Expanding luciferase reporter systems for cell-free protein expression

Abstract

Luciferases are often used as a sensitive, versatile reporter in cell-free transcription-translation (TXTL) systems, for research and practical applications such as engineering genetic parts, validating genetic circuits, and biosensor outputs. Currently, only two luciferases (Firefly and Renilla) are commonly used without substrate cross-talk. Here we demonstrate expansion of the cell-free luciferase reporter system, with two orthogonal luciferase reporters: N. nambi luciferase (Luz) and LuxAB. These luciferases do not have cross-reactivity with the Firefly and Renilla substrates. We also demonstrate a substrate regeneration pathway for one of the new luciferases, enabling long-term time courses of protein expression monitoring in the cell-free system. Furthermore, we reduced the number of genes required in TXTL expression, by engineering a cell extract containing part of the luciferase enzymes. Our findings lead to an expanded platform with multiple orthogonal luminescence translation readouts for in vitro protein expression.

Chloroplast Cell-Free Systems from Different Plant Species as a Rapid Prototyping Platform

“…To this end, we employed the NanoLuc reporter system, noted for its high sensitivity and recent application in cell-free systems. 45 To facilitate our initial experiments, we engineered a 'universal test construct', designed to be a standard tool for troubleshooting chloroplast cell-free extracts. This construct comprised genetic elements of viral origins, specifically the T7 RNA polymerase promoter, gene10 5′UTR, 46 and the Tobacco Mosaic Virus (TMV) 3′UTR, 10,47 each chosen for their proven efficacy in driving strong gene expression in chloroplasts.…”