iGEM 2025 — Team uga


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Introduction◯

Cycle 1◯

Cycle 2◯

Cycle 3◯

Cycle 4◯

📐

# Engineering

Our project followed the
**Design**,
**Build**,
**Test**,
**Learn** (DBTL)
cycle to optimize the production and
targeting of engineered extracellular vesicles (EVs) displaying the GoldenEye fusion protein.
Below, we describe the main iterative steps that guided our engineering process in the "cell
side" of the project.

4
Steps

5
Cycles

∞
Learning

[Run an iteration](#content)

Cycle 1: Establishing a Robust HEK293 Cell Platform

A) Design

* We planned to use HEK293 cells as our EV-producing system. Both adherent (HEK293T) and
  suspension (HEK293F) cultures were tested to identify the most suitable format for
  large-scale EV production.

B) Build

Two culture systems were set up:

* HEK293T (adherent, grown in DMEM)

![Engineering Figure 1](https://static.igem.wiki/teams/5825/assets/photos/schemas/engineering-figure-1.webp)

**Figure 1:** P HEK293T under an inverted microscope with
X20 magnification at the left and X40 magnification at the right. Pictures from the top half
are from 24wells culture plate, and the bottom is from T150 Flask

* HEK293F (suspension, grown in serum-free medium)

![Engineering Figure 2](https://static.igem.wiki/teams/5825/assets/photos/schemas/engineering-figure-2.webp)

**Figure 2:** HEK293F culture under an inverted microscope
with X20 magnification

C) Test

* HEK293F cultures rapidly lost viability due to insufficient agitation, leading to cell
  sedimentation and oxygen limitation. In contrast, HEK293T cells remained healthy and
  adherent with high viability.

D) Learn

* We concluded that adherent HEK293T cells were better suited for consistent transfection
  and EV production. Suspension systems were discontinued, and subsequent experiments
  focused on HEK293T.

Cycle 2: Optimizing Transfection Efficiency

A) Design

* To maximize EV production and labeling, we needed to optimize HEK293T transfection
  efficiency using different reagents and DNA:reagent ratios.

B) Build

* We tested multiple transfection reagents (Dreamfect®, ICAFectin®, PEI®, jetPEI®) using a
  GFP plasmid (pcDNA3.1-GFP) as a reporter.

C) Test

* Dreamfect® achieved the highest transfection rate (~27–30%), while other reagents gave
  poor results. We tested these conditions twice, several weeks apart, with the pcDNA3.1
  plasmid and with the plasmid encoding GoldenEye.

![Engineering Figure 3](https://static.igem.wiki/teams/5825/assets/photos/schemas/engineering-figure-3.webp)

**Figure 3:** Histogram of HEK293 cell transfection rates
depending on transfection agents, under the best conditions observed for each agent. JetPEI®
is not included here because the transfection rates obtained with this agent are zero

![Engineering Table 1](https://static.igem.wiki/teams/5825/assets/photos/schemas/engineering-table-1.webp)

**Table 1:** Effect of Dreamfect:DNA ratio on transfection
rate at different DNA quantities

D) Learn

* Dreamfect® was selected for further experiments using the pexpIDT plasmid
  ("GFP-Lamp2b.trunc-Linker4-cCPE"). The optimal condition was a
  **1:2 Dreamfect:DNA ratio**
  with 2 µg DNA per well.

Cycle 3: Testing Engineered EV-Cell Interactions

A) Design

* Evaluate whether the engineered EVs could specifically bind to or be internalized by
  pancreatic cells (BxPC3, MIA PaCa-2, and PANC-1) via the cCPE–Claudin-4 interaction.

B) Build

* Cells were incubated with modified EVs at multiple time points (30 min to 6 h) under
  continuous agitation to promote contact.

C) Test

* A slight increase in FITC fluorescence was observed at early time points, suggesting
  initial EV-cell interactions. However, prolonged agitation caused massive cell death,
  making results non-interpretable.

D) Learn

* Agitation induced mechanical stress, destroying cells and masking real interactions.
  Future iterations will use
  **gentle EDTA detachment**
  and
  **static incubation**
  to preserve
  viability and assess specific EV uptake accurately.

Cycle 4: Target Validation and Characterization

A) Design

* Confirm that the pancreatic cell lines express the target receptor, Claudin-4 (CLDN4).

B) Build

* Western blotting was performed on MIA PaCa-2, BxPC3, and PANC-1 lysates.

C) Test

* All three cell lines showed CLDN4 bands between 25–37 kDa, confirming expression and validating them as suitable target models.

D) Learn

* This confirmed that the observed EV targeting (early fluorescence shift) was biologically plausible. Future work will focus on optimizing incubation and detection to confirm specific binding.