Prolonging genetic circuit stability through adaptive evolution of overlapping genes

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biotechnology

Background:

• Maintaining synthetic biological circuit function over extended periods is a key challenge.
• Existing kill-switch circuits often suffer from instability due to mutations.
• Novel strategies are needed to enhance the evolutionary stability of engineered genetic systems.

Purpose of the Study:

• To develop a novel method for increasing the evolutionary stability of synthetic biological circuits.
• To engineer a synthetic circuit where a toxin gene is "entangled" within a non-essential gene.
• To assess the stability and functionality of the entangled circuit over evolutionary time.

Main Methods:

• Employed synthetic overlapping sequences to entangle the toxin gene relE within the essential gene ilvA.
• Optimized the ribosome-binding site of the internal relE gene for functional expression.
• Utilized adaptive laboratory evolution to select for stable circuit variants over >130 generations.

Main Results:

• The selection pressure for maintaining functional IlvA stabilized the production of the burdensome RelE toxin.
• Mutations inactivating the entangled genes were disfavored, altering the mutation landscape.
• Accumulated mutations in the regulatory region of ilvA reduced baseline relE expression, lowering circuit burden and prolonging kill-switch function.

Conclusions:

• Sequence entanglement is a powerful strategy for enhancing the evolutionary stability of burdensome synthetic circuits.
• Adaptive laboratory evolution can be effectively combined with sequence entanglement to achieve long-term circuit function.
• This approach offers a promising solution for developing more robust synthetic biological systems for various applications.