Cell-Free Protein Synthesis as a Prototyping Platform for Mammalian Synthetic Biology
| Autor: | Caoimhe Canavan, Velia Siciliano, Bruno Goetzmann, David W. McClymont, Michael A. Crone, James M. MacDonald, Paul S. Freemont, M. Kopniczky, Lorna Suckling, Kirsten Jensen |
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| Rok vydání: | 2020 |
| Předmět: |
0106 biological sciences
Transcription Genetic 0601 Biochemistry and Cell Biology 01 natural sciences chemistry.chemical_compound Synthetic biology Tissue culture RNA-POLYMERASE DESIGN 0903 Biomedical Engineering RNA polymerase CRISPR-Associated Protein 9 BINDING Protein biosynthesis CRISPR cell-free Gene Regulatory Networks Promoter Regions Genetic Cell Engineering high-throughput 0303 health sciences medicine.diagnostic_test Chemistry prototyping General Medicine Synthetic Biology Genetic Engineering Life Sciences & Biomedicine Plasmids Biochemistry & Molecular Biology Biomedical Engineering Computational biology Internal Ribosome Entry Sites Biochemistry Genetics and Molecular Biology (miscellaneous) Biochemical Research Methods VALIDATION Flow cytometry 03 medical and health sciences 010608 biotechnology medicine Escherichia coli Humans OPTIMIZATION PUF automation 030304 developmental biology Cell-free protein synthesis Science & Technology Binding Sites 0304 Medicinal and Biomolecular Chemistry Cell-Free System RECOGNITION Reproducibility of Results Internal ribosome entry site Protein Biosynthesis CYTOPLASMIC EXPRESSION CRISPR-Cas Systems HeLa Cells |
| Zdroj: | ACS synthetic biology. 9(1) |
| ISSN: | 2161-5063 |
| Popis: | The field of mammalian synthetic biology is expanding quickly, and technologies for engineering large synthetic gene circuits are increasingly accessible. However, for mammalian cell engineering, traditional tissue culture methods are slow and cumbersome, and are not suited for high-throughput characterization measurements. Here we have utilized mammalian cell-free protein synthesis (CFPS) assays using HeLa cell extracts and liquid handling automation as an alternative to tissue culture and flow cytometry-based measurements. Our CFPS assays take a few hours, and we have established optimized protocols for small-volume reactions using automated acoustic liquid handling technology. As a proof-of-concept, we characterized diverse types of genetic regulation in CFPS, including T7 constitutive promoter variants, internal ribosomal entry sites (IRES) constitutive translation-initiation sequence variants, CRISPR/dCas9-mediated transcription repression, and L7Ae-mediated translation repression. Our data shows simple regulatory elements for use in mammalian cells can be quickly prototyped in a CFPS model system. |
| Databáze: | OpenAIRE |
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