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.
['Vibhute|Mahesh A|MA|', 'Schaap|Mark H|MH|', 'Maas|Roel J M|RJM|', 'Nelissen|Frank H T|FHT|', 'Spruijt|Evan|E|0000-0003-4793-9923', 'Heus|Hans A|HA|', 'Hansen|Maike M K|MMK|0000-0001-7998-6631', 'Huck|Wilhelm T S|WTS|0000-0003-4222-5411']
D058045:Artificial Cells D002474:Cell-Free System D003593:Cytoplasm D004058:Diffusion D015870:Gene Expression D049452:Green Fluorescent Proteins D007700:Kinetics D008081:Liposomes D044085:Microfluidics D011108:Polymers D014176:Protein Biosynthesis D058615:Synthetic Biology D014158:Transcription, Genetic
Protocells macromolecular crowding cell free gene expression liposomes microfluidics