Double emulsions for analyzing gene expression dynamics in cell-free systems

“The ability to build synthetic cellular populations from the bottom-up provides the groundwork to realize minimal living tissues comprising single cells which can communicate and bridge scales into multicellular systems. Engineered systems made of synthetic micron-sized compartments and integrated reaction networks coupled with mathematical modeling can facilitate the design and construction of complex and multiscale chemical systems from the bottom-up. Toward this goal, we generated populations of monodisperse liposomes encapsulating cell-free expression systems (CFESs) using double-emulsion microfluidics and quantified transcription and translation dynamics within individual synthetic cells of the population using a fluorescent Broccoli RNA aptamer and mCherry protein reporter. CFE dynamics in bulk reactions were used to test different coarse-grained resource-limited gene expression models using model selection to obtain transcription and translation rate parameters by likelihood-based parameter estimation. The selected model was then applied to quantify cell-free gene expression dynamics in populations of synthetic cells. In combination, our experimental and theoretical approaches provide a statistically robust analysis of CFE dynamics in bulk and monodisperse synthetic cell populations. We demonstrate that compartmentalization of CFESs leads to different transcription and translation rates compared to bulk CFE and show that this is due to the semipermeable lipid membrane that allows the exchange of materials between the synthetic cells and the external environment.

gene expression dynamics using droplet microfluidics

“Variability in synthetic cell populations. (A) Schematic of the bulk inverse emulsion phase transfer method and double-emulsion microfluidics to generate liposomes or synthetic cells. (B) Synthetic cell population expressing eGFP protein from 1.17 nM pEXP5-NT/6xHis eGFP plasmid DNA generated using the bulk inverse emulsion phase transfer method. (C) Microfluidic-generated synthetic cells expressing eGFP protein from 4.5 nM pEXP5-NT/6xHis eGFP F30-2xdBroccoli plasmid DNA. (D) Merged image of the synthetic cell population expressing both eGFP and mCherry protein from two plasmids (4.5 nM pEXP5-NT/6xHis eGFP and 4.5 nM pEXP5-NT/6xHis mCherry plasmid DNA). Endpoint histograms of radius and protein RFU are plotted alongside each of the synthetic cell populations (B–D). The number of cells analyzed is 206, 106, and 85 for (B–D), respectively. Black lines are Gaussian distributions obtained by fitting mean and variance of the data. These experiments show the relative levels of expressed protein and do not refer to absolute concentrations. RFU values between the microfluidic-generated synthetic cells in (C,D), but not the inverse emulsion-made synthetic cells, are comparable, as these images were acquired using the same microscopy settings. However, CV values can be compared across all populations. All images are taken at the endpoint after 12 h of incubation at 30 °C using confocal microscopy with a 40× objective for (B) and 10× objective for (C,D). Scale bars are all 100 μm.” Reproduced under Creative Commons Attribution 4.0 International License from Gonzales, T, D., Yandarpalli, N., Robinson, T., Zechner, C., Tang, D, T-Y., Cell-free Gene Expression Dynamics in Synthetic Cell Populations. Acs. Syn. Bio. 2022: https://doi.org/10.1021/acssynbio.1c00376


Figures and the abstract are reproduced from N., Robinson, T., Zechner, C., Tang, D, T-Y., Cell-free Gene Expression Dynamics in Synthetic Cell Populations. Acs. Syn. Bio. 2022: https://doi.org/10.1021/acssynbio.1c00376 under Creative Commons Attribution 4.0 International License

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Cell-Free Gene Expression Dynamics in Synthetic Cell Populations