One bottleneck in engineering bacteria for synthetic-biology applications is the lack of organelles to compartmentalize chemical reactions. To overcome this limitation, researchers have sought to create artificial condensates in the bacterial cytosol that can isolate enzymatic pathways in a manner reminiscent of eukaryotic organelles. New work reported in Cell improves the properties, modularity and scalability of this approach by adapting an RNA-based method for liquid–liquid phase separation (LLPS) previously demonstrated in eukarotic cells. The system relies on a fusion of two RNA sequences: 47 rCAG repeats, which self-assemble into scaffold-like membrane-less compartments, and an RNA aptamer, which allows recruitment of any protein tagged with a suitable aptamer-binding domain. “Can we recreate such compartments in bacteria that are devoid of organelles in order to understand their evolution, characterize them systematically and harness them toward new chemistries?” asks Ariel B. Lindner, corresponding author of the study. The authors explore various physical properties of the condensates, dubbed TEARS (transcriptionally engineered addressable RNA solvent) droplets, including temperature sensitivity, localization in the cell, stability, and the dynamics of solute partitioning between the droplets and the cytosol. They show that the droplets largely exclude endogenous proteins and create a multilayered architecture capable of sequestering biochemical reactions and fluorescent proteins. The modular nature of TEARS makes it easy to alter the molecular composition of the droplets by changing the aptamer sequences and aptamer-binding proteins.
The authors find that the multiple phases created by TEARS allow at least two immiscible single proteins to coexist — a behavior previously uncharacterized in E. coli. They demonstrate compartmentalization that supports complex bioprocessing reactions, such as controlling metabolic branch point bioprocessing, buffering mRNA translation rates and scaffolding protein–protein interactions, hitherto only possible with organelle separations. By enabling organelle-like spatial organization in bacteria, TEARS may “overcome scaling limitations observed in protein-driven LLPS systems due to protein-fusion instability and aggregation,” says Lindner. Much of this work was initiated by a group of students co-mentored by graduate student H. Guo in Lindner’s lab as part of their International Genetically Engineered Machine competition 2017 entry.
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