PROJECT

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:

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.

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:

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.

> 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: