Controlled exchange of protein and nucleic acid signals from and between synthetic minimal cells

Abstract

Synthetic minimal cells are a class of small liposome bioreactors that have some, but not all functions of live cells. Here, we report a critical step towards the development of a bottom-up minimal cell: cellular export of functional protein and RNA products. We used cell penetrating peptide tags to translocate payloads across a synthetic cell vesicle membrane. We demonstrated efficient transport of active enzymes, and transport of nucleic acid payloads by RNA binding proteins. We investigated influence of a concentration gradient alongside other factors on the efficiency of the translocation, and we show a method to increase product accumulation in one location. We demonstrate the use of this technology to engineer molecular communication between different populations of synthetic cells, to exchange protein and nucleic acid signals. The synthetic minimal cell production and export of proteins or nucleic acids allows experimental designs that approach the complexity and relevancy of natural biological systems.

A gene expression control technology for cell‐free systems and synthetic cells via targeted gene silencing and transfection

“…Transfection, using lipid reagents to facilitate the uptake of nucleic acids, is one of the most common ways to introduce foreign genes to live cells (Chong et al, 2021; Liu et al, 2005). In liposomal synthetic minimal cells, the only way to add genetic material after the formation of cells is to induce fusion with another liposome (which causes dilution of the cell content) (Gaut et al, 2022; Peruzzi et al, 2019) or the use of cell‐penetrating peptides carrying a nucleic acid payload (which has significant payload size limitations) (Heili et al, 2022). Transfection enables the introduction of genetic material to induce or silence the expression of genes in tested cells.…”

Engineering transmembrane signal transduction in synthetic membranes using two-component systems

“…Recently, synthetic membranes have further expanded the capabilities of cell-free systems, most often acting as a compartment to concentrate and protect encapsulated components (9)(10)(11)(12)(13)). Yet increasingly, membranes have been recognized for their capacity to augment cell-free systems by incorporating functional transmembrane proteins (9), enabling protein posttranslational modification (14), lipid biosynthesis (15), and membrane permeability (16,17).…”

“…Initially used to decipher the genetic code ( 2 ), cell-free systems have now been used for applications ranging from the development of therapeutics ( 3–5 ) to distributable biosensors ( 6–8 ). Recently, synthetic membranes have further expanded the capabilities of cell-free systems, most often acting as a compartment to concentrate and protect encapsulated components ( 9–13 ). Yet increasingly, membranes have been recognized for their capacity to augment cell-free systems by incorporating functional transmembrane proteins ( 9 ), enabling protein posttranslational modification ( 14 ), lipid biosynthesis ( 15 ), and membrane permeability ( 16, 17 ).…”

A gene expression control technology for cell-free systems and synthetic cells via targeted gene silencing and transfection

“…Transfection, using lipid reagents to facilitate the uptake of nucleic acids, is one of the most common ways to introduce foreign genes to live cells. 14,15 In liposomal synthetic minimal cells, the only way to add genetic material after the formation of cells is to induce fusion with another liposome (which causes dilution of the cell content) 16,17 or the use of cell-penetrating peptides carrying a nucleic acid payload (which has significant payload size limitations) 18 . Transfection enables the introduction of genetic material to induce or silence the expression of genes in tested cells.…”