iGEM 2025 — Team vis-romania

{% extends "layout.html" %}
{% set page\_css = "css/template-pages.css" %}
{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your daily activities, experiments, discussions, and decisions.{% endblock %}
{% block page\_content %}

![Background](https://static.igem.wiki/teams/5912/icons/header-banner2.webp)

# NOTEBOOK

![Project Logo](https://static.igem.wiki/teams/5246/header/implementation-11zon.webp)

![Background Pattern](https://via.placeholder.com/3814x3648/f5f5f5/e0e0e0?text=Organic+Pattern)

[![Introduction](https://static.igem.wiki/teams/5246/svg/drop-active.svg)
Introduction](#introduction)
[![Background](https://static.igem.wiki/teams/5246/svg/drop-active.svg)
Background](#background)
[![Our Solution](https://static.igem.wiki/teams/5246/svg/drop-active.svg)
Our solution](#our-solution)
[![Conclusion](https://static.igem.wiki/teams/5246/svg/drop-active.svg)
Conclusion](#conclusion)
[![References](https://static.igem.wiki/teams/5246/svg/drop-active.svg)
Key References](#references)

## Introduction

A material that subtly binds our world together harbors a concealed dark side. Adhesives play a crucial
role in our daily lives, as they are used in a wide range of applications, from basic household repairs to
complex industrial manufacturing. The demand for adhesive products continues to rise rapidly yearly, with
the industry growing by approximately one billion USD from 2023 to 2024.

Annual global consumption is approximately 24.7 million metric tons. However, many conventional adhesives
are made from synthetic chemicals, raising severe environmental and health concerns due to their toxicity
and non-biodegradable properties. Besides their environmental impact and use in industrial structures,
adhesives are crucial in the medical industry: electrocardiogram (ECG) sensor patches, transcutaneous
electrical nerve stimulation (TENS) patches for chronic pain, negative pressure wound therapy (NPWT), and
hormonal therapy.

**Key Statistics:**

* 537 million people worldwide live with diabetes
* 1.5 million deaths annually from diabetes-related complications
* Individual insulin pump value: up to $5,000 USD

More specialized bandages, such as those used by patients with diabetes, are designed with specific
purposes in mind. However, these plasters often have the drawback of peeling off prematurely - an issue that
may seem minor but can create significant challenges for individuals managing diabetes.

![Medical device adhesives illustration](https://via.placeholder.com/600x300/4a90e2/ffffff?text=Medical+Device+Adhesives)

Figure 1: Medical devices requiring strong adhesive patches

Currently, there is no natural adhesive that minimizes the risk of allergies and skin irritations while
offering strength up to three times greater than traditional super glue. Or is there?

This is precisely where our project, **Synhesion**, takes action. We boldly address this
challenge by creating bio-based adhesives through genetically engineered bacteria. These adhesives surpass
the strength of traditional superglue and significantly reduce allergy risks, offering a revolutionary
solution.

## Background

Bio-based adhesives present a compelling opportunity to revolutionize various industries, from medical
devices to industrial manufacturing. The adhering properties of bacterial adhesives, particularly those
produced by *Caulobacter crescentus* and its close relative *Hirschia baltica*, are of
significant interest due to their exceptional strength and biocompatibility.

### Caulobacter crescentus

Renowned for producing holdfast, a natural glue alternative among the strongest known biological
adhesives.

### Polysaccharide-based

This holdfast exhibits remarkable adhesive capabilities, making it ideal for eco-friendly applications.

### Complex Life Cycle

Makes it impractical for large-scale adhesive production and biomanufacturing applications.

![Bacterial adhesive structure](https://via.placeholder.com/700x400/2ecc71/ffffff?text=Bacterial+Adhesive+Structure)

Figure 2: Bacterial adhesive production mechanism

## Our Solution

**Synhesion** targets a global challenge for 537 million diabetes patients by leveraging
*Escherichia coli* to produce and refine adhesive polysaccharides from *C. crescentus*. By
transferring and activating the relevant holdfast-producing system within *E. coli*, we could utilize
its well-established culturing and genetic manipulation techniques to achieve scalable and efficient
production.

1

#### Gene Transfer

Transfer holdfast genes from C. crescentus to E. coli

→

2

#### Production

Scale up adhesive production using E. coli systems

→

3

#### Application

Create biocompatible medical adhesives

The significance of this work lies in its potential to provide a sustainable, biodegradable alternative to
synthetic adhesives, reducing environmental impact while enhancing biocompatibility in medical applications.
By harnessing the natural adhesive properties of bacterial polysaccharides and tools of synthetic biology,
Synhesion paves the way for significant advancements in day to day lives of diabetes patients all over the
world.

## Conclusion

**Synhesion** represents a significant step towards sustainable and versatile adhesive
solutions. By exploiting the adhesive properties of bacterial glue and refining their production in *E.
coli*, this project aims to deliver innovative solutions that address the escalating demand for
eco-friendly and high-performance adhesives.

### Our Impact

Through our efforts, we strive to build a greener, more efficient future. Our team pursues to solve an
immense problem for millions of people with a patch-sized solution.

![Future vision of bio-adhesives](https://via.placeholder.com/600x350/e74c3c/ffffff?text=Future+Vision)

Figure 3: Vision for sustainable bio-adhesive applications

## Key References

1. Global Adhesives Market Report 2024. Industry Analysis and Forecasts.
2. Annual Consumption Statistics for Adhesive Products Worldwide, 2024.
3. Environmental Impact Assessment of Synthetic Adhesives. Journal of Environmental Chemistry, 2023.
4. Medical Patch Allergic Reactions: A Clinical Study. Dermatology Research, 2023.
5. International Diabetes Federation. Diabetes Atlas 10th Edition, 2021.
6. World Health Organization. Diabetes Fact Sheet, 2023.
7. Insulin Pump Technology Market Analysis, 2024.
8. Continuous Glucose Monitor Pricing Study, Medical Device Economics, 2023.
9. Bio-based Adhesives: Opportunities and Challenges. Green Chemistry Review, 2023.
10. Caulobacter crescentus Adhesive Properties. Microbiology Today, 2022.
11. Hirschia baltica: A Close Relative with Similar Adhesive Capabilities. Marine Biology, 2023.
12. Holdfast Polysaccharides: Nature's Strongest Glue. Biochemistry Journal, 2023.

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{% extends "layout.html" %} {% set page\_css = "css/engineering.css" %} {% block
title %}Engineering{% endblock %} {% block lead %}Demonstrate engineering
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# Engineering

To navigate our DBTL cycles, please click through the wheel on the left on
the pages below.

[Dry Lab](#second)
[Wet Lab](#third)

## Dry Lab Iterations

^^ Go to Top

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![build](https://static.igem.wiki/teams/5912/engineering/eng-btn-1.webp)
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![learn](https://static.igem.wiki/teams/5912/engineering/eng-btn-2.webp)

>> Go to Wet Lab Iterations

<

##

>

Iteration 1
Iteration 2
Iteration 3
Iteration 4
Iteration 5

Iteration 6
Iteration 7

## Wet Lab Iterations

[<< Go to Dry Lab Iterations](#second)

![design](https://static.igem.wiki/teams/5912/engineering/eng-btn-0.webp)
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[^^ Go to Top](#cover)

<

##

>

Iteration 1
Iteration 2
Iteration 3

{% endblock %} {% block extra\_scripts %}
{% endblock %}

{% extends "layout.html" %} {% set page\_css = "css/template-pages.css" %} {% block title %}Experiments{% endblock %} {%
block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide
sufficient information
for other teams to replicate our work.{% endblock %} {% block page\_content %}

![Background](https://static.igem.wiki/teams/5912/icons/header-banner2.webp)

# EXPERIMENTS

![Project Logo](https://static.igem.wiki/teams/5246/header/implementation-11zon.webp)

![Background Pattern](https://via.placeholder.com/3814x3648/f5f5f5/e0e0e0?text=Organic+Pattern)

[![Introduction](https://static.igem.wiki/teams/5246/svg/drop-active.svg)
Introduction](#introduction)
[![Protocols](https://static.igem.wiki/teams/5246/svg/drop-active.svg)
Protocols](#protocols)
[![Lab Notebook  ](https://static.igem.wiki/teams/5246/svg/drop-active.svg)
Lab Notebook](#lab-notebook)

## Introduction

For simplicity and wishing to avoid redundancy, our protocols section only includes protocols
which are either unique to our project, or were improved by our team during the wet lab testing
period. For an in-depth description of all
of our experiments, please refer to the Lab Notebook section, which has both embedded links to
standard protocols as well as complete written protocols.

## Protocols

Measuring internalisation of HiBiT tagged protein via TfR
or IGF-2 receptors in BV-2 cells

Whilst other researchers have proposed the use of HiBit tagging to track the
internalization of receptors, in our project the aim was slightly different: to
track the internalisation of the protein itself. Therefore, through
a series of experiments, we perfected and optimised a protocol for performing such
measurement.

**Materials**

• BV-2 cells seeded in a 96-well plate at 70% confluence

• HiBiT-tagged protein of interest purified or in culture medium

• Nano-Glo® HiBiT
Extracellular Detection System (Promega)

o LgBiT protein

o
HiBiT Extracellular Substrate

o HiBiT Extracellular Buffer

• Multichannel pipette and reservoirs

**Protocol steps:**

1. Prepare protein stock

o Dilute the HiBiT-tagged protein in complete culture medium to the
desired working concentration (based on
sensitivity and resolution of plate reader) .

o Keep an aliquot aside for the t₀ reference.

2. Apply to
cells

o Remove medium from cells.

o
Add 50 ul protein stock to each well of the 96-well plate containing BV-2 cells.

3. Timed medium removal

o Remove supernatant sequentially every 3 minutes to capture
uptake kinetics.

o Example: In our experiments, each row had a different timepoint,
therefore, we would remove Row A at 3 min (vs. t₀), Row B at 6 min, Row C at 9 min,
etc.

o When measuring 8 or more timepoints (without t0), use two or
more plates in parallel:

 Plate 1: t₀ to t₇ time points

 Plate 2: t₀ (for normalisation) and t₈→ tn,

4. Prepare
Extracellular Reagent

o Dilute LgBiT protein 1:100 and HiBiT substrate 1:50 into
Extracellular Buffer (room temperature), so that the
final detection mix’s volume is equal to the (n+4)\*V, where n is the number of wells
you are using, and V is equal to the volume of medium you remove from each well (or
keep for t0)

 Example: If you measure a total of 7 timepoints and a t0, with
each time point in triplicate, and 4 different test groups at each timepoint, you
would need a (96+4)\*50 = 5mL total Extracellular Reagent.

o Mix thoroughly in a reservoir suitable for multichannel
pipetting.

5. Add Extracellular Reagent

o Dispense into each well immediately after medium removal.

o Mix gently by pipetting up and down.

6. Incubate

o Allow plate to sit for 15 minutes at room temperature in the dark
after
adding the detection mix.

7. Read signal

o Measure luminescence using a plate reader.

o
Normalise uptake across plates using t₀ controls

**Notes:**

• Sequential removal and immediate addition of detection reagent
enables precise kinetic curves.

• Results are expressed as luminescence relative to t₀ to account
for
baseline protein exposure.

90-min SimpleStep GFP ELISA Protocol (Conditioned Medium
Samples and Low Concentrations)

This protocol is adapted from the manufacturer’s SimpleStep GFP ELISA kit ([See more info](https://www.abcam.com/en-us/products/elisa-kits/gfp-elisa-kit-ab171581?srsltid=AfmBOooUFTXYg7Xhixrr5LIxBLfP1gG3n7PvfSDR6iLFn8C4S2M2BEjQ)).
Our goal was to measure the GFP binding and internalising of our GFP-binding LYTAC
variant directly in conditioned medium rather than in purified form. This implied
several deviations from the manufacturer’s protocol,
due to low concentrations (approximated through HiBiT luminescence measurements
(**0pg/mL to 0pg/mL**7)) and incompatibility with the kit’s desired use
(recommended use is cell extraction, tissue). Therefore,
several steps were modified to account for the complexity of medium (secreted
proteins, metabolites), to ensure valid comparisons between standards and samples.

**Summary of adaptations versus standard protocol:**

• Standards: prepared in 1:1 conditioned medium from pcDNA
transfected cells and Cell Extraction Buffer instead of plain buffer,
to ensure standards and samples share the same background.

• Sample prep: conditioned medium mixed 1:1 with Cell Extraction
Buffer

• Standard curve: 2-fold dilutions
starting from ~70 pg/mL down to 0 pg/mL, rather than the manufacturer’s starting
concentration (20,000 pg/mL) were chosen because secreted protein levels were
low.

• TMB development: extended
beyond 20 minutes to achieve measurable color change, due to lower concentrations

**Materials**

• Conditioned medium samples (mixed 1:1 with Cell Extraction
Buffer)

• Nano-Glo® SimpleStep GFP ELISA kit (Antibody Cocktail, Wash
Buffer
PT, TMB Development Solution, Stop Solution)

• GFP standard provided in kit

• Multichannel pipette, reservoirs, plate shaker, 96-well plate

**Preparation of standards**
Always prepare fresh; discard after
use.

1. Reconstitute GFP standard in 1× Cell Extraction Buffer PTR (per
vial instructions) to make a 20,000 pg/mL
stock.

2. Label eight tubes: Standards 1–8.

3. Add 376 µL conditioned medium + Extraction Buffer (same as
samples) to tube 1, and 150 µL to tubes 2–8.

4.
Make a 2-fold serial dilution from stock down to Standard 7; Standard 8 is
blank.

o In our case, this yielded a 70 pg/mL → 0 pg/mL curve suitable
for the concentration of our secreted proteins.

**Protocol steps:**

1. Equilibrate reagents, standards, and samples to room
temperature.

2. Add 50 µL of each standard or sample per well (run in
duplicate).

3. Add 50 µL Antibody Cocktail to each well.

4. Seal and incubate for 1 h at room temperature on a shaker (400
rpm).

5. Wash wells 3× with
350 µL Wash Buffer PT (≥10 s contact each wash). Invert and tap plate to remove
liquid.

6. Add 100 µL TMB Development Solution to each well. Incubate in
the dark on a shaker (5-20min).

o Adaptation: extended incubation to 30 min to develop sufficient
signal.

7. Add 100 µL Stop
Solution. Shake for 1 min.

8. Record OD at 450 nm (endpoint).

**Notes**

• Standards in conditioned medium ensure results reflect the same
chemical environment as samples.

• Lower starting concentration (70 pg/mL)
prevents wasted reagents and better matches expected sample levels, allowing for
higher accuracy.

• Extended TMB incubation compensates for reduced color development
in complex medium.

• All values normalised to the conditioned medium standard curve

PEI Transfection in Starvation Media (Adherent
HEK293)

In order to properly test our logic gates, we had to produce cells which had very
limited background. It was decided that this would be done through switching from
standard media (10% FBS) to starvation media (2% FBS).
Thus, we transiently transfected HEK293 cells using PEI under low-serum
(“starvation”) conditions, minimizing serum interference with DNA uptake.

**Materials**

• HEK293 cells (adherent)

• Culture media: DMEM (or appropriate basal), with and without
serum

• Starvation medium:
basal DMEM + %2 FBS

• Plasmid DNA, high purity, no contamination

• Linear PEI (e.g. 25 kDa), stock ~1 mg/mL, pH neutralized,
sterile-filtered

•
Opti-MEM or equivalent serum-free medium (for complexation)

• 1.5 mL Eppendorf tubes

• Pipettes, sterile tips

• 6-, 12-, 24-, 96- well
plates (as needed)

• CO₂ incubator (37 °C, 5%)

• Sterile PBS (optional, for washes)

**Before Transfection (Day 1)**

1. Plate HEK293 cells so that they
will reach ~60–80% confluence on the day of transfection.

2. Use regular complete medium (DMEM + 10% FBS) until
transfection.

**Day of Transfection (Day 2)**

1. Switch to starvation medium (if needed): ~1–2 h before
transfection (or at the time of transfection), replace the culture medium with your
starvation
medium (2% FBS). This ensures that serum components don’t interfere with complex
formation or uptake.

2. Prepare DNA solution:

o
Calculate the total DNA needed per well (or per plate).

o Dilute DNA in Opti-MEM to a small volume (e.g. 50–100 µL) per
well.

3. Prepare PEI solution:

o Use a typical DNA:PEI mass ratio of 1:2.5

o
Dilute PEI in the same volume of Opti-MEM (or serum-free medium) as used for DNA (to
the same final volume).

4. Complex formation:

o
Add the PEI solution into the DNA solution, gently pipette up and down (avoid
bubbles).

o Incubate at room temperature for
~10 min (up to 15 min, min 5 min) to allow complexes to form.

5. Add complexes to cells:

o Add
the DNA–PEI complexes dropwise onto cells (e.g. along the side of the well) while
gently swirling the plate to ensure even distribution.

o
Avoid disturbing the monolayer.

6. Incubation:

o Incubate cells under standard conditions (37
°C, 5% CO₂).

o Leave the complexes on cells for 4–6 h to overnight, depending on
toxicity and cell health.

7.
Optional medium change:

o If toxicity is high, after 4–6 h you may gently replace the
medium with fresh starvation medium.
However, for tighter control, especially under stress conditions, you should not
change the medium.

**After Transfection (24–48 h)**

1. Continue culture in starvation medium or switch back to normal
complete medium (depending on your experimental design).

2.
At 24–48 h post-transfection, assay for reporter expression (in our case nanoLuc)
and cell viability.

3. Collect samples (lysates, supernatant) as required for
downstream assays.

## Lab Notebook

[See August
2025 Lab Notebook](https://static.igem.wiki/teams/5912/lab-notebook/lab-notebook-aug.pdf)

[See
September 2025 Lab Notebook](https://static.igem.wiki/teams/5912/lab-notebook/lab-notebook-sept.pdf)

{% endblock %}

{% extends "layout.html" %} {% set page\_css = "css/template-pages.css" %} {% block title %}Results{% endblock %} {% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future
plans and reflections on the impact of your project.{% endblock %} {% block page\_content %}

![Background](https://static.igem.wiki/teams/5912/icons/header-banner2.webp)

# RESULTS

![Project Logo](https://static.igem.wiki/teams/5246/header/implementation-11zon.webp)

PROMNESIS is our tau-centric therapeutic platform which combines disease-context logic with a modular effector layer, as a potential approach to Alzheimer’s Disease. Here we report the data obtained from across dry-lab modeling and wet-lab assays, culminating
in a compact, quantitative proof of concept for Promnesis.

![Background Pattern](https://via.placeholder.com/3814x3648/f5f5f5/e0e0e0?text=Organic+Pattern)

[![Logic-Gated Therapeutic Control](https://static.igem.wiki/teams/5246/svg/drop-active.svg)
Logic-Gated Therapeutic Control](#logic)
[![TRI-LYTAC](https://static.igem.wiki/teams/5246/svg/drop-active.svg)
TRI-LYTAC](#tri-lytac)
[![Quantitative extracellular clearance by ELISA ](https://static.igem.wiki/teams/5246/svg/drop-active.svg)
Quantitative extracellular clearance](#quantitative)
[![TauTuner (Optogenetic Phosphatase)](https://static.igem.wiki/teams/5246/svg/drop-active.svg)
TauTuner](#tautunner)
[![Reproducibility, Statistics and Reporting](https://static.igem.wiki/teams/5246/svg/drop-active.svg)
Reproducibility, Statistics and Reporting](#reporting)
[![Integrated Interpretation](https://static.igem.wiki/teams/5246/svg/drop-active.svg)
Integrated Interpretation](#integrated)

## Logic-Gated Therapeutic Control

The first step of testing our project was asking whether our split-GAL4 logic controller can cleanly separate healthy-like (OFF) from disease-like (ON) conditions when driving a pseudotherapeutic (NanoLuc) payload. In HEK293 cells,
the three sensors (stress-responsive promoters driving Gal4DBD, VP16AD, Cre respectively) were stimulated to model disease context, and the gate output was read on a luminometer. A first experiment set out to see whether all
the plasmids functioned appropriately. This was done by (1) using a cell culture media supplemented with fetal calf serum (FCS) which is likley to stimulate the targeted cell signaling pathways and (2) providing the three sensors
independent of extracellular environment (F1).

![](https://static.igem.wiki/teams/5912/photos/juliusimage-19.avif)

**Fig. F1**

HEK293T cells were transfected with P1, P2, P3, P4 (S), VP16Gal4, Cre, P4 (C-poz), or pcDNA3.1 (C-neg) using FCS(10%) complemented cell culture media.

The second experiment laid out to test the specificity of the gate. The “inputs” were varied in two ways: (1) genetically (only transfection of some of the sensors in extracellular environment dependent (II) or independent manner (I));
(2) by stress (where all wells were transfected with all sensors and output plasmid, and the treatment was applied or not (III)). In the first approach, the gate produced a strong ON state while either incomplete input remained close
to baseline, yielding an ON/OFF separation consistent with low leak (≤5–10% of ON). Assay quality, computed plate-wise on dedicated control wells, gave Z′ factors up to 0.67 with a typical range ≥0.50, indicating excellent dynamic
separation and reproducibility. This established that our safety layer can reliably restrict therapeutic expression to the intended context while preserving a broad signal window for downstream readouts (**F2a** ).
In the second approach, cells transfected with all detection components were either treated (with EGF for JAK-STAT pathway; Menadione for oxidative stress (ROS); tunicamycin for ER stress (CHOP)) or left untreated in low FCS (2%) complemented cell culture media.
The modest signal-to-noise ratio reflects residual leakage within certain sensors, highlighting opportunities for optimization of this proof-of-concept design.
(**F2b** ).

![](https://static.igem.wiki/teams/5912/results/f2b.webp)

**Fig. F2a:**

Z′ factor per plate with 95% CI and acceptance line at 0.5.

![](https://static.igem.wiki/teams/5912/results/results-f2a.avif)

**Fig. F2b:**

(I) Specificity of stress-independent gate, obtained by VP16Gal4 and/or Cre expressing plasmids via transfection; (II) Specificity of stress-dependent gate, obtained by selective transfection of P1-P3 together with P4;
(III) Logic gate response under stress conditions (EGF 2.56nM (JAK-STAT pathway, 5h), 10µM Menadione (Osidative Stress, 3h), 5µg/mL Tunicamycin (ER stress, 5h)), all plasmids transfected. Data
was normalized to average of the respective off states to clearly show fold.

## TRI-LYTAC

**Design and biophysical plausibility (dry lab).** We designed TRI-LYTAC as a three-arm chimera:  **AT8 scFv** (binds phospho-tau epitope S202/T205),  **HAI/TfR-binding peptide**(blood–brain barrier
shuttle), and  **IGF-2 dimer** (CI-M6PR engagement for lysosomal routing) linked by (G4S)₃ spacers. AlphaFold produced models in which each domain retained its canonical fold and the linkers remained solvent-exposed
without creating persistent steric clashes. Docking analyses supported both recognition events: IGF-2 binding poses on CI-M6PR consistent with sub-100 nM interaction affinity and an AT8-epitope engagement that positioned CDR loops as expected
for selective binding. (Figures  **F3–F4**).

**TRI-LYTAC Domain and AlphaFold Ribbon (AT8 scFv, HAI/TfR, IGF-2 dimer; (G4S)₃ linkers).**

![](https://static.igem.wiki/teams/5912/results/screenshot-2025-10-09-at-16-17-34.avif)

**Fig. F3:**

(A) Schematic comparison of the three TRI-LYTAC constructs (G1–G3, controls G-drop-TfR, G-drop-Lys, G-drop-Bind) illustrating modular differences in the tau-targeting domain while maintaining the same receptor-targeting and secretion framework.
(1) G1 (AT8 scFv TRI-LYTAC): Incorporates the AT8 single-chain variable fragment recognizing the phosphorylated tau AT8 epitope (pSer²⁰²/pThr²⁰⁵). (2) G2 (Tau-VHH TRI-LYTAC): Employs a nanobody (VHH) specific for misfolded or aggregated tau conformers,
offering broader recognition of extracellular tau seeds. (3) G3 (W-Tau control): Uses a non-pathological wild-type tau fragment as a negative-control binder to assess non-specific receptor activation. Each variant includes an N-terminal Ig κ (IGK)
signal peptide for secretion, flexible (G₄S)₃ linkers between domains, a HAIYPRH transferrin receptor (TfR)-binding motif for blood–brain barrier transcytosis, an IGF-2(F19L) dimertargeting the Insulin-like Growth Factor 2 Receptor (IGF-2R / CI-M6PR)
for lysosomal routing, and a C-terminal HiBiT tag for luminescent quantification. (B) AlphaFold2 (ColabFold) predicted tertiary structures of the TRI-LYTAC variants, colored by domain (blue = tau-binding module (AT8), grey = linkers, green = HAI/TfR motif,
pink = IGF-2 dimer, orange=HiBiT tag). All three models adopt semi-extended configurations with minimal steric interference, preserving accessibility of both the tau-binding and receptor-binding regions. The consistent fold across variants supports the
feasibility of modular replacement of the tau-recognition arm without compromising overall structural stability.

**Docking using HADDOCK, Showing TRI-LAC with TfR and IGF-2R**

![](https://static.igem.wiki/teams/5912/results/figura4.avif)

**Fig. F4:**

(A–B) Docking of TRI-LYTAC to the human transferrin receptor (TfR) using HADDOCK 2.4. The model reveals the HAIYPRH motif positioned at the receptor binding pocket, forming complementary electrostatic and van der Waals contacts while leaving the antibody and IGF-2 domains solvent-exposed. Cluster 1 (size = 43, Z = −2.0) exhibited a HADDOCK score of −79.3 ± 2.2, E\_vdW = −46.5 ± 5.9 kcal mol⁻¹, E\_elec = −140.7 ± 20.4 kcal mol⁻¹, E\_desolv = −13.2 ± 1.5 kcal mol⁻¹, and a buried surface area ≈ 1,397 ± 76 Å², supporting a stable, well-packed interface. Panel B shows the close-up of the TfR binding pocket, with hydrogen bonds and hydrophobic contacts highlighted. (C–D) Docking of the TRI-LYTAC IGF-2(F19L) dimer to the Insulin-like Growth Factor 2 Receptor (IGF-2R / CI-M6PR) domain 11. The complex reached the semi-flexible (it1) refinement stage; although water refinement failed, the pre-water model aligns accurately with the canonical IGF-2 binding pocket from the reference crystal structure (PDB 2V5P). Panel D provides a close-up view of the predicted interface. Together, these docking models validate the biophysical plausibility of TRI-LYTAC receptor engagement through both its TfR- and IGF-2R-binding modules.

**TRI-LYTAC internalization (wet lab).**The first step was to produce TRI-LYTAC conditioned media (CM) by transfecting HEK293 cells with the corresponding mammalian expression designed plasmids. As shown in figure F5a, all constructs led to extracellular secretion of HiBit tagged TRI-LYTACs. To verify the first step of the intended mechanism—target-bound uptake into the endo-lysosomal pathway - we measured over time the HiBiT luminescence in the extracellular media of BV2 microglia exposed to similar concentrations of TRI-LYTACs (approximated by RLUs). Initially, we performed a long time course experiment up to 120 minutes (F5c). We noticed a significant decrease of the signal in the first 20 minutes followed by a plateau or a partial restoration of the signal for the different LYTACs (F5b). This data might be in line with our strategy of TRI-LYTAC recycling but more data are needed to prove this hypothesis.

![](https://static.igem.wiki/teams/5912/results/figura5-a.avif)

**Fig. F5a**

Quantification of LYTAC secretion (relative luminescence units, RLU) measured at 24 and 72 hours for each construct. Values represent the mean ± standard deviation of three replicates. Secretion increased over time in all variants, with G2, G3, and G-drop-bind exhibiting exceptionally high luminescence signals at 72 h, suggesting enhanced expression or stability of the secreted construct.

![](https://static.igem.wiki/teams/5912/results/trilac1.webp)

![](https://static.igem.wiki/teams/5912/results/trilac1-1.webp)

**Fig. F5b**

Short-term internalization dynamics of TRI-LYTAC constructs (0–21 min). Each construct (G1–G3, G-drop-TfR, G-drop-lys, G-drop-bind) is shown individually and as a normalized overlay. Most variants internalized rapidly within 6–12 min, with G-drop-bind
showing the highest uptake (45%) and G2 remaining the most stable (94.9%). This timeframe captures the early endocytic phase dominated by internalization, with minimal recycling observed.

![](https://static.igem.wiki/teams/5912/results/trilac2.webp)

![](https://static.igem.wiki/teams/5912/results/trilac2-1.webp)

**Fig. F5c**

Long-term trafficking and recycling kinetics of TRI-LYTAC constructs over a 120-minute period. Each construct’s individual curve and normalized comparison plot reveal a biphasic trend: initial internalization followed by progressive
recovery, indicating recycling or re-expression at the cell surface. G-drop-lys exhibited the deepest internalization (29.6% at 15 min) and strongest recycling (46.8% recovery by 120 min), while G1 showed a comparable 45.4% recovery.
Overall, F5b illustrates the complete trafficking cycle of TRI-LYTACs, highlighting receptor-specific differences between lysosomal, binding, and TfR-only constructs.

## Quantitative extracellular clearance

Following LYTAC internalization monitoring, we further intended to investigate two stages of the process: a) attachment of the TRI-LYTAC complex – ligand (Tau or GFP) to cell surface receptors (CI-M6PR or Transferrin receptor) and b) receptor mediated endocytosis. We used a standard internalization assay as described in Figure F6. As model TRI-LYTAC, we chose GFP specific G-drop-bind and selected G3 as negative control since it presented similar internalization kinetics. We were able to detect LYTAC cell attachment at 4C, but we failed to detect GFP internalization due to the low LYTAC concentration present in CM. Further experiments should be conducted with purified recombinant LYTACs expanding the range of experimental approaches (e.g. IF, FACS etc).

![](https://static.igem.wiki/teams/5912/results/elisa1.avif)

**Fig. F6**

Schematic of our LYTAC GfP internalisation assay. Cells are serum-starved to suppress competing ligands, incubated at 4 °C for surface binding and probed at 4 °C with HiBiT extracellular reagent to detect membrane-bound TRI-LYTAC. After warming to 37°C, complexes internalise and residual surface complexes are removed by trypsination. Internalised LYTAC and GFP are quantified via HiBiT lytic luminescence and GFP ELISA, respectively.

## TauTuner (Optogenetic Phosphatase)

**In silico validation and role.** Although we did not pursue TauTuner wet-lab characterization within this cycle, we developed a detailed in silico validation to support its integration. The **LOV2–PP2A** fusion
uses LOV2’s dark-state Jα helix to cage PP2A in the absence of light, with 470 nm pulses transiently releasing the active site. COPASI models, parameterized to conservative phosphatase kinetics, predict ~80% activation of PP2A
within a 30 s pulse, a dark-state decay half-life of ~5 min, and a steady-state **30–50%** drop in AT8/total tau with a 30 s every 20 min regimen over 2–4 h. AlphaFold2 fusion models suggest feasible geometries for
both dark and light poses, while Rosetta-based scans supported a mixed (G4S/EAAAK) linker that balances basal caging with light responsiveness. In the whole-system timeline, TauTuner offers rapid, reversible intracellular adjustment
of tau phosphorylation that complements TRI-LYTAC’s slower extracellular seed clearance, with additional safety through dTAG-mediated degradation and logic-gated expression

## Reproducibility, Statistics and Reporting

All plate assays were run with  **n≥2 technical replicates**  per condition. Biological replicates were included whenever possible, though certain assays were limited by format or sample availability. Data were normalized
to matched controls, with background subtraction from media-only wells. Plate-wise **Z′** was computed from ON/OFF control wells and reported for each experiment, using **0.5** as the acceptance threshold.
Outlier handling, where needed, followed a predefined rule (Median Absolute Deviation).

## Integrated Interpretation

Together, these results support  **Promnesis** as a logic-gated, modular therapeutic architecture. The Logic Gate Module demonstrates low leak and robust ON/OFF separation in the same configuration used to gate the effector.
TRI-LYTAC displays the expected biophysical behavior and internalization profile. While lysosomal dependence was not directly assessed, preliminary results are consistent with partial extracellular clearance. TauTuner, while not
yet tested on the bench, is modeled to provide rapid and reversible intracellular action that pairs well with TRI-LYTAC’s extracellular action. The combined package: control, mechanism, and modeling, presents a clear path from
concept to proof-of-mechanism which sets clear next steps for scaling toward in-disease validation.

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