Abstract
Cell‐free expression systems enable rapid prototyping of genetic programs in vitro. However, current throughput of cell‐free measurements is limited by the use of channel‐limited fluorescent readouts. Here, we describe DNA Regulatory element Analysis by cell‐Free Transcription and Sequencing (DRAFTS), a rapid and robust in vitro approach for multiplexed measurement of transcriptional activities from thousands of regulatory sequences in a single reaction. We employ this method in active cell lysates developed from ten diverse bacterial species. Interspecies analysis of transcriptional profiles from > 1,000 diverse regulatory sequences reveals functional differences in promoter activity that can be quantitatively modeled, providing a rich resource for tuning gene expression in diverse bacterial species. Finally, we examine the transcriptional capacities of dual‐species hybrid lysates that can simultaneously harness gene expression properties of multiple organisms. We expect that this cell‐free multiplex transcriptional measurement approach will improve genetic part prototyping in new bacterial chassis for synthetic biology.
Highlights
The cell envelope is a key physical barrier that compartmentalizes cellular functions and insulates biological systems from the external environment
S. enterica yielded only small amounts of protein (< 1 ng/ll). These results demonstrate that our cell lysate preparation methods are able to generate active cell-free expression reactions from diverse bacteria with relative ease, provide a foundation for further optimizations of transcription and translation efficiency in cell-free expression systems, and demonstrate their potential for rapid characterization of genetic designs in diverse bacterial species
We developed and characterized cell-free lysates for 10 diverse bacterial species from three phyla (Proteobacteria, Firmicutes, and Actinobacteria), many of which have never been utilized in cell-free expression reactions
Summary
The cell envelope is a key physical barrier that compartmentalizes cellular functions and insulates biological systems from the external environment. Cell-free systems, made up of either individually reconstituted cellular components (Shimizu et al, 2001; Wang et al, 2012; Villarreal et al, 2018) or cell lysates (Jewett et al, 2013; Garamella et al, 2016), can support a variety of catalytic reactions in vitro when supplied with energy sources, cofactors, and ions (Calhoun & Swartz, 2005, 2007; Jewett et al, 2008) These cell-free approaches have facilitated the development of therapeutically useful natural products (Dudley et al, 2015; Maini et al, 2016) and biologics with non-standard amino acids (Martin et al, 2018) or chemical moieties otherwise challenging to synthesize (Jaroentomeechai et al, 2018). The correspondence between in vitro measurements and actual in vivo conditions in live cells remains understudied (Jewett et al, 2008; Chappell et al, 2013; Siegal-Gaskins et al, 2014)
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