iGEM 2025 — Team wlsa-shanghaiacademy

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block section %}wet-lab{% endblock %}
{% block lead %}This serves as a chronological record of our team's progress throughout the season. It documents our daily activities, experiments, discussions, and decisions.{% endblock %}
{% block header\_bg %}https://static.igem.wiki/teams/6015/headers/notebook.webp{% endblock %}
{% block page\_content %}

## Overview

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This page is a very detailed record of our activities throughout the iGEM season. It serves as a chronological log of our team's progress, documenting our daily activities, experiments, discussions, and decisions. The notebook is essential for tracking our work, ensuring transparency, and providing a comprehensive overview of our project development.

See our activities below!

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block section %}project{% endblock %}
{% block lead %}This page shows the iterations of our DBTL engineering cycle.{% endblock %}
{% block header\_bg %}https://static.igem.wiki/teams/6015/headers/project-engineering.webp{% endblock %}
{% block page\_content %}

## Abstract

Engineering applies systematic principles to efficiently design and build functional systems. Unlike basic research, engineering focuses on creating solutions that meet specific goals. In synthetic biology, this involves modular biological parts, predictive DNA circuit design, measurement of system performance, and iterative optimization based on experimental data.

Our LEGO system (Light-induced Expression of GLP-1 and Insulin) illustrates this approach. It comprises a blue light-responsive transcriptional activator and a dual-hormone expression vector. The activator integrates a light-sensitive iLID/sspB switch, transcriptional activation domains, and the DNA-binding domain of Gal4, while the expression vector contains a 5×UAS-driven hsp70 promoter and a dual-hormone expression cassette, enabling precise and reversible control of insulin and GLP-1 expression and secretion.

Like other engineering fields, engineering a biological system is a complex, iterative process described by the Design-Build-Test-Learn (DBTL) Cycle:

* Design: Engineering principles are used to specify the system’s intended function and to guide computational modeling of its components.
* Build: The desired DNA sequences encoding the system are constructed and introduced into the host organism.
* Test: The function of the engineered system is measured and compared with expectations.
* Learn: Discrepancies between desired and observed performance are analyzed. Learn informs the next design iteration, creating a continuous feedback loop.

+ (Although it appears last, learning occurs throughout the process, from initial design to testing, ensuring the system can be refined efficiently.)

## Engineering Cycle Overview

Wet Lab

Cycles 1, 2, 3

Dry Lab

Cycles 4, 5

## Cycle 1: Protein Engineering (iLID sspB, Gal4 VP64)

This DBTL cycle is about the conceptual design of the light-responsive transcriptional activation system and the initial engineering of optogenetic switches. The goal was to establish a reversibly activatable modular foundation for dual-hormone gene expression.

### Design 1:

* Selected iLID and sspB as optogenetic “switch” proteins for light-dependent dimerization.
![](https://static.igem.wiki/teams/6015/wetlab/engineering/ilid-sspb.webp)* Integrated GAL4DBD + VP64 transcriptional activation system to drive the expression of the target genes.
![](https://static.igem.wiki/teams/6015/wetlab/engineering/promoter.webp)* Ensured modularity by separating functional domains and containing flexible linker sequences.
* Integrated the dual-component system into a single vector using IRES.

### Build 1:

Generated DNA sequences for NLS-iLID-mCherry, NLS-miRFPnano3-sspB, iLID-mCherry, NLYN-miRFPnano3-sspB, NLS-iLID-VP64 and NLS-Gal4DBD-miRFPnano3-sspB.

![](https://static.igem.wiki/teams/6015/wetlab/engineering/sequence-b11.webp)
![](https://static.igem.wiki/teams/6015/wetlab/engineering/sequence-b12.webp)
![](https://static.igem.wiki/teams/6015/wetlab/engineering/sequence-b13.webp)
![](https://static.igem.wiki/teams/6015/wetlab/engineering/sequence-b14.webp)
![](https://static.igem.wiki/teams/6015/wetlab/engineering/sequence-b11.webp)

The fusion protein constructs were cloned and expressed in HEK-293T cells.

### Test 1:

We used confocal microscopy to verify two key points:

* Nuclear Localization – iLID and sspB fusion proteins with NLS successfully localized to the nucleus and were not degraded or mislocalized under prolonged blue light stimulation.
* Light-Induced Dimerization and Reversibility – Upon blue-light illumination, cytosolic iLID was recruited to the plasma membrane by sspB. The interaction was dissociated upon light removal, with subsequent restoration of cytoplasmic localization.

The imaging results clearly demonstrated correct nuclear targeting, successful dimerization under blue light, and reversibility, confirming the functionality of the optogenetic switch system.

### Learn 1:

Before: Literature review indicates that the iLID-sspB system functions as a compact blue-light-inducible dimerization tool characterized by rapid response kinetics and reversibility (Guntas et al, 2015). While it has been widely implemented in mammalian cells and model organisms (Countryman et al., 2025), its application in light-gated transcriptional regulation remains unexplored.
Studies indicate that when employing the CRY2-CIB1 optogenetic dimerization system for light-inducible transcriptional activation in mammalian cells, abnormal nuclear clearance of CRY2 occurs after 18 hours of blue light exposure (Pathak et al., 2017). This observation prompted our team to design parallel experiments to validate the sustained nuclear localization capability of the iLID-sspB system in mammalian cells.

After: From these experiments, we learned that the nuclear localization of iLID-sspB in mammalian cells remains unaffected by blue light stimulation. The iLID-sspB system responds to blue light reliably, rapidly, and reversibly in live cells. This provides a strong foundation for the next cycle, where we will use endogenous bioluminescence to trigger the system.

### Recap of Cycle 1

In this first engineering cycle, we successfully designed, built, and experimentally validated the light-sensitive protein system iLID-sspB. The confocal microscopy experiments confirmed nuclear localization and light-controlled dimerization, and the system exhibited reversible control.

## Cycle 2: Bioluminescence-Triggered Activation

This cycle introduces the luciferase NanoLuc (Nluc) as a photoswitch trigger, where NanoLuc interacts with its substrate FFz to generate localized blue light in situ, thereby testing the corresponding iLID-sspB response induced by bioluminescence.

### Design 2:

The NLuc is fused within the optogenetic activator to produce localized blue light. The iLID-sspB dimerization system responds to this internal light source, thereby enabling non-invasive control.

![](https://static.igem.wiki/teams/6015/wetlab/engineering/gal4-bind.webp)

### Build 2:

Generated DNA sequences for Nluc-iLID-mCherry and NLS-Nluc-iLID-VP64.

![](https://static.igem.wiki/teams/6015/wetlab/engineering/sequence-b21.webp)
![](https://static.igem.wiki/teams/6015/wetlab/engineering/sequence-b22.webp)

The fusion protein constructs were cloned and expressed in HEK-293T cells.

### Test 2:

FFz + NanoLuc generates sufficient blue light to trigger iLID-sspB dimerization.
The system demonstrated a rapid response to endogenously generated bioluminescence upon FFz addition.

### Learn 2:

Before: Our initial design relied on external blue light delivery for system activation. However, through early-stage expert consultation with Professor Xu from Zhejiang University, we were alerted to the significant challenge of limited tissue penetration depth of external blue light, which would severely restrict the potential for future in vivo therapeutic applications. This critical feedback prompted us to explore alternative activation strategies. Literature research indicates that NanoLuc is a blue-light-emitting luciferase characterized by exceptionally high luminescence efficiency and intensity (England et al., 2016). It has been successfully applied to drive various optogenetic systems and is suitable for transcriptional activation control (Li et al., 2021; Yang et al., 2016). We hypothesized that replacing external light with this intrinsic bioluminescence source could enable non-invasive, in situ control of our iLID-sspB system.

After: The experimental results from Test2 robustly validated our hypothesis. The bioluminescence generated by the Nluc-FFz pair was indeed sufficient to trigger iLID-sspB dimerization effectively. More importantly, this shift from an external to an internal light source represents a pivotal learning point in our engineering journey. It directly addresses the penetration limitation issue raised during the expert interview, confirming the feasibility of non-invasive, chemically-induced control of our optogenetic system. Bioluminescence effectively triggers iLID-sspB, validating non-invasive, in situ control. This supports further integration with hormone expression systems and indicates potential for in vivo applications, guiding our efforts toward a more clinically relevant implementation of the LEGO system.

### Recap of Cycle 2

In this cycle, we successfully engineered and validated a bioluminescence-triggered activation mechanism for our optogenetic switch. By integrating NanoLuc, we overcame the limitation of external light penetration, establishing a foundation for non-invasive control of therapeutic hormone expression via oral administration of the small molecule FFz, laying the groundwork for in vivo bioluminescence-activated dual-hormone expression and future therapeutic applications.

## Cycle 3: Dual Hormone Expression Design

This cycle involved the design, construction, and validation of a dual-hormone expression cassette containing insulin and GLP-1 sequences, enabling the secretion of both hormones in various mammalian cell types beyond pancreatic β-cells, and ensuring the “single activation, dual effect” principle.

### Design 3:

The processing of native human insulin relies on pancreatic β-cell-specific prohormone convertases PC1/3 and PC2 for post-translational modification and precise C-peptide excision. To engineer a universally processable insulin sequence compatible with diverse mammalian cell types, we replaced the native PC1/3 and PC2 cleavage sites with a Furin cleavage motif (RRKR) while retaining the N-terminal signal peptide, thereby ensuring efficient secretion.

![](https://static.igem.wiki/teams/6015/wetlab/engineering/liver.webp)

The active fragment of human GLP-1 (7-37) was engineered with an A8G substitution to enhance resistance to DPP-IV enzymatic hydrolysis, and fused with the secretion signal peptide of Exendin-4 (Parsons et al., 2007), thereby enabling efficient expression and secretion of bioactive GLP-1 in mammalian cells.

![](https://static.igem.wiki/teams/6015/wetlab/engineering/chain.webp)

IRES sequence ensures both proteins are translated independently from a single mRNA transcript.

A mCherry fluorescent protein sequence, serving as a reporter gene, was inserted downstream of the dual-hormone expression cassette under the guidance of an IRES sequence.

The dual-hormone expression cassette is driven by a 5×UAS-Hsp70 promoter, where the 5×UAS sequence serves as the targeted binding motif for Gal4DBD, providing a binding scaffold for the transcriptional activator.

![](https://static.igem.wiki/teams/6015/wetlab/engineering/cell-release.webp)

### Build 3:

The engineered insulin construct consists of an insulin signal peptide followed by Chain B-Furin-Chain C-Furin-Chain A, with its interchain A-B disulfide bond sites predicted by Disulfide by Design 2.0 and confirmed to be identical to those in native human insulin (Craig et al., 2013):
![](https://static.igem.wiki/teams/6015/wetlab/engineering/disulfide.webp)
The engineered GLP-1 construct: Extendin-4EX leader sequence-Furin-GLP-1(7-37)(A8G).
![](https://static.igem.wiki/teams/6015/wetlab/engineering/sequence.webp)
The binding targeting of the NLS-Gal4DBD-miRFPnano3-sspB module within the optogenetic activator to the 5×UAS motif was simulated and analyzed using AlphaFold 3 (Abramson et al., 2024).
![](https://static.igem.wiki/teams/6015/wetlab/engineering/alphafold.webp)
Constructs cloned into mammalian vectors and transfected into HEK-293T cells for light-responsive co-expression of insulin and GLP-1.
![](https://static.igem.wiki/teams/6015/wetlab/engineering/sequence-b31.webp)

### Test 3:

Confocal microscopy was used to quantify reporter fluorescence intensity in HEK-293T cells transfected with the LEGO system under blue-light stimulation or dark conditions, demonstrating blue-light-dependent activation of target gene expression.

Quantitative PCR was employed to assess the transcriptional levels of the two engineered hormone genes and the reporter gene in HEK-293T cells under blue-light stimulation or dark conditions. The results demonstrated significantly elevated transcriptional levels of the target genes in light-stimulated cells, validating light-inducible expression.

Live-cell confocal microscopy imaging of PI3K-Akt signaling pathway activation was performed to validate the bioactivity of insulin present in the supernatant of HEK-293T cells following blue-light stimulation.

### Learn 3:

Before: We evaluated multiple strategies for reconstituting native insulin and bioactive GLP-1 in common mammalian cells such as HEK-293T, and ultimately selected the engineered approach described in this cycle.

After: The engineered insulin and GLP-1 were successfully co-expressed under optogenetic activation, and our designed universally applicable insulin for mammalian cells was demonstrated to be functionally active.

### Recap of Cycle 3

Successful design, build, and validation of light-responsive insulin + GLP-1 co-expression with functional bioactivity, providing a strong foundation for light-triggered hormone therapy.

## Cycle 4: Design and Iteration of the Software

This cycle focused on developing the computational core that translates clinical needs into executable commands for the LEGO system. The goal was to create a reliable, accurate and adaptable software model for calculating hormone doses and their corresponding light parameters.

### Design 4:

We designed a software architecture to perform a two-step translation. First, it calculates therapeutic hormone doses (insulin or insulin/GLP-1) from patient-specific physiological parameters (e.g., body weight, blood glucose), using adapted pharmacological models. Second, it converts these biological doses into precise light parameters required to activate the LEGO system *in vivo*

### Build 4:

We built a web-based application with a modular structure, implementing key modules including a user profile manager, a dose calculation engine, and a dose-to-light translator. The initial version provided a functional interface for data input and result display.

### Test 4:

We verified the software's logical consistency and output accuracy by comparing software results with manual calculations across a wide range of simulated patient scenarios. We also ensured that the generated light parameters matched the effective ranges established in prior Wetlab experiments.

### Learn 4:

The initial model calculations were functionally correct. However, testing revealed the need for greater flexibility to accommodate clinical variability. This informed the critical design addition of an adaptive rule configurator, allowing user adjustment of key variables (e.g., insulin sensitivity factor) to enhance personalization and clinical applicability.

### Recap of Cycle 4

Cycle 4 yielded a robust software engine capable of generating personalized light control parameters for the LEGO system. The key learning was the importance of user-adjustable parameters, which increased versatility. The final output of this cycle—a defined set of light parameters (intensity, duration, interval and repetition)—served as the primary input requirement for the Hardware design in Cycle 5.

## Cycle 5: Design and Iteration of the Hardware

This cycle focused on building the physical device to reliably deliver the light regimens defined by the Software. The goal was to build a wearable, user-friendly, and precise blue-light delivery system based on the specifications generated in Cycle 4.

### Design 5:

Informed by the software's outputs, we specified a hardware device with the following core specifications: a wearable form factor, precise PWM-controlled blue LEDs (465-470 nm), Wi-Fi connectivity for receiving commands, and the ability to execute complex, time-based light profiles ("animations") generated by the software's calculations.

### Build 5:

We constructed a prototype centered on an ESP32 microcontroller, integrating an LED array, a Li-Po battery, and basic firmware. The initial firmware enabled manual adjustment of light intensity and duration via a simple built-in web server hosted on the device.

### Test 5:

We measured the prototype's light output stability and spectrum using a spectrometer. We tested the reliability of the Wi-Fi connection and the accuracy of command execution during prolonged operation periods.

### Learn 5:

This cycle produced a robust hardware device capable of faithfully executing the complex light profiles defined by the Software from Cycle 4. Importantly, the completion of this cycle marks the establishment of a fully integrated, end-to-end therapeutic pipeline: from patient data input in the Software, to precise light regimen execution by the Hardware, culminating in LEGO system activation for hormone production.

### Recap of Cycle 5

Cycle 5 delivered a fully functional hardware controller and demonstrated its seamless integration with the software, thereby completing the operational backbone of our proposed therapy. The key learning is that while the integrated control system is functionally operational, its ultimate validation through biological testing is the essential next step.

## References

Abramson, J., Adler, J., Dunger, J., Evans, R., Green, T., Pritzel, A., Ronneberger, O., Willmore, L., Ballard, A. J., Bambrick, J., Bodenstein, S. W., Evans, D. A., Hung, C. C., O'Neill, M., Reiman, D., Tunyasuvunakool, K., Wu, Z., Žemgulytė, A., Arvaniti, E., Beattie, C., … Jumper, J. M. (2024). Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature, 630(8016), 493–500. <https://doi.org/10.1038/s41586-024-07487-w>

Countryman, A. D., Doherty, C. A., Herrera-Perez, R. M., & Kasza, K. E. (2025). Endogenous OptoRhoGEFs reveal biophysical principles of epithelial tissue furrowing. Nature communications, 16(1), 7665. <https://doi.org/10.1038/s41467-025-62483-6>

Craig, D. B., & Dombkowski, A. A. (2013). Disulfide by Design 2.0: a web-based tool for disulfide engineering in proteins. BMC bioinformatics, 14, 346. <https://doi.org/10.1186/1471-2105-14-346>

England, C. G., Ehlerding, E. B., & Cai, W. (2016). NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence. Bioconjugate chemistry, 27(5), 1175–1187. <https://doi.org/10.1021/acs.bioconjchem.6b00112>

Guntas, G., Hallett, R. A., Zimmerman, S. P., Williams, T., Yumerefendi, H., Bear, J. E., & Kuhlman, B. (2015). Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins. Proceedings of the National Academy of Sciences of the United States of America, 112(1), 112–117. <https://doi.org/10.1073/pnas.1417910112>

Li, T., Chen, X., Qian, Y., Shao, J., Li, X., Liu, S., Zhu, L., Zhao, Y., Ye, H., & Yang, Y. (2021). A synthetic BRET-based optogenetic device for pulsatile transgene expression enabling glucose homeostasis in mice. Nature communications, 12(1), 615. <https://doi.org/10.1038/s41467-021-20913-1>

Parsons, G. B., Souza, D. W., Wu, H., Yu, D., Wadsworth, S. G., Gregory, R. J., & Armentano, D. (2007). Ectopic expression of glucagon-like peptide 1 for gene therapy of type II diabetes. Gene therapy, 14(1), 38–48. <https://doi.org/10.1038/sj.gt.3302842>

Pathak, G. P., Spiltoir, J. I., Höglund, C., Polstein, L. R., Heine-Koskinen, S., Gersbach, C. A., Rossi, J., & Tucker, C. L. (2017). Bidirectional approaches for optogenetic regulation of gene expression in mammalian cells using Arabidopsis cryptochrome 2. Nucleic acids research, 45(20), e167. <https://doi.org/10.1093/nar/gkx260>

Yang, J., Cumberbatch, D., Centanni, S., Shi, S. Q., Winder, D., Webb, D., & Johnson, C. H. (2016). Coupling optogenetic stimulation with NanoLuc-based luminescence (BRET) Ca++ sensing. Nature communications, 7, 13268. <https://doi.org/10.1038/ncomms13268>

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block section %}wet-lab{% endblock %}
{% block lead %}This page describes the research, experiments, and protocols we used in our project. It is designed to provide
sufficient information for other teams to replicate our work.{% endblock %}
{% block header\_bg %}https://static.igem.wiki/teams/6015/headers/experiment.webp{% endblock %}
{% block page\_content %}

## Experiment Overview

1 - Plasmid Preparation

The optogenetic activator plasmid was constructed by assembling the fragments into the pLVX-Hygro vector
digested by XhoI/XmaI. The dual-hormone expression plasmid was synthesized and constructed by Tsingke
Biotechnology using pGL3basic vector. Plasmids were extracted from E. coli cultures using the alkaline lysis
method, followed by purification via silica-column chromatography. DNA concentrations were quantified via
spectrophotometry, yielding high-purity plasmid preparations ranging from 1246.1 to 1311 ng/µL.

![](https://static.igem.wiki/teams/6015/experiments/top1.webp)

2 - Cell Culture & Transfection

HEK-293T cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% (V/V) fetal
bovine serum (FBS) and 1% (V/V) penicillin/streptomycin solution. Cells are digested and passaged using
0.05% trypsin digestion solution when reaching 80% confluence. It was ensured that no more than 10 passages
of HEK-293T cells were used in the experiments.

![](https://static.igem.wiki/teams/6015/experiments/top2.webp)

3 - Plasmid Transfection

HEK-293T cells were transfected with plasmids using Lipofectamine 3000 according to the manufacturer's protocol.

4 - Blue light LEDs activation

Transfected cells were incubated in dark or illuminated with 480 nm pulsatile blue light (1 s ON, 5 s OFF;
light intensity: ~ 500 μW as measured with a Thorlabs PM160T Wireless Power Meter) by using a custom
designed light-emitting diode (LED) array.

![](https://static.igem.wiki/teams/6015/experiments/top3.webp)

5 - Confocal Imaging

Cells for confocal imaging were plated into 35 mm glass bottom dish. All confocal images were acquired on an
Olympus FV3000 confocal laser scanning microscope equipped with a ×60 NA 1.42 oil immersion objective and
405/488/561/640 nm lasers. For live-cell imaging, cells were maintained at 37℃ and 5% CO2 in a
humidified chamber.

![](https://static.igem.wiki/teams/6015/experiments/top4.webp)

6-Total RNA extraction and RT-qPCR

Total RNA was isolated with Trizol reagent and quantified. Reverse transcription was conducted as follows
with the Fast All-in-One RT Kit, according to the manufacturer’s instructions. Quantitative PCR of cDNA was
performed using Realtime PCR Super mix SYBR green on the Light Cycler 480 real-time PCR instrument (Roche
Life Science). The program for qPCR amplifications was as follows: 95℃ for 5 min, followed by 40 cycles at
95℃ for 10s, 60℃ for 30s. All samples were normalized to the housekeeping gene *RPLP0* as the
endogenous control and determined by the standard 2-ΔΔCt method to obtain relative mRNA
expression levels.

![](https://static.igem.wiki/teams/6015/experiments/top5.webp)

## Protocol

### PCR Amplification

**Purpose:** to amplify the target DNA fragment from an existing plasmid.

**Steps:**

1. **Reaction mixture preparation:**
   10×PCR buffer: 5 μL
   2.5mM MgSO₄: 3 μL
   2mM dNTP: 5 μL
   Forward primer (10μM): 1.5 μL
   Reverse primer (10μM): 1.5 μL
   Template plasmid: 1 μg
   KOD plus: 1 μL
   ddH₂O: 32 μL
2. **Initial denaturation:** 94°C for 2 minutes
3. **Amplification Cycles** (repeat 35 times):
   * Denaturation: 98°C for 10 seconds
   * Annealing: Tm for 30 seconds
   * Extension: 68°C for 30 seconds per kb
4. **Final Extension:** 68°C for 7 minutes
5. **Hold:** 4°C indefinitely

### Vector Digestion

**Purpose:** to linearize the vector plasmid by restriction enzyme digestion at specific cleavage sites.

**Steps:**

1. **Reaction mixture preparation:**
   rCutSmart buffer: 5 μL
   Template plasmid: 2 μg
   Restriction Enzyme 1 (XhoI): 1 μL
   Restriction Enzyme 2 (XmaI): 1 μL
   ddH₂O: 41 μL
2. Digest at 37°C for 1 hour

### DNA Gel Electrophoresis

**Purpose:** to confirm the size of DNA fragments such as PCR products and separate DNA molecules by their molecular weight.

**Steps:**

1. Prepare a 1% agarose gel by dissolving 0.3 g of agarose in 30 mL of 1× TAE buffer, then add 3 μL of Gel Red. Heat the mixture for 1 minute using microwave heating, pour it into a casting mold, and allow it to solidify at room temperature
2. Add 6× Loading Buffer to the DNA samples, place the agarose gel in the electrophoresis tank, and load the samples into the wells
3. Run the gel at 120 V for 30 minutes

### DNA Gel Extraction

**Purpose:** to extract high-purity DNA fragments from the gel following electrophoresis.

**Steps:**

1. Excise the DNA band from the gel and transfer it to a 1.5 mL microcentrifuge tube
2. Add 700 μL of Buffer GM and incubate at 50°C until the gel slice is completely dissolved
3. Transfer the solution to a spin column and centrifuge at 12,000 rpm for 1 minute
4. Discard the flow-through, add 700 μL of Buffer WB, and centrifuge at 12,000 rpm for 1 minute
5. Repeat step 4
6. Discard the flow-through and centrifuge at 12,000 rpm for 1 minute
7. Place the spin column into a new microcentrifuge tube and air-dry for 1 minute
8. Add 20 μL of ddH₂O preheated to 50°C, incubate at 50°C for 5 minutes, then centrifuge at 12,000 rpm for 1 minute
9. Quantify the DNA concentration using a microvolume spectrophotometer

### Homologous Recombination

**Purpose:** to assemble one or multiple DNA fragments with a vector into a new plasmid.

**Steps:**

1. **Reaction mixture preparation:** combine 5 μL of 2× Basic Assembly Mix with insert fragments and linearized backbone at a 1:5 molar ratio, then adjust the total volume to 10 μL with ddH₂O
2. Incubate at 50°C for 15 minutes, then store at 4°C

### Transformation and Plating

**Purpose:** to introduce the homologous recombination product into competent cells and culture them on bacterial plates.

**Steps:**

1. Thaw 50 μL of DH5α competent cells on ice, add 2 μL of the homologous recombination product, and incubate on ice for 30 minutes
2. Heat shock at 42°C for 30 seconds, then immediately transfer to ice for 2 minutes
3. Plate the competent cell mixture
4. Incubate at 37°C for 16 hours, then store at 4°C

### Plasmid Extraction

**Purpose:** to isolate high-purity plasmid DNA from E. coli.

**Steps:**

1. Centrifuge the bacterial culture at 4°C and 4500 rpm for 30 minutes, discard the supernatant, and invert the tube to drain
2. Add 8 mL of RES buffer (+RNase) to resuspend the pellet, then add 8 mL of LYS buffer and invert the tube 5 times. Allow the mixture to stand at room temperature for 5 minutes
3. Add 12 mL of EQU buffer along the wall of the spin column
4. Add 8 mL of NEU buffer to the mixture from step 2, then shake and mix until the solution becomes colorless. Transfer the mixture to spin column and allow it to drain completely by gravity flow
5. Add 5 mL of EQU buffer along the inner wall of the spin column, and allow it to drain completely by gravity flow
6. Remove the spin column, add 8 mL of WASH buffer to the adsorption column, and allow it to drain completely by gravity flow
7. Add 5 mL of ELU buffer preheated to 50°C to the adsorption column, and collect the eluate in a 15 mL centrifuge tube
8. Add 3.5 mL of isopropanol to the centrifuge tube, shake vigorously to mix thoroughly, and incubate at -20°C for 20–30 minutes
9. Centrifuge the tube at 4°C and 4500 rpm for 30 minutes
10. Carefully remove the supernatant, wash the precipitated plasmid DNA with 70% ethanol, and transfer it to a 1.5 mL microcentrifuge tube
11. Centrifuge the plasmid pellet at 4°C and 12,000 rpm for 1 minute, discard the supernatant, and air-dry the pellet
12. Add 200 μL of ddH₂O and resuspend the plasmid by pipetting
13. Quantify the concentration of the plasmid solution using a microvolume spectrophotometer, adjusted to 1000 ng/μL with ddH₂O, and stored at 4°C

### HEK-293T Cells Transfection

**Purpose:** to deliver the target plasmid into the cells

**Steps:**

1. For 12-well plates and 35 mm glass-bottom dishes, dilute 2.25 μL of Lipofectamine 3000 in 75 μL of Opti-MEM
2. Dilute 1.5 μg of plasmid and 3 μL of P3000 in 75 μL of Opti-MEM
3. Combine the solutions from steps 1 and 2, mix thoroughly by pipetting, and incubate at room temperature for 10-15 minutes
4. Add the mixture to the cell culture medium and gently swirl to ensure even distribution

### Confocal Microscopy

**Purpose:** to observe the fluorescence-labeled protein dynamics in living cells

**Steps:**

1. Clean lenses with ethanol, apply immersion oil
2. Place the 29 mm glass-bottom dish at the center of the stage
3. Focus cells using coarse and fine knobs, select target cells
4. Capture single images or time-series images

### Total RNA Extraction

**Purpose:** to extract high-purity total RNA from HEK-293T cells

**Steps:**

1. For 12-well plates, aspirate the culture medium, rinse the cells with ice-cold PBS, add 400 μL of TRIzol lysis reagent to each well, swirl the plate to ensure uniform coverage
2. Incubate at room temperature for 5 minutes to complete lysis, mix the lysate by pipetting and transfer it to 1.5 mL microcentrifuge tubes
3. Add 80 μL of chloroform, invert the tube vigorously for 15 seconds, and incubate at room temperature for 3 minutes
4. Centrifuge at 12,000 rpm for 15 minutes at 4°C
5. Pipette 180 μL of the upper aqueous phase into a new 1.5 mL microcentrifuge tube, add 200 μL of isopropanol, mix gently, and incubate at -20°C overnight
6. Centrifuge at 12,000 rpm for 10 minutes at 4°C
7. Discard the supernatant, add 400 μL of 75% ethanol solution prepared with DEPC-treated water, and centrifuge at 7500 rpm for 5 minutes at 4°C
8. Remove as much supernatant as possible, then air-dry the pellet at room temperature for 5–10 minutes
9. Add 20 μL of DEPC-treated water and mix thoroughly by pipetting
10. Quantify the RNA concentration using a microvolume spectrophotometer and store the solution at -80°C

### RNA Reverse Transcription

**Purpose:** to reverse transcribe RNA into cDNA

**Steps:**

1. Take 500-1000 ng of total RNA, add 4 μL of DNase, and adjust the volume to 16 μL with DEPC-treated water. Mix thoroughly by pipetting
2. Incubate at room temperature for 5 minutes
3. Add 4 μL of 5× RT Mix and mix thoroughly by pipetting
4. Incubate at 42°C for 15 minutes, then dilute the product 10-fold with ddH₂O, and stored at 4°C

### Quantitative Real-Time PCR

**Purpose:** to quantify the expression level of target genes in samples

**Steps:**

1. **Reaction mixture preparation:**
   2×SYBR Green qPCR Mix: 10 μL
   cDNA: 1.5 μL
   Forward primer (10μM): 0.4 μL
   Reverse primer (10μM): 0.4 μL
   ddH₂O: 7.7 μL
2. **Initial denaturation:** 95°C for 5 minutes
3. **40 cycles:**
   * Denaturation: 95°C for 10 seconds
   * Annealing/Extension: 60°C for 30 seconds
   * Melting curve

**For full protocols, see our Lab Notebook.**

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{% block title %}Results{% endblock %}
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{% block lead %}This page shows the results of our project, along with a detailed analysis and discussion of their significance. Future plans and reflections on the impact of our project are also outlined here.{% endblock %}
{% block header\_bg %}https://static.igem.wiki/teams/6015/headers/project-result.webp{% endblock %}
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## Overview

We present LEGO (Light-induced Expression of GLP-1 and Insulin for Obesity and diabetes treatment), an innovative optogenetic gene therapy system. LEGO is designed for the simultaneous, precise co-expression of insulin and GLP-1 within the body. Through systematic design and validation, we have created a robust toolkit for light-regulated hormone delivery with therapeutic potential.

On this page, we provide a comprehensive description of all experimental results obtained during the design and validation of the LEGO system, along with corresponding analysis and discussion. While we encountered certain challenges and limitations during implementation, we remain committed to continuously improving the LEGO system to unlock its full potential for future *in vivo* therapeutic applications.

## Design and Simulation of LEGO Components

Through literature research, we identified the iLID-sspB system as the optogenetic switch for LEGO (**Figure 1A**) (Guntas et al, 2015), with NanoLuc serving as a potential endogenous activation trigger (England et al., 2016). The transcriptionally inactive Gal4DBD was employed as the DNA-targeting module, 5×UAS as the binding motif (**Figure 1B**), and VP64 as the highly efficient transcriptional activation module (**Figure 1C**).

![](https://static.igem.wiki/teams/6015/results/1.webp)

The light-responsive activator plasmid was designed using the most streamlined configuration (**Figure 2**).

![](https://static.igem.wiki/teams/6015/results/2.webp)

We subsequently designed the amino acid and DNA sequences of human insulin and GLP-1 optimized for secretion by HEK-293T cells. The processing of native human insulin relies on pancreatic β-cell-specific prohormone convertases PC1/3 and PC2 for post-translational modification and precise C-peptide excision (**Figure 3A**). To engineer a universally processable insulin sequence compatible with diverse mammalian cell types, we replaced the native PC1/3 and PC2 cleavage sites with a Furin cleavage motif (RRKR) while retaining the N-terminal signal peptide, thereby ensuring efficient secretion (**Figure 3B**).

![](https://static.igem.wiki/teams/6015/results/3.webp)

To thoroughly validate the design feasibility, we employed the protein disulfide bond prediction tool Disulfide by Design 2.0 to analyze the inter/intra-chain disulfide bonds of the engineered human insulin (Craig et al., 2013). Comparative analysis with native human insulin confirmed complete conservation of all disulfide bond positions (**Figure 4**).

![](https://static.igem.wiki/teams/6015/results/4.webp)

The active fragment of human GLP-1 (7-37) was engineered with an A8G substitution to enhance resistance to DPP-IV enzymatic hydrolysis, and fused with the secretion signal peptide of Exendin-4 (Parsons et al., 2007) (**Figure 5**), thereby enabling efficient expression and secretion of bioactive GLP-1 in mammalian cells.

![](https://static.igem.wiki/teams/6015/results/5.webp)

Subsequently, we integrated the two engineered hormone genes using an IRES element to create a dual-hormone expression cassette for mammalian cell expression. A reporter gene encoding the red fluorescent protein mCherry was incorporated to visualize expression dynamics and secretion competence. The dual-hormone expression cassette is driven by a 5×UAS-Hsp70 promoter, where the 5×UAS sequence serves as the targeted binding motif for Gal4DBD, providing a binding scaffold for the transcriptional activator. (**Figure 6**)

![](https://static.igem.wiki/teams/6015/results/6.webp)

To confirm the feasibility of targeted binding between the Gal4DBD domain of the optogenetic activator protein and the UAS motif, we utilized AlphaFold 3 to predict their interaction interface, validating the reliability of Gal4DBD's specific binding to UAS (**Figure 7**) (Abramson et al., 2024).

![](https://static.igem.wiki/teams/6015/results/7.webp)

The light-responsive activator and dual-hormone expression vector, as two core components, constitute the LEGO system. Through rigorous design principles and minimalist configuration, we have established a streamlined toolkit for light-controlled engineered hormone expression regulation.

## Plasmid Construction

The optogenetic activator plasmid was constructed by assembling the fragments into the pLVX-Hygro vector digested by XhoI/XmaI. We successfully PCR-amplified the insert fragments—NLS-Gal4DBD (538 bp), VP64 (193 bp), miRFPnano3-sspB (839 bp), IRES (634 bp), NLS-Nluc (609 bp), and iLID (470 bp)—from existing plasmids and confirmed their sizes by agarose gel electrophoresis (**Figure 8**). The vector plasmid pLVX-Hygro was digested with restriction enzymes XhoI and XmaI, and the digestion products were also verified by agarose gel electrophoresis (**Figure 8**).

![](https://static.igem.wiki/teams/6015/results/8.webp)

Subsequently, we ligated the insert fragments with the linearized vector using homologous recombination. The recombinant products were transformed into DH5α competent cells and plated. After 16 hours of incubation at 37°C, scattered single colonies were observed on the bacterial plates, indicating successful transformation (**Figure 9**). We selected six colonies for inoculation in 3 mL LB medium and cultured them with shaking at 37°C for 5 hours. Plasmid sequencing was performed using primers CMV-F and PGK-R, and bidirectional sequencing results confirmed that the plasmid sequences were consistent with the expected designs, validating the successful construction of the optogenetic activator plasmid.

![](https://static.igem.wiki/teams/6015/results/9.webp)

The plasmids used for validation and the dual-hormone expression vector plasmid were synthesized and constructed by Tsingke Biotechnology. All plasmids were extracted from E. coli cultures using the alkaline lysis method, followed by purification via silica-column chromatography. DNA concentrations were quantified via spectrophotometry, yielding high-purity plasmid preparations ranging from 1246.1 to 1311 ng/µL.

## Validation of the Feasibility and Reversibility of the Light-Responsive System

Before validating the overall functionality of the LEGO system, we systematically characterized the basic parts within the composite modules. This included validation of the optogenetic iLID-sspB system's nuclear localization stability, light responsiveness, reversible dimerization kinetics, and feasibility of NanoLuc-mediated activation.

To validate whether the two core optogenetic components of the LEGO system, iLID and sspB, exhibit stable and consistent nuclear localization under blue-light illumination in HEK-293T cells (Pathak et al., 2017), we designed and constructed plasmids NLS-Nluc-iLID-mCherry and NLS-miRFPnano3-sspB, each fused with a nuclear localization sequence (NLS) and a fluorescent protein tag. After 24 hours of co-transfection of these two validation plasmids into HEK-293T cells, confocal microscopy imaging revealed that both under short-term (200 s) continuous blue-light stimulation and long-term (18 h) pulsed blue-light illumination, the components exhibited clear and sustained nuclear localization. These results confirm the nuclear localization stability and reliability of the light-responsive system (**Figure 10**).

![](https://static.igem.wiki/teams/6015/results/10.webp)

To visually validate the blue light-responsive interaction between the optogenetic components, we monitored their spatial redistribution in real time. The sspB protein was anchored to the plasma membrane via a membrane localization sequence (NLYN), while iLID remained freely diffusible in the cytoplasm. Both components were tagged with fluorescent proteins and encoded in the plasmids Nluc-iLID-mCherry and NLYN-miRFPnano3-sspB. After 24 hours of co-transfection in HEK-293T cells, live-cell confocal imaging demonstrated that upon brief blue light illumination (100 s), iLID was rapidly recruited to the plasma membrane. Following light withdrawal, iLID quickly reverted to its diffuse cytoplasmic distribution under dark conditions (**Figure 11**), confirming the rapid light responsiveness and excellent reversibility of this optogenetic module.

![](https://static.igem.wiki/teams/6015/results/11.webp)

## Validation of Bioluminescence-Induced Activation of the Light-Responsive System

We next sought to determine whether bioluminescence generated by NanoLuc (Nluc) could activate the iLID-sspB interaction. Upon adding 40μM of the Nluc substrate fluorofurimazine (FFz) to the culture medium, we observed robust recruitment of iLID to the plasma membrane, closely mirroring the localization pattern induced by blue light illumination (**Figure 12**). These results validate the feasibility of using intracellular bioluminescence to activate the light-responsive components within the LEGO system.

![](https://static.igem.wiki/teams/6015/results/12.webp)

## Validation of Light-Activated Co-expression of Insulin and GLP-1

We next proceeded to evaluate the functionality of the LEGO system's composite parts. Using confocal live-cell imaging, we validated both the feasibility and light-responsive characteristics of target gene activation by the LEGO system. Following 24 hours of pulsed blue-light stimulation, a marked enhancement in co-expressed mCherry fluorescence was observed compared to the dark-treated control group (**Figure 13A**). Statistical analysis further confirmed reliable significance (**Figure 13B**), thereby validating the successful blue-light-responsive activation of the dual-hormone expression.

![](https://static.igem.wiki/teams/6015/results/13.webp)

We also measured the transcript levels of insulin, GLP-1, and mCherry in HEK-293T cells by RT-qPCR. Consistently, the mRNA levels of insulin (INS), GLP-1, and mCherry were markedly upregulated compared to the dark control group (**Figure 14**). These results demonstrate that the LEGO system enables robust, blue light-dependent co-expression of Insulin and GLP-1.

![](https://static.igem.wiki/teams/6015/results/14.webp)

## Validation of Engineered Human Insulin Activity

Since the engineered insulin was independently designed, it was essential to characterize its bioactivity. Native human insulin initiates a precise signaling cascade upon binding to insulin receptors on target cells, ultimately leading to the activation of key signaling molecule Akt, which translocates to the plasma membrane during this process. To confirm the secretion and bioactivity of insulin produced by LEGO system, we monitored the activation of the PI3K-Akt signaling pathway using live-cell confocal microscopy in response to conditioned medium from HEK-293T cells expressing the LEGO system. Culture medium collected from cells exposed to either blue light stimulation or dark incubation was applied to reporter cells expressing AktPH-EGFP (the PH domain of Akt fused with EGFP). We observed significant translocation of AktPH to the plasma membrane in cells treated with medium from blue light-stimulated conditions, confirming blue light-dependent production, secretion, and bioactivity of the insulin generated by the engineered LEGO gene circuits (**Figure 15A and 15B**). To exclude potential effects of cellular heterogeneity, the same reporter cells were sequentially treated with conditioned medium from the dark-incubated group followed by that from the blue-light-stimulated group. Results showed that PI3K-Akt pathway activation occurred only after treatment with the blue-light-stimulated conditioned medium (**Figure 15C**), further validating both the light responsiveness of the LEGO system and the functional activity of the secreted products.

![](https://static.igem.wiki/teams/6015/results/15.webp)

## Discussion

The successful establishment of the LEGO system represents a significant advancement in optogenetic gene therapy. Our modular design—integrating the iLID/sspB optogenetic switch, NanoLuc bioluminescence activation, and engineered insulin/GLP-1 expression cassettes—demonstrates robust spatiotemporal control over metabolic hormone production. The system's tight regulation, evidenced by minimal basal leakage and strong light-induced responses, along with the confirmed bioactivity of the engineered insulin through PI3K/Akt pathway activation, positions LEGO as a reliable platform for future translational applications.

Despite these results, several limitations warrant attention. Though basal leakage is low, further optimization of promoter strength and target gene design could enhance the dynamic range. the translational potential depends on efficient and safe delivery methods for both genetic constructs and the FFz substrate in vivo, which presents technical and regulatory hurdles.

Looking forward, the LEGO system holds substantial promise for diabetes treatment and beyond. Its modular architecture allows straightforward adaptation to express other therapeutic proteins, enabling broad applications in hormone replacement therapies. Furthermore, the bioluminescent activation mechanism could be adapted for autonomous feedback-controlled therapy, where hormone secretion is triggered by physiological cues detected by engineered sensors. By addressing these challenges and leveraging its unique capabilities, the LEGO platform may ultimately pave the way for a new generation of smart, precise, and personalized gene therapies for metabolic and other diseases.

## References

Abramson, J., Adler, J., Dunger, J., Evans, R., Green, T., Pritzel, A., Ronneberger, O., Willmore, L., Ballard, A. J., Bambrick, J., Bodenstein, S. W., Evans, D. A., Hung, C. C., O'Neill, M., Reiman, D., Tunyasuvunakool, K., Wu, Z., Žemgulytė, A., Arvaniti, E., Beattie, C., … Jumper, J. M. (2024). Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature, 630(8016), 493–500.
<https://doi.org/10.1038/s41586-024-07487-w>

Craig, D. B., & Dombkowski, A. A. (2013). Disulfide by Design 2.0: a web-based tool for disulfide engineering in proteins. BMC bioinformatics, 14, 346.
<https://doi.org/10.1186/1471-2105-14-346>

England, C. G., Ehlerding, E. B., & Cai, W. (2016). NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence. Bioconjugate chemistry, 27(5), 1175–1187.
<https://doi.org/10.1021/acs.bioconjchem.6b00112>

Guntas, G., Hallett, R. A., Zimmerman, S. P., Williams, T., Yumerefendi, H., Bear, J. E., & Kuhlman, B. (2015). Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins. Proceedings of the National Academy of Sciences of the United States of America, 112(1), 112–117.
<https://doi.org/10.1073/pnas.1417910112>

Parsons, G. B., Souza, D. W., Wu, H., Yu, D., Wadsworth, S. G., Gregory, R. J., & Armentano, D. (2007). Ectopic expression of glucagon-like peptide 1 for gene therapy of type II diabetes. Gene therapy, 14(1), 38–48.
<https://doi.org/10.1038/sj.gt.3302842>

Pathak, G. P., Spiltoir, J. I., Höglund, C., Polstein, L. R., Heine-Koskinen, S., Gersbach, C. A., Rossi, J., & Tucker, C. L. (2017). Bidirectional approaches for optogenetic regulation of gene expression in mammalian cells using Arabidopsis cryptochrome 2. Nucleic acids research, 45(20), e167.
<https://doi.org/10.1093/nar/gkx260>

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