Engineering Success

Engineering Success: Applying the iGEM Cycle to Optimize GFP Expression in Yeast

In our project, we aimed to create a yeast model stably expressing GFP as a chassis for testing antisense oligonucleotides (ASOs). Following the iGEM Engineering Cycle, we iteratively improved our system from a low-expressing initial version to a robust, high-yield GFP-expressing strain.

Engineering Cycle
Figure 1: Applying the iGEM Engineering Cycle to Engineer GFP-Expressing Yeast
Top: The iGEM Engineering Cycle (Design → Build → Test → Learn) guided our iterative approach to optimize GFP expression in Saccharomyces cerevisiae. Bottom Left: Wild-type yeast colonies on agar plate without any fluorescence. Bottom Right: Engineered S. cerevisiae colonies stably expressing GFP under the GAL10 promoter show bright green fluorescence, indicating successful genomic integration and expression of the GFP-degron construct.

First Iteration

Design & Build:

We used Gibson Assembly to construct a plasmid containing a human-optimized GFP gene under a constitutive promoter. The plasmid was designed for genomic integration into the ADE1 locus of S. cerevisiae W303. The insert was amplified from HEK293 GFP plasmid to match our mammalian model.

Test:

After transforming the plasmid into yeast and selecting with hygromycin B, fluorescence was confirmed under a microscope. However, plate reader analysis revealed a very low GFP signal, similar to the negative control.

Learn:

We concluded that despite correct integration and transcription, codon usage and promoter strength were likely limiting factors for translation efficiency and protein accumulation in yeast.

Second Iteration - Redesign & Optimization

Redesign:

We redesigned the construct with three major changes:

  1. Codon optimization: The first 8 codons of the GFP gene were optimized for yeast expression.
  2. Stronger promoter: We replaced the original promoter with GAL10, a strong galactose-inducible promoter shown to work even without induction in certain strains [1].
  3. Degron addition: To enable controlled degradation in future ASO experiments, we added an auxin-inducible degron downstream of GFP.

Build:

The new GFP-degron fusion was synthesized and assembled into a linearized vector backbone. Colony PCR, minipreps, and sequencing confirmed the correct construct. We linearized the plasmid with SrfI and integrated it into yeast as before.

Test:

Four colonies were cultured in glucose and galactose media. In galactose, all showed substantially higher GFP expression, confirmed by plate reader analysis. Colony 4 showed the best signal and was selected for downstream ASO testing.

Learn:

Our results validated the importance of host-specific optimization (codon usage + regulatory elements) and demonstrated how the engineering cycle can be applied in synthetic biology to systematically troubleshoot and enhance construct performance.

🧪 Summary of Improvements

Feature First Iteration Second Iteration
Promoter Weak constitutive Strong GAL10 (galactose-inducible)
Codon optimization Human-optimized Partially yeast-optimized
GFP detection Microscope only (low) Strong signal by plate reader
Degron None Auxin-inducible degron (future control)
Integration locus ADE1 (both) ADE1 (both)

🔄 How We Used the iGEM Engineering Cycle

  1. Design: Selected GFP and designed constructs for genomic integration.
  2. Build: Assembled constructs using Gibson Assembly.
  3. Test: Transformed into yeast and measured fluorescence (microscope + plate reader).
  4. Learn: Identified bottlenecks and redesigned using promoter and codon optimization.
  5. Repeat: Successfully implemented improvements and achieved desired expression levels.

ASO Design

References

[1] M.-J. Kim, B. H. Sung, H.-J. Park, J.-H. Sohn, and J.-H. Bae, “A new platform host for strong expression under GAL promoters without inducer in Saccharomyces cerevisiae,” Biotechnol. Rep., vol. 36, p. e00763, Dec. 2022, doi: 10.1016/j.btre.2022.e00763.
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