Linear DNA for Rapid Prototyping of Synthetic Biological Circuits in an Escherichia coli Based TX-TL Cell-Free System

Abstract

Accelerating the pace of synthetic biology experiments requires new approaches for rapid prototyping of circuits from individual DNA regulatory elements. However, current testing standards require days to weeks due to cloning and in vivo transformation. In this work, we first characterized methods to protect linear DNA strands from exonuclease degradation in an Escherichia coli based transcription-translation cell-free system (TX-TL), as well as mechanisms of degradation. This enabled the use of linear DNA PCR products in TX-TL. We then compared expression levels and binding dynamics of different promoters on linear DNA and plasmid DNA. We also demonstrated assembly technology to rapidly build circuits entirely in vitro from separate parts. Using this strategy, we prototyped a four component genetic switch in under 8 h entirely in vitro. Rapid in vitro assembly has future applications for prototyping multiple component circuits if combined with predictive computational models.

Streptomyces venezuelae TX‐TL – a next generation cell‐free synthetic biology tool

“…2A and 2B). For comparison, in E. coli Rosetta TX-TL extracts, saturation requires 10-15 nM [17]. In addition, for S. venezuelae TX-TL, the fluorescence signal for sfGFP was observed to decay after approximately 4 h of incubation.…”

“…TX-TL can provide a tool to rapidly prototype the cellular machinery of synthetic biology hosts [17]. Herein, we provide evidence for the development of a high-activity S. venezuelae TX-TL system utilizing the kasOp* promoter as a standard for cell-extract optimization [18].…”

Synthetic biology devices for in vitro and in vivo diagnostics

“…In this approach, commercially available cell-free transcription/translation systems (bacterial or mammalian) are freeze-dried onto paper or other porous substrates to create poised genetic regulatory networks that are stable for long-term storage at room temperature and are activated by rehydration. This work follows on other efforts in cell-free synthetic biology, which have provided important dynamic and mechanistic insight on gene regulatory networks and allowed for rapid "build-test" cycles for prototyping engineered gene circuits and biomanufacturing pathways (36)(37)(38)(39)(40)(41). The in vitro nature of these systems resolves the challenge faced by cell-based approaches of importing biomolecular components into the intracellular space, making these cell-free environments easily modified and excellent platforms for engineering.…”

Modeling the effects of compositional context on promoter activity in an E. coli extract based transcription-translation system

“…Thus, to isolate the effects of compositional context from these sources of variability, we chose an in vitro expression assay based on a BL21 Rosetta 2 E. coli S30 extract transcriptiontranslation (TX-TL) system developed in [19]. The TX-TL system was also demonstrated as a biomolecular breadboard environment for rapid high-throughput prototyping of novel biocircuits [4]. Hence, any additional insight we gain about compositional context also informs prototyping efforts in the TX-TL system.…”

“…These applications often involve the implementation of synthetic networks of genes, also known as biocircuits. Recently, advancements in DNA synthesis techniques [2], [3] coupled with the simultaneous drop in sequencing and prototyping costs [4], [5] have enabled rapid development of new biocircuits. In principle, design iterations for a complex biocircuit can be performed in a matter of hours [4], leading to circuit synthesis in a matter of weeks.…”