Genetic circuit performance under conditions relevant for industrial bioreactors

Area of Science:

• Synthetic biology
• Genetic engineering
• Industrial biotechnology

Background:

• Synthetic genetic circuits are crucial for industrial applications.
• These circuits must maintain fidelity in diverse and complex environments.
• Previous research has focused on circuit design, but performance under industrial conditions requires further investigation.

Purpose of the Study:

• To evaluate the performance of two synthetic transcriptional circuits (AND and NOR gates) in Escherichia coli under industrially relevant conditions.
• To compare the robustness and reliability of these circuits across different media, strains, and growth rates.
• To assess the scalability of synthetic circuits from laboratory to pilot-scale fermentation.

Main Methods:

• Testing of AND and NOR genetic circuits in Escherichia coli strains DH10B and DS68637.
• Evaluation across varying media compositions (minimal vs. supplemented) and growth rates.
• Performance assessment at three scales: 100 mL shake flask, 1 mL microtiter plate reactor, and 10 L bioreactor.
• Utilizing a GFP reporter plasmid for quantitative comparison of circuit performance.

Main Results:

• The AND gate's activity was dependent on media composition and strain, being inactive in minimal media for E. coli DH10B but functional in E. coli DS68637.
• The NOR gate demonstrated robustness, functioning consistently across different media and strains.
• AND gate performance varied significantly with scale, deteriorating during fermentation due to resource depletion and growth rate changes.
• One output state of the AND gate failed in the bioreactor, limiting its responsiveness.
• Cells with the AND gate exhibited reduced biomass accumulation.

Conclusions:

• Synthetic genetic circuits, particularly the AND gate, face significant challenges in maintaining fidelity under industrial fermentation conditions.
• Environmental factors like media composition and growth rate dynamics critically impact circuit performance.
• The observed scale-dependent performance and failure modes highlight the need for modified strategies in developing and characterizing genetic circuits for industrial biotechnology.
• The NOR gate presents a more robust option for industrial synthetic biology applications compared to the tested AND gate.