Figure 1.
Multiple orthogonally translating (OT) organelles for equipping cells with multiple orthogonal expanded genetic codes
(A) Our experimental design: forming two fully orthogonal OT organelles in one cell equips the host with two orthogonal genetic codes. Only selected mRNAs are translated with the respective expanded genetic code and hence only selected proteins contain the specific ncAAs. An assembler is a synthetic-organelle-forming unit like a protein condensation functionality. An RNA-binding domain (RBD) is chosen to bind its cognate RNA motif (RM), which is fused to the mRNA of the protein of interest (POI) in the 3′ untranslated region.
(B) In order to construct multiple OT organelles, three criteria need to be fulfilled: independent assembly, selective RNA recruitment, and distinct ncAA specificity.
(C) Schematic representation of membranes targeted for the development of OT organelles.
(D–G) Overview of the classes of synthetic organelles in this work. To develop OT organelles on the cytoplasmic surface of cellular membranes, PylRS and MCP or λN22 peptides were fused to the phase-separating assembler FUS as well as to membrane-targeting domains.