Multiplex transcriptional characterizations across diverse and hybrid bacterial cell-free expression systems

Abstract

Cell-free expression systems enable rapid prototyping of genetic programs in vitro.However, current throughput of cell-free measurements is often limited by the use of single-channel reporter assays. 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 employed this method in active cell lysates developed from ten diverse bacterial species. Interspecies analysis of transcriptional profiles from >1,000 diverse regulatory sequences revealed functional differences in gene expression that could be predictively modeled. Finally, we constructed and examined the transcriptional capacities of dual-species "hybrid" cell lysates that can simultaneously harness gene expression properties of multiple organisms. We expect that this cell-free multiplex transcriptional measurement approach will improve genetic circuit prototyping in new bacterial chassis for synthetic biology.

Deconstructing Cell-Free Extract Preparation for in Vitro Activation of Transcriptional Genetic Circuitry

“…Because the open environment enables flexibility for optimizing extract and reaction conditions and is amenable to high-throughput automation, cell-free gene expression (CFE) technology has found great utility in a wide range of contexts. Since their first application in deciphering the genetic code, , cell-free systems have been successfully applied for the bulk production of model − and therapeutic proteins. − Beyond protein synthesis, CFE technologies have evolved more generally to enable complex and diverse functions, including prototyping cellular metabolism − and glycosylation, − expressing minimal synthetic cells, virus-like particles, and bacteriophages, ,− portable on-demand manufacturing of pharmaceuticals, , incorporation of noncanonical amino acids within proteins, − prototyping of genetic circuitry, − and sensing nucleic acids and small molecules through rapid, low-cost, and field-deployable molecular diagnostics. − Most progress has occurred in CFE systems generated from Escherichia coli strains engineered for protein production, largely due to the bacterium’s well-characterized genetics and metabolism . However, there has been recent progress in adapting CFE protocols to make lysates from eukaryotic and nonmodel organisms, including yeast, , Gram-positive bacteria, , plants, , and mammalian cells. − CFE technology is therefore at the point of expanding beyond specialist laboratories and becoming a major toolbox throughout synthetic biology research, application, and education. ,, …”