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 Re...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2022-07-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-022-15624-6 |
| _version_ | 1811224090290159616 |
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| author | Wakana Sato Melanie Rasmussen Christopher Deich Aaron E. Engelhart Katarzyna P. Adamala |
| author_facet | Wakana Sato Melanie Rasmussen Christopher Deich Aaron E. Engelhart Katarzyna P. Adamala |
| author_sort | Wakana Sato |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-12T08:43:04Z |
| format | Article |
| id | doaj.art-b865d5d369ac4745baa1bbf5919d2d99 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-12T08:43:04Z |
| publishDate | 2022-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-b865d5d369ac4745baa1bbf5919d2d992022-12-22T03:39:47ZengNature PortfolioScientific Reports2045-23222022-07-0112111010.1038/s41598-022-15624-6Expanding luciferase reporter systems for cell-free protein expressionWakana Sato0Melanie Rasmussen1Christopher Deich2Aaron E. Engelhart3Katarzyna P. Adamala4Department of Genetics, Cell Biology and Development, University of MinnesotaDepartment of Genetics, Cell Biology and Development, University of MinnesotaDepartment of Genetics, Cell Biology and Development, University of MinnesotaDepartment of Genetics, Cell Biology and Development, University of MinnesotaDepartment of Genetics, Cell Biology and Development, University of MinnesotaAbstract 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.https://doi.org/10.1038/s41598-022-15624-6 |
| spellingShingle | Wakana Sato Melanie Rasmussen Christopher Deich Aaron E. Engelhart Katarzyna P. Adamala Expanding luciferase reporter systems for cell-free protein expression Scientific Reports |
| title | Expanding luciferase reporter systems for cell-free protein expression |
| title_full | Expanding luciferase reporter systems for cell-free protein expression |
| title_fullStr | Expanding luciferase reporter systems for cell-free protein expression |
| title_full_unstemmed | Expanding luciferase reporter systems for cell-free protein expression |
| title_short | Expanding luciferase reporter systems for cell-free protein expression |
| title_sort | expanding luciferase reporter systems for cell free protein expression |
| url | https://doi.org/10.1038/s41598-022-15624-6 |
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