Phase 4: Promoter-Terminator Pairing and Expression Simulation (Asimov Kernel)

Initial Design Strategy

I first assembled a promoter library containing 20 plant promoters with different reported expression strengths, together with a smaller library of seven plant terminators.

The initial strategy was to generate multiple promoter–terminator combinations for each CODH gene and then computationally simulate their predicted expression behavior using the Asimov Kernel platform. This simulation step was intended to help compare the different expression architectures before final construct selection.

The design process was based on several important principles:

• Stronger genes or more critical proteins should receive stronger promoters
• Structural subunits should maintain relatively balanced stoichiometry
• Strong promoters should generally be paired with stronger terminators
• Construct size should remain compatible with cloning and synthesis workflows
• Extremely high expression should be avoided when possible to reduce metabolic stress and instability risks

Using these principles, multiple candidate expression sets were generated for both the structural genes and the maturation genes.

Structural Gene Expression Sets

The structural construct contains the three genes directly forming the CODH enzyme complex:

• coxL —> the large catalytic subunit
• coxM —> the electron transfer medium subunit
• coxS —> the iron-sulfur small subunit

Set 1 — High Balanced Expression (Primary Candidate)

This configuration was designed to maximize structural gene expression while maintaining relatively balanced production between the three subunits.

Set 2 — Medium-High Balanced Expression (Alternative)

This set provided a more moderate expression profile. Although weaker than Set 1, it was expected to reduce cellular burden and lower the risks associated with excessive transgene expression.

Set 3 — Very High Expression Configuration

This configuration aimed to maximize expression output. However, because of the extremely strong promoters involved, it also carried higher risks of stoichiometric imbalance, metabolic stress, transcriptional instability, and possible silencing effects.

Maturation Gene Expression Sets

The maturation construct contains four accessory genes involved in CODH assembly and activation:

coxD coxE coxF coxG

Unlike the structural genes, these proteins are not part of the final catalytic complex itself but are essential for proper enzyme maturation, sulfur insertion, and cofactor incorporation.

Special attention was given to coxD because it plays a central role in active-site maturation.

Set 4 — Balanced Maturation Expression

This set was designed to maintain balanced maturation-protein production while prioritizing coxD expression because of its importance in catalytic-site activation.

Set 5 — Lower Expression Configuration

This configuration represented a weaker-expression alternative intended to minimize cellular burden and reduce possible stress associated with transgene overexpression.

The original plan for this phase was to computationally simulate all designed expression sets using the Asimov Kernel platform.

The objective of these simulations was to:

• Predict relative expression behavior
• Evaluate stoichiometric balance between genes
• Identify potential bottlenecks in the pathway
• Detect excessive or insufficient expression levels
• Refine promoter–terminator combinations before DNA synthesis

At the current stage of the project, access to the Asimov Kernel platform is still pending. To avoid delaying the workflow, provisional promoter–terminator combinations were selected manually based on promoter strength, expected biological balance, construct compactness, and cloning feasibility.

If access to Asimov Kernel becomes available later, the selected systems will still be computationally validated, and additional adjustments may be introduced if simulation results suggest improved expression architectures.

Final Selected Expression Systems

Final Structural Construct Selection

For the structural genes, Set 2 was selected as the final configuration.

Although Set 1 and Set 3 could potentially generate stronger expression, Set 2 was considered more biologically balanced and technically safer. The moderate promoter strengths reduce the likelihood of excessive chloroplast burden, instability, or transcriptional silencing while still maintaining relatively balanced subunit expression.

Final structural configuration:

Final Maturation Construct Selection

For the maturation genes, a modified version of Set 4 was selected.

Initially, the promoter PTSB1 was assigned to coxD because of its relatively strong expression profile. However, this promoter was approximately 1.5 kb long, which significantly increased construct size and cloning complexity.

To maintain a more compact and synthesis-friendly construct, PTSB1 was replaced with D100 while preserving the overall balanced-expression strategy.

Final maturation configuration:

This final configuration aimed to preserve balanced maturation-gene expression while improving construct compactness and compatibility with downstream Gibson Assembly and DNA synthesis workflows.