ArticleMicrobial cell factories2024
Thermostable in vitro transcription-translation compatible with microfluidic droplets.
Article in Microbial cell factories, 2024. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 4 papers.
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Who cites it
4 citing papers in PubMed.
- Microbial cell-free protein synthesis and its progression toward industrial use.Microbiology (Reading, England) · 2026Review
- Cell-Free-Based Thermophilic Biocatalyst for the Synthesis of Amino Acids from One-Carbon Feedstocks.ACS synthetic biology · 2025Article
- Extreme thermal environments: reservoirs of industrially important thermozymes.Frontiers in microbiology · 2025Review
- Cell-free protein synthesis with technical additives - expanding the parameter space of in vitro gene expression.Beilstein journal of organic chemistry · 2024Article
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Authors and funding
13 authors.
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Abstract
backgroundIn vitro expression involves the utilization of the cellular transcription and translation machinery in an acellular context to produce one or more proteins of interest and has found widespread application in synthetic biology and in pharmaceutical biomanufacturing. Most in vitro expression systems available are active at moderate temperatures, but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures.
objectivesDevelop an in vitro expression system operating at high temperatures compatible with enzymatic assays and with technologies that enable ultrahigh-throughput protein expression in reduced volumes, such as microfluidic water-in-oil (w/o) droplets.
resultsWe produced cell-free extracts from Thermus thermophilus for in vitro translation including thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable or superior to other thermostable in vitro expression systems, while the preparation procedure is much simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time.
conclusionsCell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput.
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