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 the 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.
| Original language | English (US) |
|---|---|
| Article number | 11489 |
| Journal | Scientific reports |
| Volume | 12 |
| Issue number | 1 |
| DOIs | |
| State | Published - Dec 2022 |
Bibliographical note
Funding Information:We thank Dr. Arjun Khakhar for providing insights and information on the fungal luciferase pathway. We thank Dr. Daniel Voytas and Dr. Arjun Khakhar for the gift of the plasmid containing five enzymes for the fungal luciferase pathway. We thank Dr. Vincent Noireaux for the gift of T7 RNA polymerase plasmid. We thank Evan Kalb for the discussion of the HiBiT/LgBiT luciferase system. This work was supported by NIH award 5R01MH114031 RNA Scaffolds for Cell Specific Multiplexed Neural Observation, NASA award 80NSSC18K1139 Center for the Origin of Life—Translation, Evolution And Mutualism, NSF award 1807461 SeMiSynBio Very Large scale genetic circuit design and automation, NSF award 1840301 RoL:FELS:RAISE Building and Modeling Synthetic Bacterial Cells, and the Funai Foundation for Information Technology.
Publisher Copyright:
© 2022, The Author(s).