Cogenerating Synthetic Parts toward a Self-Replicating System

To build replicating systems with new functions, the engineering of existing biological machineries requires a sensible strategy. Protein synthesis Using Recombinant Elements (PURE) system consists of the desired components for transcription, translation, aminoacylation and energy regeneration. PURE might be the basis for a radically alterable, lifelike system after optimization. Here, we regenerated 54 E. coli ribosomal (r-) proteins individually from DNA templates in the PURE system. We show that using stable isotope labeling with amino acids, mass spectrometry based quantitative proteomics could detect 26 of the 33 50S and 20 of the 21 30S subunit r-proteins when coexpressed in batch format PURE system. By optimizing DNA template concentrations and adapting a miniaturized Fluid Array Device with optimized feeding solution, we were able to cogenerate and detect at least 29 of the 33 50S and all of the 21 30S subunit r-proteins in one pot. The boost on yield of a single r-protein in coexpression pool varied from ∼1.5 to 5-fold compared to the batch mode, with up to ∼2.4 μM yield for a single r-protein. Reconstituted ribosomes under physiological condition from PURE system synthesized 30S r-proteins and native 16S rRNA showed ∼13% activity of native 70S ribosomes, which increased to 21% when supplemented with GroEL/ES. This work also points to what is still needed to obtain self-replicating synthetic ribosomes in situ in the PURE system.