Leveraging Active Learning to Establish Efficient In Vitro Transcription and Translation from Bacterial Chromosomal DNA.

Gene expression is a fundamental aspect in the construction of a minimal synthetic cell, and the use of chromosomes will be crucial for the integration and regulation of complex modules. Expression from chromosomes in vitro transcription and translation (IVTT) systems presents limitations, as their large size and low concentration make them far less suitable for standard IVTT reactions. Here, we addressed these challenges by optimizing lysate-based IVTT systems at low template concentrations. We then applied an active learning tool to adapt IVTT to chromosomes as template DNA. Further insights into the dynamic data set led us to adjust the previous protocol for chromosome isolation and revealed unforeseen trends pointing at limiting transcription kinetics in our system. The resulting IVTT conditions allowed a high template DNA efficiency for the chromosomes. In conclusion, our system shows a protein-to-chromosome ratio that moves closer to in vivo biology and represents an advancement toward chromosome-based synthetic cells.

Transcription and Translation in Cytomimetic Protocells Perform Most Efficiently at Distinct Macromolecular Crowding Conditions.

The formation of cytomimetic protocells that capture the physicochemical aspects of living cells is an important goal in bottom-up synthetic biology. Here, we recreated the crowded cytoplasm in liposome-based protocells and studied the kinetics of cell-free gene expression in these crowded containers. We found that diffusion of key components is affected not only by macromolecular crowding but also by enzymatic activity in the protocell. Surprisingly, size-dependent diffusion in crowded conditions yielded two distinct maxima for protein synthesis, reflecting the differential impact of crowding on transcription and translation. Our experimental data show, for the first time, that macromolecular crowding induces a switch from reaction to diffusion control and that this switch depends on the sizes of the macromolecules involved. These results highlight the need to control the physical environment in the design of synthetic cells.

Biomolecular Chemistry in Liquid Phase Separated Compartments.

Biochemical processes inside the cell take place in a complex environment that is highly crowded, heterogeneous, and replete with interfaces. The recently recognized importance of biomolecular condensates in cellular organization has added new elements of complexity to our understanding of chemistry in the cell. Many of these condensates are formed by liquid-liquid phase separation (LLPS) and behave like liquid droplets. Such droplet organelles can be reproduced and studied in vitro by using coacervates and have some remarkable features, including regulated assembly, differential partitioning of macromolecules, permeability to small molecules, and a uniquely crowded environment. Here, we review the main principles of biochemical organization in model membraneless compartments. We focus on some promising in vitro coacervate model systems that aptly mimic part of the compartmentalized cellular environment. We address the physicochemical characteristics of these liquid phase separated compartments, and their impact on biomolecular chemistry and assembly. These model systems enable a systematic investigation of the role of spatiotemporal organization of biomolecules in controlling biochemical processes in the cell, and they provide crucial insights for the development of functional artificial organelles and cells.

Macromolecularly Crowded Protocells from Reversibly Shrinking Monodisperse Liposomes.

The compartmentalization of cell-free gene expression systems in liposomes provides an attractive route to the formation of protocells, but these models do not capture the physical (crowded) environment found in living systems. Here, we present a microfluidics-based route to produce monodisperse liposomes that can shrink almost 3 orders of magnitude without compromising their stability. We demonstrate that our strategy is compatible with cell-free gene expression and show increased protein production rates in crowded liposome protocells.