iGEM 2025 — Team nis-kazakhstan

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}A chronological record of our team's progress, documenting daily activities, experiments, and decisions.{% endblock %}
{% block page\_content %}

# Lab Notebook

![Arrival & Safety](https://static.igem.wiki/teams/6025/timeline/timeline1.avif)

15.09.2025

### Arrival & Safety

During the general tour, the Safety Officer introduced us to the biosafety rules of the National Center for Biotechnology. The Research Technician conducted a tour of the stem cell laboratory and explained the main procedures and equipment used there.

![Start & Equipment Training](https://static.igem.wiki/teams/6025/timeline/timeline2.avif)

16.09.2025

### Start & Equipment Training

Received instructions for using the equipment and performed stem cell thawing.

![Cell Culturing](https://static.igem.wiki/teams/6025/timeline/timeline3.avif)

17.09.2025

### Cell Culturing

Attended a lecture on molecular biology and cell culture methods using VR tools; cultivated cells; performed replating; changed culture medium; performed cell counting; monitored viability.

![Sample Preparation](https://static.igem.wiki/teams/6025/timeline/timeline4.avif)

18.09.2025

### Sample Preparation for Western Blot

Performed cell lysis; added protease inhibitors; conducted an MTT assay; measured absorbance; determined protein concentration.

![SDS-PAGE Experiment](https://static.igem.wiki/teams/6025/timeline/timeline5.avif)

18.09.2025

### SDS-PAGE Experiment

Prepared gels; loaded samples; carried out electrophoresis; checked separation quality using a molecular weight marker.

![Transfer & Immunoblot](https://static.igem.wiki/teams/6025/timeline/timeline6.avif)

18-21.09.2025

### Transfer & Immunoblot

Transferred proteins from the gel to a membrane; assembled the sandwich; blocked nonspecific binding; incubated with primary and secondary antibodies.

![Completion](https://static.igem.wiki/teams/6025/timeline/timeline7.avif)

22.09.2025

### Completion

Analyzed MTT assay results, summarized findings, and completed notebook entries.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrating engineering success through iterative design cycles and systematic problem-solving.{% endblock %}
{% block page\_content %}

# Engineering

* [Design](#design)
* [Build](#build)
* [Test & Learn](#test-learn)

## Design

In the design phase, we wanted to create a mammalian cell-based biosensor that could detect oxidative and electrophilic stress caused by harmful cosmetic ingredients such as hydroquinone. To design our biosensor, we drew inspiration from the **Keap1-Nrf2 signaling mechanism**, a key cellular defense pathway against oxidative and electrophilic stress. Under normal conditions, Nrf2 is bound by Keap1 and targeted for degradation; however, during oxidative stress, Keap1's cysteine residues are modified by electrophiles and ROS, releasing Nrf2 to translocate into the nucleus. There, it binds to specific DNA sequences known as **Antioxidant Response Elements (AREs)** located upstream of stress-responsive genes (Figure 1).

We harnessed this mechanism by placing an ARE enhancer sequence upstream of a minimal CMV promoter to create an inducible transcriptional system (Figure 2). When Nrf2 binds to the ARE, it initiates transcription of our EGFP reporter gene, producing a measurable fluorescence signal in response to oxidative stress (Figure 3).

![Keap1-Nrf2 signaling mechanism](https://static.igem.wiki/teams/6025/engineering/engineering/engfigure1.avif)

**Figure 1.** Keap1-Nrf2 signaling mechanism under normal and oxidative stress conditions

![Initial plasmid construct plan](https://static.igem.wiki/teams/6025/engineering/engineering/engfigure2.avif)

**Figure 2.** The initial plan of our plasmid construct

![Biosensor proof-of-concept](https://static.igem.wiki/teams/6025/engineering/engineering/screenshot-2025-10-09-at-11-10-32.avif)

**Figure 3.** The proof-of-concept of our biosensor

## Build

We built our construct *in silico* using SnapGene. We modified the existing CMV enhancer-promoter part by removing the enhancer region and inserting the ARE sequence to create the ARE-minCMV-EGFP construct. The designed fragment was then virtually assembled into the pGGAselect mammalian vector.

![SnapGene construct assembly](https://static.igem.wiki/teams/6025/engineering/engineering/engfigure4.avif)

**Figure 4.** *In silico* assembly of ARE-minCMV-EGFP construct in SnapGene

![pGGAselect vector assembly](https://static.igem.wiki/teams/6025/engineering/engineering/engfigure5.avif)

**Figure 5.** Final construct in pGGAselect mammalian vector

## Test & Learn

Due to time and resource limitations, we were unable to complete the full transfection and fluorescence-based validation of our ARE-minCMV-EGFP construct. However, we performed preliminary experiments to validate the cellular stress response system that our biosensor is designed to detect.

We conducted Western blot analysis and MTT assays using HEK293T cells exposed to hydroquinone to confirm that our target cell line responds appropriately to oxidative stress. While we could not obtain Nrf2-specific antibodies, we successfully demonstrated HSP70 upregulation, an orthogonal stress marker that validates the biological conditions under which our ARE-driven reporter would be activated.

We also validated our design through *in silico* modeling and literature review. Prior studies have shown that Nrf2 activation by oxidative agents increases transcription of ARE-driven genes, supporting the expected functionality of our construct.

Through this process, we learned about the complexity of mammalian gene regulation, the importance of modular design, and biosafety considerations in synthetic biology. These insights will guide the next stage, where we plan to complete the transfection experiments and directly measure EGFP expression through fluorescence microscopy to fully validate our biosensor system.

{% endblock %}


{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Our research journey, constraints, and proof-of-concept validations.{% endblock %}
{% block page\_content %}

# Results

* [Research Journey](#journey)
* [pGGA-ARE Construct](#pgga-are)
* [Western Blot](#western-blot)
* [MTT Assay](#mtt)

## Research Journey

What we love about iGEM is that it values not only results, but the effort, creativity,
and persistence behind every step of the scientific journey. It reminds us that even the smallest
contribution to science matters, especially when it comes from young researchers who are still
learning how the world of synthetic and molecular biology works. We understood from the beginning
that conducting advanced research within a few months is highly ambitious for any high-school team.
In many regions, including ours, this challenge is compounded by the limited access to laboratories,
lack of funding, and the general unfamiliarity of high-school research programs among scientists.

When we first started this project, our goal was to go beyond in-silico design and develop a complete
experimental system. We aimed to construct the biosensor plasmid through Golden Gate Assembly,
transfect it into mammalian cells, and analyse the reporter response experimentally. Our original
plan included observing EGFP expression through fluorescence microscopy to confirm successful t
ransfection and activation of the ARE-driven reporter, alongside Western blot analysis of our
key oxidative-stress proteins NRF2 for deep analysis.

However, as a high-school team working with limited funding, time, and access to reagents, we
faced significant challenges in obtaining certain antibodies and materials. In particular,
NRF2 antibodies were unavailable or prohibitively expensive, and delays in shipping prevented
us from accessing many specialized reagents before our experimental deadline.

Given these constraints, we decided to focus on feasible, resource-based validation methods.
The HSP70 antibody was available to us, and because hydroquinone exposure is known to trigger
both oxidative and proteotoxic stress, we used HSP70 as an orthogonal marker to confirm that our
treatment induced a general cellular stress response. Although this approach does not directly
measure Nrf2 activation, it allows us to demonstrate the biological relevance of our system and
support the stress conditions under which our reporter construct would normally be activated.

In essence, our experiments serve as a proof of concept - we designed the plasmid construct
in silico using the Golden Gate Assembly framework and validated the cellular stress response
pathway through Western blot and MTT assays. This establishes the foundation for the next stage
of our project. In the future, we aim to complete the physical assembly of the plasmid, transfect
HEK293T cells, visualize EGFP expression through fluorescence microscopy, and perform Western blot
analysis with Nrf2-specific antibodies to fully validate the ARE-driven biosensor system.

## pGGA-ARE construct

In our design plan, we used basic parts from the Asimov Mammalian Toolkit, a collection of well-characterized
parts for mammalian synthetic biology available through the iGEM Registry. Specifically, we built our construction
using the following parts: BBa\_J433000, BBa\_J433045, BBa\_J433021, BBa\_J428076 and added our new basic part: BBa\_2508GVGJ
(the ARE sequence).

We modified the full CMV enhancer-promoter (BBa\_J433000) to create a minimal CMV promoter by removing its enhancer
region. We then added the ARE enhancer upstream of the minCMV promoter. The complete construct includes regulatory
components such as a 5' UTR, Kozak sequence, EGFP reporter gene, 3' UTR, and bGH Poly(A) signal for proper expression
in mammalian cells.

![pGGA-ARE Construct Design](https://static.igem.wiki/teams/6025/results/res-figure1.avif)

![Construct Assembly](https://static.igem.wiki/teams/6025/results/res-figure2.avif)

## Western Blot

Our original plan was to analyze the Nrf2 protein, but because we did not have the specific antibody, we adapted our method and chose to detect HSP70, another well-known protein upregulated under stress conditions.

We prepared our cell lysates, quantified the protein concentrations, and loaded them onto SDS-PAGE gels. After transferring the proteins onto membranes, we performed immunoblotting using the HSP70 antibody.

![Western Blot HSP70 Results](https://static.igem.wiki/teams/6025/results/res-figure3.avif)

As you can see from the diagram above, we did not observe an immediate increase in HSP70 protein levels following the initial exposure to the hydroquinone-containing solution. However, after one hour, faint protein bands began to appear, suggesting the early activation of the stress response pathway. This indicates that hydroquinone activates a gradual cellular adaptation rather than an immediate reaction. After two hours, we detected a clear increase in HSP70 expression, which remained consistently elevated up to eight hours of exposure. This prolonged expression pattern illustrates that cells continuously respond to ongoing oxidative or proteotoxic stress induced by hydroquinone. After 18 hours, we observed a slight decline in HSP70 levels. We think this is reflecting cellular exhaustion or the natural attenuation of the stress response after long hours of stimulation. We used actin as the loading control to confirm equal protein loading across samples to ensure that the observed changes represent true variations in HSP70 expression.

![Western Blot Analysis](https://static.igem.wiki/teams/6025/results/res-figure4.avif)

## MTT Assay

Another additional experiment we conducted was the MTT assay. We used this experiment to test whether our HEK293T cells could respond to oxidative stress caused by hydroquinone (HQ), since we couldn't yet test our ARE-minCMV-EGFP plasmid in the lab. The MTT test shows how many cells are alive and active after being exposed to a chemical. Living cells can turn the yellow MTT solution into a purple color, and we measure that color at 570 nm using a plate reader. The darker the purple color, the higher the absorbance, which means more living cells.

![MTT Assay Results](https://static.igem.wiki/teams/6025/results/res-figure5.avif)

In our experiment, the control group (cells without hydroquinone)
showed the highest absorbance value, meaning the cells were healthy.
When we added hydroquinone, the absorbance dropped depending on how strong the concentration was.
At higher concentrations, the absorbance was much lower, showing that more cells were damaged/dead.
As we diluted the hydroquinone, the absorbance increased again, meaning more cells survived.
This result shows that hydroquinone is toxic to cells and that our HEK293T cells can sense and
respond to changes in their environment. Even though we couldn't test our biosensor plasmid yet,
this MTT experiment confirmed that the cells react to oxidative stress, the same signal our plasmid
is designed to detect by expressing the EGFP.

{% endblock %}