Impact of Porous Matrices and Concentration by Lyophilization on Cell-Free Expression

Abstract

Cell-free expression systems have drawn increasing attention as a tool to achieve complex biological functions outside of the cell. Several applications of the technology involve the delivery of functionality to challenging environments, such as fieldforward diagnostics or point-of-need manufacturing of pharmaceuticals. To achieve these goals, cell-free reaction components are preserved using encapsulation or lyophilization methods, both of which often involve an embedding of components in porous matrices like paper or hydrogels. Previous work has shown a range of impacts of porous materials on cell-free expression reactions. Here, we explored a panel of 32 paperlike materials and 5 hydrogel materials for the impact on reaction performance. The screen included a tolerance to lyophilization for reaction systems based on both cell lysates and purified expression components. For paperlike materials, we found that (1) materials based on synthetic polymers were mostly incompatible with cell-free expression, (2) lysate-based reactions were largely insensitive to the matrix for cellulosic and microfiber materials, and (3) purified systems had an improved performance when lyophilized in cellulosic but not microfiber matrices. The impact of hydrogel materials ranged from completely inhibitory to a slight enhancement. The exploration of modulating the rehydration volume of lyophilized reactions yielded reaction speed increases using an enzymatic colorimetric reporter of up to twofold with an optimal ratio of 2:1 lyophilized reaction to rehydration volume for the lysate system and 1.5:1 for the purified system. The effect was independent of the matrices assessed. Testing with a fluorescent nonenzymatic reporter and no matrix showed similar improvements in both yields and reaction speeds for the lysate system and yields but not reaction speeds for the purified system. We finally used these observations to show an improved performance of two sensors that span reaction types, matrix, and reporters. In total, these results should enhance efforts to develop field-forward applications of cell-free expression systems.

Assessment of Colorimetric Reporter Enzymes in the PURE System

“…Eye-readable colorimetric reporters are highly attractive for sensing applications in resource-limited environments, such as developing economies or remote locations. In recent years, cell-free gene expression (CFE) systems expressing such reporters have been leveraged for this reason while also offering shelf stability, , low cost, incorporation into paper and other materials, − and application-relevant sensitivities and specificities. Recent studies have demonstrated the detection of a variety of analytes, including pathogens, ,− biomarkers, ,, explosive precursors, vital nutrients, and water contaminants. − …”

Inexpensive and colorimetric RNA detection by E. coli cell-free protein synthesis platform at room temperature

“…For -complementation, LacZ subunit gene expression was placed under the control of endogenous E. coli promoters (Figure S7B and Table S1). To test the complemented LacZ activity, 2 mM (final or 12 µg/µL) Chlorophenol Red-β-D-galactopyranoside (CPRG) was used as a substrate to give an expected color change from yellow to reddish-purple (we note that the precise color change is heavily dependent on the solid support) 32 .…”

Self-assembled nanoparticle-enzyme aggregates enhance functional protein production in pure transcription-translation systems

“…Cell-free protein synthesis systems (CFPS), also referred to as transcription-translation systems (TX-TL), produce RNA and proteins outside of the confines of a cell using cellular lysate or purified components [ 1 – 6 ]. The products of CFPS can be used as end-products themselves ( e .…”

“…g ., antibodies, enzymes, etc .) [ 2 , 7 – 9 ] or be used for different applications such as biosensing [ 1 , 10 – 12 ], bioremediation [ 13 ], or chemical biosynthesis [ 9 , 14 ]. CFPS offer an alternative to in vivo biosynthetic systems with distinct advantages conveyed by their non-living nature.…”