Thrombin-Mediated Transcriptional Regulation Using DNA Aptamers in DNA-Based Cell-Free Protein Synthesis

Abstract

Realizing the potential of cell free systems will require development of ligand sensitive gene promoters that control gene expression in response to a ligand of interest. Here, we describe an approach to designing ligand sensitive transcriptional control in cell free systems that is based on the combination of a DNA aptamer that binds thrombin and the T7 bacteriophage promoter. Placement of the aptamer near the T7 promoter, and using a primarily single stranded template, results in up to a five-fold change in gene expression in a ligand concentration dependent manner. We further demonstrate that the sensitivity to thrombin concentration and the fold change in expression can be tuned by altering the position of the aptamer. The results described here pave the way for the use of DNA aptamers to achieve modular regulation of transcription in response to a wide variety of ligands in cell free systems.

A cost-effective polyphosphate-based metabolism fuels an all E. coli cell-free expression system

“…Linear or circular DNA programs are now executed in cell-free transcription-translation (TX-TL) mixtures for biomedical applications (He and Taussig, 2001), nanotechnology (Daube and Bar-Ziv, 2013), synthetic biology (Chappell et al, 2013;Iyer and Doktycz, 2014;Lentini et al, 2013;Niederholtmeyer et al, 2013;Shin and Noireaux, 2012;Sokolova et al, 2013;Sun et al, 2013) and metabolic engineering (Bujara et al, 2010;Ye et al, 2012). A direct consequence is the high demand for novel, efficient and cost-effective systems.…”

Effective Use of Linear DNA in Cell-Free Expression Systems

“…In a few cases, these obstacles have been overcome or circumvented sufficiently to allow DNA aptamers to be successfully used in CFEs. For example, DNA aptamers integrated into plasmids have successfully allowed transcriptional regulation in CFEs ( Iyer and Doktycz 2014 ; Wang et al, 2017 ). This confirms that the environment of CFEs (in these examples, a commercially available extract) can accommodate proper aptamer secondary structure formation to allow for both target analyte binding and subsequent conformational changes.…”

Protocell Arrays for Simultaneous Detection of Diverse Analytes

“…The embedding of CFE reactions in ATPS-formed protocells is uniquely poised to meet multiple key challenges in biosensing applications, as this platform can be customized to detect diverse sets of analytes merely by reprogramming the DNA sequence of plasmids used as the basis for their sensing. Previous work has demonstrated that CFE can be used to detect metabolites via incorporation of genetically encoded metabolic transducers 11,44 , proteins via genetically encoded aptamers 45,46 , and nucleic acids via genetically encoded synthetic regulators 7,8,31 . These different sensing systems may require differently optimized CFE reactions (e.g., lysate preparations or macromolecular reaction additives), but these individually optimized sub-systems can all be used together in parallel to measure different targets in the same sample; of note, the protocells sensing nucleic acids in Figure 5 use different lysate preparations than the other protocells in Figure 5 .…”