Traditional protocols and optimization methods lead to absent expression in a mycoplasma cell-free gene expression platform.

Cell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species. To generate a CFE system compatible with recently established minimal cell constructs, we attempted to optimize a bacterium-based CFE system using lysates of the genome-minimized cell JCVI-syn3A (Syn3A) and its close phylogenetic relative (Mcap).