterial transformation following the High Efficiency Transformation protocol, employing 50 µL of E. coli DH5α as the host strain. Chemocompetent E. coli DH5α were prepared using Competent Cell Preparation protocol. One hundred microliters of the transformed bacterial suspension were plated onto LB agar plates supplemented with the appropriate antibiotic. For toehold switches, kanamycin was used at a concentration of 50 µg/mL, while for TU1, ampicillin was used at 100 µg/mL. Plates were incubated overnight at 37°C. A single colony from each construct was selected for plasmid isolation. Each colony was picked using a pipette tip and inoculated into 3 mL of liquid LB medium containing the corresponding antibiotic. The cultures were incubated overnight at 37°C with shaking at 180 RPM. Plasmid isolation was performed according to the Plasmid Mini protocol, and plasmid concentration and purity were assessed using a Nanodrop spectrophotometer.

To facilitate construct generation, the TU1 vector was modified by inserting the LacZα sequence (BBa\_25DTJWTR) into the site originally designated for the toehold switch with eGFP, thereby enabling blue-white screening. To obtain this modified construct, a PCR reaction was first performed using the previously synthesized L2TU2 construct (BBa\_259T9MVI), which contains the LacZα sequence, as a template. Primers specific to both ends of the LacZα cassette, containing overhangs, allowing us to use this PCR product directly for cloning, were used. The PCR product was subjected to agarose gel electrophoresis, and the band of the expected size was excised and purified according to the DNA Gel Extraction protocol.

Both the TU1 backbone and the LacZα insert flanked with cloning-compatible sequences were subjected to restriction digestion in separate reactions using the BsaI enzyme, according to the Restriction Digestion – BsaI protocol. The digested products were then ligated following the T4 DNA Ligase Protocol and 1 µL of the product was used for bacterial transformation following the High Efficiency Transformation protocol with E. coli DH5α. One hundred microliters of the transformed culture were plated on LB agar supplemented with 100 µg/mL ampicillin, 1 mM IPTG, 40 µg/mL X-Gal and incubated overnight at 37°C. A blue colony was selected and used for plasmid isolation. The colony was inoculated into 3 mL of LB medium containing 100 µg/mL ampicillin and incubated overnight at 37°C with shaking at 180 RPM. Plasmid isolation was carried out using the Plasmid Mini protocol, and plasmid concentration and purity were verified with a Nanodrop spectrophotometer. Samples were then prepared for sequencing according to Genomed’s recommendations. This procedure yielded a functional platform for testing the designed toehold switches.

Cloning of toehold switches into TU1 was performed using the Golden Gate assembly method following the Golden Gate Assembly - BsaI protocol. 5 µL of the product was used for bacterial transformation following the High Efficiency Transformation protocol with E. coli DH5α. One hundred microliters of the transformed culture were plated on LB agar supplemented with 100 µg/mL ampicillin, 1 mM IPTG, 40 µg/mL X-Gal and incubated overnight at 37°C. Selected white colonies were collected with a pipette tip and used to perform colony PCR to determine the correctness of cloning. The results of the cloning procedure are described in the Results section.

##### Distribution kit

**Introduction**

The iGEM collection represents a remarkably rich repository of genetic parts, enabling research across a wide range of biological systems. When designing projects based on mammalian cellular systems, an especially valuable source of genetic components is the Asimov Mammalian Parts Collection, which provides elements such as promoters, 5' UTRs, 3' UTRs, poly(A) sequences, and coding sequences for fluorescent proteins.

The aim of this experiment is to obtain plasmid stocks of Asimov Mammalian Part Collection that we wanted to use for the cloning in our project.

**Materials & Methods**

Lyophilised DNA from the Distribution Kit was resuspended in 10 µL of Mili-Q water. It was then used for the transformation of both E. coli strains DH5α and DH10β (with the same plasmids, 50 µL of chemocompetent bacteria per transformation) according to the High Efficiency Transformation protocol. Chemocompetent E. coli DH5α and DH10β were prepared using Competent Cell Preparation protocol. Initially 1 µL of plasmid was used for transformation and after almost no colonies were obtained, transformations were repeated on both strains, this time using 2 µL of plasmid. As a positive control of transformation we used 1 ng of pUC19 vector (1 ng/µL) from New England Biolabs and as a negative we used 1 µL of Mili-Q water.

The obtained colonies were used to inoculate 3 mL of LB supplemented with corresponding antibiotic. Cultures where set overnight in 37°C with 180 RPM shaking. Then, they were centrifuged for 2 minutes at 6000 RCF and plasmid DNA was isolated from bacterial pellet using Plasmid Isolation - Plasmid Mini protocol. Isolated plasmids were sent to the Sanger sequencing in Genomed to confirm the sequence with specific primer binding around 100 nt upstream from the desired inserts.

##### Assembly of dual-reporter construct (TU1-eGFP + TU2-RFP)

**Introduction**

Red fluorescent proteins (RFPs) are a class of coral-derived proteins that emit red light when illuminated with light of a suitable wavelength. RFPs are highly valuable in a range of biological applications, from fluorescence microscopy and protein localization studies to in vivo imaging and molecular biotechnology. The compatibility of RFPs with other fluorescent proteins enables multi-color imaging and facilitates a deeper understanding of complex cellular processes.

The aim of this experiment is to obtain the construct with both the transcription unit 1 having insert site for introducing toehold switches (part ID) with eGFP and transcription unit 2 constitutively expressing RFP. We wanted to use it first as a control if the lack of GFP signal from AFP negative cells comes from the lack of transfection or the lack of translation. Secondly, it would also allow us to normalize the expression of eGFP in the population of cells, since every cell would be transfected with a slightly different amount of pDNA.

**Materials & Methods**

Transcription unit 2 (TU2) containing RFP was delivered by IDT as 3 linear gBlocks™ from IDT containing 20 nt overlaps. To assembly them together and obtain full transcription unit for further cloning, we performed HiFi cloning.

|  |  |
| --- | --- |
| DNA Molar Ratio | TU2.1:TU2.2:TU2.3=1:1:1 |
| Total Amount of | 0,19 pmol |
| Fragments | TU2.1 0.97 µL  TU2.2 1 µL  TU2.3 1.96 µL |
| NEBuilder HiFi DNA Assembly Master Mix | 10 µL |
| Nuclease-free Water | 6,07 µL |
| Total Volume | 20 µL |

Reaction mixture was then cleaned using Clean-up Concentrator kit, to remove reaction components. Then we performed the Golden Gate Assembly using Backbone for assembly of 2 transcription units (BBa\_259T9MVI), TU2 - Mammalian constitutive RFP reporter (BBa\_25VE60AA) and TU1 with cloning site for 5' UTR and CDS (BBa\_25SH329F).

| **Reagent** | **Assembly reagents** |
| --- | --- |
| Destination vector: Backbone for assembly of 2 transcription units (BBa\_259T9MVI) | 3 nM (final concentration), 2.34 µL |
| Amplicon inserts:  TU1  TU2 | 3 nM each amplicon (final concentration)  TU1 - 2.45 µL  TU2 - 4.67 µL |
| SapI (NEB #R0569), 10 U/µL | 1.5 µL (15 units) |
| T4 DNA Ligase (NEB #M0202), 2000 U/µL | 0.25 µL (500 units) |
| T4 DNA Ligase Buffer (NEB #B0202) (10X) | 2 µL |
| Nuclease-free H2O (NEB #B1500) | 6.79 µL |

Reaction was performed using Golden Gate Assembly - SapI protocol. 2 µL of the reaction mix was used for bacterial transformation following the High Efficiency Transformation protocol with E. coli DH5α. Chloramphenicol was used as a selective antibiotic. Plates were incubated overnight at 37 °C. The distinguished white colonies were used for colony PCR according to Colony PCR protocol, which enabled us to check construct presence. According to PCR results, we picked the colonies with constructs and inoculated them into 5 mL of LB medium containing 25 µg/mL chloramphenicol and incubated overnight at 37°C with shaking at 200 RPM. Subsequently we used growth cultures for plasmid isolation with use of Plasmid Mini protocol, and plasmid concentration and purity were assessed using a Nanodrop spectrophotometer. Samples were then prepared for Sanger sequencing in Genomed. Results showed us the cloning was unsuccessful.

For analysis, we selected additional white colonies that appeared on the plate. The colony PCR was repeated, followed by culture initiation, plasmid isolation, and sample preparation for sequencing. The sequencing results once again indicated unsuccessful assembly.

We hypothesized that the amount of correct product following the HiFi reaction was insufficient. Therefore, we performed PCR on the reaction mixture purified using the Clean-up Concentrator protocol, employing primers specific to the ends of correctly assembled construct. We obtained bands at the expected positions, which were subsequently isolated from the gel using the DNA Gel Extraction protocol. In the next step, we performed a second Golden Gate reaction employing the Backbone for assembly of two transcription units, TU1 and TU2, according to the Golden Gate Assembly - SapI protocol. 2 µL of the reaction mix was used for bacterial transformation following the High Efficiency Transformation protocol with E. coli DH5α. Chloramphenicol was used as a selective antibiotic. Plates were incubated overnight at 37 °C. The distinguished white colonies were used for colony PCR according to Colony PCR protocol, which enabled us to check construct presence. We obtained bands at the expected positions. On positive colonies we performed culture initiation, plasmid isolation, and sample preparation for sequencing. Sequencing results did not detect RFP presence.

To obtain the TU2 construct in a stable plasmid first, we performed PCR using the TU1 plasmid as a template and modified primers containing 5′ overhang sequences designed to convert the TU1 backbone into a vector suitable for TU2 insertion using HiFi cloning. Agarose gel electrophoresis confirmed the presence of the expected amplification product, which was subsequently purified from the gel using the DNA Gel Extraction protocol. The purified fragment was then used in a HiFi Assembly reaction together with the TU2 insert to generate the complete construct. 5 µL of the reaction mix was used for bacterial transformation following the High Efficiency Transformation protocol with E. coli DH5α. Plates were incubated overnight at 37 °C. Unfortunately, we obtained no colonies. Only positive control, which was pUC19 yielded robust colony growth. We repeated the HiFi assembly reaction, increasing the ratio of inserts to the backbone. 5 µL of the reaction mix was used for bacterial transformation following the High Efficiency Transformation protocol with E. coli DH5α. Plates were incubated overnight at 37 °C. We again obtained no colonies. Only positive control, which was pUC19 yielded robust colony growth.

**Summary**

We successfully obtained and verified key components of our system, including the TU1 construct, the toehold switch collection, and the modified TU1-LacZα vector enabling blue–white screening. Plasmids from the Asimov Mammalian Parts Collection were also isolated, though sequencing revealed inconsistencies within the distribution kit.

Attempts to assemble the dual-reporter construct (TU1-eGFP + TU2-RFP) using HiFi and Golden Gate cloning were unsuccessful despite protocol optimization. Nonetheless, the established constructs provided a solid foundation for further development and troubleshooting of the toehold-based expression system.

Optimization of transfection
+

##### Introduction

Our project leverages toehold switch reporter constructs encoded on plasmids to selectively detect and respond to the presence of alpha-fetoprotein (AFP) inside mammalian cells. We engineered 20 distinct plasmid variants, each bearing a different toehold switch.

To assess the specificity and efficacy of our toehold switch designs we will need to perform transfection and cytometry to quantify eGFP expression. Therefore we need to optimize our transfection method for best usage of our resources.

Due to this, we performed two transfection methods: forward and reverse in four conditions: (-) control, (+) GFP expressing control, 0.15 μl of lipofectamine, 0.3 μl of lipofectamine on four human cell lines: HepG2, WI38, A549, HEK293T.

By comparing fluorescent signals from post-transfection cells, we aim to decide and optimize the transfection method for performing toehold switch screening.

##### Hypothesis

There is no significant difference between the two tested transfection methods for each cell line used in the experiment.

##### Materials

**Cell lines**

* HepG2 (human hepatocellular carcinoma cell line)
* HEK293T (human embryonic kidney cell line)
* WI38 (human embryonic lung fibroblast cell line)
* A549 (human lung epithelium carcinoma cell line)

**Plasmids**

* plasmids containing toehold switch constructs
* control plasmids: mammalian vectors for constitutive GFP expression

**Reagents and solutions**

* DMEM, high glucose (Gibco)
* Trypsine
* Opti-MEM™ Reduced Serum Medium
* Lipofectamine™ 3000 (Thermo Fisher)
* P3000™ reagent (Thermo Fisher)
* Sterile nuclease-free water
* Sterile PBS (Phosphate-Buffered Saline)

**Plastics and consumables**

* 96-well tissue culture plate
* 1.5 mL microcentrifuge tubes (Eppendorf type)
* Sterile serological pipettes (5, 10, 25 mL)
* Sterile pipette tips (P10, P200, P1000 filter tips)

**Equipment**

* Biological safety cabinet (laminar flow hood)
* CO₂ incubator (37°C, humidified atmosphere with CO₂ control)
* Vortex mixer
* Micropipettes (P10, P200, P1000)
* Microscope (fluorescent)

**General lab supplies**

* Personal protective equipment (lab coat, gloves, protective eyewear)
* Waste containers for biohazard disposal

##### Transfection

**Protocol used: Cell culture protocols, Transfection**

**Day 1 (Preparations)**

Collect cells from cell cultures (HepG2, WI38, A549, HEK293T), prepare for seeding in concentration 70 000 cells/mL.

Seeding will be performed on a 96-well plate, 7000 cells per well (100 μl of 70 000 cells/mL), like on the graphic below. 32 wells total (8 wells of HepG2, 8 wells of WI38, 8 wells of A549, 8 wells of HEK293T).

![Diagram of cells seeding into 24-well plates](https://static.igem.wiki/teams/5865/experiments/cell-plating-for-optimising-transfections.webp)

Figure: Cell seeding layout for HepG2 and HEK293T cells (3 technical replicates per condition).

**Transfection forward**

Seed cells: 100 μL = 7 000 cells (per one well)

(Transfection will be performed next day when confluency reaches 70-80%)

**Transfection reverse**

Prepare transfection mixes according to the table below (amounts for 1 well). For each variant prepare 2 eppendorfs (one with diluted DNA and another with diluted lipofectamine), mix them well and then mix them together. Vortex and set for 15 minutes for lipofectamine-DNA complex to form.

![Diagram of cells seeding into 24-well plates](https://static.igem.wiki/teams/5865/experiments/transfection-reagents.webp)

Seed cells: 100 μL = 7 000 cells (per one well), add 10 μL of transfection mix to the well.

Put the plate into the incubator.

**Day 2**

**Transfection forward**

Prepare transfection mixes according to the Table 1. For each variant prepare 2 eppendorfs (one with diluted DNA and another with diluted lipofectamine), mix them well and then mix them together. Vortex and set for 15 minutes for lipofectamine-DNA complex to form.

Change medium in wells and then add 10 μL of transfection mix to each well. Put the plate into the incubator.

**Transfection reverse**

Replace medium in the wells with a fresh one.

**Day 3**

**Transfection forward**

Replace medium in the wells with a fresh one.

**Transfection reverse**

Observations using fluorescent microscope.

**Day 4**

**Transfection forward**

Observations using fluorescent microscope

Verification of AFP expression in selected human cell lines
+

##### Introduction

Since alpha-fetoprotein (AFP) is a marker of hepatocellular carcinoma and acts as the activating factor for the toehold switches designed in our study, we needed to verify whether AFP expression is elevated in other human cell lines besides HepG2.

This also served the purpose of selecting a cell line for the subsequent experiment involving transfection and flow cytometry, which was crucial for identifying the activity and selectivity of the toehold switches. We tested HEK293T and WI-38 cell lines, both derived from fetal cells, which could potentially exhibit elevated expression of alpha-fetoprotein, a hypothesis verified during this experiment. Additionally, we tested HeLa and A549 cell lines, as cancer cells may undergo neo-expression of inactive genes (Tronik-Le Roux et al., 2024).

Initially, total RNA was isolated from these cell lines, followed by reverse transcription and quantitative PCR (qPCR). GAPDH was used as a reference gene for normalization in the qPCR analysis.

##### Hypothesis

We hypothesize that alpha-fetoprotein (AFP) expression will be significantly elevated in HepG2 cells, a hepatocellular carcinoma cell line, compared to other tested human cell lines such as HEK293T, WI-38, HeLa, and A549. Furthermore, due to their fetal origin, HEK293T and WI-38 cells may also exhibit higher baseline AFP expression relative to other non-fetal cell lines.

##### Materials

**Reagents and Solutions for RT-qPCR**

* Luna Universal One-Step Reaction Mix (2X)
* Luna WarmStart RT Enzyme Mix (20X)
* Forward and reverse primers (10 μM) for target genes (AFP, GAPDH)
* Nuclease-free water

**Plastics and Consumables**

* Total RNA Mini Kit
* Optical 96- or 384-well plates or qPCR tubes with sealing film/caps
* Pipette tips (sterile, filtered)
* Fiber-free tissues

**Equipment**

* Microcentrifuge
* Incubator (50°C)
* NanoDrop UV-Vis spectrophotometer
* Real-time PCR instrument (SYBR/FAM detection)

**General Lab Supplies**

* Autoclaved nuclease-free water
* Pipettes
* Personal protective equipment (gloves, lab coat)

##### Workflow of experiment

Total RNA was isolated from HEK293T, WI-38, HepG2, and A549 cell lines using the Total RNA Mini Kit (A&A Biotechnology) following the RNA Isolation - Total RNA Mini protocol. For each isolation approximately 1×106 cells were collected.

Additionally, HeLa total RNA was provided by the Laboratory of the Molecular Biology of Cancer at the Centre of New Technologies, University of Warsaw to supplement the set of tested cell lines. RNA concentration and purity were measured using a NanoDrop spectrophotometer, ensuring accurate quantification and confirming sample quality (A260/280 ≈ 1.9-2.0).

For gene expression analysis, RT-qPCR was performed using the Luna Universal One-Step RT-qPCR Kit (New England Biolabs). This system enables reverse transcription and quantitative PCR amplification within a single reaction, minimizing contamination risk and improving reproducibility. Reaction mixtures (20 μL total volume) consisted of: 10 μL 2× Reaction Mix, 1 μL 20× RT Enzyme Mix, 0.8 μL each of forward and reverse primers (final 0.4 μM), 1–2 μL RNA (≤1 μg), and nuclease-free water to the final volume.

Before proceeding to the main experiment, primer standardization was performed using RNA from HepG2 cells at two annealing temperatures (57°C and 60°C) to determine optimal amplification conditions for AFP, GAPDH, and GSDMD-NT. The best amplification curves and specific single melting peaks were obtained at 60°C, which was subsequently used for all further analyses.

The standardized RT-qPCR reactions were then prepared using RNA isolated from all cell lines. Samples were pipetted into a 96-well plate as shown in the experimental layout pictured below. For the negative control, nuclease-free water was used instead of RNA or plasmid DNA. The reactions were run on a LightCycler instrument using the thermal profile summarized in the table below.

![Diagram of cells seeding into 24-well plates](https://static.igem.wiki/teams/5865/experiments/afp.webp)

After amplification, melting curve analysis was performed to confirm reaction specificity. The obtained amplification plots and melting curves were further analyzed, and detailed results are presented in the Results section.

First verification of toeholds
+

##### Introduction

Our project leverages toehold switch constructs encoded on plasmids to selectively detect and respond to the presence of alpha-fetoprotein (AFP) inside mammalian cells. We engineered 20 distinct plasmid variants, each bearing a different toehold switch design, and transfected them into two human cell lines: HepG2 and HEK293T.

HepG2 is a hepatocellular carcinoma cell line known to express high levels of AFP, while HEK293T exhibits negligible AFP expression under the same conditions. The toehold switches are designed such that upon recognition of intracellular AFP mRNA, they trigger expression of a lethal effector (pyroptosis-inducing cascade), thereby causing cell death. Thus, in cells with high AFP (HepG2) the toehold should be activated, whereas in cells low in AFP (HEK293T) it should remain inert.

By comparing cell survival in these two cell lines post-transfection, we aim to assess the specificity and efficacy of our toehold designs in recognizing endogenous AFP transcripts and inducing selective cytotoxicity.

##### Hypothesis

If our toehold switch plasmids are functioning as intended, then transfection into HepG2 cells will lead to activation of the toehold sensor by AFP mRNA, triggering the downstream effector and resulting in reduced viability (increased cell death) compared to controls. In contrast, HEK293T cells, with negligible AFP expression, should show minimal to no cell death, indicating specificity of the system.

##### Materials

**Cell lines**

* HepG2 (human hepatocellular carcinoma cell line)
* HEK293T (human embryonic kidney cell line)

**Plasmids**

* 20 plasmids containing toehold switch constructs (Plasmid 1–20)
* Control plasmids: GFP (midi prep)
* Mock DNA control (no DNA)

**Reagents and solutions**

* Opti-MEM™ Reduced Serum Medium
* Lipofectamine™ 3000 (Thermo Fisher)
* P3000™ reagent (Thermo Fisher)
* Sterile nuclease-free water (control condition)

**Plastics and consumables**

* 24-well tissue culture plates (at least 6)
* 15 mL conical Falcon tubes
* 1.5 mL microcentrifuge tubes (Eppendorf type)
* Sterile serological pipettes (5, 10, 25 mL)
* Sterile pipette tips (P10, P200, P1000 filter tips)

**Equipment**

* Biological safety cabinet (laminar flow hood)
* CO₂ incubator (37°C, humidified atmosphere with CO₂ control)
* Benchtop centrifuge (suitable for 1.5 mL and 15 mL tubes)
* Vortex mixer
* Micropipettes (P10, P200, P1000)
* Hemocytometer or automated cell counter (for seeding accuracy)
* Flow cytometer (for viability analysis)

**General lab supplies**

* Personal protective equipment (lab coat, gloves, protective eyewear)
* Waste containers for biohazard disposal
* Ice bucket

##### Cells seeding

**Protocol used: Cell culture protocol**

Seed six 24-well plates, 80000 cells per well (200 000 cells/mL), like on the graphic below. 132 wells total (66 wells of HEK293T and 66 wells of HepG2). This allows 3 technical repetitions per measurement.

![Diagram of cells seeding into 24-well plates](https://static.igem.wiki/teams/5865/experiments/cell-seeding.webp)

Figure: Cell seeding layout for HepG2 and HEK293T cells (3 technical replicates per condition).

##### Transfection

**Protocol used: Cell culture protocol, Transfection**

Transfect the cells with appropriate plasmid numbers. Additionally make two controls: one well per plate as a control with water instead of DNA and one well per plate as a control with no transfection.

Divide the transfection of the plates in two parts (part 1: plate1-3, part 2: plate 4-6) and conduct them with two hours break in between to ensure maximum credibility.

|  | Concentration | Volume containing 500 ng (for 1 well) | Volume containing 1.65 µg (for 3 wells with 10% excess) |
| --- | --- | --- | --- |
| Plasmid 1 | 235.7 | 2.12 | 7.00 |
| Plasmid 2 | 1254 | 0.40 | 1.32 |
| Plasmid 3 | 882 | 0.57 | 1.87 |
| Plasmid 4 | 1393.8 | 0.36 | 1.18 |
| Plasmid 5 | no plasmid obtained | | |  |  |
| Plasmid 6 | 612.8 | 0.82 | 2.69 |
| Plasmid 7 | 1122 | 0.45 | 1.47 |
| Plasmid 8 | 852.2 | 0.59 | 1.94 |
| Plasmid 9 | 1194 | 0.42 | 1.38 |
| Plasmid 10 | 839.4 | 0.60 | 1.97 |
| Plasmid 11 | 1142.3 | 0.44 | 1.44 |
| Plasmid 12 | 988.4 | 0.51 | 1.67 |
| Plasmid 13 | no plasmid obtained | | |  |  |
| Plasmid 14 | 1011 | 0.49 | 1.63 |
| Plasmid 15 | 994 | 0.50 | 1.66 |
| Plasmid 16 | 771.9 | 0.65 | 2.14 |
| Plasmid 17 | 679.2 | 0.74 | 2.43 |
| Plasmid 18 | 1063.5 | 0.47 | 1.55 |
| Plasmid 19 | 473.5 | 1.06 | 3.48 |
| Plasmid 20 | 1270 | 0.39 | 1.30 |
| Plasmid 21 | 1007 | 0.50 | 1.64 |
| (midi) GFP | 263.5 | 1.90 | 6.26 |
| Mock (no DNA) | 0 | 1 µL of H₂O | 3.3 µL of H₂O |
| No transfection | 0 | 1 µL of H₂O | 3.3 µL of H₂O |

Prepare two Eppendorfs of each sample containing 1,65 µg of DNA (for both parts of transfection).

Master mix preparations of P3000™ for all wells:
132 x 25 µL x 115% = 3795 µL of Opti-MEM
132 x 1 µL x 115% = 151.8 µL of P3000™

Master mix preparations of Lipofectamine™ 3000 dilution for all wells:
132 x 25 µL x 115% = 3795 µL of Opti-MEM
132 x 1.5 µL x 115% = 227.7 µL of Lipofectamine™ 3000

|  | Solution 1 | | | Solution 2 | |
| --- | --- | --- | --- | --- | --- |
| Reagent | Opti-MEM | P3000™ | DNA | Opti-MEM | Lipofectamine™ 3000 |
| Volume per 1 well | 25 µL | 1 µL | 500 ng | 25 µL | 1.5 µL |
| x6.6 | 165 µL | 6.6 µL | 3.3 µg | 165 µL | 9.9 µL |

###### Transfection Part 1.

To each Eppendorf containing 1,65 µg of DNA add 85,8 µL from Master mix of P3000™. Vortex and spin down.

To the solution of DNA and P3000™ add 87,45 µL from Master mix of Lipofectamine™ 3000 dilution. Vortex, spin down, incubate for 15 min.

Add each solution to the appropriate wells on the plates 1-3.
Amount: (volume of DNA + 173,25)/3,3 µL per well.

###### Transfection Part 2.

Start two hours after Transfection Part 1.

To each Eppendorf containing 1,65 µg of DNA add 85.8 µL from the Master mix of P3000™. Vortex and spin down.

To the solution of DNA and P3000™ add 87.45 µL from the Master mix of Lipofectamine™ 3000 dilution. Vortex, spin down, incubate for 15 min.

Add each solution to the appropriate wells on the plates 4-6.
Amount: (volume of DNA + 173.25)/3.3 µL per well.

##### Flow cytometry

Flow cytometric analysis was performed using a Beckman Coulter CytoFLEX SRT flow cytometer equipped with a 488 nm blue laser. GFP fluorescence was detected using the standard 525/40 nm channel, optimal for fluorescent proteins with an emission maximum around 509 nm.

**Sample preparation and data acquisition**

HepG2 (AFP+) and HEK293T (AFP–) cells containing toehold riboswitch constructs fused to the GFP gene were prepared according to standard protocols. Each cell suspension, centrifuged at 500g for 5min and suspended in 1mLl PBS medium, was analyzed. Each measurement included up to 5,000 single-cell events or continued until the sample was exhausted, whichever occurred first.

**Calibration and quality control**

Prior to analysis, quality control was performed using CytoFLEX Ready to Use Daily QC Fluorospheres-3 μm fluorescent microspheres dedicated to the CytoFLEX system. The acceptance criterion for the relative coefficient of variation (rCV) was set at a maximum of 5%. A standard gating strategy was applied, starting with the selection of single cells based on FSC-A/FSC-H and SSC-A/SSC-W parameters to exclude doublets and cell debris.

**Cleaning procedures**

After data acquisition, the cytometer was cleaned using BD Clean reagent for 3 minutes, followed by Contrad 70 solution for another 3 minutes, in accordance with the manufacturer’s protocols. This procedure ensured the removal of biological residues and prevented carry-over between samples.

**Data analysis**

GFP fluorescence was measured in the FITC channel (525/40 nm) with spillover compensation between channels performed according to standard procedures. Expression values were reported as relative fluorescence units (RFU). Data were analyzed using FCS Express software, with additional statistical analyses. Comparison of GFP expression levels between AFP+ and AFP– cell lines was based on mean fluorescence intensity (MFI)

**Results**

We analyzed a total of 144 samples using flow cytometry. The results and conclusions of the experiment are presented in the "Results" section.

#### Sequences

**Functional elements**

The table includes genetic elements and constructs designed for the production of the final product - a functional transcriptional unit containing the selected toehold switch.
It presents individual components as well as the complete vector intended for the cloning of specific toehold switches. The basic elements comprise sequences encoding fluorescent reporter proteins, which are used for direct testing of toehold switch specificity, as well as the sequence of the α-peptide of β-galactosidase (LacZ), enabling blue-white screening.

| Part Name | ID | Description | Sequences |
| --- | --- | --- | --- |
| Mammalian optimized eGFP | BBa\_25N3AWWS | Encodes eGFP protein, which emits green fluorescence when being excited with wavelengths between 450-490 nm. | ATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAA |
| Mammalian constitutive RFP reporter | BBa\_25VE60AA | Encodes full mammalian transcription unit, that lead to constitutive high expression of RFP in transfected mammalian cells. | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACATCATAAGGTTACGATAGGAGAGCAAGCCACCATGGCCGAGGGCTCCGTCGCCAGACAGCCTGACCTGCTGACCTGCGACGACGAGCCCATCCACATCCCTGGCGCTATCCAGCCCCATGGCCTGCTGCTGGCTCTGGCTGCCGACATGACAATCGTGGCTGGCTCCGACAACCTGCCTGAGCTGACCGGCCTGGCCATCGGCGCCCTGATCGGCAGATCTGCCGCCGATGTGTTCGACTCCGAAACCCACAACCGGCTGACCATCGCCCTCGCTGAGCCTGGCGCCGCCGTGGGCGCTCCTATCACAGTCGGCTTCACCATGAGAAAGGACGCCGGCTTCATCGGATCTTGGCACCGGCACGACCAGCTGATCTTCCTGGAACTGGAACCTCCTCAGCGGGACGTGGCCGAGCCCCAGGCCTTCTTCAGACGGACCAACTCCGCCATCAGACGGCTGCAGGCCGCTGAAACCCTGGAATCTGCTTGTGCTGCTGCCGCTCAAGAGGTGCGGAAGATCACCGGATTTGATAGAGTGATGATCTACAGATTCGCCAGCGACTTCTCCGGCTCTGTGATCGCCGAGGACAGATGCGCCGAAGTGGAATCCAAGCTGGGCCTGCACTACCCTGCCTCTTTCATCCCTGCTCAGGCTAGACGGCTGTACACCATCAATCCTGTGCGGATCATCCCAGACATCAACTACCGGCCCGTGCCTGTGACCCCTGATCTGAACCCCGTCACAGGCAGACCTATCGACCTGTCCTTTGCCATCCTGCGGTCCGTGTCTCCTAACCACCTGGAGTTCATGCGGAACATCGGCATGCACGGCACCATGTCCATCTCCATCCTGAGAGGCGAGCGGCTGTGGGGCCTCATCGTGTGCCACCACAGAACCCCTTACTACGTGGACCTGGACGGCCGGCAGGCCTGCGAGCTGGTGGCCCAGGTGCTGGCCTGGCAGATCGGCGTGATGGAAGAGTGACCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |
| TU1 with cloning site for 5' UTR and CDS | BBa\_25SH329F | Encodes transcription unit containing CMV promoter, strong 3' UTR and rabbit β-globin poly(A) signal. It contains cloning site for inserting 5' UTR and CDS using BsaI. Designed to be used to measure the translation under designed 5' UTR in mammalian cells. | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACGGAGACCGGTCTCTCCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |
| TU1 with cloning site for 5' UTR and CDS - LacZ | BBa\_254JMBEU | Encodes transcription unit containing CMV promoter, strong 3' UTR and rabbit β-globin poly(A) signal. It contains cloning site containing LacZ cassette for inserting 5' UTR and CDS and white-blue screening of colonies. Designed to be used to measure the translation under designed 5' UTR in mammalian cells. | GCTCTTCAAGGGCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACGGAGACCGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACGGTCTCTCCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACAACCTGAAGAGC |
| Toehold switch testing cassette | BBa\_25V2E0HW | Encodes a cassette for testing toehold switch activity in mammalian cells. Contain two transcription units - one with the cloning site for inserting toehold switch with the reporter protein of choice and another constitutively expressing RFP, as a control of transfection. | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACGGAGACCGGTCTCTCCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACAACCGCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACATCATAAGGTTACGATAGGAGAGCAAGCCACCATGGCCGAGGGCTCCGTCGCCAGACAGCCTGACCTGCTGACCTGCGACGACGAGCCCATCCACATCCCTGGCGCTATCCAGCCCCATGGCCTGCTGCTGGCTCTGGCTGCCGACATGACAATCGTGGCTGGCTCCGACAACCTGCCTGAGCTGACCGGCCTGGCCATCGGCGCCCTGATCGGCAGATCTGCCGCCGATGTGTTCGACTCCGAAACCCACAACCGGCTGACCATCGCCCTCGCTGAGCCTGGCGCCGCCGTGGGCGCTCCTATCACAGTCGGCTTCACCATGAGAAAGGACGCCGGCTTCATCGGATCTTGGCACCGGCACGACCAGCTGATCTTCCTGGAACTGGAACCTCCTCAGCGGGACGTGGCCGAGCCCCAGGCCTTCTTCAGACGGACCAACTCCGCCATCAGACGGCTGCAGGCCGCTGAAACCCTGGAATCTGCTTGTGCTGCTGCCGCTCAAGAGGTGCGGAAGATCACCGGATTTGATAGAGTGATGATCTACAGATTCGCCAGCGACTTCTCCGGCTCTGTGATCGCCGAGGACAGATGCGCCGAAGTGGAATCCAAGCTGGGCCTGCACTACCCTGCCTCTTTCATCCCTGCTCAGGCTAGACGGCTGTACACCATCAATCCTGTGCGGATCATCCCAGACATCAACTACCGGCCCGTGCCTGTGACCCCTGATCTGAACCCCGTCACAGGCAGACCTATCGACCTGTCCTTTGCCATCCTGCGGTCCGTGTCTCCTAACCACCTGGAGTTCATGCGGAACATCGGCATGCACGGCACCATGTCCATCTCCATCCTGAGAGGCGAGCGGCTGTGGGGCCTCATCGTGTGCCACCACAGAACCCCTTACTACGTGGACCTGGACGGCCGGCAGGCCTGCGAGCTGGTGGCCCAGGTGCTGGCCTGGCAGATCGGCGTGATGGAAGAGTGACCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |
| Backbone for assembly of 2 transcription units | BBa\_259T9MVI | This is a vector providing backbone for Golden Gate Assembly of 2 transcription units. Contains lacZ cassette allowing white-blue colony screening with LacZ and IPTG and chloramphenicol resistance gene. | AGGCTAGGTGGAGGCTCAGTGATGATAAGTCTGCGATGGTGGATGCATGTGTCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCAGAGGGCACAATCCTATTCCGCGCTATCCGACAATCTCCAAGACATTAGGTGGAGTTCAGTTCGGCGTATGGCATATGTCGCTGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAAATGGGTAGGGGGCTTCAAATCGTCCCCCCATACGATATAAGTTGTTACTAGTGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTGATACCTGAAACAAAACCCATCGTACGGCCAAGGAAGTCTCCAATAACTGTGATCCACCACAAGCGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTCATGCATAATCCGCACGCATCTGGAATAAGGAAGTGCCATTCCGCCTGACCTAGGTGAAGAGCGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACGCTCTTCACGA |
| LacZ cassette | BBa\_25DTJWTR | Encodes cassette expressing LacZ gene in the presence of IPTG in E. coli, what results in blue color of the colonies grown in the presence of X-Gal derived from BBa\_J433047. Modified with overhangs allowing direct cloning of it into BBa\_25LFVVO5. | CATGCAACGGAGACCGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACGGTCTCTCCCAGTAC |

**Toehold switches**

Table presents the sequences of twenty-one designed toehold switches, each engineered for the selective binding of the alpha-fetoprotein (AFP) transcript. Under physiological conditions, the transcripts of these sequences adopt a secondary structure that, upon interaction with the cognate agonist, unfolds to expose the Kozak sequence, thereby enabling translation initiation.

| Part Name | ID | Description | Sequences |
| --- | --- | --- | --- |
| AFP sensing toehold switch 1 | BBa\_25ZHOR52 | Encodes toehold switch, designed to form secondary structure, which blocks translation (OFF state). In the presence of AFP mRNA, it binds to the toehold switch and unwinds it, leading to the release of start codon and initiation of translation (ON state). | GCAGTAAGGTGGAATTATTTTGTCGTAGTGAGTAGTTTCCGGACCATTATTAAACACGGAAATTATTTATTACGCCACC |
| AFP sensing toehold switch 2 | BBa\_250CZ1N4 | CTAAAGAAGAATTGTAGGTGTATATGGGGGGGGATGTTCCGCACTAATTACACCCACGGAATATTCTCTCTCAGCCACC |
| AFP sensing toehold switch 3 | BBa\_25CBV2KZ | TGGTTGTTAGTTGTTTTGTTATTGGTAGTGGTGGAAGTTGCTACCTCAATAAACCTGCAATTTCTATCACTACGCCACC |
| AFP sensing toehold switch 4 | BBa\_253W34O5 | TGTTTTGTTGTTTGTAGTGTTATATCTTGAGTTTGGTGTCCTCACCCTACTAACCCGGACATTAAATTCAAGAGCCACC |
| AFP sensing toehold switch 5 | BBa\_258PVAE5 | CTGCATGAATTATACGTTGACCATGTTTTAGTGTGGTTGCTCCAAACACACCCAACAGCAATTACATTAAGACGCCACC |
| AFP sensing toehold switch 6 | BBa\_25LTGU9W | GTTTCTTTTTTATGGCAAAGTTCTTTTAGGAGGGTGGGGTCAAATCAATTAAACCAGACTTCATTCTTCTAAAGCCACC |
| AFP sensing toehold switch 7 | BBa\_25KMR72M | CACGCCGAATGAAAGACTTGTTTTGTTTTTTCTTCCTTTCCACACTCAGAATAACAGGAAAGGGAGAAAGAGCGCCACC |
| AFP sensing toehold switch 8 | BBa\_25EK59Z3 | CTTTTTTATGGCAAAGTTCTTTTAGGGGGGTGGGTAGTCCCTCAATCTTACACACTGGGACTATTTACTTCTCGCCACC |
| AFP sensing toehold switch 9 | BBa\_2536A8PW | GTGAGATGGGATTGTTTTGTTATAGTGAGTAGTTTGTCGCACCAACACAACCGACGAATTACTTACGCCACC |
| AFP sensing toehold switch 10 | BBa\_252Z2H1Q | GTGAGATGGGATTGTTTTGTTATAGTGAGTAGTTTGTGCTAACCCATCCACGCACGAATTATTTACGCCACC |
| AFP sensing toehold switch 11 | BBa\_25ZTWO4B | GAGTGAATTTGTTGTTAGAGAATGTAGGAGGGATATGTGCGGACCAATTAATTAAACGCGTATATTTCTCTTAGCCACC |
| AFP sensing toehold switch 12 | BBa\_25MXPZ2S | GTAGTTTGTCCTTATTGAGTTGGCGACGGGTGGTTGTTATTCACCATCCACAATCAAATAATAGTTACTCGTCGCCACC |
| AFP sensing toehold switch 13 | BBa\_257KS0RB | GTAGTTTGTTTTTATTGAGTTGGCGGCAGGTGGTTGTTGGGTTAATAACACACACTCCCAATAATTATTTGTCGCCACC |
| AFP sensing toehold switch 14 | BBa\_25RHES30 | GGACGTGATTTTTTTTCTATTTTGTAGATAATTTAGTGGACCACCAAACCCACAAAGTCCATTAAATTATTTAGCCACC |
| AFP sensing toehold switch 15 | BBa\_25Q1KWBC | TGTTGTTTTTTTGGTGATGGTCATTGGTTCTGATGGCGGTCACTTATCACTCCGCTATTAGAATCAGCCACC |
| AFP sensing toehold switch 16 | BBa\_25OJUJ4Z | GAAGGGTTGTAGGTGCATATAGGGGGGGGTGCTTTCGGCACCATTATCTAAGCCGGGAGCACTTTCGCCACC |
| AFP sensing toehold switch 17 | BBa\_25LKZVOU | TTCAGTAAAGTTAATTTTGGTAAATTTTTGATTTAGTTGACTACCACATCAATACTGTCAATTAAATTAAAGAGCCACC |
| AFP sensing toehold switch 18 | BBa\_250L4JGE | AGTTAAAGTTAGAGAGAAAAGTTTATATTGAATGAAGGCGCAAACCATCAAACAACGCGTCTTTATTTAATATGCCACC |
| AFP sensing toehold switch 19 | BBa\_25QCCD17 | CAATAGTGTTCGTGTATATGGGCTATATTCGGGATTCCCAAACAACAACCAGGGAATTTCGAATATGCCACC |
| AFP sensing toehold switch 20 | BBa\_25ZKTTE9 | TTCTTCAATAACTTCTGGTATTTTTTAGTGATTTTTAGGGCCATTCATAAACACACGCCTTGGGAATCATTAAGCCACC |
| AFP sensing toehold switch 21 | BBa\_25W42RKX | GTGTTTTAAAATGTCTGTGAATAGAGAATTGAATAAGGGCCCACAATCAATCACCAGGCCTTTATTTAATTCTGCCACC |

**Toehold switch sequences associated with the coding region of the eGFP**

The table presents toehold switch sequences associated with the coding region of the enhanced Green Fluorescent Protein (eGFP), whose product serves as a reporter protein for the direct evaluation of toehold switch performance. Under physiological conditions, toehold switches adopt secondary structures that prevent the expression of eGFP. Upon the appearance of an agonist in the form of an alpha-fetoprotein (AFP) transcript, the hairpin structure unfolds, thereby enabling the initiation of eGFP translation.

| Part Name | ID | Description | Sequences |
| --- | --- | --- | --- |
| AFP sensing toehold switch 1 + eGFP | BBa\_255KXS8E | Encodes toehold switch, designed to form secondary structure, which blocks translation of eGFP (OFF state). When AFP mRNA is present, it binds to the toehold switch and unwinds it, leading to the release of start codon and initiation of translation of eGFP (ON state). | GCAGTAAGGTGGAATTATTTTGTCGTAGTGAGTAGTTTCCGGACCATTATTAAACACGGAAATTATTTATTACGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAA |
| AFP sensing toehold switch 2 + eGFP | BBa\_25OHWS9U | CTAAAGAAGAATTGTAGGTGTATATGGGGGGGGATGTTCCGCACTAATTACACCCACGGAATATTCTCTCTCAGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAA |
| AFP sensing toehold switch 3 + eGFP | BBa\_25P40TUI | TGGTTGTTAGTTGTTTTGTTATTGGTAGTGGTGGAAGTTGCTACCTCAATAAACCTGCAATTTCTATCACTACGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAA |
| AFP sensing toehold switch 4 + eGFP | BBa\_25WKB8EQ | TGTTTTGTTGTTTGTAGTGTTATATCTTGAGTTTGGTGTCCTCACCCTACTAACCCGGACATTAAATTCAAGAGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAA |
| AFP sensing toehold switch 5 + eGFP | BBa\_25DE4PM1 | CTGCATGAATTATACGTTGACCATGTTTTAGTGTGGTTGCTCCAAACACACCCAACAGCAATTACATTAAGACGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAA |
| AFP sensing toehold switch 6 + eGFP | BBa\_25LLAQMP | GTTTCTTTTTTATGGCAAAGTTCTTTTAGGAGGGTGGGGTCAAATCAATTAAACCAGACTTCATTCTTCTAAAGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAA |
| AFP sensing toehold switch 7 + eGFP | BBa\_258EGC7I | CACGCCGAATGAAAGACTTGTTTTGTTTTTTCTTCCTTTCCACACTCAGAATAACAGGAAAGGGAGAAAGAGCGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAA |
| AFP sensing toehold switch 8 + eGFP | BBa\_25M4C8KD | CTTTTTTATGGCAAAGTTCTTTTAGGGGGGTGGGTAGTCCCTCAATCTTACACACTGGGACTATTTACTTCTCGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAA |
| AFP sensing toehold switch 9 + eGFP | BBa\_25JN4KH4 | GTGAGATGGGATTGTTTTGTTATAGTGAGTAGTTTGTCGCACCAACACAACCGACGAATTACTTACGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAA |
| AFP sensing toehold switch 10 + eGFP | BBa\_25CAXBKO | GTGAGATGGGATTGTTTTGTTATAGTGAGTAGTTTGTGCTAACCCATCCACGCACGAATTATTTACGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAA |
| AFP sensing toehold switch 11 + eGFP | BBa\_25LVR6GD | GAGTGAATTTGTTGTTAGAGAATGTAGGAGGGATATGTGCGGACCAATTAATTAAACGCGTATATTTCTCTTAGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAA |
| AFP sensing toehold switch 12 + eGFP | BBa\_25T247XK | GTAGTTTGTCCTTATTGAGTTGGCGACGGGTGGTTGTTATTCACCATCCACAATCAAATAATAGTTACTCGTCGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAA |
| AFP sensing toehold switch 13 + eGFP | BBa\_25C53YTI | GTAGTTTGTTTTTATTGAGTTGGCGGCAGGTGGTTGTTGGGTTAATAACACACACTCCCAATAATTATTTGTCGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAA |
| AFP sensing toehold switch 14 + eGFP | BBa\_256TOU7T | GGACGTGATTTTTTTTCTATTTTGTAGATAATTTAGTGGACCACCAAACCCACAAAGTCCATTAAATTATTTAGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAA |
| AFP sensing toehold switch 15 + eGFP | BBa\_25X549TG | TGTTGTTTTTTTGGTGATGGTCATTGGTTCTGATGGCGGTCACTTATCACTCCGCTATTAGAATCAGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAA |
| AFP sensing toehold switch 16 + eGFP | BBa\_25XWVEXZ | GAAGGGTTGTAGGTGCATATAGGGGGGGGTGCTTTCGGCACCATTATCTAAGCCGGGAGCACTTTCGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAA |
| AFP sensing toehold switch 17 + eGFP | BBa\_25W6KU7P | TTCAGTAAAGTTAATTTTGGTAAATTTTTGATTTAGTTGACTACCACATCAATACTGTCAATTAAATTAAAGAGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAA |
| AFP sensing toehold switch 18 + eGFP | BBa\_25KWUOMK | AGTTAAAGTTAGAGAGAAAAGTTTATATTGAATGAAGGCGCAAACCATCAAACAACGCGTCTTTATTTAATATGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAA |
| AFP sensing toehold switch 19 + eGFP | BBa\_25YFIIUQ | CAATAGTGTTCGTGTATATGGGCTATATTCGGGATTCCCAAACAACAACCAGGGAATTTCGAATATGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAA |
| AFP sensing toehold switch 20 + eGFP | BBa\_25M17XDK | TTCTTCAATAACTTCTGGTATTTTTTAGTGATTTTTAGGGCCATTCATAAACACACGCCTTGGGAATCATTAAGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAA |
| AFP sensing toehold switch 21 + eGFP | BBa\_25NVO2ZB | GTGTTTTAAAATGTCTGTGAATAGAGAATTGAATAAGGGCCCACAATCAATCACCAGGCCTTTATTTAATTCTGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAA |
| 5UTR13 + eGFP | BBa\_253UBWIF | Encodes eGFP under the control of strong 5' UTR. | ATCATAAGGTTACGATAGGAGAGCAAGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAA |

**qPCR primers**

The table presents primers used in qPCR reactions designed to evaluate the expression levels of alpha-fetoprotein (AFP) and gasdermin D (GSDMD) in the analyzed cell lines. AFP serves as a tumor marker and functions as an agonist for the designed toehold switches. Gasdermin D, on the other hand, is an effector protein that induces pyroptosis, with its expression regulated through a corresponding toehold switch. The table also includes primers for a reference gene, which was used to normalize and determine the relative expression levels of the analyzed genes.

| Part Name | ID | Description | Sequences |
| --- | --- | --- | --- |
| AFP\_F | BBa\_25QPUUES | Forward primer designed to amplify the AFP gene. The primer is 20 nucleotides long, with a melting temperature (Tm) of approximately 58.0 °C and a GC content of 50.0%. It exhibits very low self-complementarity (2.0) and no 3′ self-complementarity (0.0), minimizing the risk of primer-dimer formation. This primer is expected to specifically anneal to the AFP target sequence under standard PCR conditions, ensuring efficient and reliable amplification of the gene fragment. | TTACACAAAGAAAGCCCCCC |
| AFP\_R | BBa\_2527BQKZ | Reverse primer designed to amplify the AFP gene. The primer is 22 nucleotides long, with a melting temperature (Tm) of approximately 59.3 °C and a GC content of 50.0%. It demonstrates moderate self-complementarity (5.0) and low 3′ self-complementarity (1.0), minimizing the risk of primer-dimer formation and ensuring stable binding during amplification. This reverse primer is expected to specifically anneal to the complementary strand of the AFP target sequence under standard PCR conditions, enabling efficient and accurate amplification of the gene fragment in combination with the AFP forward primer. | CGATAATAATGTCAGCCGCTCC |
| GSDMD-NT\_F | BBa\_25GRWIBZ | The GSDMD-NT forward primer is designed to specifically anneal to the N-terminal region of the Gasdermin D (GSDMD) gene, enabling its efficient amplification in PCR-based assays. This region encodes the pore-forming domain responsible for pyroptosis activation. The primer’s optimal thermodynamic properties and low self-complementarity ensure high specificity and stable binding, making it suitable for use in gene expression studies, functional assays, and molecular analyses investigating the role of GSDMD in inflammatory cell death pathways. | CTGATAGATTCCGCTGCTTCC |
| GSDMD-NT\_R | BBa\_25CKF5M2 | The GSDMD-NT reverse primer is designed to specifically anneal to the complementary strand of the N-terminal region of the Gasdermin D (GSDMD) gene, enabling precise and efficient amplification of the target fragment in PCR assays. With optimal thermodynamic parameters and minimal self-complementarity, this primer ensures stable binding and reduces the risk of primer-dimer formation. When used together with the corresponding forward primer, it allows accurate detection and quantification of GSDMD-NT expression, supporting studies focused on pyroptosis, inflammatory signaling, and cell death mechanisms at the molecular level. | GTTCATCTAACCACTTGTCCCC |
| GAPDH\_F | BBa\_25GHP2EI | The GAPDH forward primer is designed to specifically bind to the housekeeping gene GAPDH, enabling stable and reproducible amplification of its mRNA (after reverse transcription) or genomic fragment in PCR assays. With a length of 21 nucleotides, a melting temperature (Tm) of about 59.65 °C, and a GC content of 47.62%, the primer ensures a strong yet balanced binding affinity to the target sequence. Its low self-complementarity score (3.0) and zero 3′ self-complementarity (0.0) minimize the risk of primer-dimer formation or internal secondary structure interference, particularly at the critical 3′ end. These favorable thermodynamic features help maintain efficient and specific annealing, making this primer suitable for quantitative real-time PCR (qRT-PCR) and gene expression normalization in diverse samples and experimental settings, as described in the referenced study. | AAGGTCGGAGTCAACGGATTT |
| GAPDH\_R | BBa\_25M1Z082 | The GAPDH reverse primer, reported in PMC9192978, anneals to the complementary strand of the GAPDH gene to enable accurate amplification in PCR and qRT-PCR assays. With a length of 21 nucleotides, Tm of 57.99 °C, and GC content of 47.62%, it ensures stable and efficient binding. Its low self-complementarity (3.0) and 3′ self-complementarity (2.0) reduce dimer formation, supporting reliable use in gene expression normalization and reference gene analysis. | AGATGATGACCCTTTTGGCTC |

**Cloning Primers**

The table lists the primers used for the isolation of genetic elements, their modification with appropriate flanking sequences facilitating the cloning process, and for the verification of the resulting genetic constructs.

| Part Name | ID | Description | Sequences |
| --- | --- | --- | --- |
| Colony\_PCR\_F | BBa\_25YFS6TE | Forward primer for colony PCR binding to the part of rabbit β-globin poly(A) sequence. Used for colony PCR to check the presence of mammalian transcription units and if the insert is in them or not. | CGTTTAGTGAACCGTCAGATC |
| Colony\_PCR\_R | BBa\_25CMDKAL | Reverse primer for colony PCR binding to the part of CMV promoter sequence. Used for colony PCR to check the presence of mammalian transcription units and if the insert is in them or not. | GTGGTATTTGTGAGCCAGGG |
| RFPseq | BBa\_25JMQ293 | Primer for sequencing and colony PCR specific to RFP containing transcription unit. Tested on the colonies with transcription unit with RFP. | GTTAGGAGACACGGACCGC |
| LacZ\_F | BBa\_2586BTCP | This primer was designed to amplify lacZ cassette, used for blue-white screening of *E. coli* colonies, from plasmid BBa\_259T9MVI. It contains overhang allowing PCR product to be cloned into BBa\_252RGHOZ using BsaI digestion and ligation. | CATGCAACGGAGACCGCAGCTGGCACGACAGGTTTC |
| LacZ\_R | BBa\_25928ERZ | This primer was designed to amplify lacZ cassette, used for blue-white screening of *E. coli* colonies, from plasmid BBa\_259T9MVI. It contains overhang allowing PCR product to be cloned into BBa\_252RGHOZ using BsaI digestion and ligation. | GTACTGGGAGAGACCGTCACAGCTTGTCTGTAAGCG || TU2\_F | BBa\_258AF7WU | Primer specific to the beginning of BBa\_25VE60AA allowing amplification of it. | GCTCTTCAACCGCTTAGTAATC |
| TU2\_R | BBa\_252JUY03 | Primer specific to the end of BBa\_25VE60AA allowing amplification of it. | GCTCTTCATCGTGTAATCTCC |
| TU2\_OV\_F | BBa\_256US3FC | Primer to amplify pTwist Amp High Copy vector with 20-nt overhangs on the ends, complementary to the beginning of BBa\_25VE60AA. This allows for it to serve as a destination vector for BBa\_25VE60AA during HiFi cloning. | TTACTAAGCGGTTGAAGAGCAGGTCAGGCGGAATGGCACTTC |
| TU2\_OV\_R | BBa\_252XEY4S | Primer to amplify pTwist Amp High Copy vector with 20-nt overhang on the ends, complementary to the end of BBa\_25VE60AA. This allows for it to serve as a destination vector for BBa\_25VE60AA during HiFi cloning. | GAGATTACACGATGAAGAGCAGGCTAGGTGGAGGCTCAGTG |

**Transcriptional units incorporating toehold switches positioned upstream of eGFP**

The table presents functional transcriptional units incorporating toehold switches positioned upstream of eGFP (BBa\_25N3AWWS). Each construct also includes a promoter sequence (BBa\_J433000), a 3' UTR (BBa\_J433018), and a poly(A) sequence (BBa\_J433023). These constructs constitute a ready-to-use platform for eGFP expression regulated by the specific characteristics of the toehold switch.

| Part Name | ID | Description | Sequence |
| --- | --- | --- | --- |
| AFP sensing toehold switch eGFP TU 1 | BBa\_25AFWI65 | Encodes toehold switch containing transcriptional unit, designed to form secondary structure, which blocks translation of eGFP (OFF state). When AFP mRNA is present, it binds to the toehold switch and unwinds it, leading to the release of start codon and initiation of translation of eGFP (ON state). | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACGCAGTAAGGTGGAATTATTTTGTCGTAGTGAGTAGTTTCCGGACCATTATTAAACACGGAAATTATTTATTACGCCACCATGATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAACCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |
| AFP sensing toehold switch eGFP TU 2 | BBa\_258INMBJ | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACCTAAAGAAGAATTGTAGGTGTATATGGGGGGGGATGTTCCGCACTAATTACACCCACGGAATATTCTCTCTCAGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAACCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |
| AFP sensing toehold switch eGFP TU 3 | BBa\_253ILJRI | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACTGGTTGTTAGTTGTTTTGTTATTGGTAGTGGTGGAAGTTGCTACCTCAATAAACCTGCAATTTCTATCACTACGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAACCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |
| AFP sensing toehold switch eGFP TU 4 | BBa\_250VNI4E | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACTGTTTTGTTGTTTGTAGTGTTATATCTTGAGTTTGGTGTCCTCACCCTACTAACCCGGACATTAAATTCAAGAGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAACCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |
| AFP sensing toehold switch eGFP TU 5 | BBa\_259WW1OO | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACCTGCATGAATTATACGTTGACCATGTTTTAGTGTGGTTGCTCCAAACACACCCAACAGCAATTACATTAAGACGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAACCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |
| AFP sensing toehold switch eGFP TU 6 | BBa\_25PU5Q27 | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACGTTTCTTTTTTATGGCAAAGTTCTTTTAGGAGGGTGGGGTCAAATCAATTAAACCAGACTTCATTCTTCTAAAGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAACCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |
| AFP sensing toehold switch eGFP TU 7 | BBa\_25X1NIS4 | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACCACGCCGAATGAAAGACTTGTTTTGTTTTTTCTTCCTTTCCACACTCAGAATAACAGGAAAGGGAGAAAGAGCGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAACCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |
| AFP sensing toehold switch eGFP TU 8 | BBa\_2584PTI1 | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACCTTTTTTATGGCAAAGTTCTTTTAGGGGGGTGGGTAGTCCCTCAATCTTACACACTGGGACTATTTACTTCTCGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAACCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |
| AFP sensing toehold switch eGFP TU 9 | BBa\_252V0MM7 | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACGTGAGATGGGATTGTTTTGTTATAGTGAGTAGTTTGTCGCACCAACACAACCGACGAATTACTTACGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAACCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |
| AFP sensing toehold switch eGFP TU 10 | BBa\_25GC8TPI | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACGTGAGATGGGATTGTTTTGTTATAGTGAGTAGTTTGTGCTAACCCATCCACGCACGAATTATTTACGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAACCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |
| AFP sensing toehold switch eGFP TU 11 | BBa\_25LY4UR8 | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACGAGTGAATTTGTTGTTAGAGAATGTAGGAGGGATATGTGCGGACCAATTAATTAAACGCGTATATTTCTCTTAGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAACCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |
| AFP sensing toehold switch eGFP TU 12 | BBa\_25ESVXUM | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACGTAGTTTGTCCTTATTGAGTTGGCGACGGGTGGTTGTTATTCACCATCCACAATCAAATAATAGTTACTCGTCGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAACCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |
| AFP sensing toehold switch eGFP TU 13 | BBa\_25X4CT6Z | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACGTAGTTTGTTTTTATTGAGTTGGCGGCAGGTGGTTGTTGGGTTAATAACACACACTCCCAATAATTATTTGTCGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAACCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |
| AFP sensing toehold switch eGFP TU 14 | BBa\_25W6GSVV | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACGGACGTGATTTTTTTTCTATTTTGTAGATAATTTAGTGGACCACCAAACCCACAAAGTCCATTAAATTATTTAGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAACCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |
| AFP sensing toehold switch eGFP TU 15 | BBa\_25O0IG16 | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACTGTTGTTTTTTTGGTGATGGTCATTGGTTCTGATGGCGGTCACTTATCACTCCGCTATTAGAATCAGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAACCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |
| AFP sensing toehold switch eGFP TU 16 | BBa\_25UN9KGQ | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACGAAGGGTTGTAGGTGCATATAGGGGGGGGTGCTTTCGGCACCATTATCTAAGCCGGGAGCACTTTCGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAACCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |
| AFP sensing toehold switch eGFP TU 17 | BBa\_250PAGEP | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACTTCAGTAAAGTTAATTTTGGTAAATTTTTGATTTAGTTGACTACCACATCAATACTGTCAATTAAATTAAAGAGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAACCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |
| AFP sensing toehold switch eGFP TU 18 | BBa\_25W81Z1F | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACAGTTAAAGTTAGAGAGAAAAGTTTATATTGAATGAAGGCGCAAACCATCAAACAACGCGTCTTTATTTAATATGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAACCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |
| AFP sensing toehold switch eGFP TU 19 | BBa\_250I00Z9 | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACCAATAGTGTTCGTGTATATGGGCTATATTCGGGATTCCCAAACAACAACCAGGGAATTTCGAATATGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAACCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |
| AFP sensing toehold switch eGFP TU 20 | BBa\_25OE6E8R | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACTTCTTCAATAACTTCTGGTATTTTTTAGTGATTTTTAGGGCCATTCATAAACACACGCCTTGGGAATCATTAAGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAACCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |
| AFP sensing toehold switch eGFP TU 21 | BBa\_254JLP5R | GCTTAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCAACGTGTTTTAAAATGTCTGTGAATAGAGAATTGAATAAGGGCCCACAATCAATCACCAGGCCTTTATTTAATTCTGCCACCATGGTGTCAAAGGGCGAAGAACTCTTTACTGGTGTTGTCCCCATCCTGGTCGAGCTCGACGGTGATGTCAACGGTCACAAGTTTTCTGTTTCCGGCGAGGGAGAGGGAGACGCTACGTACGGAAAACTCACGCTTAAGTTTATATGCACAACTGGTAAGCTTCCAGTGCCCTGGCCAACCCTTGTAACTACACTTACATATGGAGTGCAATGCTTTTCAAGATACCCTGACCATATGAAACAGCATGATTTCTTCAAGTCAGCCATGCCCGAGGGTTATGTCCAAGAAAGAACAATCTTCTTTAAAGATGATGGCAACTATAAAACTCGAGCAGAAGTTAAGTTCGAAGGGGACACCCTCGTGAACCGTATTGAGTTGAAGGGAATTGATTTCAAGGAAGATGGAAATATCCTTGGCCATAAGCTGGAGTATAACTACAATTCACACAATGTCTATATCATGGCGGATAAGCAAAAGAATGGAATTAAGGTCAACTTCAAGATCCGGCATAACATCGAGGATGGTTCCGTGCAACTCGCCGACCATTATCAACAAAATACCCCGATAGGTGATGGCCCAGTTCTGTTACCCGATAATCACTATTTGTCCACCCAATCCGCCCTCAGTAAGGATCCAAATGAGAAACGGGACCATATGGTCCTCTTAGAGTTCGTGACGGCCGCGGGAATTACATTAGGGATGGATGAATTGTATAAGTAATAACCCACCACTGGTTTCTACATTTACGAGTATGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATTACA |

#### References

In this section we list the main sources and protocols that we used as references when designing and carrying out our experiments. These documents provided us with standardized procedures and ensured reproducibility of our work. However, our final protocols are not simple copies-they are the result of adapting these resources through our own laboratory experience, discussions with experts (Human Practices), and guidance from the research groups and laboratories where we were trained. In this way, our protocols combine established methodologies with insights gained from practical, hands-on work. Access to all online resources was confirmed on 08 August 2025.

1. ##### Bacterial protocols

   1. 5 Minute Transformation Protocol.
      New England Biolabs.
      [https://www.neb.com/.../5-minute-transformation-protocol-c2987](https://www.neb.com/en/protocols/0001/01/01/5-minute-transformation-protocol-c2987)
   2. 14 Minute Transformation Protocol.
      New England Biolabs.
      [https://www.neb.com/.../14-minute-transformation-protocol](https://www.neb.com/en/protocols/2015/05/19/14-minute-transformation-protocol-c2987h-c2987i?pdf=true)
   3. High Efficiency Transformation Protocol.
      New England Biolabs.
      [https://www.neb.com/.../high-efficiency-transformation](https://www.neb.com/en-sg/protocols/2012/05/29/high-efficiency-transformation-protocol-for-competent-e-coli?pdf=true)
   4. Using Colony PCR to Identify Positive Clones (supplemental protocol, NEB #M0689).
      New England Biolabs.
      [https://www.neb.com/.../colony-pcr-positive-clones](https://www.neb.com/en-us/protocols/2023/08/11/supplemental-protocol-2-using-colony-pcr-to-identify-positive-clones-neb-m0689)
   5. How to Make Competent Cells: Protocols and Tips.
      Zymo Research.
      [https://zymoresearch.eu/.../competent-cells](https://zymoresearch.eu/blogs/blog/how-to-make-competent-cells-protocols-and-tips)
2. ##### Cell culture protocols

   1. Cell screening, maintenance, freezing and thawing procedures (in-house). Laboratory of Epitranscriptomics, Faculty of Biology, University of Warsaw; adapted based on our practical experience.
   2. General Transfection Protocol for Mammalian Cells (Lipofectamine 3000).
      Thermo Fisher Scientific.
      [https://www.thermofisher.com/.../jc-cells-protocol.html](https://www.thermofisher.com/us/en/home/references/protocols/cell-culture/transfection-protocol/jc-cells-protocol.html)

   ###### Flow cytometry – cell preparation

   1. Cossarizza A, Chang HD, Radbruch A, *et al.* Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition).
      *Eur J Immunol.* 2021;51(12):2708–3145. doi:[10.1002/eji.202170126](https://doi.org/10.1002/eji.202170126)
   2. Abrahamsen I, Lorens JB. Evaluating Extracellular Matrix influence on adherent cell signaling by Cold Trypsin Phosphorylation-specific Flow Cytometry.
      *BMC Cell Biol* 2013;14:36.
      doi:[10.1186/1471-2121-14-36](https://doi.org/10.1186/1471-2121-14-36)
3. ##### Molecular biology protocols

   ###### Golden Gate Assembly – BsaI

   1. Golden Gate 2–4 Fragment Assembly Protocol.
      New England Biolabs.
      [https://www.neb.com/.../golden-gate-24-fragment-assembly](https://www.neb.com/en/protocols/2018/06/05/golden-gate-24-fragment-assembly-protocol)
   2. Bird JE, Marles-Wright J, Giachino A. A user’s guide to Golden Gate cloning methods and standards.
      *ACS Synth Biol.* 2022;11(11):3551–3563.
   3. Engler C, Marillonnet S. Golden Gate cloning. In: *DNA Cloning and Assembly Methods.* Totowa, NJ: Humana Press; 2013:119–131.

   ###### Golden Gate Assembly – SapI

   1. 1–3 Fragment Golden Gate Assembly with SapI.
      New England Biolabs.
      [https://www.neb.com/.../golden-gate-assembly-with-sapi](https://www.neb.com/en/protocols/2021/01/11/13-fragment-golden-gate-assembly-with-sapi)

   ###### Other enzymatic protocols

   1. NEBuilder HiFi DNA Assembly – Reaction Protocol.
      New England Biolabs.
      [https://www.neb.com/.../nebuilder-hifi-dna-assembly](https://www.neb.com/en/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol)
   2. Restriction Digest – BsaI.
      New England Biolabs.
      [https://www.neb.com/.../restriction-digest-protocol](https://www.neb.com/en/protocols/2018/07/30/restriction-digest-protocol)
   3. T4 DNA Ligase Protocol.
      New England Biolabs.
      [https://www.neb.com/.../dna-ligation-with-t4-dna-ligase](https://www.neb.com/en/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202?srsltid=AfmBOooAF0JgF3rlipeQ8bvTreHcAYvR4bzyVur--mWU_R2LA2-MYSR3)
   4. HiScribe T7 High Yield RNA Synthesis Kit Manual (E2040).
      New England Biolabs.
      [manualE2040.pdf](https://www.neb.com/en/-/media/nebus/files/manuals/manuale2040.pdf)
   5. PCR using Q5® High-Fidelity DNA Polymerase (M0491).
      New England Biolabs.
      [https://www.neb.com/.../pcr-using-q5-high-fidelity](https://www.neb.com/en/protocols/2013/12/13/pcr-using-q5-high-fidelity-dna-polymerase-m0491)
   6. Luna Universal One-Step RT-qPCR Kit (E3005).
      New England Biolabs.
      [https://www.neb.com/.../e3005-luna-universal-one-step-rt-qpcr](https://www.neb.com/en/products/e3005-luna-universal-one-step-rt-qpcr-kit)
   7. Oligo handling and primer treatment.
      Thermo Fisher Scientific.
      [https://www.thermofisher.com/.../oligo-protocols.html](https://www.thermofisher.com/pl/en/home/life-science/oligonucleotides-primers-probes-genes/custom-dna-oligos/oligo-technical-resources/oligo-protocols.html)
4. ##### Nucleic acids purification and quantification

   1. Clean-up Concentrator.
      A&A Biotechnology.
      <https://www.aabiot.com/en/clean-up-concentrator>
   2. DNA concentration measurement.
      Addgene.
      <https://www.addgene.org/protocols/dna-quantification/>
   3. DNA gel electrophoresis.
      Addgene.
      <https://www.addgene.org/protocols/gel-electrophoresis/>
   4. Gel-Out – DNA Gel Extraction.
      A&A Biotechnology.
      <https://www.aabiot.com/gel-out>
   5. Plasmid Midi AX Endotoxin-Free.
      A&A Biotechnology.
      <https://www.aabiot.com/en/plasmid-midi-ax-endotoxin-free>
   6. Plasmid Mini.
      A&A Biotechnology.
      <https://www.aabiot.com/plasmid-minii>
   7. RNA concentration measurement.
      Thermo Fisher Scientific.
      [https://www.thermofisher.com/.../quantitating-rna.html](https://www.thermofisher.com/pl/en/home/references/ambion-tech-support/rna-isolation/tech-notes/quantitating-rna.html)
   8. Total RNA Mini – RNA Isolation.
      A&A Biotechnology.
      <https://www.aabiot.com/total-rna-mini>

{% endblock %}

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![Results graphic](https://static.igem.wiki/teams/5865/banners/h-results.avif)

# Results

![ikona 1](https://static.igem.wiki/teams/5865/icons/results1.webp)

Successfully constructed and validated toehold switches through a Golden Gate cloning approach.

![ikona 2](https://static.igem.wiki/teams/5865/icons/results2.webp)

Optimized mammalian cell culture protocols enabled reliable testing of our constructs in both AFP-positive and AFP-negative cell lines.

![ikona 3](https://static.igem.wiki/teams/5865/icons/results3.webp)

Our first flow cytometry and qPCR experiments provided promising indications that selected toehold switches may function as intended, laying the groundwork for further validation.

* [**CHAPTER I: CLONING**](#ch1-chapter)
* [TOEHOLD SWITCH ASSEMBLY](#ch1-1)
  + [Introduction](#ch1-1-intro)
  + [Aim of this subproject](#ch1-1-aim)
  + [Key Steps and results](#ch1-1-steps)
  + [Discussion](#ch1-1-discussion)
* [DISTRIBUTION KIT](#ch1-2)
  + [Introduction](#ch1-2-intro)
  + [Aim of this subproject](#ch1-2-aim)
  + [Key Steps and results](#ch1-2-steps)
  + [Discussion](#ch1-2-discussion)
* [ASSEMBLY OF DUAL-REPORTER CONSTRUCT](#ch1-3)
  + [Introduction](#ch1-3-intro)
  + [Aim of this subproject](#ch1-3-aim)
  + [Key Steps and results](#ch1-3-steps)
  + [Discussion](#ch1-3-discussion)
* [**CHAPTER II: qPCR AFP RESULTS**](#ch2-1-chapter)
  + [Introduction](#ch2-1-intro)
  + [Results](#ch2-1-results)
  + [Discussion](#ch2-1-discussion)
* [**CHAPTER III: TRANSFECTION**](#ch3-chapter)
* [BACKGROUND INFORMATION](#ch3-2)
* [INTRODUCTION](#ch3-3-intro)
* [HYPOTHESIS](#ch3-4)
* [OBSERVATIONS - FLUORESCENT MICROSCOPE](#ch3-5)
  + [HepG2](#ch3-5-1)
  + [HEK293T](#ch3-5-2)
  + [WI38](#ch3-5-3)
  + [A549](#ch3-5-4)
* [RESULTS](#ch3-6)
* [CONCLUSIONS](#ch3-7)
* [**CHAPTER IV: CYTOMETRY**](#ch4-chapter)
* [INTRODUCTION](#ch4-1-intro)
* [FLOW CYTOMETRY RESULTS](#ch4-2-results)
* [DISCUSSION](#ch4-3-discussion)

▼ Scroll for more

#### Chapter I: Cloning

#### Toehold Switch Assembly

##### Introduction

Toehold switches are advanced tools in synthetic biology that enable precise regulation of gene expression at the translational level. They are artificially engineered single-stranded RNAs that function as sensors within the 5′ untranslated region (5′ UTR) of a transcript. In their inactive state, a toehold switch adopts a complex secondary structure that occludes the ribosome binding site and the start codon, thereby preventing the initiation of translation. Only in the presence of a specific RNA sequence (the so-called trigger) does the toehold switch undergo a conformational change, allowing the ribosome to initiate protein synthesis.

##### Aim of this subproject

The objective of this experiment is to obtain 21 constructs, each consisting of Transcription Unit 1 (TU1) (BBa\_25SH329F) and a genetic element comprising one of the twenty-one toehold switches placed upstream of the eGFP coding sequence. The prepared constructs will provide an effective platform for testing all of the designed toehold switches.

##### Key Steps and results

In the first stage, 21 genetic parts were ordered from Twist Biosciences, each consisting of a single toehold switch placed upstream of an eGFP coding sequence (Toehold switches collection, Parts). Each part was additionally flanked by sequences recognized by the restriction enzyme BsaI, enabling the generation of sticky ends suitable for cloning. Twist Biosciences also provided us TU1 (BBa\_25SH329F) which contains sequences flanked by BsaI recognition sites, designed to allow the insertion of parts carrying toehold switches. All ordered elements were transformed into E. coli and subsequently plated on a selective medium containing the appropriate antibiotic. The resulting colonies were used for plasmid isolation, thus providing purified genetic parts.

Following unsuccessful cloning of TU2 (BBa\_25VE60AA), we decided to simplify the process of constructing test systems for toehold switches. Using the restriction enzyme BsaI and T4 ligase, we inserted the gene encoding the α-peptide of β-galactosidase (LacZ, BBa\_25DTJWTR) with elements enabling expression in a bacterial system into the TU1 region designated for toehold switches with eGFP (Fig. 1). To enable this, a PCR was performed on the L2TU2 construct (BBa\_259T9MVI), which contains the LacZ cassette. We used modified primers containing additional nucleotides at their 5' ends, providing flanking sequences recognized by the BsaI restriction enzyme, thereby enabling the insertion of the element into TU1 (Fig. 2). This modification enabled us to perform blue-white screening.

![](https://static.igem.wiki/teams/5865/images/results-cloning-fig1.webp)

**Fig. 1.** Process of creating the TU1 + LacZ construct, which enables blue-white screening.

![](https://static.igem.wiki/teams/5865/images/results-cloning-fig10.webp)

**Fig. 2.** PCR on purified LacZ, using primers with appropriate overhangs on their 5’ ends.

Cloning of constructs containing toehold switches into TU1 was performed using the Golden Gate assembly system with the restriction enzyme BsaI (Fig. 3). The reaction mixture was transformed into E. coli and plated on a selective medium with the appropriate antibiotic. The white colonies were used for colony PCR to confirm construct attendace (Fig. 4). Subsequently confirmed colonies were used for plasmid isolation, thus providing purified genetic parts.

![](https://static.igem.wiki/teams/5865/images/results-cloning-fig2.webp)

**Fig. 3.** Process of creating the TU1 + toehold switch final construct.

![](https://static.igem.wiki/teams/5865/images/results-cloning-fig3.webp)

**Fig. 4.** Colony PCR confirming the presence of certain TU1 + toehold switch constructs. Correctly assembled constructs are marked with black frames.

##### Discussion

We have obtained 20 genetic constructs that enable us to test the specificity of each designed toehold switch in a eukaryotic system based on eGFP expression.

#### Distribution kit

##### Introduction

The iGEM collection represents a remarkably rich repository of genetic parts, enabling research across a wide range of biological systems. When designing projects based on mammalian cellular systems, an especially valuable source of genetic components is the Asimov Mammalian Parts Collection, which provides elements such as promoters, 5' UTRs, 3' UTRs, poly(A) sequences, and coding sequences for fluorescent proteins.

##### Aim of this subproject

To obtain plasmid stocks of Asimov Mammalian Part Collection that we wanted to use for the cloning in our project.

##### Key Steps and results

To obtain plasmid stocks of Asimov Mammalian Part Collection we performed transformation of E. coli. Despite positive and negative controls being ok we barely saw any colonies on the plates. We minipreped them and sent them to Sanger sequencing to confirm the sequence fidelity.
We obtained two clones of P3 (supposed to be Inert/synthetic1 3UTR, BBa\_J433018) and one clone of H3 (supposed to be CMV\_v1, BBa\_J433000). Surprisingly, clone H3 and one of P3 clones turned out to be Inert/synthetic1 3UTR (Photo 1), but the second clone from P3 turned out to be bGH (BBa\_J433021) (Photo 2). It indicates that it is a problem with a plate, because cross-contamination was impossible, since we never opened the well that was supposed to have bGH plasmid (well L5) (Photo 3)

![](https://static.igem.wiki/teams/5865/images/results-cloning-fig4.webp)

**Photo 1.** Sequencing results of the clone H3 and one of the clones P3 using L4440 primer. The coverage corresponds to Inert/synthetic1 3UTR part.

![](https://static.igem.wiki/teams/5865/images/results-cloning-fig5.webp)

**Photo 2.** Sequencing results of the clone P3 using L4440 primer. The coverage corresponds to the bGH part.

![](https://static.igem.wiki/teams/5865/images/results-cloning-fig6.webp)

**Photo 3.** Plate 1 of Distribution Kit 2025 with unopened L5 well.

##### Discussion

Our observations indicate an incorrect distribution of genetic components on the supplied plate. The lack of colonies may be in that case the result of using the wrong antibiotic or possible damage to the plasmids.

#### Assembly of dual-reporter construct (TU1‑eGFP + TU2‑RFP)

##### Introduction

Red fluorescent proteins (RFPs) are a class of coral-derived proteins that emit red light when illuminated with light of a suitable wavelength. RFPs are highly valuable in a range of biological applications, from fluorescence microscopy and protein localization studies to in vivo imaging and molecular biotechnology. The compatibility of RFPs with other fluorescent proteins enables multi-color imaging and facilitates a deeper understanding of complex cellular processes.

##### Aim of this subproject

To obtain the construct with both the transcription unit 1 having insert site for introducing toehold switches with eGFP and transcription unit 2 constitutively expressing RFP (BBa\_25V2E0HW). We wanted to use it first as a control if the lack of GFP signal from AFP negative cells is from the lack of transfection or the lack of translation. Secondly, it would also allow us to normalize the expression of eGFP in the population of cells, since every cell would be transfected with a slightly different amount of pDNA.

##### Key Steps and results

We ordered from IDT the TU1 on a cloning vector pTwist Amp High Copy, ready for Golden Gate Assembly. We also wanted to order an already assembled TU2, but the sequence was too complex for the company to synthesize. We decided to split it into 3 parts with 20 nt overhangs and use HiFi cloning to assemble them and then use this product for Golden Gate Assembly. To do so we first tested a non-classical approach without placing an insert in a vector but it failed, probably due to the low amount of correctly assembled product. We used primers specific to ends to amplify it from the reaction mixture and we obtained a band with corresponding length that we extracted and sequenced (Fig. 5). We used it in the Golden Gate Assembly of TU1, TU2 and backbone, but it continuously failed, even after modifying protocol. We also tried preparing a HiFi reaction with backbone, but we obtained no colonies. At this point we decided to omit this cloning step due to time constraints and perform flow cytometry experiment without this additional control.

![](https://static.igem.wiki/teams/5865/images/results-cloning-fig11.webp)

**Fig. 5.** PCR on HiFi reaction mixture. PCR was performed with primers corresponding to correctly assembled TU2 constructs.

##### Discussion

The construct containing the RFP coding sequence could not be obtained due to significant issues with the cloning reaction. This might have resulted from the linear nature of our assembly, since it cannot self replicate as stably as a plasmid. In the future we plan to first fully sequence the transcript and order new primers to extend TU2 more, since currently the SapI recognition site is close to the end of dsDNA, which may decrease the restriction digestion efficiency. We also ordered a fully assembled TU2 from Ansa Biotechnologies cloned into a vector, so it may increase efficiency of our next attempts.

#### Chapter II: qPCR AFP Results

##### Introduction

Reverse transcription quantitative PCR (**RT-qPCR**) is a technique used to quantify mRNA levels within cells. We used it to measure **AFP** expression in 5 cell lines (HEK293T, HepG2, WI-38, A549, and HeLa), since AFP served as the trigger for our toehold switches, and we needed AFP-negative cells to assess switch activity. Our main concern was that the fetal-origin cell lines (HEK293T and WI-38) might express this fetal transcript, and that the cancer cell lines (A549 and HeLa) might show aberrant AFP expression due to dysregulated gene expression in cancer. Therefore, we performed this experiment to ensure that our chosen negative control truly lacked AFP expression. Additionally, we prepared standard curves to ensure our primers are efficient.

##### Results

Standard curves were obtained for AFP and GAPDH primers using 7 serial dilutions of both pairs.

![](https://static.igem.wiki/teams/5865/images/results-cloning-fig12.avif)

**Fig. 6.** Standard curve obtained for GAPDH primers.

![](https://static.igem.wiki/teams/5865/images/results-cloning-fig13.avif)

**Fig. 7.** Standard curve obtained for AFP primers.

Primers efficiency was calculated with the formula: *E = 10−1/slope*

For GAPDH: *E = 10−1/−3.14 = 2.082*
For AFP: *E = 10−1/−3.13 = 2.087*

The delta-delta Ct (**ΔΔCt**) method developed by Kenneth Livak and Thomas Schmittgen in 2001
was used for the analysis of relative gene expression in this experiment.

The AFP mRNA levels were normalized to a reference gene (GAPDH) and expressed as a relative
change compared to the HepG2 cell line, which was used as the calibrator, applying the
*2−ΔΔCt* method. The results are presented as mean ± SD from three independent
replicates, and statistical significance was calculated using the t-student test.
The results are presented in the graph below (Figure 8.).

![](https://static.igem.wiki/teams/5865/images/results-cloning-fig14.avif)

**Fig. 8.** Endogenous AFP mRNA levels in comparison with HepG2 cell line.

##### Discussion

Obtained standard curves are correct R2 > 0.99 and primers efficiency are in the acceptable range of 1.9-2.1 for RT-qPCR (Rogers-Broadway et al., 2015), confirming that primers design and protocol are optimal.

AFP measurements revealed that in all cell lines we compared with HepG2 the expression of AFP is a few levels of magnitude lower and it is most likely just a slight background expression of the transcriptionally inactive genes, which will not be a problem, since toehold switch - mRNA binding is stoichiometric. It confirms that we can use either of those cell lines as a negative AFP(-) control for our experiment. We decided to proceed with HEK293T, as they exhibited the lowest AFP expression levels and are one of the most commonly used and well-characterized mammalian cell lines in synthetic biology, facilitating reproducibility and standardization within the iGEM framework.

#### Chapter III: Transfection

#### Background information

Transfection is the process of introducing nucleic acids such as DNA or RNA into eukaryotic cells to study gene function or protein expression. One of the most commonly used chemical approaches is lipofection, which relies on positively charged lipids forming complexes with negatively charged nucleic acids. These lipid-nucleic acid complexes, called lipoplexes, facilitate cellular uptake through endocytosis or membrane fusion. A widely used reagent for this purpose is Lipofectamine, a lipid-based formulation that ensures efficient delivery while maintaining high cell viability. This method enables both transient and stable gene expression across a wide range of mammalian cell types, making it ideal for testing the functionality of our toehold constructs.

#### Introduction

Our project leverages toehold switch reporter constructs encoded on plasmids to selectively detect and respond to the presence of alpha-fetoprotein (AFP) inside mammalian cells. We engineered 20 distinct plasmid variants, each bearing a different toehold switch.
To assess the specificity and efficacy of our toehold switch designs we will need to perform transfection and cytometry to quantify eGFP expression. Therefore we need to optimize our transfection method for best usage of our resources.
Due to this, we performed two transfection methods: forward and reverse in four conditions: (-)control, (+)GFP expressing control, 0.15 μL of lipofectamine, 0.3 μL of lipofectamine on four human cell lines: HepG2, WI38, A549, HEK293T.
By comparing fluorescent signals from post-transfection cells, we aim to decide and optimize the transfection method for performing toehold switch screening.
Protocol used: Cell culture protocols, Transfection

#### Hypothesis

There is no significant difference between the two tested transfection methods for each cell line used in the experiment.

#### Observations - Fluorescent microscope

##### HepG2

We can observe green signals from (+) GFP expressing control, 0.15 μL of lipofectamine, 0.3 μL of lipofectamine, and there is no signal on (-) control. There is more signal spotted on samples transfected with 0.3 μL of lipofectamine than in samples transfected with 0.15 μL of lipofectamine.

There was no significant difference observed in signal between the two tested transfection methods.

![](https://static.igem.wiki/teams/5865/images/results-transfection-fig11.webp)
![](https://static.igem.wiki/teams/5865/images/results-transfection-fig12.webp)
![](https://static.igem.wiki/teams/5865/images/results-transfection-fig13.webp)
![](https://static.igem.wiki/teams/5865/images/results-transfection-fig14.webp)

**Fig. 9.**Fluorescence microscopy image of HepG2 cells transfected with toehold-eGFP construct

##### HEK293T

Similarly like with the HepG2 cell line, green signals were spotted from (+) GFP expressing control, 0.15 μL of lipofectamine, 0.3 μL of lipofectamine, and there is no signal on (-) control. There is more signal spotted on samples transfected with 0.3 μL of lipofectamine than in samples transfected with 0.15 μL of lipofectamine.

There was no significant difference spotted in signal between the two tested transfection methods.

However, there was more signal observed for the HEK293T cell line than for the HepG2 cell line.

![](https://static.igem.wiki/teams/5865/images/results-transfection-fig21.webp)
![](https://static.igem.wiki/teams/5865/images/results-transfection-fig22.webp)

**Fig. 10** Fluorescence microscopy image of HEK293T cells transfected with toehold-eGFP construct

##### WI38

Similarly like with the other cell lines, green signals were spotted from (+) GFP expressing control, 0.15 μL of lipofectamine, 0.3 μL of lipofectamine, and there is no signal on (-) control. There is more signal spotted on samples transfected with 0.3 μL of lipofectamine than in samples transfected with 0.15 μL of lipofectamine.

There was no significant difference spotted in signal between the two tested transfection methods.

The signal observed for the WI38 cell line was higher than the HepG2 cell line, but lower than the HEK293T cell line.

![](https://static.igem.wiki/teams/5865/images/results-transfection-fig3.webp)

**Fig. 11.**Fluorescence microscopy image of A549 cells transfected with toehold-eGFP construct

##### A549

Similarly like with the previously observed cell lines, green signals were spotted from (+) GFP expressing control, 0.15 μL of lipofectamine, 0.3 μL of lipofectamine, and there is no signal on (-) control. There is more signal spotted on samples transfected with 0.3 μL of lipofectamine than in samples transfected with 0.15 μL of lipofectamine.

There was no significant difference spotted in signal between the two tested transfection methods.

The signal observed for the A549 cell line was similar to the WI38 cell line, higher than the HepG2 cell line, but lower than the HEK293T cell line.

![](https://static.igem.wiki/teams/5865/images/results-transfection-fig4.webp)

**Fig. 12.**Fluorescence microscopy image of WI-38 cells transfected with toehold-eGFP construct

#### Results

![](https://static.igem.wiki/teams/5865/images/results-transfection-fig51.webp)

**Fig. 13.** Comparison of reverse transfection efficiency between cell lines (HEK293T, HepG2, WI-38, A549) using 0.15 µL and 0.3 µL of Lipofectamine 3000 per well.

![](https://static.igem.wiki/teams/5865/images/results-transfection-fig52.webp)

**Fig. 14.** Comparison of forward transfection efficiency between cell lines (HEK293T, HepG2, WI-38, A549) using 0.15 µL and 0.3 µL of Lipofectamine 3000 per well.

There was no visible difference between the two tested transfection methods. Both had sufficient efficiency for our experiments.
The transfection with 0.3 μL of lipofectamine had higher efficiency than the transfection with 0.15 μL of lipofectamine.
The transfection efficiency was the highest for the HEK293T cell line and the lowest, but relatively good, for the HepG2 cell line, which is considered to be hard to transfect.

#### Conclusions

As expected, there was no clear difference in transfection efficiency between the two tested methods - forward and reverse transfection. Considering its shorter duration and simpler workflow, the reverse transfection protocol was chosen as the preferred method for future large-scale experiments. A noticeable difference was observed between the two tested reagent volumes: transfections performed with 0.3 µL of Lipofectamine per well resulted in visibly higher efficiency than those using 0.15 µL, and this condition was therefore selected for further work.
It should be noted that this experiment was performed only once, without technical or biological replicates. The aim of this test was exploratory - to obtain a general impression of which transfection approach and reagent amount appeared more effective - rather than to generate quantitative or validated data. Future experiments with additional replicates and quantitative analysis are planned to confirm these initial observations and establish a more reliable protocol.

#### Chapter IV: Cytometry

#### Introduction

Toehold switches are sophisticated single-stranded RNA sensors that precisely regulate gene expression at the translational level. In their default, inactive state, they form a secondary structure that blocks the ribosome binding site, preventing protein synthesis. The switch is activated only when it binds to a specific trigger RNA sequence, causing a conformational change that exposes the binding site and initiates translation. Motivated by the urgent need for effective therapeutic strategies against advanced Hepatocellular Carcinoma, we vowed to develop highly selective and adaptable therapies, using tools of synthetic biology. Understanding that HCC is often associated with the high expression of Alpha-fetoprotein (AFP) mRNA-a fetal protein re-expressed in HCC tumours, which became the marker for the disease, as it is not found in cells outside of fetal development. We decided to target this characteristic to design toehold switches to activate, should the sensor find the trigger sequence uniquely associated with the mRNA, expressing the protein of interest, which will help eradicate the cancer cells.
The primary objective of this project was to test unique toehold switches, their expression levels, sensitivity and specificity, after delivering them to cells of choice. This was achieved by developing 21 distinct genetic constructs, each integrating one toehold switch positioned upstream of the eGFP coding sequence. This library allows for the systematic evaluation of each switch's function, using eGFP fluorescence as a quantifiable output.

#### Flow Cytometry Results

To compare the performance of these constructs, a total of 144 samples of HepG2 (AFP+) and HEK293T (AFP-) cells were analyzed using flow cytometry. GFP fluorescence, which measures the expression driven by the toehold switches, was detected in the FITC channel after standard quality control and single-cell gating. The experiment's core data involved quantifying the mean fluorescence intensity (MFI) of GFP to compare the expression levels of the toehold riboswitch constructs between the AFP+ and AFP- cell lines. Two lines, each of 3 repetitions were transfected with a plasmid containing.
The results were then scaled to correctly convey the effectiveness of each toehold, regardless of transfection efficiency. The results were then compared with the maximum possible expression in the cells, which was the positive control, transfected with TU1 plasmids containing the eGFP sequence without a toehold. The result of the analysis can be seen in the figure below. The toeholds 15 and 16 were excluded due to mistakes during experiment process.

![](https://static.igem.wiki/teams/5865/images/results-cytometry-fig1.webp)

**Fig. 15.** Mean expression of toeholds, scaled with regard to a positive control and transfection efficiency.

As the mammalian cells are a challenging environment for riboswitches, due to different translation initiation mechanisms and else, we can see that a significant portion of the toeholds, has low specificity, despite targeting the sequence specific only for the AFP. However among many failed toeholds, we may see that there are few with expression levels higher for cells of interest. However, before any conclusion is drawn, we performed statistical analysis to ensure the significance of the results. In the table below the statistically significant toeholds are listed, which returned the p-value of less than 0.1 for the t-student’s test between the expression levels between the cell lines for each toehold.

**Table 1.** Table of parameters of toeholds with statistically significant difference between the mean values of expression between HepG2 and HEK cells

| Toehold | P-value | Fold Change | Relative expression in HepG2 |
| --- | --- | --- | --- |
| 21 | 0.009 | 31.9 | 0.4 |
| 2 | 0.049 | 14.4 | 0.195 |
| 10 | 0.073 | 3.1 | 1.19 |
| 14 | 0.074 | 3.3 | 1.13 |

To analyse the reason for the success of these toeholds, we tried to correlate their results with parameters available to us.

![](https://static.igem.wiki/teams/5865/images/results-cytometry-fig2.webp)

**Fig. 16.** Correlation matrix of computed toehold parameters and experimentally acquired data.

#### Discussion

The results confirmed GFP expression within the transfected cells, indicating that the construct is functional. The differences in expression between the toehold variants also suggest that they have a significant impact on expression levels in the human cell environment.

Despite the lower efficiency of riboswitches in mammalian cells documented in the literature, we were able to find a **statistically significant difference** in expression among the toehold variants between cells containing alpha-fetoprotein mRNA, indicating the constructs' sensitivity in recognizing the target sequence. However, in many cases, this specificity is absent, and variances in expression make it impossible to draw conclusions about the constructs' effectiveness in target sequence recognition. This was further complicated by significant differences in **transfection efficiency**. While both positive controls fluoresced, the difference in the amount of plasmid taken up by HEK cells compared to HepG2 cells was many times greater (approx. 100-fold), causing the results to be scaled relative to expression.

Correlating parameters related to the toeholds, although we do not have strong correlations between the results, we can note the strongest ones. The average fluorescence in HepG2 cells is positively correlated with the **co-fold energy** of the toeholds and moderately negatively correlated with the **ON/OFF ratio**. The second characteristic can be interpreted simply: as the riboswitch leaks and loses specificity, the average expression increases.

The **co-fold energy** in toeholds signifies the stability of the hybridized state of the toehold molecule and its trigger sequence. The RNA-RNA structure they form is considered more stable the lower the co-fold energy is, which can be interpreted as another way of calculating free energy. Interestingly, because the energy of this bond is negative, a positive correlation between protein expression and this energy means that the stronger the binding and the toehold-trigger duplex, the lower the expression. This result is not necessarily intuitive, as opening the toehold and keeping it open does not necessarily mean higher expression. Toeholds with the lowest co-fold energy had very low expression in both HEK and HepG2 cells, suggesting an increase in specificity at the cost of the construct's efficiency. The final negative correlation between the proportion of **GC pairs** in the trigger sequence and the co-fold energy points to a relatively simple conclusion that the stability of the hybridization between the toehold and the recognition sequence is directly dependent on the number of hydrogen bonds between the bases.

Analyzing the individual toeholds, the results immediately draw attention to **toehold 21**, which has very good statistics in every aspect. Its high specificity and accuracy give it excellent parameters and a high ON/OFF ratio; however, this result should be treated with some caution due to the low OFF value. Nevertheless, this structure and result indicate a very good candidate for further experiments and for various modifications for optimization.

**Toehold 2** has twice the expression of toehold 21, which is also reflected in its ON/OFF ratio, yet it is also a satisfactory result of our experiment. Worthy of note are **toeholds 10 and 14**, with sufficiently good statistical significance; they are an example of high sensitivity but lower specificity. Understanding the mechanisms causing their parameters would be important for the further design of riboswitches used in mammalian cells.

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