iGEM 2025 — Team tsinghua-m

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![TEAM](https://static.igem.wiki/teams/5805/imgs/team.webp)

# WET LAB

![notebook](https://static.igem.wiki/teams/5805/imgs/wetlab/notebook.webp)

We have documented our wetlab Notebook below, in which our wetlab process is displayed in detail.

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![TEAM](https://static.igem.wiki/teams/5805/imgs/team.webp)

# PROJECT

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-0.webp)

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-0-1.webp)

The overall objective of our project is not merely to construct a three-node oscillator, but rather to develop a modular framework that enables programmable, multidimensional regulation in yeast cell factories. The oscillator serves as a representative implementation to demonstrate this principle. Guided by the concept of modular design, we divide the system into three functional modules — stress sensing and resistance module, degradation degron module, and repressor circuit module — which collectively establish the foundation for robust, precise, and programmable dynamic control.

Stress Sensing and Resistance Module

The stress-sensing and resistance module aims to equip yeast with the ability to sense and respond to diverse environmental stresses.

We began by assembling a parts library that includes stress-responsive inducible promoters and a set of stress-tolerance genes (for heat, osmotic stress, and reactive oxygen species). These promoters and genes that documented to have the strongest responses to high temperature, high osmolarity and reactive oxygen species were then tested in yeast to prove their functions. From these efforts we chose six genetic elements in total (three inducible promotors and three stress-tolerance genes) as the best candidates for downstream use in the cell factory to ensure robust sensing and conditional control. Specifically, we selected UAS-CYC1 as the high-temperature stress-responsive promotor, GPD1p as the hyperosmotic stress-responsive promoter and SOD2p as the oxidative stress-responsive promoter. As for the regulatory genes, we have come to the conclusion that SHSP is the proper gene to cope with high-temperature stress, HOG1 is the suitable gene to deal with hyperosmotic stress, while SOD1 being the ideal gene to handle oxidative stress.

The parts we selected for the stress sensing and resistance module can be concluded by the following table:
[![click icon](https://static.igem.wiki/teams/5805/imgs/click-this.webp)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D)

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-0-2.webp)

Degradation Degron Module

To achieve tight temporal control of protein pulses while conserving cellular resources, we implemented a proteolytic control strategy using degradation degrons.

Based on a literature survey, we selected six ubiquitin–proteasome–dependent degradation tags for testing. These degrons had previously been used as fusion protein components. We chose ubiquitin–proteasome–dependent degrons for two main reasons. First, they are straightforward to apply, since simply appending the degron to the N terminus of the target protein is sufficient to trigger degradation. Second, their half-lives can be conveniently tuned: altering just a few amino acids can modify the degron’s structural stability, thereby adjusting protein half-life, which is advantageous for controlling oscillation periods.

To further expand the regulatory dimension of degradation control, we introduced a temperature-sensitive degron, J6. This degron consists of Ub–Arg–DHFRts. Upon synthesis of the fusion protein, the Ub moiety is cleaved, leaving an unstable Arg residue at the N terminus. At low temperature (23 °C), DHFRts folds into a partially stable conformation that shields the N-terminal Arg from recognition by the E3 ubiquitin ligase. At high temperature (37 °C), DHFRts undergoes conformational changes that expose the Arg residue, enabling E3 recognition, polyubiquitylation, and rapid degradation of the target protein by the 26S proteasome.

For degron performance testing, we employed inducible promoters driving the expression of fluorescent reporter proteins. Different N-terminal degrons were fused to the reporter, and the resulting protein lifetimes were compared.

Following induction, both fluorescence and OD600 were continuously monitored to generate fluorescence time courses and evaluate the efficiency of each degron. The genetic map below portraits one of the six degrons that we tested.
[![click icon](https://static.igem.wiki/teams/5805/imgs/click-this.webp)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D)

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-1.webp)

Repressor Circuit Module

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-2.webp)

The core oscillator is a three-node circuit built from three repressor proteins. We sourced well-characterized repressors validated in prokaryotic systems, which are LacI, TetR, λCI and their variants, and adapted them for expression in yeast to form a circular negative-feedback loop. To ensure these parts function well in yeast, we make a set of routine molecular engineering steps to ensure correct expression, folding, subcellular localization and functional activity.

LacI acts as a temperature-sensitive repressor that inhibits transcription of target genes by binding to operator sites at low temperatures, but loses its ability at higher temperatures due to conformational changes.

TetR is the repressor that regulates tetracycline resistance by binding to the *tetO* operator, blocking transcription of resistance genes unless bound by tetracycline, which induces a conformational change.

λCI is a repressor protein from bacteriophage λ that maintains the lysogenic state by binding to specific operator regions, inhibiting transcription of lytic genes and autoregulating its own expression. Together these three core genes and their variants can fulfill our demand for precise and rhythmic regulation.

Moreover, it's worth mentioning that nuclear localization signals (NLS) play a significant role in adapting genes derived from prokaryotes for yeast expression systems. In eukaryotic cell biology research, precise control of protein subcellular localization is crucial for deciphering their functions. NLS serves as a key molecular code. As a short peptide sequence composed of specific basic amino acids, NLS can be recognized and bound by specific receptors in the cytoplasm. Subsequently, the formed protein complex is actively transported through the nuclear pore complex into the nucleoplasm. By adding nuclear localization signal sequence at the C-terminus, we ensure the repressors can function well in yeast.

At the early stage of building our repressor circuit module, we separately tested the function of every core parts. The following genetic maps shows the plasmids we constructed for the testing of TetR, and the other two repressors were tested in the same way:

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-3.webp)

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-4.webp)

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-5.webp)

To make a circuit, each repressor protein inhibits the promoter driving the next repressor in the oscillator. To make the circuit responsive to stress inputs, we placed proteins that relieve repression (hereafter referred to as anti-repressors) under the control of stress-responsive promoters. Under defined stress conditions, corresponding anti-repressor protein is expressed and inserted into the ring circuit, which relieves repression of the downstream node, initiating a cascade of expression change that propagates around the ring and can return to baseline as the oscillator proceeds.

By pairing small-molecule-inducible promoters and their chemical inducers as triggers, we expect the following results: expression of a given repressor blocks the downstream fluorescent signal, while induction of the anti-repressor restored that signal. Under normal OD600, both repression and anti-repression were observable by changes in fluorescence; when the anti-repressor was instead driven by a stress-responsive promoter, stress exposure effectively mimicked the small-molecule trigger.

To make our project more well-rounded, we also replicated an oscillator recorded in a recently published paper, further exploring the various possibilities of biological oscillators. The system consists of three main components: GP-McsB, LacI, and antiMcsB. The mechanism operates as follows:

1. GP-McsB specifically degrades LacI, reducing its intracellular level.

2. Once LacI is degraded, repression on antiMcsB expression is lifted, allowing antiMcsB to be produced.

3. AntiMcsB then binds to and inhibits GP-McsB activity, establishing a negative feedback loop.

This negative feedback loop enables periodic fluctuations in mKate2 abundance, producing oscillatory behavior.

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-6.webp)

> Li, Z., Qiao, G., Wang, X. et al. De novo designed protein guiding targeted protein degradation. Nat Commun 16, 6598 (2025). https://doi.org/10.1038/s41467-025-62050-z

We replicated this design, replacing mKate2 with mScarlet, and modifying some proteins to adapt to yeast expression. Through real-time monitoring of changes in mScarlet fluorescence intensity, the oscillation status of the oscillator can be directly reflected. The following genetic maps shows the plasmids we constructed imitating the design of the paper:
[![click icon](https://static.igem.wiki/teams/5805/imgs/click-this.webp)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D)

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-7.webp)

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-8.webp)

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-9.webp)

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-10.webp)

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-11.webp)

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-12.webp)

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-13.webp)

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-14.webp)

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-15.webp)

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-15-1.webp)

These three modules — stress sensing/resistance, degradation degrons, and the repressor circuit — are integrated to produce the complete three-node oscillator. The resulting system is intended to translate multidimensional environmental information into programmable temporal signature that can be used for conditional control of metabolic functions in yeast cell factories. This architecture balances robustness via selected stress-tolerance genes, temporal precision via degron-mediated turnover, and programmable logic via the three-node circuit, providing an integrated platform for downstream engineering.

The oscillator can play a vital role in various fields like boosting production in microbial cell factories and duel-strain synergy, just to name a few.

Pulsed Anti-Stress Control in Cell Factories

A pulsed anti-stress control strategy integrates stress-responsive promoters with N-terminal degrons to produce transient, tightly controlled pulses of stress-tolerance proteins. The key advantage lies in alleviating the metabolic burden caused by the sustained expression of stress-resistant proteins.

In this scheme, stress-sensing promoters drive expression of stress-resistance proteins whose N-termini are fused to validated degrons; the degrons shorten protein lifetime so that expression produces a defined pulse rather than long-lived accumulation, promoting energy utilization efficiency. The engineered cassette can be introduced into production strains and subjected to handle multi-factor stresses. By monitoring OD600 and product titer, we can quantify its effects on cell survival and process yield. The expected outcome is that, after integration, both the strain’s robustness and its production capacity can improve under fluctuating stress.

Oscillatory Anti-Stress Control in Cell Factories

An oscillatory anti-stress control scheme couples the three-node oscillator to a set of stress-resistance genes to create coordinated, periodic expression of protective functions. It's advantage lies in the ability to artificially program the response threshold to complex stress.

As an example, we introduced the three-node oscillator as well as three stress-tolerance genes into a β-carotene producing strain. Through continuous measurement of OD600 and β-carotene concentration, we evaluated how dynamic, time-structured protection affects product formation. The hypothesis is that rhythmic expression of protective genes reduces steady-state burden while providing timely defense, and thereby increases overall β-carotene yield.

The following genetic maps shows the plasmids we constructed to achieve oscillatory anti-stress control:

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-16.webp)

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-17.webp)

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-18.webp)

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-19.webp)

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-20.webp)

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-21.webp)

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-22.webp)

Division-of-Labor Strategy Using AHL–LuxR Communication

In our design, we divide the yeast population into two functional specialists: a doctor strain and a worker strain. The doctor strain is dedicated to sensing environmental stresses and converting those signals into chemical messages, while the worker strain focuses on production and stress response. This approach minimizes the regulatory burden on yeast metabolism, thus greatly improves energy utilization efficiency.

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-23.webp)

This division-of-labor is coordinated through the AHL-LuxR quorum-sensing system, a well-established cell-to-cell communication mechanism. In this system, the doctor strain expresses a LuxI synthase that continuously or conditionally produces acyl-homoserine lactone (AHL) molecules. These small, membrane-permeable molecules diffuse freely into the surrounding medium and are taken up by neighboring cells. In the worker strain, AHL molecules bind to a LuxR transcriptional regulator to form an AHL-LuxR complex, which then activates specific LuxR-responsive promoters. This design tightly couples stress detection in the doctor strain to rapid induction of protective genes in the worker strain, effectively turning environmental information into a population-wide, coordinated response.

![description](https://static.igem.wiki/teams/5805/imgs/project/project-engineering-24.webp)

The benefits of this two-strain collaboration are significant. By offloading sensing and signal-processing functions to the doctor strain, the worker strain can dedicate more of its cellular resources to production, improving metabolic efficiency and product yield. In addition, population-level communication ensures that all cells in the consortium respond synchronously, preventing performance variability caused by stochastic single-cell responses. This architecture is modular, allowing sensing and production to be optimized independently and replaced or upgraded without redesigning the entire circuit.

Finally, we extend this framework to incorporate three orthogonal AHL-LuxR systems. Orthogonality is achieved by selecting AHL synthases and LuxR regulators with minimal cross-reactivity, meaning they each produce and sense a distinct AHL molecule. This allows three independent communication channels to coexist in the same culture: one for heat stress, one for oxidative stress, and one for osmotic stress. Because each channel operates without interfering with the others, the consortium can integrate multiple environmental inputs simultaneously and trigger a coordinated, multi-layered stress response, offering fine-grained and programmable control over the system’s behavior.

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## What Should this Page Contain?

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Provide comprehensive, step-by-step protocols for all experiments conducted. Include a list of materials, reagents, and equipment used. Specify concentrations, volumes, incubation times, temperatures, and other critical parameters. Document any modifications or optimizations made to standard protocols.

Explain the purpose of each experiment and its relevance to your project goals. Describe the experimental design, including controls and replicates. Provide a clear rationale for the chosen methods and approaches.

Document any troubleshooting steps taken and optimizations made during the experimental process. Share any lessons learned that could benefit future teams.

## Inspirations

---

* [2024 Heidelberg](https://2024.igem.wiki/heidelberg/experiments)
* [2024 Marburg](https://2024.igem.wiki/marburg/experiments)
* [2024 EPFL](https://2024.igem.wiki/epfl/experiments)
* [2023 Estonia-TUIT](https://2023.igem.wiki/estonia-tuit/experiments)

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![TEAM](https://static.igem.wiki/teams/5805/imgs/team.webp)

# WET LAB

![notebook](https://static.igem.wiki/teams/5805/imgs/wetlab/results.webp)

To validate the feasibility of our modular system for enabling dynamic, precise stress adaptation in yeast cell factories, we separately characterized the performance of three core functional modules—**stress sensing & resistance**, **degradation degron**, and **repressor circuit**—and confirmed their ability to fulfill key technical roles in the overall stress-responsive framework.

Each module targets a critical need: The **stress sensing & resistance module** provides specific "stress recognition-execution" genetic elements. The **degradation degron module** achieves tight temporal control of protein expression to reduce metabolic burden. And the **repressor circuit module** constructs the rhythmic core for oscillatory regulation. Collectively, the characterization results of these three modules lay a solid foundation for integrating the complete system and realizing adaptive stress regulation in industrial yeast strains.

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/stress-sensing-resistance-module.webp)

I. Theory and Mechanism

The core theory of this experiment lies in using synthetic biology approaches to enhance the tolerance of cell factories towards environmental stresses, thereby stabilizing or increasing the yield of target products. The intrinsic mechanism is as follows: when cells encounter specific stresses (such as high temperature, hyperosmotic pressure, oxidative stress, etc.), the corresponding intracellular signaling pathways are activated. We clone promoters that respond to these specific stresses and splice them with functional genes that can enhance cellular stress resistance (such as genes for synthesizing compatible solutes, molecular chaperones, or antioxidant enzymes), thereby constructing a "stress-sensing and resistance-enhancing" genetic circuit. The design logic of this circuit is to maintain low-level expression under normal conditions to conserve cellular resources; once stress is encountered, the promoter is activated, driving the high-efficiency expression of the resistance genes to help the cell counteract the stress, maintain normal metabolic flux, and ultimately ensure the efficient synthesis of the target product. Additionally, a degradation tag is appended to the gene to help the cell promptly downregulate the circuit's expression after the stress subsides.

II. Experimental Design and Methods

Our experimental work is divided into three main steps:
**1. Genetic Circuit Construction and Preliminary Screening:

    Plasmid Construction:**

We selected the following promoters responsive to heat shock (pHSP82, pHSP104, pUAS-CYC1), high salt (pGPD1, pGpp2, pGRE2), and ROS stress (pGPX, pSOD, pTRX), along with the following target genes with potential resistance to high temperature (SHSP, THIIF, HSF1), high osmolarity (GPD1, RCK2, HOG1), and ROS (GRX1, GRX2, SOD1). Using molecular cloning techniques, these were combined to construct various expression plasmids.

(For specific gene sequences and information, please refer to our Parts page.[![click icon](https://static.igem.wiki/teams/5805/imgs/click-this.webp)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D) )

**Validation of Promoter and Resistance Gene Function in Yeast:**

After amplifying the aforementioned expression plasmids in E. coli, each constructed plasmid was separately introduced into model yeast cells. Our control group utilized yeast cells not transformed with the plasmid to fairly compare the efficacy of different circuits.

Validation of Promoter Response to Stress: The introduced plasmids contained a fluorescent protein (mScarlet) linked downstream of the promoter. Fluorescence intensity of yeast under stress conditions was measured using a microplate reader to assess promoter strength. The high-temperature group was screened by measuring fluorescence intensity during cultivation at 40°C, while the high osmolarity and ROS groups were tested across concentration gradients of 0, 0.2, 0.4, 0.6, 0.8, 1 mol/L NaCl and 0, 2, 4, 6, 8 mM H₂O₂, respectively, measuring yeast fluorescence at different gradients. The experimental results are as follows:

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-perceptionandresistance-1.webp)

Fig. 1 High-temperature promoter

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-perceptionandresistance-2.webp)

Fig. 2 High osmotic promoter

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-perceptionandresistance-3.webp)

Fig. 3 ROS promoter fluorescence

Validation of Resistance Genes: The test conditions were set up similarly to the method described above, except that the plasmids used constitutive promoters driving the resistance genes. The OD600 value of the culture was monitored during the process to characterize yeast growth. Partial results are shown below:

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-perceptionandresistance-4.webp)

Fig. 4 Anti-high temperature gene

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-perceptionandresistance-5.webp)

Fig. 5 Anti-high osmotic gene

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-perceptionandresistance-6.webp)

Fig. 6 Anti-ROS gene

Finally, we screened the most effective components: the anti-high temperature promoter – pUAS-CYC1, the anti-high temperature gene – SHSP; the anti-hyperosmotic promoter – pGPD1, the anti-hyperosmotic gene – HOG1; the anti-ROS promoter – pTRX, the anti-ROS gene – SOD1.
 **2. Integration of the Optimal Circuit:**

The best-performing promoter-gene pairs screened in the previous step were assembled with the optimal degradation tag using homologous recombination technology and integrated into the genome of the carotenoid-producing yeast cell factory, constructing stable engineered strains.
 **3. Performance Verification:**

In shake flasks, parallel fermentation was conducted comparing the engineered strains harboring the integrated optimal circuit with control strains (the original strain without the integrated resistance circuit). The integrated yeast strains were fermented at 37°C. During the fermentation process, carotenoid concentration (represented by OD450) and cell density (represented by OD600) were measured daily.

III. Results and Analysis

Analysis of the experimental data yielded the following results: In the preliminary screening experiments, yeast strains carrying specific promoter-gene combinations showed significantly better growth under the corresponding stress conditions compared to the control group. The optimal combinations identified were: for high-temperature resistance: pUAS-CYC1---SHSP; for hyperosmotic resistance: pGPD1---HOG1; for ROS resistance: pTRX---SOD1.

To evaluate the thermo-protective effect of the composite stress-resistance parts, engineered strains (G9, G6) and control strains (β04) were cultivated in YPD medium at 30°C and 37°C for 72 hours. Cell growth (OD₆₀₀) and β-carotene production (OD₄₅₀) were monitored at 24-hour intervals.

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-perceptionandresistance-7.webp)

Fig. 7 Fermentation result of β-carotene producing control strain

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-perceptionandresistance-8.webp)

Fig. 8 Fermentation result of β-carotene producing, G9

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-perceptionandresistance-9.webp)

Fig. 9 Fermentation result of β-carotene producing, G6

In the final cell factory validation, the engineered strain with the integrated optimal circuit showed an increased yield of the target product compared to the control strain after being subjected to stress. The introduction of this resistance circuit effectively mitigated the impact of stress on cell growth and production, enhancing the stability of yeast cell production under stressful conditions. These results confirm that the constructed genetic circuit functions as designed, significantly improving the robustness and production capacity of the cell factory under adverse conditions.

IV. Discussion

The successful validation of our engineered yeast strains underscores the efficacy of a synthetic biology approach in bolstering the resilience of cell factories. The core of our strategy was the design of a dynamic, stress-responsive genetic circuit that remains largely silent under optimal conditions but is rapidly activated upon stress perception. The data presented confirm that this design logic functions as intended.

The most significant finding is that the integrated optimal circuit (pUAS-CYC1-SHSP, pGPD1-HOG1, pTRX-SOD1) conferred a marked advantage under sub-optimal fermentation temperatures (37°C). The engineered strains (G9, G6) achieved a higher yield of the target product, β-carotene (OD450). This directly demonstrates that the circuit successfully decouples stress survival from product synthesis. Instead of merely diverting resources to endure the stress, the engineered cells appear to maintain a more robust metabolic state, thereby preserving the flux towards the desired biosynthetic pathway.

The enhanced stability of production observed throughout the fermentation timeline is a key indicator of the circuit's success for industrial application. Bioprocesses are often plagued by productivity losses due to cumulative stress from substrates, products, and process parameters. Our engineered strains demonstrate a clear path towards more consistent and reliable biomanufacturing, reducing process volatility.

However, several considerations warrant further investigation. First, while the circuit functioned effectively against thermal stress, its performance under other stressors for which it was designed (e.g., high osmolarity, direct oxidative shock) should be explicitly quantified in the production host. Second, the potential metabolic burden imposed by the circuit, even with its degradation tag and stress-responsive nature, needs to be more thoroughly evaluated over extended fermentation periods and in different media. Finally, exploring more complex circuit architectures, such as those incorporating logic gates to fine-tune the response to multiple simultaneous stresses, could be the next step in further optimizing performance.

In conclusion, this work provides compelling evidence that synthetic stress-sensing and response circuits are a powerful tool for enhancing the robustness and productivity of microbial cell factories. By engineering a proactive defense mechanism, we have moved beyond simple tolerance and towards creating resilient yeast chassis that can maintain high-level production under industrially relevant adverse conditions.

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/degradation-degron-module.webp)

Validation of Degradation Tags

To achieve precise temporal control of protein pulses while conserving cellular resources, we implemented a proteolytic control strategy based on degrons. This degradation mechanism can not only rapidly degrade useless proteins and recycle resources when stress is eliminated or switched, but also reduce interference between the regulatory processes of different stresses during stress switching. In this way, it achieves the goal of intelligent stress-resistant regulation desired in the yeast SMART system.Through a literature survey, we selected six ubiquitin-proteasome-dependent degradation tags for testing. These degrons had previously been used as components of fusion proteins. We chose ubiquitin-proteasome-dependent degrons primarily for three reasons: first, the ubiquitin-proteasome degradation mechanism is relatively well-characterized. With the cooperation of ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3, the small protein ubiquitin is added to specific lysine (Lys) residues of unstable proteins. Subsequent polyubiquitin chains are recognized as "degradation signals" and targeted for degradation by the proteasome; second, these degrons are straightforward to apply—simply appending them to the N-terminus of the target protein is sufficient to trigger degradation; third, their half-lives can be easily regulated—altering just a few amino acids can change the structural stability of the degron, thereby adjusting the half-life of the target protein, a feature that offers significant advantages for controlling oscillation periods.

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-degron-1.webp)

Fig. 1 Working principle of ubiquitin degradation
> Yu H, Matouschek A. Recognition of Client Proteins by the Proteasome. Annu Rev Biophys. 2017 May 22;46:149-173. doi: 10.1146/annurev-biophys-070816-033719. Epub 2017 Mar 9. PMID: 28301771.

For degron performance testing, we used the inducible DDI2 promoter to drive the expression of fluorescent reporter proteins. Different N-terminal degrons were fused to the reporter proteins. We set up a negative control group (4741) without fluorescent protein, a positive control group (J0) with fluorescent protein but no degradation tag, and experimental groups (J1[1] J2[2] J3[3] J4[3] J5[3] J6[4]) with fluorescent proteins tagged with six different degrons. All groups were given the same nutritional conditions and inducer concentration. At 30°C, we continuously measured fluorescence intensity and optical density at 600 nm (OD600) to generate fluorescence intensity changes and growth curves, thereby evaluating the degradation efficiency of each degron.

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-degron-2.webp)

Fig. 2 The ratio of fluorescence intensity to OD600 over time at 30°C

Preliminary experiments were conducted at 30°C, the optimal growth temperature for Saccharomyces cerevisiae. Under these conditions, all fusion proteins exhibited extremely high stability with excessively long half-lives (t½), making reliable discrimination between different degrons impossible. Inspired by reports that UPS-mediated degradation is temperature-sensitive, we next subjected the strains to a temperature gradient ranging from 30°C to 40°C. A clear temperature dependence was observed: increasing the culture temperature shortened the half-life (t½) of all fusion proteins, with 37°C providing the optimal balance between degron activity and cell fitness. Among the six candidate degrons, degron 6 resulted in the shortest half-life (t½) for the fusion protein. Based on this, we obtained an efficient degradation tag in S. cerevisiae, which was subsequently applied in the ternary oscillator.

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-degron-3.webp)

Fig. 3 The ratio of fluorescence intensity to OD600 over time at 37°C

Reference

[1] Meyn MA 3rd, Melloy PG, Li J, Holloway SL. The destruction box of the cyclin Clb2 binds the anaphase-promoting complex/cyclosome subunit Cdc23. Arch Biochem Biophys. 2002 Nov 15;407(2):189-95. doi: 10.1016/s0003-9861(02)00467-8. PMID: 12413490.

[2] Kornitzer D, Raboy B, Kulka RG, Fink GR. Regulated degradation of the transcription factor Gcn4. EMBO J. 1994 Dec 15;13(24):6021-30. doi: 10.1002/j.1460-2075.1994.tb06948.x. PMID: 7813440; PMCID: PMC395579.

[3] Suzuki T, Varshavsky A. Degradation signals in the lysine-asparagine sequence space. EMBO J. 1999 Nov 1;18(21):6017-26. doi: 10.1093/emboj/18.21.6017. PMID: 10545113; PMCID: PMC1171667.

[4] Dohmen RJ, Varshavsky A. Heat-inducible degron and the making of conditional mutants. Methods Enzymol. 2005;399:799-822. doi: 10.1016/S0076-6879(05)99052-6. PMID: 16338396.

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/repressor-circuit-module.webp)

Mechanism

There is a class of DNA-binding proteins that can recognize and bind to specific operator sequences, block the binding of RNA polymerase to promoters, and thereby inhibit the transcription of downstream genes—these proteins are referred to as repressors in this study. Repressors enable transcriptional regulation of yeast gene expression, acting as "on/off switches" for gene expression. In the expression of stress-resistant genes, repressors allow for precise regulation of stress-resistant gene expression, saving intracellular resources and achieving "intelligent stress resistance" in yeast, i.e., yeast SMART.

The tripartite oscillator in yeast SMART is a genetic oscillation circuit composed of repressors, degradation degrons, and other genetic elements. Structurally, the oscillator consists of a closed-loop negative feedback circuit formed by three repressor proteins (arranged in a "head-to-tail" manner). To enable the oscillator to respond to stress, anti-repressors (proteins that can relieve the inhibitory effect of repressors) are also incorporated into the oscillator.

The team identified 3 repressor proteins, their mutants, their operator sequences and their corresponding anti-repressors through research. The mechanisms of different repressors and anti-repressors vary, with specific correspondences and mechanisms shown in the table below:

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-repressors-1.webp)

Additionally, yeast is a eukaryote, and its genome is localized in the nucleus. Since repressors used in this study are derived from prokaryotic systems, they need to enter the nucleus to bind to operator sequences. Therefore, a nuclear localization signal (NLS) was fused to the C-terminus or middle region of all repressors and anti-repressors in the experiment. The NLS guides proteins into the nucleus via nuclear transport signals, ensuring effective binding between repressors and DNA operator sequences—this is a critical prerequisite for prokaryotic repressors to function in yeast.

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-repressors-2.webp)

Fig. 1 Addition of nuclear localization signal (NLS) to the C-terminus of repressors and anti-repressors (taking LacI as an example)

Experiments

To construct the tripartite oscillator in yeast, the experiment was carried out in three phases: "Monopartite Validation of Oscillator Components → Design and Testing of Closed-Loop Tripartite Oscillator → Construction and Detection of Tripartite Oscillator with Yeast Stress-Resistant Genes".

**[Phase 1: Monopartite Validation of Oscillator Components]**

This phase aimed to verify whether individual repressors can be normally expressed in yeast, enter the nucleus, bind to target sequences, and inhibit downstream gene transcription.
The core of this phase was to validate repressors using a small-molecule inducible promoter: The promoter of the repressor was replaced with a melamine-inducible promoter; subsequently, the repressor’s operator sequence was inserted into a constitutive promoter, with a red fluorescent protein (mScarlet) integrated downstream. In the absence of melamine, the repressor is not expressed, and the downstream red fluorescent protein is normally expressed; upon adding melamine, the repressor is induced to express, binds to the operator sequence, and inhibits red fluorescent protein expression.

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-repressors-3.webp)

Fig. 2 Mechanism of monopartite validation for oscillator components

**Step 1: Plasmid Construction**

For each set of "repressor-operator sequence-anti-repressor", 3 recombinant plasmids were constructed: ① Plasmid containing the "melamine-inducible promoter - repressor (with NLS)" cassette; ② Plasmid containing the "constitutive promoter inserted with repressor operator sequence - mScarlet red fluorescent protein gene" cassette; ③ Plasmid containing the "galactose-inducible promoter - anti-repressor" cassette. A total of 12 plasmids were constructed using this method.
[![click icon](https://static.igem.wiki/teams/5805/imgs/click-this.webp)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)
 **Step 2: Yeast Transformation**

The 12 plasmids were separately transformed into competent *Saccharomyces cerevisiae* cells.
 **Step 3: Induction Treatment and Detection**

All strains were inoculated in the same YPD medium and cultured for 18–20 hours to reach the logarithmic phase. Then, melamine was added to a final concentration of 0.5 mM (the same dosage for all experimental groups). The OD600 (cell density) and mScarlet fluorescence intensity (excitation wavelength: 579 nm; emission wavelength: 616 nm) were continuously monitored, with measurements recorded every 15 minutes for 24 hours.

Experimental results are as follows:

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-repressors-4.webp)

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-repressors-5.webp)

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-repressors-6.webp)

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-repressors-7.webp)

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-repressors-8.webp)

Fig. 3 Results of monopartite validation for oscillator components

As shown in Fig. 3, when the OD600 curves (indicating cell density) of all groups overlapped (ruling out the influence of cell growth differences on fluorescence intensity), the induced expression groups of LacI^ts, TetR, and λCI exhibited significantly inhibited fluorescence expression compared to the control groups. In contrast, LacI and CI857 showed unsatisfactory inhibitory effects.

These results indicate that LacI^ts, TetR, and λCI can be normally expressed under melamine induction. The fused NLS can effectively guide these repressors into the nucleus. These repressors specifically bind to their respective operator sequences (LacO, TetO, OR) and significantly inhibit mScarlet transcription. Thus, LacI^ts, TetR, and λCI are compatible with the yeast expression system and can be used as core components of the tripartite oscillator.
 **[Phase 2: Design and Testing of Closed-Loop Tripartite Oscillator]**

This phase aimed to verify whether LacI^ts, λCI, and TetR can form a closed-loop negative feedback circuit ("Repressor A → Repressor B → Repressor C → Repressor A") and achieve rhythmic transcriptional regulation.

After successful monopartite validation, the team constructed these three repressors into a closed-loop negative feedback circuit. To detect its function via fluorescence oscillation, the operator sequence of the previous repressor was inserted into the promoter upstream of each repressor (for LacI^ts/λCI/TetR). Additionally, mScarlet expression was coupled with TetR, allowing the observation of mScarlet oscillation over time (fluorescence intensity reflects TetR expression, indirectly indicating the oscillation state of the circuit).

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-repressors-9.webp)

Fig. 4 Schematic design of the closed-loop tripartite oscillator

Four recombinant plasmids were constructed to form the closed-loop circuit:

① Plasmid with LacO inserted into the promoter of λCI (LacI^ts binds to LacO to inhibit λCI transcription); ② Plasmid with OR inserted into the promoter of TetR (λCI binds to OR to inhibit TetR transcription); ③ Plasmid with TetO inserted into the promoter of LacI^ts (TetR binds to TetO to inhibit LacI^ts transcription); ④ Plasmid with TetO inserted into the promoter of mScarlet (fluorescence intensity reflects TetR expression, indirectly indicating circuit oscillation).
[![click icon](https://static.igem.wiki/teams/5805/imgs/click-this.webp)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)
 **Step 2: Yeast Transformation**

The four plasmids were separately transformed into competent *Saccharomyces cerevisiae* cells, resulting in a total of 7 strains.
 **Step 3: Fluorescence Detection**

The 7 strains were cultured at a constant temperature of 30°C (no inducer added). Changes in mScarlet fluorescence intensity were continuously monitored for 136 hours, with measurements recorded every 15 minutes.

The results are as follows:

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-repressors-10.webp)

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-repressors-11.webp)

Fig. 5 Results of closed-loop testing for the tripartite oscillator

As shown in Fig. 5:

All 7 strains exhibited periodic changes in mScarlet fluorescence intensity, confirming that the closed-loop circuit was functional (Fig. 5a). After superimposing and averaging the results of the 7 strains, the system’s oscillation became more intuitive (Fig. 5b). Further denoising processing made the oscillation pattern even clearer (Fig. 5c).

The oscillation period of the tripartite oscillator was approximately 21 hours. These results verify the establishment of the "LacI^ts → λCI → TetR → LacI^ts" closed-loop negative feedback logic: The three repressors restrict each other to form rhythmic expression, indicating the successful construction of the tripartite oscillator.

To further explore the application potential of biological oscillators, the team also reproduced an oscillator design reported in a literature study.

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-repressors-12.webp)

Fig. 6 Tripartite oscillator proposed by Li et al.
> Li, Z., Qiao, G., Wang, X. et al. De novo designed protein guiding targeted protein degradation. Nat Commun 16, 6598 (2025). https://doi.org/10.1038/s41467-025-62050-z

The core design of the literature-derived oscillator relies on a negative feedback loop between three components to achieve periodic regulation: GP-McsB (targets and degrades LacI); LacI (inhibits antiMcsB expression); antiMcsB (inhibits GP-McsB function).

Specifically, GP-McsB targets and degrades LacI, leading to a decrease in LacI levels; Reduced LacI levels relieve its inhibition of antiMcsB expression; Expressed antiMcsB inhibits GP-McsB function by targeted binding, thereby reducing LacI degradation; LacI levels rise again, re-inhibiting antiMcsB expression—forming a periodic negative feedback cycle that ultimately regulates the oscillation of mKate2 abundance.

The team replaced mKate2 with mScarlet and constructed 9 plasmids according to the literature design, which were then transformed into yeast. The 9 plasmids included: ① Plasmid for constitutive expression of LacI; ② Plasmid with LacO inserted into the promoter of mScarlet (fluorescent protein); ③ Plasmid with LacO inserted into the promoter of antiMcsB; ④ Plasmid for constitutive expression of GP-McsB; ⑤ Plasmid for constitutive expression of the degradation protein ClpP; ⑥ Plasmid for constitutive expression of the chaperone protein ClpC (ClpC provides "substrate recognition, unfolding, and delivery" services for ClpP and activates ClpP’s degradation activity); ⑦ Plasmid for constitutive expression of McsA (a protein that activates McsB); ⑧–⑨ Plasmids containing selection markers.
[![click icon](https://static.igem.wiki/teams/5805/imgs/click-this.webp)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-repressors-13.webp)

Fig. 7 Design and working mechanism of the literature-derived oscillator in yeast

The strains were cultured at a constant temperature of 30°C (no inducer added). Changes in mScarlet fluorescence intensity were continuously monitored for 48 hours, with measurements recorded every 15 minutes.

However, no fluorescence fluctuations were detected. The core issue is likely the poor compatibility between prokaryotic components and the yeast eukaryotic system. The literature-derived oscillator relies on prokaryotic degradation enzymes (ClpP/ClpC) to degrade repressors. However, yeast lacks a Clp protease system homologous to that of prokaryotes, leading to the accumulation of repressor proteins. Accumulated repressors continuously inhibit downstream genes, disrupting the oscillation balance.
 **[Phase 3: Construction and Detection of Tripartite Oscillator with Yeast Stress-Resistant Genes]**

To achieve dynamic, periodic stress resistance in industrial yeast while minimizing metabolic burden, we designed an oscillatory anti-stress control scheme—coupling the validated three-node oscillator (LacI^ts → λCI → TetR) to a set of yeast endogenous stress-resistance genes. This design enables coordinated, periodic expression of protective functions: instead of constitutively overexpressing stress-resistance genes (which wastes cellular resources), the oscillator drives these genes to “turn on/off” rhythmically, matching the timing of stress challenges in industrial fermentation processes.

A core advantage of this scheme is the ability to artificially program the response threshold to complex stress: by tuning the oscillator’s period and the repressors’ DNA-binding affinities, we can precisely control when and how strongly stress-resistance genes are expressed, allowing adaptation to diverse fermentation environments (e.g., fluctuating temperature, osmolarity, or oxidative stress).

As a proof-of-concept, we targeted a β-carotene producing *Saccharomyces cerevisiae* strain: we introduced both the three-node oscillator and three key stress-tolerance genes into this host. Each stress-tolerance gene is regulated by one of the oscillator’s repressors, and their functions are:

    shsp (Small Heat Shock Protein, enhancing resistance to heat stress.)

    SOD1 (Superoxide Dismutase 1, mitigating oxidative stress by scavenging reactive oxygen species.)

    HOG1 (Mitogen-Activated Protein Kinase Hog1, mediating osmotic stress response and promotes cell survival under high-osmolarity conditions.)

By rhythmically expressing these genes, we hypothesize that yeast can achieve “timely defense” against multiple stresses while reducing steady-state metabolic burden—ultimately boosting β-carotene yield.
 **Step 1: Plasmid Construction for Stress-Resistant Oscillator**
Based on the three-node oscillator and stress-resistance gene regulation logic, we constructed three 3 recombinant plasmids: ① Plasmid with LacO inserted into the promoter of shsp; ② Plasmid with OR inserted into the promoter of SOD1; ③ Plasmid with TetO inserted into the promoter of HOG1.
[![click icon](https://static.igem.wiki/teams/5805/imgs/click-this.webp)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)
 **Step 2: Yeast Transformation and Strain Construction**

A β-carotene producing *Saccharomyces cerevisiae* strain was used as the host. We sequentially introduced genetic modules into this strain.
 **Step 3: Fermentation and Continuous Detection**

Engineered strains (with oscillatory stress-resistance modules) and control strains (β-carotene producer without the oscillator or oscillatory stress-resistance gene expression) were inoculated into YPD medium and cultured at both 30°C and 37℃. Over 72 hours (3 days), we continuously monitored two key parameters, cell density (reflected by OD600) and β-carotene concentration (reflected by OD450). Measurements were recorded every 24 hours to capture both cell growth dynamics and effects of oscillatory stress protection on β-carotene productivity.

The results are as follows:

![results](https://static.igem.wiki/teams/5805/imgs/wetlab/results-repressors-14.webp)

Fig. 8 Comparison of β-Carotene Production in Engineered vs. Control Strains

Analysis: we assessed β-carotene production in engineered and control strains at 30°C and 37°C over 3 days via quantitative OD₄₅₀/OD₆₀₀ ratio measurement (reflecting β-carotene yield relative to cell density).

For the engineered strain, the OD₄₅₀/OD₆₀₀ ratio at 37°C increased over time and even surpassed the 30°C group on Day 2 and Day 3. Conversely, the control strain exhibited a consistently lower OD₄₅₀/OD₆₀₀ ratio at 37°C compared to 30°C across all time points, with no upward trend throughout fermentation.

In summary, the engineered strain not only tolerates heat stress but also maintains or enhances β-carotene productivity—while the control strain lacks such adaptability and suffers reduced production under heat stress. This direct comparison validates that our stress-resistant oscillator effectively improve both thermal tolerance and β-carotene yield in industrial-relevant conditions.

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