Cell-Free Gene Expression Dynamics in Synthetic Cell Populations

Area of Science:

• Synthetic biology
• Biophysics
• Chemical engineering

Background:

• Bottom-up construction of synthetic cellular populations enables the creation of minimal living tissues.
• Engineered micron-sized compartments with integrated reaction networks facilitate the design of complex chemical systems.

Purpose of the Study:

• To generate populations of synthetic cells encapsulating cell-free expression systems (CFESs).
• To quantify transcription and translation dynamics within individual synthetic cells.
• To analyze the impact of compartmentalization on gene expression.

Main Methods:

• Utilized double-emulsion microfluidics to produce monodisperse liposomes encapsulating CFESs.
• Employed fluorescent Broccoli RNA aptamer and mCherry protein reporters to monitor gene expression dynamics.
• Applied mathematical modeling, including coarse-grained resource-limited gene expression models and likelihood-based parameter estimation, to analyze CFE dynamics.

Main Results:

• Quantified transcription and translation dynamics in individual synthetic cells.
• Determined transcription and translation rate parameters using model selection and parameter estimation.
• Demonstrated that compartmentalization within liposomes alters gene expression rates compared to bulk reactions.

Conclusions:

• Compartmentalization of CFESs within synthetic cells results in different transcription and translation rates than in bulk reactions.
• The semipermeable nature of the lipid membrane influences gene expression by allowing material exchange.
• This study provides a statistically robust analysis of CFE dynamics in both bulk and synthetic cell populations.