iGEM 2021 — Team NUDT_CHINA

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NUDT CHINA/Notebook

NUDT\_CHINA/Home

![](https://static.igem.org/mediawiki/2021/b/be/T--NUDT_CHINA--logo.png)

1. # project
2. # Labwork
3. # Parts
4. # Human Practices
5. # Teamwork

↑

![](https://static.igem.org/mediawiki/2021/b/be/T--NUDT_CHINA--logo.png)

![](https://static.igem.org/mediawiki/2021/7/72/T--NUDT_CHINA--notebook.jpg)

Notebook

Monthly Progress & Milestones

***2020***

*December*

    ●Hold a biology contest to select team members for NUDT\_CHINA.

***2021***

*January*

    ●2021 NUDT\_CHINA set-up. Team members split into 4 groups: Wet Lab, Human Practice, Art&design, Model.

*February*

    ●Brainstorm for 2020 team project.

    ●Learn the molecular biology and basic experimental technologies.

*March*

    ●Discuss the 2020 iGEM project in depth and determine the direction of the 2020 iGEM project, consider to turn the switch of our Predator system from small molecule induced pairs to blue light induced ones.

    ●Conduct preliminary Proof of Concept design and plasmid design.

*April*

    ●The first offline collective meeting to discuss the 2020iGEM project and anchor blue light-controlled protein degradation system as the field to anchor our project.

*May*

    ●Collection and study of relevant literature on the application of our project.

    ●Choose cyclinD1 and cyclinE1 to as the targeting module of Predator system to further demonstrate our project.

*June*

    ●Offline meetings of the experimental group for literature reading, reporting the progress of the project and arranging the follow-up tasks.

    ●Promotional video production.

*July*

    ●Online meetings of the experimental group for literature reading and plasmid design.

    ●Team uniform design.

*August*

    ●Conducting relevant experimental basic learning, including software design and skill training online.

    ●Carry out specific experimental operations related to the project under the guidance of our instuctors.

    ●Participation of CCiC communication online to observe the presentation of other projects and look for the possibility of cooperation within experiment and HP.

    ●Combine the advice and the feedback from Model to redesign the Proof of Concept to achieve higher experimental stability.

*September*

    ●Interview experts and receive optimization suggestions.

    ●Successfully complete the Proof of Concept and carry out the experimental design of Demonstration.

    ●Complete the Demonstration experiment, feed the results back to Model group, and carry out the optimization design of experiment.

    ●The optimized Demonstration experiment obtain better effect.

*October*

    ●Taking family photos.

    ●Conduct offline Communication meetings with CSU\_CHINA and reach consensus on deep cooperation in collaboration, interview and science communication.

    ●Conduct expert interviews, and consider changing experimental targets and regulation methods based on advice.

    ●Wiki construction.

    ●Presentation Video Production.

*November*

    ●Participation of Giant Jamboree.

![](https://static.igem.org/mediawiki/2021/2/20/T--NUDT_CHINA--footer.png)

Project

* [Description](https://2021.igem.org/Team%3ANUDT_CHINA/Description)
* [Design](https://2021.igem.org/Team%3ANUDT_CHINA/Design)
* [Proof Of Concept](https://2021.igem.org/Team%3ANUDT_CHINA/Proof_Of_Concept)
* [Improvement](https://2021.igem.org/Team%3ANUDT_CHINA/Improvement)
* [Model](https://2021.igem.org/Team%3ANUDT_CHINA/Model)
* [Engineering](https://2021.igem.org/Team%3ANUDT_CHINA/Engineering)

Labwork

* [Results](https://2021.igem.org/Team%3ANUDT_CHINA/Results)
* [Experiment](https://2021.igem.org/Team%3ANUDT_CHINA/Experiment)
* [Notebook](https://2021.igem.org/Team%3ANUDT_CHINA/Notebook)
* [Safety](https://2021.igem.org/Team%3ANUDT_CHINA/Safety)
* [Hardware](https://2021.igem.org/Team%3ANUDT_CHINA/Hardware)

Parts

* [Parts Overview](https://2021.igem.org/Team%3ANUDT_CHINA/Parts_Overview)
* [Basic Parts](https://2021.igem.org/Team%3ANUDT_CHINA/Basic_Parts)
* [Composite Parts](https://2021.igem.org/Team%3ANUDT_CHINA/Composite_Parts)
* [Parts Collection](https://2021.igem.org/Team%3ANUDT_CHINA/Parts_Collection)

Human Practices

* [HP Overview](https://2021.igem.org/Team%3ANUDT_CHINA/HP_Overview)
* [Integrated HP](https://2021.igem.org/Team%3ANUDT_CHINA/Human_Practices)
* [Education](https://2021.igem.org/Team%3ANUDT_CHINA/Education)
* [Science Communication](https://2021.igem.org/Team%3ANUDT_CHINA/Communication)
* [Implementation](https://2021.igem.org/Team%3ANUDT_CHINA/Implementation)
* [Contribution](https://2021.igem.org/Team%3ANUDT_CHINA/Contribution)

Team

* [Team Members](https://2021.igem.org/Team%3ANUDT_CHINA/Team_Members)
* [Attributions](https://2021.igem.org/Team%3ANUDT_CHINA/Attributions)
* [Collaborations](https://2021.igem.org/Team%3ANUDT_CHINA/Collaborations)
* [Partnership](https://2021.igem.org/Team%3ANUDT_CHINA/Partnership)

Contact Us

📧Email: nudt\_china2021@126.com

No. 109 Deya Road,Kaifu District,
Changsha,
Hunan Province 410073

P.R.China

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NUDT CHINA/Engineering

NUDT\_CHINA/Home

![](https://static.igem.org/mediawiki/2021/b/be/T--NUDT_CHINA--logo.png)

1. # project
2. # Labwork
3. # Parts
4. # Human Practices
5. # Teamwork

↑

![](https://static.igem.org/mediawiki/2021/b/be/T--NUDT_CHINA--logo.png)

![](https://static.igem.org/mediawiki/2021/5/53/T--NUDT_CHINA--engineer.png)

Engineering

Introduction

In our iGEM 2021 project, tremendous amount of engineering efforts has been made to bring the light responsive features into the system we kept working on for years. This year, by evaluating the degradation effects and interviewing some experts for improvement advice, we constructed a light-inducible protein degradation system that works in high efficiency. Here in this Engineering Success page, we will discuss the design and build, test and learn, and re-design process during the development of this system.

![](https://static.igem.org/mediawiki/2021/f/f1/T--NUDT_CHINA--engineering_fig1.png)

***Figure 1.** Engineering flow chart of constructing LiPrePro system.*

Design and Build

In 2018 and 2019, we have demonstrated the PREDATOR system, an effective endogenous protein degradation system that allows for direct regulation of target protein degradation. In 2020, we have improved it as PREDATOR PRO system to achieve direct signal responsiveness by designing a proper interface to rewire the signal induced protein-protein interaction or other biochemical reactions. Moreover, in iGEM2021, we stepped forward upon a protein degradation system directly controlled by blue light.

According to the experience over the past few years of the mechanism of the interaction of Trim21 and targeted protein, we intended to replace the core module of PREDATOR PRO system into light sensitive Cry2/CIB1 pairs to develop a light inducible PREDATOR (LiPrePro) system. In this system, truncated Trim21 protein was linked with Cry2 with a GGGSG protein linker, targeting module was liked to CIB1 in the similar manner. In order to simplify the test and optimization procedure, GFP was used as the target protein, therefore a simple fluorescent imaging could be sufficient to evaluate the degradation efficiency . In this way, we build a light inducible PREDATOR system.

Test and Learn

To evaluate the light responsiveness of such system, HEK-293T cells were co-transfected with GFP expressing plasmid and LiPrePro plasmid/empty vector. Fluorescent imaging were captured 48 h post blue light illumination. Images showed a slight decrease (~5%) of GFP fluorescence in LiPrePro expressing group comparing to the control group kept under dark condition (Fig. 1B and 1C), indicating that our system couldn’t work as expected.

![](https://static.igem.org/mediawiki/2021/b/b5/T--NUDT_CHINA--Engineering_fig2WM.png)

***Figure 2. First trial of light inducible PREDATOR system.** (A) Schematic representation showing the design of Blue light-inducible Predator Pro (LiPrePro) system. (B) Representative fluorescence images of the LiPrePro transfected group and negative control group transfected with an empty vector. (C) Quantification of fluorescent intensity in (B).For (B-C), cells were illuminated with 3mW/cm2 of 405nm blue light, the illumination was programmed as the repeat of [2 s ON/58 s OFF] cycle for indicated hours. Statistical significance was calculated via student t test. \*\*\* p ＜ 0.001.*

In the [interview we had with Mirta Viviani](https://2021.igem.org/Team%3ANUDT_CHINA/Hp_Overview#MV), a phD student focusing on mammalian cell synthetic biology in Westlake University, we were informed that the potential problem could be related to the linker length between protein domains, which may dramatically affect protein folding.

Redesign and Retest

Building on the advice from Mirta, we redesigned our plasmids, replacing the 1x GS linker into 3x or even 5x GS linkers(Fig. 2A). After cells were transfected with these new plasmids, much lower GFP level were gladly observed. Indicating significant reduction of GFP levels in cells transfected with either LiPrePro (3x GS Linker) or LiPrePro (5x GS linker) comparing to the EGFP transfected control cells under 24, 48 and 72 h of blue light illumination, suggesting an improved protein degradation ability of these designs (Fig. 2B and C).

![](https://static.igem.org/mediawiki/2021/1/15/T--NUDT_CHINA--Engineering_fig3WM.png)

***Figure 3. Optimization of light inducible PREDATOR system.** (A) Schematic representation showing the design of 3x and 5x GS linkers. (B) Representative fluorescence images of the LiPrePro (3x GS) transfected group, LiPrePro(5x GS) transfected group and negative control group transfected with an empty vector. (C) Quantification of fluorescent intensity in (B). For (B-C), cells were illuminated with 3mW/cm2 of 405nm blue light, the illumination was programmed as the repeat of [2 s ON/58 s OFF] cycle for indicated hours. Statistical significance was calculated via student t test. \*\*\* p ＜ 0.001.*

With such iteration process, we successfully developed a light inducible protein degradation system with decent degradation efficiency.

![](https://static.igem.org/mediawiki/2021/2/20/T--NUDT_CHINA--footer.png)

Project

* [Description](https://2021.igem.org/Team%3ANUDT_CHINA/Description)
* [Design](https://2021.igem.org/Team%3ANUDT_CHINA/Design)
* [Proof Of Concept](https://2021.igem.org/Team%3ANUDT_CHINA/Proof_Of_Concept)
* [Improvement](https://2021.igem.org/Team%3ANUDT_CHINA/Improvement)
* [Model](https://2021.igem.org/Team%3ANUDT_CHINA/Model)
* [Engineering](https://2021.igem.org/Team%3ANUDT_CHINA/Engineering)

Labwork

* [Results](https://2021.igem.org/Team%3ANUDT_CHINA/Results)
* [Experiment](https://2021.igem.org/Team%3ANUDT_CHINA/Experiment)
* [Notebook](https://2021.igem.org/Team%3ANUDT_CHINA/Notebook)
* [Safety](https://2021.igem.org/Team%3ANUDT_CHINA/Safety)
* [Hardware](https://2021.igem.org/Team%3ANUDT_CHINA/Hardware)

Parts

* [Parts Overview](https://2021.igem.org/Team%3ANUDT_CHINA/Parts_Overview)
* [Basic Parts](https://2021.igem.org/Team%3ANUDT_CHINA/Basic_Parts)
* [Composite Parts](https://2021.igem.org/Team%3ANUDT_CHINA/Composite_Parts)
* [Parts Collection](https://2021.igem.org/Team%3ANUDT_CHINA/Parts_Collection)

Human Practices

* [HP Overview](https://2021.igem.org/Team%3ANUDT_CHINA/Hp_Overview)
* [Integrated HP](https://2021.igem.org/Team%3ANUDT_CHINA/Human_Practices)
* [Education](https://2021.igem.org/Team%3ANUDT_CHINA/Education)
* [Science Communication](https://2021.igem.org/Team%3ANUDT_CHINA/Communication)
* [Implementation](https://2021.igem.org/Team%3ANUDT_CHINA/Implementation)
* [Contribution](https://2021.igem.org/Team%3ANUDT_CHINA/Contribution)

Team

* [Team Members](https://2021.igem.org/Team%3ANUDT_CHINA/Team_Members)
* [Attributions](https://2021.igem.org/Team%3ANUDT_CHINA/Attributions)
* [Collaborations](https://2021.igem.org/Team%3ANUDT_CHINA/Collaborations)
* [Partnership](https://2021.igem.org/Team%3ANUDT_CHINA/Partnership)

Contact Us

📧Email: nudt\_china2021@126.com

No. 109 Deya Road,Kaifu District,
Changsha,
Hunan Province 410073

P.R.China

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NUDT CHINA/Experiments

NUDT\_CHINA/Home

![](https://static.igem.org/mediawiki/2021/b/be/T--NUDT_CHINA--logo.png)

1. # Project
2. # Labwork
3. # Parts
4. # Human Practices
5. # Teamwork

↑

![](https://static.igem.org/mediawiki/2021/b/be/T--NUDT_CHINA--logo.png)

![](https://static.igem.org/mediawiki/2021/9/96/T--NUDT_CHINA--HOME-background.jpg)

Light-mediated control of cell cycle

in mammalian cells

Promotion video

[
](https://static.igem.org/mediawiki/2021/a/a4/T--NUDT_CHINA--PromotionVideo.mp4)

BRIEF INTRODUCTION

Optogenetic tools provide essential
approaches to control designer cell function. Our project intends
to optimize Predator Pro, a modularized protein degradation system we’ve been working on in the past
few years, to achieve light mediated degradation of endogenous cyclin D1, a key regulator protein in
cell cycle, thereby manipulating cell cycle in mammalian cells. Integrating with a raspberry pie based
blue light illumination device, our system enables convenient control of cell cycle synchronization and
remote spatiotemporal control of cell division. Our work may provide novel tools for fundamental
research and therapeutic purposes, including dealing with complex diseases like superficial cancer,
melanoma, psoriasis or chronic skin wounds, which are all caused by cell abnormal division.

![](https://static.igem.org/mediawiki/2021/2/20/T--NUDT_CHINA--footer.png)

Project

* [Description](https://2021.igem.org/Team%3ANUDT_CHINA/Description)
* [Design](https://2021.igem.org/Team%3ANUDT_CHINA/Design)
* [Proof of Concept](https://2021.igem.org/Team%3ANUDT_CHINA/Proof_Of_Concept)
* [Improvement](https://2021.igem.org/Team%3ANUDT_CHINA/Improvement)
* [Model](https://2021.igem.org/Team%3ANUDT_CHINA/Model)
* [Engineering](https://2021.igem.org/Team%3ANUDT_CHINA/Engineering)

Labwork

* [Results](https://2021.igem.org/Team%3ANUDT_CHINA/Results)
* [Experiment](https://2021.igem.org/Team%3ANUDT_CHINA/Experiment)
* [Notebook](https://2021.igem.org/Team%3ANUDT_CHINA/Notebook)
* [Safety](https://2021.igem.org/Team%3ANUDT_CHINA/Safety)
* [Hardware](https://2021.igem.org/Team%3ANUDT_CHINA/Hardware)

Parts

* [Parts Overview](https://2021.igem.org/Team%3ANUDT_CHINA/Parts_Overview)
* [Basic Parts](https://2021.igem.org/Team%3ANUDT_CHINA/Basic_Parts)
* [Composite Parts](https://2021.igem.org/Team%3ANUDT_CHINA/Composite_Parts)
* [Parts Collection](https://2021.igem.org/Team%3ANUDT_CHINA/Parts_Collection)

Human Practices

* [HP Overview](https://2021.igem.org/Team%3ANUDT_CHINA/HP_Overview)
* [Integrated HP](https://2021.igem.org/Team%3ANUDT_CHINA/Human_Practices)
* [Education](https://2021.igem.org/Team%3ANUDT_CHINA/Education)
* [Science Communication](https://2021.igem.org/Team%3ANUDT_CHINA/Communication)
* [Implementation](https://2021.igem.org/Team%3ANUDT_CHINA/Implementation)
* [Contribution](https://2021.igem.org/Team%3ANUDT_CHINA/Contribution)

Team

* [Team Members](https://2021.igem.org/Team%3ANUDT_CHINA/Team_Members)
* [Attributions](https://2021.igem.org/Team%3ANUDT_CHINA/Attributions)
* [Collaborations](https://2021.igem.org/Team%3ANUDT_CHINA/Collaborations)
* [Partnership](https://2021.igem.org/Team%3ANUDT_CHINA/Partnership)

Contact Us

📧Email: nudt\_china2021@126.com

No. 109 Deya Road,Kaifu District,
Changsha,
Hunan Province 410073

P.R.China

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NUDT CHINA/Results

NUDT\_CHINA/Home

![](https://static.igem.org/mediawiki/2021/b/be/T--NUDT_CHINA--logo.png)

1. # project
2. # Labwork
3. # Parts
4. # Human Practices
5. # Teamwork

↑

![](https://static.igem.org/mediawiki/2021/b/be/T--NUDT_CHINA--logo.png)

![](https://static.igem.org/mediawiki/2021/e/ee/T--NUDT_CHINA--result.png)

Results

Preliminary Test
*Constructing Blue Light Illumination System & Selecting Protein Dimerization Pairs*

The first step in constructing the Cycle Blue system is to build a blue light illumination system which can manually adjust the light intensity and illumination time. The device was built with 4 sets of blue light LEDs (6 LEDs each, 405nm, 1-3W) glued on an aluminum heatsink. The illumination device was covered by an acrylic light shield, with non-transparent surroundings and a clear top. Four standard cell culture plates could be placed on the top of the illumination device. Each LED sets were controlled independently with electromagnetic relays under control of a Raspberry Pi single-board computer (Fig.1A). The Raspberry Pi was loaded with ubuntu system and a python-based control script, allowing us to control the on/off time of each set of LEDs conveniently [(Click here to see our Hardware page and know more about the blue light illumination system design)](https://2021.igem.org/Team%3ANUDT_CHINA/Hardware)(Fig.1B)

![](https://static.igem.org/mediawiki/parts/8/84/T--NUDT_CHINA--Result_fig1.png)

***Figure 1.Construction of blue light illumination system.** (A) Schematic representation of the system design. (B) Pictures of the blue light illumination system installed in the incubator.*

During our discussion with Professor Yang regarding the light-inducible protein pairs, we were suggested to use online databases (like https://www.optobase.org/) to identify potential optoswitches that could function as core module (interface: in IGEM 2020 NUDT\_CHINA) to generate a set of light controlled protein degradation tools. Herein we chose Cry2/CIB1, a protein pair that dimerize under blue light, and other two protein pairs that dissociate under blue light: LOV2/zdk1 and PixD/PixE. To verify the fitness of these interaction pairs to our illumination device and our chassis, we inserted the candidate protein pairs into a tetR-based system. Whiles one member of the protein pair was linked to a tetR DNA binding domain, the other member of the protein pair was linked to a nuclear localized transcriptional activation domain VP64. Upon co-transfection of these plasmids with a tetO7 promoter driven SEAP reporter, we would be able to evaluate the binding affinity and light responsiveness of the interaction pairs by measuring SEAP activity in the culture medium (Fig.2A).

As expected, HEK-293T cells co-transfected with tetR-Cry2, CIB1-VP64 and tetO7-SEAP expressing plasmids showed significantly increased SEAP production upon 24h or 48 h of blue light illumination comparing to control cells transfected with the same plasmids placed in a dark incubator (p＜0.001, Fig.2B). Also, in consistent to previous reports, cells co-transfected with tetR-Lov2, Zdk-VP64 and tetO7-SEAP plasmids showed significant reduction of SEAP production upon either 24 h or 48 h of blue light illumination comparing to the control cells (p＜0.001, Fig.2C). To our surprise, low SEAP production was detected in cells co-transfected with tetR-PixD, CIB1- PixE and tetO7-SEAP expressing plasmids under either light or dark conditions (p＜0.001, Fig.2D). Considering the absolute level of SEAP production was way higher in Cry2/CIB1 pair comparing to the lov2/zdk pair, these results indicated that Cry2/CIB1 suits our illumination hardware and parameter the best. Therefore, Cry2/CIB1 pair was then used in our next phase of experiments.

![](https://static.igem.org/mediawiki/2021/d/d5/T--NUDT_CHINA--Res2WM.png)

***Figure 2. Validation of the blue light-induced pairs.** (A) Schematic representation showing the function of light inducible protein pairs and design of Pairs-TetON system. (B) SEAP activity in the culture medium collect from cells transfected with tetR-Cry2, CIB1-VP64 and tetO7-SEAP expressing plasmids under either blue light illumination or dark condition. (C) SEAP activity in the culture medium collect from cells transfected with tetR-lov2, zdk-VP64 and tetO7-SEAP expressing plasmids under either blue light illumination or dark condition. (D) SEAP activity in the culture medium collect from cells transfected with tetR-PixD, PixE-VP64 and tetO7-SEAP expressing plasmids under either blue light illumination or dark condition. For (B-D), cells were illuminated with 3 mW/cm2 of 405nm blue light, the illumination was programed as the repeat of [2 s ON /58 s OFF] cycle for 48 hours. BDL represents below detection limits, Data represent mean ± s.e.m. (n=3 biological replicates); \*\*\*P ＜ 0.001; two-tailed unpaired Student’s t-test.*

A-Side: Blue
*Construction and Optimization of Blue Light Inducible PREDATOR System*

To develop a light inducible PREDATOR (LiPrePro) system, we intended to replace the core module of PREDATOR PRO system into Cry2/CIB1 pairs. In order to simplify the test and optimization procedure, GFP was used as the target protein, therefore a simple fluorescent imaging could be sufficient to evaluate the degradation efficiency (Fig.3 A). To begin with, truncated Trim21 protein was linked with Cry2 with a GGGSG protein linker, GFPnanobody was liked to CIB1 in the similar manner. As a validation of such system, HEK-293T cells were co-transfected with GFP expressing plasmid and LiPrePro plasmid/empty vector. Fluorescent imaging showed slight decrease (~5%) of GFP fluorescence in LiPrePro expressing group comparing to the control group (Fig. 3B and 3C) under 48 h blue light illumination, which was unsatisfying.

During our discussion with Mirta Viviani, a phD student working on synthetic biology in Westlake University, we mentioned the preliminary failure in generating satisfying protein degradation under blue light control. She pointed out that we could try optimizing our system structure, such as adjusting the linker length between modules to help proteins folding correctly. Therefore, we changed the length of linker between Cry2 and GFPnano and the linker between CIB1 and truncated Trim21 from 1x GS linker into 3x or even 5x GS linkers (Fig.3D) [(Click here to see our design page for more information)](https://2021.igem.org/Team%3ANUDT_CHINA/Design#conopt). Results showed significant reduction of GFP levels in cells transfected with either LiPrePro (3x GS Linker) or LiPrePro (5x GS linker) comparing to the EGFP transfected control cells under 24, 48 and 72 h of blue light illumination, indicating an improved protein degradation ability of these designs. (Fig. 3E and F).

![](https://static.igem.org/mediawiki/2021/d/d5/T--NUDT_CHINA--Res3WM.png)

***Figure 3. Construction and Optimization of light inducible PREDATOR system.** (A) Schematic representation showing the design of Blue light-inducible Predator Pro (LiPrePro) system. (B) Representative fluorescence images of the LiPrePro transfected group and negative control group transfected with an empty vector. (C) Quantification of fluorescent intensity in (B). (D) Schematic representation showing the design of LiPrePro with 3x and 5x GS linkers. (E) Representative fluorescence images of the LiPrePro (3x GS) transfected group, LiPrePro(5x GS) transfected group and negative control group transfected with an empty vector. (F) Quantification of fluorescent intensity in (E). For (B-C, E-F), cells were illuminated with 3 mW/cm2 of 405nm blue light, the illumination was programed as the repeat of [2 s ON /58 s OFF] cycle for indicated hours. Data represent mean ± s.e.m. (n=3 biological replicates); \*\*\*P ＜ 0.001; two-tailed unpaired Student’s t-test.*

B-Side: Cycle
*Targeting Cyclin and Control Cell Proliferation*

As intracellular antibodies and ScFvs are the most obvious approaches for specific recognition of target protein, we first focused on finding the published ScFvs targeting specific cyclin protein. To simplify the experiments, we kept the original DocS/Coh2 core module of the PREDATOR system. With this constitutively binding core module, we expect to see significant reduction of cell proliferation in Cyclin PREDATOR transfected cells with either microscopic imaging or CCK8 assay (Fig.4A).

With the Cyclin E specific ScFv sequence published by Y Wu, we designed multiple ScFv or split ScFv based Cyclin E PREDATORs[(Click here to see our design page for more information)](https://2021.igem.org/Team%3ANUDT_CHINA/Design#TarCyc). To validate these designs, HEK-293T cells were transfected with PREDATOR expressing plasmids, CCK8 viability assay was performed 36 h post transfection to evaluate the cell proliferation. While results showed significant reduced cell proliferation in most of these designs (Fig. 4B and C) the reduction of cell proliferation was unsatisfying.

In the meantime, two other targeting modules were designed under the suggestions from Prof. Chun Meng[(Click here to see our design page for more information)](https://2021.igem.org/Team%3ANUDT_CHINA/Design#TarCyc). Herein, we obtained the αCHelix region of CDK4 and Rb, which were the endogenous binding proteins of Cyclin D1. Using the tandem repeats of these Cyclin D1 binding regions, we designed new Cyclin D1 PREDATORs. Results showed significantly reduced proliferation in cells transfected with either designs, indicating tandem repeats of CDK4 αCHelix or Rb αCHelix could be considered as an optimal targeting module.

![](https://static.igem.org/mediawiki/2021/3/32/T--NUDT_CHINA--Res4WM.png)

***Figure 4. Optimalization of Cyclin-targeting module.** (A) Schematic representation of the experimental process of cyclin PREDATOR validation. (B,D) Representative microscopic images of HEK-293T cells transfected with indicated plasmids. (C,E) CCK8 cell proliferation assay of HEK-293T cells transfected with indicated plasmids. For (C and E), data represent mean ± s.e.m. (n=3 biological replicates); \*\*\*P ＜ 0.001; two-tailed unpaired Student’s t-test.*

Finale: CycleBlue
*Light-mediated control of mammalian cell cycle*

With the optimal protein-protein pairs, optimization of linkers and Cyclin-targeting module, we then build a blue light inducible Predator system to mediate the regulation and intervention of the cell cycle as a proof of our concept. Integrating with a raspberry pie-based blue light illumination device, light-controlled dimerization of protein pairs may occur and subsequently ternary degradation complex formed, leading to the degradation of Cyclin (Fig.5A). Herein, we designed two CycleBlue systems namely CycleBlue-1 and CycleBlue-2, using CDK4 αCHelix or Rb αCHelix as targeting module, respectively.

For characterization, HEK-293T cells were transfected with either CycleBlue-1 or CycleBlue-2 expressing plasmids and illuminated with blue light 24h post transfection. Cyclin levels and cell proliferation were analyzed by Western Blotting and CCK8 assay, respectively. Intriguingly, under blue light illumination, western blotting showed significantly reduced cyclin D1 level in cells transfected with CycleBlue-1 comparing to the control group, while the reduction on cyclin D1 level in cells transfected with CycleBlue-2 was not observed (Fig. 5B). Interestingly, CCK8 assay(Fig. 5C) showed significant reduction of cell proliferation under blue light illumination in both CycleBlue-1 and CycleBlue-2 transfected cells. (Fig.5C, right panel, p ＜ 0.001), while the effect of CycleBlue-1 and CycleBlue-2 transfection in dark condition was insignificant (Fig.5C, left panel, p ＜ 0.001).

In general, these results showed that our CycleBlue system can successfully degrade Cyclin D1 and regulate cell proliferation under the control of blue light with decent performance. The engineering success of these designs also showed the potential of CycleBlue system to be further developed into a novel synthetic biology toolbox for controlled protein degradation.

![](https://static.igem.org/mediawiki/2021/b/bd/T--NUDT_CHINA--Res5WM.png)

***Figure 5. CycleBlue: Blue-light controllable cell growth.** (A) Schematic representation showing the design of CycleBlue system. (B) Representative Western blotting determining the Cyclin abundance in HEK-293T cells 48 h post transfected with CycleBlue-1 plasmid and CycleBlue-2 plasmid under blue light illumination condition. (C) CCK8 cell proliferation assay on CycleBlue-1/ CycleBlue-2 transfected HEK-293T cells under dark or blue light illuminated conditions. For (B-C, E-F), cells were illuminated with 3 mW/cm2 of 405nm blue light, the illumination was programed as the repeat of [2 s ON /58 s OFF] cycle for indicated hours. Data represent mean ± s.e.m. (n=3 biological replicates); \*\*\*P ＜ 0.001; two-tailed unpaired Student’s t-test.*

![](https://static.igem.org/mediawiki/2021/2/20/T--NUDT_CHINA--footer.png)

Project

* [Description](https://2021.igem.org/Team%3ANUDT_CHINA/Description)
* [Design](https://2021.igem.org/Team%3ANUDT_CHINA/Design)
* [Proof Of Concept](https://2021.igem.org/Team%3ANUDT_CHINA/Proof_Of_Concept)
* [Improvement](https://2021.igem.org/Team%3ANUDT_CHINA/Improvement)
* [Model](https://2021.igem.org/Team%3ANUDT_CHINA/Model)
* [Engineering](https://2021.igem.org/Team%3ANUDT_CHINA/Engineering)

Labwork

* [Results](https://2021.igem.org/Team%3ANUDT_CHINA/Results)
* [Experiment](https://2021.igem.org/Team%3ANUDT_CHINA/Experiment)
* [Notebook](https://2021.igem.org/Team%3ANUDT_CHINA/Notebook)
* [Safety](https://2021.igem.org/Team%3ANUDT_CHINA/Safety)
* [Hardware](https://2021.igem.org/Team%3ANUDT_CHINA/Hardware)

Parts

* [Parts Overview](https://2021.igem.org/Team%3ANUDT_CHINA/Parts_Overview)
* [Basic Parts](https://2021.igem.org/Team%3ANUDT_CHINA/Basic_Parts)
* [Composite Parts](https://2021.igem.org/Team%3ANUDT_CHINA/Composite_Parts)
* [Parts Collection](https://2021.igem.org/Team%3ANUDT_CHINA/Parts_Collection)

Human Practices

* [HP Overview](https://2021.igem.org/Team%3ANUDT_CHINA/Hp_Overview)
* [Integrated HP](https://2021.igem.org/Team%3ANUDT_CHINA/Human_Practices)
* [Education](https://2021.igem.org/Team%3ANUDT_CHINA/Education)
* [Science Communication](https://2021.igem.org/Team%3ANUDT_CHINA/Communication)
* [Implementation](https://2021.igem.org/Team%3ANUDT_CHINA/Implementation)
* [Contribution](https://2021.igem.org/Team%3ANUDT_CHINA/Contribution)

Team

* [Team Members](https://2021.igem.org/Team%3ANUDT_CHINA/Team_Members)
* [Attributions](https://2021.igem.org/Team%3ANUDT_CHINA/Attributions)
* [Collaborations](https://2021.igem.org/Team%3ANUDT_CHINA/Collaborations)
* [Partnership](https://2021.igem.org/Team%3ANUDT_CHINA/Partnership)

Contact Us

📧Email: nudt\_china2021@126.com

No. 109 Deya Road,Kaifu District,
Changsha,
Hunan Province 410073

P.R.China