Reducing metabolic burden in the PACEmid evolver system by remastering high-copy phagemid vectors

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biotechnology

Background:

• Orthogonal transcription factors are crucial for constructing complex genetic circuits in synthetic biology.
• Previous work engineered cIλ transcription factor variants using the PACEmid system, but high-copy phagemid vectors caused significant metabolic burden in host cells.
• This burden negatively impacted cellular functions, such as growth, limiting the utility of these tools.

Purpose of the Study:

• To engineer low-burden phagemid vectors for housing cIλ transcription factor variants.
• To maintain the functionality of both the phagemid system and the transcription factors after burden reduction.
• To provide improved tools for synthetic biology applications and future research.

Main Methods:

• Directed evolution using the PACEmid system to engineer cIλ transcription factor variants.
• Modification of phagemid backbones to reduce the metabolic load on host cells (Escherichia coli).
• Assessment of host cell growth recovery and retention of transcription factor activity in remastered vectors.

Main Results:

• Substantial reduction in metabolic burden was achieved by remastering phagemid backbones.
• Host cell growth (Escherichia coli) significantly recovered after implementing the low-burden vectors.
• The remastered phagemids retained functionality within the PACEmid system, and cIλ transcription factors remained active.
• The improved phagemid versions are now available on the Addgene repository.

Conclusions:

• Reducing metabolic burden in genetic circuit components is critical for efficient synthetic biology.
• The remastered low-burden phagemids enhance the practicality of PACEmid-based evolution and genetic circuit construction.
• Understanding and mitigating metabolic burden should be a key consideration in future synthetic biology design.