iGEM 2025 — Team nudt-china

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![Art Gallery Header](https://static.igem.wiki/teams/5819/notebook/notebook-1-100.avif)

![logo](https://static.igem.wiki/teams/5819/test/duihui.webp)

* [March](#March)
* [April](#April)
* [May](#May)
* [June](#June)
* [July](#July)
* [August](#August)
* [September](#September)
* [October](#October)
* [Back to the top](#top)

# March 2025

We gathered a group of students interested in synthetic biology and organized a life sciences competition among them. Through written tests and interviews, we comprehensively evaluated the participants to select outstanding talents in various aspects, preparing for the project.

![](https://static.igem.wiki/teams/5819/notebook/march.webp)

# April 2025

![](https://static.igem.wiki/teams/5819/notebook/april.webp)

Establishment of team NUDT-CHINA 2025. Team members from different majors and grade levels chose their preferred subgroups based on their interests and strengths, including wet lab, human practices, art and design, etc.

# May 2025

Brainstorm for 2025 team project.

We divided the team members into multiple groups to propose topics and design solutions, cultivating synthetic biology thinking and developing initial project ideas.

The team trained new iGEMers. They learned basic molecular biology experimental techniques, making preliminary preparations for subsequent wet lab experiments.

Determin the direction for the 2025 iGEM project, considering the use of a photosensitive system and a small molecule system to achieve a dissociation process.

Design questionnaire content and launched a public survey on preferences for different types of science outreach activities, initiating the education process.

![](https://static.igem.wiki/teams/5819/notebook/may.webp)

# June 2025

![](https://static.igem.wiki/teams/5819/notebook/june.webp)

Construct plasmids for the PhoCl family of photolyase proteins.

Preliminarily design and manufactur hardware for illumination.

In cell experiments, the constructed plasmids were transfected into cells. The cells were then exposed to light of different wavelengths and durations. After a certain period, the levels of indicator proteins secreted by the cells were detected. Changes in these indicators and controls were used to verify if the system was functioning properly.

Unfortunately, after repeated attempts, it was found that the constructed system consistently failed to work properly. With guidance from instructors, we realized this was because the ultraviolet light energy was too high, causing significant damage to the cells during irradiation, resulting in low cell survival rates and thus preventing our system from functioning well. To address these limitations, we switched to light of other colors, and the system was optimized accordingly.

Simultaneously, we held our first educational lecture titled *"Why is Synthetic Biology Important to You?"* at Zhucheng No.1 High School; held the second lecture *"Why is Synthetic Biology Important to You?"* at Changsha No.1 High School; and hosted a debate on *"Is Gene Editing Beneficial to Humanity?"* at Zhucheng No.1 High School.

Additionally, we made efforts in early childhood education by conducting a series of fun interactive classes titled *"Exploring Magical 'Tiny Lives'"*, using activities like handicrafts and small experiments to explore the structures of cells, viruses, and DNA with children.

# July 2025

Construct mMaple photocleavable plasmids.
Construct CarH photo-separating protein plasmids.
The constructed plasmids were transfected into cells. The cells were then exposed to light of different wavelengths and durations. After a certain period, the levels of indicator proteins secreted by the cells were detected. Changes in these indicators and controls were used to verify if the system was functioning properly.
It worked,but we didn't stop here.
Interview Shaowei Zhang to gather ideas regarding the hardware (SPARKbox). Begin optimizing the existing hardware to enable it to emit four wavelengths of light, prevent light leakage, ensure ventilation, and remain user-friendly. Interviewed Jiawei Shao to understand the value of our project and wet lab design in the field of synthetic biology.

![](https://static.igem.wiki/teams/5819/notebook/notebookjuly1.avif)

# August 2025

![](https://static.igem.wiki/teams/5819/notebook/august.webp)

Construct small molecule system plasmids.
The plasmids were transfected into cells, and then the cells were stimulated with Gra. After a certain period, the levels of indicator proteins secreted by the cells were detected. Changes in these indicators and controls were used to verify if the system was functioning properly.
It still worked.
Promotion video is finished.
Participate in the Conference for China iGEMer Community (CCiC), present the project poster and deliver the presentation; engage in project exchanges with teams from across the country and establish multi-faceted collaborations, ultimately achieving second place.
Organize an online meeting with Jilin University\_China (Jilin-China) to share respective projects and exchange experiences.
Hold an online meeting with Beijing Institute of Technology (BIT-CHINA) to discuss and divide tasks for the *"Coordinated Development Handbook of Synthetic Biology and Sustainable Development Goals (SDGs)"*.

# September 2025

Finish the initiative *From Static to Dynamic Regulation* with Jilin-China,HZAU-China,HainanU-China.

Finish the mini-game.

Attend 3i Engagement online conference with PekingHSC, NJU-China,XJTLU-CHINA,Tongji-China,Tsinghua-China.

Finish the picture book with CAFA-Beijing and CJUH-JLU-China.

Safety form is submitted.

Finish *Trust in Dialogue: A Handbook for Building Warm and Effective Doctor-Patient* with SUSTECH-MED and SMU-Union-China.

Attend science popularization fair with CSU-CHINA and HNU-China.

![](https://static.igem.wiki/teams/5819/notebook/september.webp)

# October 2025

![](https://static.igem.wiki/teams/5819/notebook/notebookwiki.avif)

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![logo](https://static.igem.wiki/teams/5819/test/duihui.webp)

* [Introduction](#Introduction)
* [Stage I: Secretion switch based on Photolytic protein](#Stage-I)
* [Stage II: Secretion switch based on signal-inducible protein disassociation](#Stage-II)
* [Conclusion](#Conclusion)
* [References](#references)
* [Back to the top](#top)

# Introduction

Our goal was to engineer a signal-controlled protein secretion switch in mammalian cells. Following multiple Design-Build-Test-Learn (DBTL) cycles (**Figure 1**), we developed two modular systems:

* OptoSPARK - light-inducible protein secretion
* ChemSPARK - small-molecule-inducible protein secretion

Both switches regulate secretion post-translationally, enabling responses that are faster and more sensitive than conventional transcriptional control. Below, we detail the iterative DBTL cycles that led from vesicle anchoring to light and drug control, then to performance optimization and benchmarking.

![长图内容](https://static.igem.wiki/teams/5819/engineering/engihb-1-2x.webp)

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**Figure 1.** A schematic Diagram of the Engineering process (you can scroll horizontally to see more information).

# Stage I: Secretion switch based on Photolytic protein

## Secretion switch foundation: vesicle anchoring with LifeAct

### Design:

To block constitutive secretion and create a controllable "release" step, we first needed to retain secretory vesicles carrying the protein of interest (POI). We fused the POI to a furin site-containing transmembrane domain (TMD) so the cargo is released into the vesicle lumen after Golgi trafficking, and then added LifeAct, a 17-aa peptide that binds F-actin with high specificity [1,2](#references). The design tethers vesicles to the actin cytoskeleton (**Figure 2**). NanoLuc (Nluc), fused to an IgK signal peptide, served as a quantitative secreted reporter.

![Vesicle-anchoring module schematic](https://static.igem.wiki/teams/5819/watermarking/1115engineering2.avif)

**Figure 2.** Schematic of the vesicle-anchoring module.

### Build & Test:

HEK293T cells were transfected with pNC25038 (PCMV-IgK-Nluc-FS-TMD-LifeAct) or pNC25039 (PCMV-IgK-Nluc-FS-TMD-mCherry). Results showed that cells expressing pNC25038 showed a significant decrease in NanoLuc secretion compared with controls (**Figure 3A**), confirming effective vesicle retention. Electron microscopy of cells expressing pNC25085 (EGFP-TMD-LifeAct) showed vesicle accumulation intracellularly (**Figure 3B**). Similarly, live-cell fluorescence imaging also demonstrated clear co-localization of EGFP-TMD-LifeAct with mCherry-LifeAct along filamentous actin (**Figure 4**), confirming that vesicles were immobilized on the cytoskeleton.

![LifeAct fusion results](https://static.igem.wiki/teams/5819/watermarking/1115engineering3.avif)

**Figure 3.** LifeAct fusion enables robust retention of secretory vesicles in mammalian cells

(A) Supernatant NanoLuc levels in HEK293T cells transfected with either pNC25038 (PCMV-IgK-Nluc-FS-TMD-LifeAct) or pNC25039 (PCMV-IgK-Nluc-FS-TMD-mCherry). NanoLuc activity was measured 48 h post-transfection. Data represent mean ± SD from n = 4 independent experiments; \*\*\*P<0.001, two-tailed unpaired t-test.
(B) Representative transmission electron microscopy images of HEK293T cells expressing POI-TMD-LifeAct, showing vesicle accumulation near the actin cytoskeleton. Samples were collected 48 hours post transfection. Scale bars, 2 μm (left) and 1 μm (right).

![Fluorescence protein localization](https://static.igem.wiki/teams/5819/engineering/engifig4.webp)

**Figure 4.** Confocal micrograph of fluorescence protein localization.

Representative live-cell fluorescence imaging of HEK293T cells co-transfected with pNC25086 (PCMV-LifeAct-mCherry) and either pNC25087 (PCMV-EGFP-TMD; upper panels) or pNC25085 (PCMV-EGFP-TMD-LifeAct; lower panels). Fluorescent images were taken 24 hours post transfection Scale bars, 10 μm (upper) and 10 μm (lower).

### Learn:

LifeAct reliably terminates constitutive secretion by anchoring vesicles, providing a solid foundation for signal-induced release in subsequent cycles.

## OptoSPARK V0.5: photolytic release (mMaple3)

### Design:

We next tested whether light-induced disruption of the anchoring complex could trigger secretion. We built POI-TMD-mMaple3-LifeAct, leveraging mMaple3, a photoconvertible FP that turns green-to-red upon 405 nm illumination and undergoes a cleavage-like structural change near aa 66-67 [3,4](#references), expected to release anchored vesicles **(Figure 5)**.

![Secretion system genetic switch schematic](https://static.igem.wiki/teams/5819/watermarking/1115engineering5.avif)

**Figure 5.** Schematic diagram illustrating the operation of the secretion system genetic switch in HEK-293T cells under violet light induction

### Build & Test:

HEK293T cells were transfected with pNC25088 (PCMV-IgK-Nluc-FS-TMD-mMaple3-LifeAct) and illuminated using our custom 405 nm light device (10 s ON / 15 s OFF, 10 min). Illumination induced strong red fluorescence, confirming mMaple3 photoconversion (**Figure 6A**), and a modest but reproducible increase in NanoLuc secretion compared with dark controls (**Figure 6B**). However, control cells expressing pNC25089 (PCMV-IgK-Nluc) showed a strong reduction in NanoLuc activity and visible stress under the same illumination, indicating that violet-light exposure was cytotoxic (**Figure 6C-D**).

![mMaple Photolytic protein results](https://static.igem.wiki/teams/5819/watermarking/1115engineering6.avif)

**Figure 6.** Engineering of Secretion switch based on mMaple Photolytic protein.

(A) Cellular Red Fluorescence / Green Fluorescence intensity. Representative fluorescence images of *pNC25088 (PCMV-IgK-Nluc-FS-TMD-mMaple3-LifeAct)* HEK293T cells maintained in the dark (control) or stimulated with violet light (Light). Fluorescent images were taken 48 hours post transfection. Scale bar, 50 µm. Quantification of single-cell red/green fluorescence ratios was performed using ImageJ. \*\*\*P<0.001, two-tailed unpaired t-test.
(B) NanoLuc secretion levels under different stimulation durations. HEK293T cells were transfected with pNC25088 (PCMV-IgK-Nluc-FS-TMD-mMaple3-LifeAct). 48 hours post-transfection, cells were illuminated with pulsed violet light (405 nm; 10 s ON/15 s OFF). Supernatants were collected before stimulation (0 min) and at 5 and 10 min after light exposure to quantify NanoLuc activity. Data represent mean ± SD from n = 4 independent experiments; p-value obtained by two-tailed unpaired t-test.
(C) Effect of violet-light stimulation on constitutive protein secretion. HEK293T cells transfected with pNC25089 (PCMV-IgK-Nluc) were illuminated with pulsed violet light (405 nm; 10 s ON/15 s OFF) 48 h post-transfection. NanoLuc activity in culture supernatants was measured before stimulation at 5, 10, and 15min after illumination. Data represent mean ± SD from n = 4 independent experiments.
(D) Representative phase-contrast images of HEK293T cells transfected with pNC25088 (PCMV-lgK-Nluc-FS-TMD-mMaple3-LifeAct) maintained in the dark or after violet-light stimulation (405 nm; 10 s ON/15 s OFF) for 15 min. Scale bar, 50 µm.

### Learn:

The experiment validated the concept of light-induced vesicle release but revealed that violet light causes significant cytotoxicity. Building on these results and the input we obtained from human practice activities (see our [Human Practices](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D) page for more details), we decided to pursue longer-wavelength, dissociation-based mechanisms for improved biocompatibility.

# Stage II: Secretion switch based on signal-inducible protein dissociation

## OptoSPARK V1.0: long-wavelength photodissociation (CarH and BphP1-PpsR2)

### Design:

Inspired by feedback from our **Human Practices** discussions, we aimed to design a system responsive to **longer wavelengths** of light, which are safer for cells and penetrate tissue more effectively. Also, we decided to focus on **light-induced protein dissociation** rather than photolysis.

Two photoreactive protein pairs were selected:

* **CarH** (green light, 540 nm): oligomerizes in the dark with vitamin B₁₂ but dissociates upon illumination[5](#references).
* **BphP1-PpsR2** (near-infrared light, 740 nm): dissociates upon photoconversion of BphP1 from the Pfr to the Pr state[6](#references).

In both systems, dissociation breaks the LifeAct anchoring, triggering vesicle release (**Figure 7**).

![Light-responsive secretion switch schematics](https://static.igem.wiki/teams/5819/watermarking/1115engineering7.avif)

**Figure 7.** Schematic illustration of the near-infrared (NIR)-responsive secretion switch and the green-light-responsive secretion switch.

### Build & Test:

* **NIR-light system:** Cells were co-transfected with pNC25054 (PCMV-PpsR2), pNC25056 (PCMV-BphP1-LifeAct), and pNC25057 (PCMV-IgK-Nluc-FS-TMD-BphP1) in a 1:1:1 ratio and illuminated with 740 nm light (30 s ON / 210 s OFF, 2 h), producing similar light-dependent secretion increases (**Figure 8A**).
* **Green-light system:** HEK293T cells were transfected with pNC25041 (PCMV-IgK-Nluc-FS-TMD-2×GS-CarH) and pNC25043 (PCMV-LifeAct-CarH) in a 1:6 ratio. Cells illuminated with 540 nm light (30 s ON / 30 s OFF, 2 h) showed increased NanoLuc secretion compared with dark controls (**Figure 8B**).

![Photosynthetic dissociation protein results](https://static.igem.wiki/teams/5819/watermarking/1115engineering8.avif)

**Figure 8.** Characterization experiments of the two types of switches based on Photosynthetic dissociation protein.

(A) NanoLuc secretion of HEK293T cells transfected with pNC25054 (PCMV-PpsR2), pNC25056 (PCMV-BphP1-LifeAct), and pNC25057 (PCMV-IgK-Nluc-FS-TMD-BphP1) in a 1:1:1 ratio. Cells were illuminated with pulsed NIR light (740 nm; 30 s ON / 210 s OFF) 48 hours post transfection, NanoLuc activity was measured immediately after illumination. Data represent mean ± SD from n = 4 independent experiments. \*\* 0.001<P<0.01, two-tailed unpaired t-test.
(B) NanoLuc secretion of HEK293T cells transfected with pNC25041 (PCMV-IgK-Nluc-FS-TMD-2×GSlinker-CarH) and pNC25043 (PCMV-LifeAct-CarH) in a 1:6 ratio. Cells were illuminated with pulsed green light (540 nm; 30 s ON / 30 s OFF) 48 hours post transfection, NanoLuc activity was measured immediately after illumination. Data represent mean ± SD from n = 4 independent experiments. \* 0.01<P<0.05, two-tailed unpaired t-test.

### Learn:

OptoSPARK V1.0 demonstrated effective secretion under low-energy light without detectable cytotoxicity. This confirmed that light-induced protein dissociation is a viable strategy for biocompatible, optical control of protein secretion. However, we also observed that homodimerization of anchor proteins could reduce retention efficiency, leading us to develop a chemically inducible version of the switch.

## ChemSPARK V1.0: chemically induced dissociation (Binder/NS3a)

### Design:

To expand SPARK into a clinically relevant platform, we engineered a small-molecule-responsive switch using the interaction between Binder (Apo-NS3a reader) and the HCV protease NS3aH1. These proteins form a stable complex that can be rapidly dissociated by grazoprevir, an FDA-approved NS3a inhibitor[7](#references).

In this system (**Figure 9**), NS3a was fused to the TMD (vesicle side) and Binder to LifeAct (anchoring side). Grazoprevir treatment disrupts the Binder-NS3a complex, releasing vesicles and inducing secretion.

![Grazoprevir-induced disassociation schematic](https://static.igem.wiki/teams/5819/watermarking/1115engineering9.avif)

**Figure 9.** Schematic diagram of the secretion switch design that based on the Grazoprevir-induced disassociation between NS3a and Binder.

### Build & Test:

HEK293T cells co-transfected with pNC25091 (PCMV-Binder-LifeAct) and pNC25090 (PCMV-Nluc-TMD-NS3a) were treated with DMSO or grazoprevir. Grazoprevir induced a mild but consistent increase in NanoLuc secretion compared with controls (**Figure 10**).

![Chemical signal-induced protein disassociation results](https://static.igem.wiki/teams/5819/watermarking/1115engineering10.avif)

**Figure 10.** The construction of the secretion switch based on chemical signal-induced protein disassociation in HEK-293T cells.

NanoLuc secretion of HEK293T cells co-transfected with pNC25091 (PCMV-Binder-LifeAct) and pNC25090 (PCMV-Nluc-TMD-NS3a). Cells were treated with DMSO or grazoprevir at the indicated concentrations 48 hours post transfection, Nanoluc levels were quantified 2 hours post induction. Data represent mean ± SD from n = 4 independent experiments measured 2 h after stimulation. Data represent mean ± SD from n = 4 independent experiments.

### Learn:

ChemSPARK achieved drug-induced secretion, confirming the feasibility of chemical control. However, the initial response amplitude was modest, motivating further optimization.

## ChemSPARK V2.0: optimization and benchmarking

### Design:

To enhance chemSPARK's performance, we optimized two key parameters (**Figure 11**):

1. Copy numbers of Nluc and NS3a within expression constructs
2. Transfection ratio between the anchoring (Binder-LifeAct) and secretion (Nluc-TMD-NS3a) modules

![chemSPARK gene circuit](https://static.igem.wiki/teams/5819/watermarking/1115engineering11.avif)

**Figure 11.** The gene circuit of chemSPARK.

### Build & Test:

* **Copy number optimization:** Increasing Nluc copy number enhanced total secretion, while increasing NS3a reduced basal secretion, improving fold-change response (**Figure 12A**).
* **Transfection ratio optimization:** A 3:1 ratio of secretion to anchoring modules provided the best balance between expression strength and responsiveness (**Figure 12B**).
* **Dose dependence:** NanoLuc secretion increased up to **40.1×** in a grazoprevir dose-dependent manner (0-10 µM, 2 h post-induction; **Figure 12C**).
* **Kinetic comparison:** ChemSPARK responded in less than an hour, whereas a transcriptional control system (**pNC25046**, **pNC25048**, **pNC25098**) required **>12 hours** to produce detectable secretion (**Figure 12D**).

![chemSPARK characterization results](https://static.igem.wiki/teams/5819/engineering/1009engifig11-4x.avif)

**Figure 12.** The characterization of the secretion switch based on chemical signal-induced protein disassociation in HEK-293T cells.

(A) Effect of varying Nluc and NS3a copy numbers on secretion efficiency. HEK293T cells were transfected with either pNC25093 (PCMV-IgK-Nluc-TMD-NS3a-Binder-LifeAct), pNC25094 (PCMV-IgK-(Nluc)2-TMD-NS3a-Binder-LifeAct), pNC25095 (PCMV-IgK-(Nluc)3-TMD-NS3a-Binder-LifeAct), pNC25096 (PCMV-IgK-Nluc-TMD-(NS3a)2-Binder-LifeAct), or pNC25097 (PCMV-IgK-Nluc-TMD-(NS3a)3-Binder-LifeAct). Cells were treated with DMSO or grazoprevir 48 h post-transfection, and NanoLuc levels were quantified 2 h after stimulation. Data represent mean ± SD from n = 4 independent experiments. \*\* 0.001<P<0.01, two-tailed unpaired t-test.
(B) Optimization of transfection ratio between anchoring and secretion modules. HEK293T cells were co-transfected with pNC25092 (PCMV-IgK-(Nluc)3-TMD-(NS3a)2) and either pNC25086 (PCMV-LifeAct-mCherry; control) or pNC25091 (PCMV-Binder-LifeAct). Cells were treated with DMSO or grazoprevir 48 h post-transfection, and NanoLuc levels were quantified 2 h after stimulation. Data represent mean ± SD from n = 4 independent experiments. Data represent mean ± SD from n = 4 independent experiments. \*\* 0.001<P<0.01, two-tailed unpaired t-test.
(C) Dose-dependent secretion response to Grazoprevir treatment (0–10 µM). Data presents mean ±SD, n = 3 independent experiments, fitted to a symmetric sigmoid curve.
(D) Kinetics of grazoprevir-inducible NanoLuc expression comparing chemSPARK with a transcriptional activation system. For the transcriptional system, HEK293T cells were co-transfected with pNC25046 (PCMV-Binder-TetR-NLS-VP64), pNC25048 (PCMV-IgK-Fluc-TMD-NS3a), and pNC25098 (PTCE-IgK-Nluc). For the secretion system, cells were co-transfected with pNC25092 (PCMV-(Nluc-FS)3-TMD-(NS3a)2) and pNC25091 (PCMV-LifeAct-mCherry-Binder). In both systems, 10 µM grazoprevir was added at the indicated time points (6 h post-transfection for transcriptional and 48 h for secretion), and NanoLuc levels were monitored over the following 48 h and 12 h, respectively. Data represent mean ± SD of fold changes calculated as the ratio of NanoLuc levels in grazoprevir-treated versus untreated samples (n = 3 independent experiments).

### Learn:

ChemSPARK achieved rapid, tunable, and dose-dependent secretion with activation kinetics far exceeding transcriptional regulation.

Through Human Practices, Prof. Jiawei Shao (Zhejiang University School of Medicine) suggested future development of closed-loop, endogenous signal-responsive systems (e.g., calcium-responsive secretion) to enable autonomous physiological regulation---an exciting next step beyond iGEM.

# Conclusion

Through iterative DBTL cycles, we engineered a family of signal-controlled secretion systems in mammalian cells:

* LifeAct anchoring enabled reliable vesicle retention.
* Light-induced photodissociation achieved safe, rapid optical control.
* Drug-induced dissociation produced a clinically compatible, tunable switch.

Together, optoSPARK and chemSPARK demonstrate a robust, modular framework for post-translational regulation of secretion, achieving fast, programmable, and biocompatible control of protein output.

## References

▼

[1] Riedl, J. *et al.* Lifeact: a versatile marker to visualize F-actin. *Nat Methods* **5**, 605-607 (2008). https://doi.org/10.1038/nmeth.1220

[2] Courtemanche, N., Pollard, T. D. & Chen, Q. Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins. *Nat Cell Biol* **18**, 676-683 (2016). https://doi.org/10.1038/ncb3351

[3] Mizuno, H. *et al.* Photo-induced peptide cleavage in the green-to-red conversion of a fluorescent protein. *Mol Cell* **12**, 1051-1058 (2003). https://doi.org/10.1016/s1097-2765(03)00393-9

[4] Hoi, H. *et al.* A monomeric photoconvertible fluorescent protein for imaging of dynamic protein localization. *J Mol Biol* **401**, 776-791 (2010). https://doi.org/10.1016/j.jmb.2010.06.056

[5] Jost, M. *et al.* Structural basis for gene regulation by a B12-dependent photoreceptor. *Nature* **526**, 536-541 (2015). https://doi.org/10.1038/nature14950

[6] Bellini, D. & Papiz, M. Z. Structure of a bacteriophytochrome and light-stimulated protomer swapping with a gene repressor. *Structure* **20**, 1436-1446 (2012). https://doi.org/10.1016/j.str.2012.06.002

[7] Cunningham-Bryant, D. *et al.* A Chemically Disrupted Proximity System for Controlling Dynamic Cellular Processes. *J Am Chem Soc* **141**, 3352-3355 (2019). https://doi.org/10.1021/jacs.8b12382

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### Molecular Experiments

#### Agarose Gel Electrophoresis

#### Double Enzyme DNA Digestion & Agarose Gel Electrophoresis

#### Gel Purification

#### Infusion

#### Overlap PCR & Agarose Gel Electrophoresis

#### PCR & Agarose Gel Electrophoresis – Colony PCR

#### PCR & Agarose Gel Electrophoresis

#### Plasmid Conservation

#### Plasmid Extraction

#### Protocol for LB medium preparation

#### Transformation

## Molecular Experiments

![Molecular Experiments Cover](https://static.igem.wiki/teams/5819/experiments/molecular.webp)

Select a molecular experiment to view details

← Back to Cover

### Cell Experiments

#### Protocol for Cell Transfection

#### Protocol for Induction Stimulation

#### Protocol for Cell Culture and Subculture

#### Protocol for SEAP Assay

#### Protocol for Nluc Assay

#### Protocol for Light Illumination and Fluorescence Imaging

## Cell Experiments

![Cell Experiments Cover](https://static.igem.wiki/teams/5819/experiments/cell.webp)

Select a cell experiment to view details

← Back to Cover

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![logo](https://static.igem.wiki/teams/5819/test/duihui.webp)

* [Proof of concept](#LifeAct)
* [optoSPARK V0.5](#Photolytic cleavage of the retention domain)
* [optoSPARK V1.0](#Light-controlled protein dissociation)
* [chemSPARK V1.0](#chemSPARK)
* [chemSPARK V2.0](#chemSPARK-dynamics)
* [References](#references)
* [Back to the top](#top)

# LifeAct fusion enables robust retention of secretory vesicles in mammalian cells

Before establishing signal responsiveness within the constitutive secretion pathway, we first needed to prevent secretory vesicles containing the protein of interest (POI) from being released constitutively. To achieve this, the POI was fused to a furin site-containing transmembrane domain (TMD), which permits proteolytic release of the cargo into the vesicle lumen following trafficking through the Golgi apparatus. The TMD was further fused to LifeAct, a 17-amino acid peptide that binds F-actin with high specificity1, enabling vesicles carrying the POI to tether to the actin cytoskeleton (**Figure 1A**).

To evaluate the anchoring efficiency of LifeAct, HEK293T cells were transfected with either pNC25038 (PCMV-IgK-Nluc-FS-TMD-LifeAct) or pNC25039 (PCMV-IgK-Nluc-FS-TMD-mCherry), where the secreted reporter NanoLuc served as the POI. Notably, cells expressing POI-TMD-LifeAct showed a marked reduction in extracellular NanoLuc activity compared with controls, indicating effective intracellular retention of the reporter (**Figure 1B**).

To directly visualize vesicle localization, we performed electron microscopy and live-cell fluorescence colocalization analyses. Electron microscopy of cells transfected with pNC25085 (PCMV-EGFP-TMD-LifeAct) revealed abundant vesicular structures accumulated near the actin cytoskeleton, consistent with vesicle retention (**Figure 1C**). Consistent to these findings, cells co-transfected with pNC25086 (PCMV-LifeAct-mCherry) and pNC25087 (PCMV-EGFP-TMD) showed green fluorescence distributed on cell membrane and secretory pathway, reflecting normal vesicle mobility and secretion. In contrast, cells co-transfected with pNC25086 and pNC25085 exhibited strong colocalization of green and red fluorescence along filamentous actin, indicating that vesicles were immobilized on the cytoskeleton (**Figure 1D**).

Together, these results demonstrate that LifeAct functions as a robust anchoring domain that effectively retains secretory vesicles in mammalian cells, thereby establishing a foundation for subsequent signal-responsive secretion control.

![Survey Results](https://static.igem.wiki/teams/5819/watermarking/1115result1.avif)

**Figure 1.**LifeAct fusion enables robust retention of secretory vesicles in mammalian cells.

(A) Schematic illustration of the retention design. LifeAct functions as an anchoring peptide that tethers secretory vesicles to the actin cytoskeleton, preventing constitutive release.
(B) Supernatant NanoLuc levels in HEK293T cells transfected with either pNC25038 (PCMV-IgK-Nluc-FS-TMD-mCherry-LifeAct) or pNC25039 (PCMV-IgK-Nluc-FS-TMD-mCherry). NanoLuc activity was measured 48 h post-transfection. Data represent mean ± SD from n = 4 independent experiments; \*\*\*P < 0.001, two-tailed unpaired t-test.
(C) Representative transmission electron microscopy images of HEK293T cells expressing POI-TMD-LifeAct, showing vesicle accumulation near the actin cytoskeleton. Samples were collected 48 hours post transfection. Scale bars, 2 μm (left) and 1 μm (right).
(D) Representative live-cell fluorescence imaging of HEK293T cells co-transfected with pNC25086 (PCMV-LifeAct-mCherry) and either pNC25087 (PCMV-EGFP-TMD; upper panels) or pNC25085 (PCMV-EGFP-TMD-LifeAct; lower panels). Fluorescent images were taken 24 hours post transfection Scale bars, 10 μm (upper) and 10 μm (lower).

# Photolytic cleavage of the retention domain enables mild light-inducible protein secretion

Having established that LifeAct effectively retains secretory vesicles, we next examined whether signal-induced disruption of the retention complex could trigger controlled protein secretion. To test this, we generated a POI-TMD-mMaple3-LifeAct construct, in which mMaple3, a photoconvertible fluorescent protein, serves as a light-responsive element. mMaple3 emits green fluorescence under basal conditions but undergoes irreversible photoconversion to a red-emitting form upon illumination with 405 nm light, accompanied by a cleavage at the 66-67 amino acid2,3 that were expected to release the tethered vesicles (**Figure 2A**).

To evaluate this design, HEK293T cells were transfected with pNC25088 (PCMV-IgK-Nluc-FS-TMD-mMaple3-LifeAct) and subjected to optical stimulation using a custom-built illumination system (**see [Hardware page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27hardware%27%29%20%7D%7D) for details; Figure 2B**). Cells exposed to 405 nm light at a cycle of 10 s ON and 15 s OFF for 10 minutes exhibited markedly increased red fluorescence intensity relative to non-illuminated controls, confirming efficient photoconversion (**Figure 2C**). Reporter assays showed a modest but reproducible increase in NanoLuc secretion following 10 min light stimulation compared with dark controls (**Figure 2D**).

![Survey Results](https://static.igem.wiki/teams/5819/watermarking/1115result2.avif)

**Figure 2.**Photolysis of the mMaple3 retention module enables mild light-inducible protein secretion.

(A) Schematic illustration of the mMaple3-based secretion switch. Upon violet-light illumination (405 nm), mMaple3 undergoes photolysis, disrupting the LifeAct-mediated vesicle anchoring and triggering secretion.
(B) Exploded view of the custom optical stimulation device used for controlled light induction.
(C) Cellular Red Fluorescece/ Green Fluorescence intensity. Representative fluorescence images of pNC25088-transfected HEK293T cells maintained in the dark (control) or stimulated with violet light (Light). Fluorescent images were taken 48 hours post transfection. Scale bar, 50 µm. Quantification of single-cell red/green fluorescence ratios was performed using ImageJ. \*\*\*P < 0.001, two-tailed unpaired t-test.
(D) NanoLuc secretion levels under different stimulation durations. HEK293T cells were transfected with pNC25019 (PCMV-IgK-Nluc-mMaple3-LifeAct). 48 hours post-transfection, cells were illuminated with pulsed violet light (405 nm; 10 s ON/15 s OFF). Supernatants were collected before stimulation (0 min) and at 5 and 10 min after light exposure to quantify NanoLuc activity. Data represent mean ± SD from n = 4 independent experiments; p-value obtained by two-tailed unpaired t-test.

Notably, despite cells remaining morphologically intact, illumination caused a significant reduction in NanoLuc activity in cells transfected with the control construct pNC25089 (PCMV-IgK-Nluc) (**Figure 3A-B**), indicating that 405 nm light exposure was cytotoxic under these conditions.

Collectively, these findings suggest that while violet-light illumination introduces measurable cytotoxicity, photolysis of the mMaple3-LifeAct retention module enables mild, light-inducible secretion of the target protein, demonstrating proof-of-concept for signal-controlled post-translational secretion.

![Survey Results](https://static.igem.wiki/teams/5819/watermarking/1115result3.avif)

**Figure 3.**Violet-light illumination induces cytotoxicity and decreases constitutive secretion efficiency.

(A) Effect of violet-light stimulation on constitutive protein secretion. HEK293T cells transfected with pNC25089 (PCMV-IgK-Nluc) were illuminated with pulsed violet light (405 nm; 10 s ON/15 s OFF) 48 h post-transfection. NanoLuc activity in culture supernatants was measured before stimulation (0 min) and at 5 and 10 min after illumination. Data represent mean ± SD from n = 4 independent experiments.
(B) Representative phase-contrast images of HEK293T cells transfected with pNC25088 (PCMV-IgK-Nluc-FS-TMD-mMaple3-LifeAct) maintained in the dark or after violet-light stimulation (405 nm; 10 s ON/15 s OFF) for 15 min. Scale bar, 50 µm.

# Light-controlled protein dissociation enables green- and near-infrared light-inducible secretion in optoSPARK

Given that violet-light illumination caused cytotoxicity, we next sought to engineer a secretion system responsive to longer-wavelength light, which penetrates tissue more effectively and minimizes phototoxic effects. Guided for insights from *Dr. Human Practices* investigations (see Human Practices page for details ), we reasoned that while most photolytic proteins respond to short-wavelength light, light-inducible protein dissociation could serve as an alternative regulatory mechanism. Based on this principle, we developed optoSPARK, a modular system that couples vesicle release to signal-induced protein dissociation under green or near-infrared (NIR) light (see Engineering page for design details).

For the NIR-responsive design, we replaced mMaple3 with the BphP1-PpsR2 interaction module, in which BphP1 homodimers dissociate upon illumination and subsequently bind to PpsR24. For green-light activation, we incorporated the CarH module, which aggregates in the dark and dissociates upon 540 nm light exposure5. In both systems, dissociation of the photoreactive proteins disrupts the LifeAct-mediated anchoring, leading to signal-induced vesicle release (**Figure 4A, 4C**).

Experimentally, HEK293T cells expressing either the NIR- or green-light system were illuminated under pulsed conditions for 2 hours (740 nm, 30 s ON / 210 s OFF for NIR; 540 nm, 30 s ON / 30 s OFF for green light). In both cases, NanoLuc secretion increased significantly compared with dark controls, indicating efficient light-triggered protein release (**Figure 4B, 4D**).

Together, these results demonstrate that protein dissociation-based control enables rapid and programmable secretion under low-energy light, effectively overcoming the cytotoxic limitations of violet-light-dependent systems. The optoSPARK framework thus establishes a versatile foundation for biocompatible, light-inducible post-translational regulation in mammalian cells.

![Survey Results](https://static.igem.wiki/teams/5819/watermarking/1115result4.avif)

**Figure 4.**Signal-controlled protein dissociation enables green- and near-infrared light-inducible secretion in mammalian cells.

(A) Schematic illustration of the near-infrared (NIR)-responsive secretion switch. Upon 740 nm illumination, BphP1 homodimers dissociate and bind to PpsR2, releasing vesicles from LifeAct-mediated anchoring.
(B) NanoLuc secretion of HEK293T cells transfected with pNC25054 (PCMV-PpsR2), pNC25056 (PCMV-BphP1-LifeAct), and pNC25057 (PCMV-IgK-Nluc-FS-TMD-BphP1) in a 1:1:1 ratio. Cells were illuminated with pulsed NIR light (740 nm; 30 s ON / 210 s OFF) 48 hours post transfection, NanoLuc activity was measured immediately after illumination. Data represent mean ± SD from n = 4 independent experiments. \*\* 0.001 < P < 0.01, two-tailed unpaired t-test.
(C) Schematic illustration of the green-light-responsive secretion switch. CarH aggregates in the dark and dissociates upon 540 nm illumination, leading to vesicle release.
(D) NanoLuc secretion of HEK293T cells transfected with pNC25041 (PCMV-IgK-Nluc-FS-TMD-2×GSlinker-CarH) and pNC25043 (PCMV-LifeAct-CarH) in a 1:6 ratio. Cells were illuminated with pulsed green light (540 nm; 30 s ON / 30 s OFF) 48 hours post transfection, NanoLuc activity was measured immediately after illumination. Data represent mean ± SD from n = 4 independent experiments. \* 0.01< P <0.05, two-tailed unpaired t-test.

# Drug-responsive protein dissociation enables chemically inducible secretion in chemSPARK

Building on the success of light-inducible secretion via protein dissociation, we next sought to establish a system responsive to chemical inducers, enabling versatile and clinically translatable control of protein secretion. To achieve this, we designed chemSPARK, a modular secretion system regulated by drug-induced protein dissociation.

The design is based on the interaction between Binder (Apo-NS3a reader) and the hepatitis C virus protease NS3aH16,7. Previous studies have shown that this interaction can be rapidly disrupted by small-molecule inhibitors such as grazoprevir, which competes for the NS3a active site, leading to complex dissociation within minutes. Leveraging this dynamic behavior, we constructed a secretion switch driven by Binder-NS3a-mediated vesicle anchoring (**Figure 5A**).

To test this design, HEK293T cells were co-transfected with the pNC25091 (PCMV-Binder-LifeAct), and pNC25090 (PCMV-Nluc-TMD-NS3a). and treated with either DMSO (control) or grazoprevir 48 hours post-transfection. Compared with DMSO-treated controls, cells exposed to grazoprevir showed a mild but reproducible increase in NanoLuc secretion, confirming drug-inducible release (**Figure 5B**).

We next optimized the system by varying the copy numbers of Nluc and NS3a within the expression constructs. As expected, increasing the copy number of Nluc markedly enhanced grazoprevir-induced secretion, likely due to the accumulation of additional reporter protein within secretory vesicles (**Figure 5C**). Conversely, increasing the copy number of NS3a reduced basal secretion levels, thereby improving the overall fold induction. With the optimized copy configuration, we further evaluated whether the ratio between the actin-anchoring and secretion modules affected circuit performance. HEK293T cells were co-transfected with pNC25092 (PCMV-IgK-(Nluc)3-TMD-(NS3a)2) and either pNC25086 (PCMV-LifeAct-mCherry; control) or pNC25091 (PCMV-Binder-LifeAct). Optimal secretion was achieved at a 3:1 ratio, which provided a balance between expression strength and responsiveness (**Figure 5D**).

Together, these results demonstrate that chemSPARK enables chemically induced, post-translational control of protein secretion in mammalian cells. By employing a drug-responsive dissociation mechanism, this system provides a robust, modular, and clinically compatible platform for small-molecule-regulated therapeutic protein release.

![Survey Results](https://static.igem.wiki/teams/5819/watermarking/1115result5.avif)

**Figure 5.**Chemical signal-induced protein dissociation enables drug-responsive secretion in chemSPARK.

(A) Schematic illustration of the secretion switch design based on grazoprevir-induced dissociation between NS3a and Binder. Under basal conditions, the NS3a-Binder interaction anchors secretory vesicles to the cytoskeleton, preventing release. Upon grazoprevir treatment, the complex dissociates, triggering vesicle release and protein secretion.
(B) NanoLuc secretion of HEK293T cells co-transfected with pNC25091 (PCMV-Binder-LifeAct) and pNC25090 (PCMV-Nluc-TMD-NS3a). Cells were treated with DMSO or grazoprevir at the indicated concentrations 48 hours post transfection, Nanoluc levels were quantified 2 hours post induction. Data represent mean ± SD from n = 4 independent experiments measured 2 h after stimulation.
(C) Effect of varying Nluc and NS3a copy numbers on secretion efficiency. HEK293T cells were transfected with either pNC25093 (PCMV-IgK-Nluc-TMD-NS3a-Binder-LifeAct), pNC25094 (PCMV-IgK-(Nluc)2-TMD-NS3a-Binder-LifeAct), pNC25095 (PCMV-IgK-(Nluc)3-TMD-NS3a-Binder-LifeAct), pNC25096 (PCMV-IgK-Nluc-TMD-(NS3a)2-Binder-LifeAct), or pNC25097 (PCMV-IgK-Nluc-TMD-(NS3a)3-Binder-LifeAct). Cells were treated with DMSO or grazoprevir 48 h post-transfection, and NanoLuc levels were quantified 2 h after stimulation. Data represent mean ± SD from n = 4 independent experiments. \*\* 0.001 < P < 0.01, two-tailed unpaired t-test.
(D) Optimization of transfection ratio between anchoring and secretion modules. HEK293T cells were co-transfected with pNC25092 (PCMV-IgK-(Nluc)3-TMD-(NS3a)2) and either pNC25086 (PCMV-LifeAct-mCherry; control) or pNC25091 (PCMV-Binder-LifeAct). Cells were treated with DMSO or grazoprevir 48 h post-transfection, and NanoLuc levels were quantified 2 h after stimulation. Data represent mean ± SD from n = 4 independent experiments. \*\* 0.001< P < 0.01, two-tailed unpaired t-test.

# ChemSPARK exhibits rapid and dose-dependent secretion dynamics with faster activation than transcriptional control

Having established that chemSPARK enables chemically inducible secretion through drug-responsive protein dissociation, we next characterized its dose sensitivity and temporal dynamics to assess suitability for downstream applications.

To characterize chemSPARK, HEK293T cells were co-transfected with pNC25091 (PCMV-LifeAct-mCherry-Binder) and pNC25092 (PCMV-(Nluc-FS)3-TMD-(NS3a)2) at a 3:1 ratio, and treated 48 h post-transfection with varying concentrations of grazoprevir (0-10 µM) or an equivalent volume of DMSO as control. NanoLuc levels were measured 2 h after drug addition. Results showed a clear dose-dependent induction of NanoLuc secretion, reaching a 40.1-fold increase at 10 µM grazoprevir (**Figure 6A**). Time-course analysis further revealed a rapid and sustained activation of the optimized system, with NanoLuc levels elevated 2.7-fold, 12.7-fold, and 12.1-fold at 1, 2, and 4 h post-stimulation, respectively (P = 0.000082 at 1 h; P <0.000001 at 2 and 4 h). These findings demonstrate that chemSPARK responds rapidly and proportionally to drug stimulation, achieving both high sensitivity and tunable control within hours of induction.

In contrast, a conventional transcriptional activation system---comprising pNC25046 (PCMV-Binder-TetR-NLS-VP64), pNC25048 (PCMV-IgK-Fluc-TMD-NS3a), and pNC25098 (PTCE-IgK-Nluc), exhibited delayed protein secretion, with NanoLuc induction detectable only 12 h post-stimulation. This clear difference underscores the superior speed and responsiveness of chemSPARK as a post-translational genetic switch, capable of near-instantaneous small-molecule-controlled protein release (**Figure 6B**).

![Survey Results](https://static.igem.wiki/teams/5819/watermarking/1115result6.avif)

**Figure 6.**chemSPARK exhibits rapid and dose-dependent secretion dynamics compared with a transcriptional activation system.

(A) Dose-dependent secretion response to Grazoprevir treatment(0-10 µM). Data presents mean ±SD, n = 3 independent experiments, fitted to a symmetric sigmoid curve.
(B) Kinetics of grazoprevir-inducible NanoLuc expression comparing chemSPARK with a transcriptional activation system. For the transcriptional system, HEK293T cells were co-transfected with pNC25046 (PCMV-Binder-TetR-NLS-VP64), pNC25048 (PCMV-IgK-Fluc-TMD-NS3a), and pNC25098 (PTCE-IgK-Nluc). For the secretion system, cells were co-transfected with pNC25092 (PCMV-(Nluc-FS)3-TMD-(NS3a)2) and pNC25091 (PCMV-LifeAct-mCherry-Binder). In both systems, 10 µM grazoprevir was added at the indicated time points (6 h post-transfection for transcriptional and 48 h for secretion), and NanoLuc levels were monitored over the following 48 h and 12 h, respectively. Data represent mean ± SD of fold changes calculated as the ratio of NanoLuc levels in grazoprevir-treated versus untreated samples (n = 3 independent experiments).

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