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.
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