Multiplex transcriptional characterizations across diverse bacterial species using cell‐free systems

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.

Bottom-Up Construction of Complex Biomolecular Systems With Cell-Free Synthetic Biology

“…In recent years the number of cell-free transcription-translation (TX-TL) systems from different organisms has grown rapidly (Zemella et al, 2015;Perez et al, 2016;Gregorio et al, 2019). The most common lysate systems include E. coli, insect, yeast, Chinese hamster ovary, rabbit reticulocyte, wheat germ, and human HeLa cells; and newly emerging systems include B. subtilis (Kelwick et al, 2016;Yim et al, 2019), V. natriegens (Failmezger, 2018;Yim et al, 2019), and P. putida (Wang et al, 2018;Yim et al, 2019), among others (Yim et al, 2019). Hybrid systems composed from multiple sources have also recently emerged (Anastasina et al, 2014;Panthu et al, 2018;Yim et al, 2019).…”

Biological Materials: The Next Frontier for Cell-Free Synthetic Biology

“…Foundational discoveries in functional genomics, cellular metabolism and natural product synthesis are also important, because they might inspire novel biosynthetic pathway designs for biological materials production. In synthetic biology, cell-free metabolic engineering (CF-ME) approaches can reconstitute entire biosynthetic pathways using either cell extracts from diverse species, engineered cells and/or cellfree synthesized recombinant enzymes (Karim and Jewett, 2018;Martin et al, 2018;Yim et al, 2019;Bowie et al, 2020) (Figure 1). Also, cell-free protein synthesis and cell extract biotransformation reactions can be combined to create more complex cell-free reactions (Karim and Jewett, 2018;Kelwick et al, 2018).…”

“…Indeed, several studies have successfully utilized acoustic liquid handling robots to rapidly setup large-scale, low-volume (≤10 µl) prototyping cell-free reactions in a 384 well plate format (Moore et al, 2018;Kopniczky et al, 2020). Microfluidic (Swank et al, 2019), droplet array or multiplex (Yim et al, 2019) strategies have also been used to enable high-throughput cell-free experiments. These approaches enable the testing of large numbers of regulatory elements or enzymes, which could potentially inform material biosynthetic pathway optimization.…”

The SAGE genetic toolkit enables highly efficient, iterative site-specific genome engineering in bacteria

“…Accordingly, we developed a method to rapidly characterize large libraries of genomeintegrated promoters that combines the efficiency of SAGE integration with a modified version of the high-throughput sequencing (HTS) transcription profiling method described by Yim, et al 57 . We apply this method, described briefly below, to characterize a collection of 287 synthetic and natural promoters (Supplementary File T1) in P. fluorescens (Fig.…”