Do the Shuffle: Expanding the Synthetic Biology Toolkit for Shufflon-like Recombination Systems

Area of Science:

• Molecular Biology
• Synthetic Biology
• Genetics

Background:

• Naturally occurring DNA inversion systems are crucial for prokaryotic genetic variation and adaptation.
• Shufflon invertase (SI) Rci from plasmid R64 is a synthetic biology tool, but its moderate recombination rates limit applications.
• Wider use of SIs is hindered by the limited availability of enzymes with optimal recombination efficiencies.

Purpose of the Study:

• To identify and characterize novel shufflon invertase (SI) genes and their corresponding sfx sites.
• To develop a robust assay for quantifying SI inversion rates and assessing enzyme cross-recognition.
• To engineer SIs with significantly improved recombination rates for synthetic biology applications.

Main Methods:

• Bioinformatic identification of 14 novel SI genes and their sfx sites from public databases.
• Development of a single-molecule sequencing-based assay for quantitative measurement of DNA inversion rates.
• Determination of enzyme cross-recognition to identify orthogonal SI/sfx pairs for precise genetic manipulation.

Main Results:

• 14 previously untested SI genes and their sfx sites were identified.
• A single-molecule sequencing assay was established to quantify inversion rates.
• Engineered SI enzymes demonstrated substantially improved shuffling rates when inducibly expressed in E. coli.

Conclusions:

• The identified and engineered SIs offer enhanced recombination rates for synthetic biology.
• These improved SIs facilitate the creation of synthetic shufflons for generating millions of in vivo sequence variants.
• Applications include DNA barcoding, experimental selection, and advancing engineering biology.