Cell-Free Synthetic Biology Platform for Engineering Synthetic Biological Circuits and Systems

Synthetic biology brings engineering disciplines to create novel biological systems for biomedical and technological applications. The substantial growth of the synthetic biology field in the past decade is poised to transform biotechnology and medicine. To streamline design processes and facilitate debugging of complex synthetic circuits, cell-free synthetic biology approaches has reached broad research communities both in academia and industry. By recapitulating gene expression systems in vitro, cell-free expression systems offer flexibility to explore beyond the confines of living cells and allow networking of synthetic and natural systems. Here, we review the capabilities of the current cell-free platforms, focusing on nucleic acid-based molecular programs and circuit construction. We survey the recent developments including cell-free transcription– translation platforms, DNA nanostructures and circuits, and novel classes of riboregulators. The links to mathematical models and the prospects of cell-free synthetic biology platforms will also be discussed.11Yscopu

Engineering Encapsulated Synthetic Cytoskeletal Dynamics via Nucleic Acid Nanotechnology and Genetic Circuits

Programmable, synthetic cells have applications in sensing and drug-delivery. Currently, development of synthetic cell components focuses on compartmentalization, and on developing the minimal machinery to carry out different cellular processes. In native cells, cytoskeletal filaments are a key structure for cell division, motility, and intra-cellular transport. Harnessing these filaments for use in synthetic systems is limited by the complexity of the dynamic behavior of the filaments. Alternatively, synthetic tile-based DNA nanotubes are comparable in length and stiffness to cytoskeletal filaments and can be engineered to demonstrate dynamic behavior using few components. To use DNA nanotubes as cytoskeletons in synthetic systems requires resilience to degrading enzymes found within cells, and the dynamic behavior must be automated and characterized in compartments.Minimal cell systems execute tasks using transcription–translation (TXTL) machinery adapted from native cells. As other DNA nanotechnology degrades rapidly in vivo, I assayed the robustness of DNA nanotubes in an Escherichia coli cell-free TXTL system. TXTL recapitulates physiological conditions as well as strong linear DNA degradation through the RecBCD complex. I demonstrated that chemical modifications of the tiles composing DNA nanotubes and the addition of a Chi-site dsDNA, an inhibitor of the RecBCD complex, extend nanotube viability in TXTL for more than 24 hours. These complementary approaches are a first step towards engineering resilient DNA nanotubes for application in active environments.To demonstrate autonomous control of assembly and disassembly processes of nanotubes in cell-sized environments, I implemented a DNA-RNA hybrid nanotube design inside of water-in-oil droplets. In this design, DNA tiles are activated by the presence of a trigger RNA molecule, which can be produced by and degraded by distinct enzymes. A pulse of nanotube assembly-disassembly occurs when both transcribing and degrading components are present with inactive tiles in droplets. Notably, the encapsulated system requires lower concentration of gene to trigger assembly than bulk solution. Varying the concentration of gene and degrading enzymes affects both the kinetics of assembly-disassembly and the morphology of the nanotubes. These methods can be employed to develop more complex dynamics and functionalities of DNA nanotubes as synthetic cytoskeletal filaments in minimal cells

Dynamic self-assembly of compartmentalized DNA nanotubes

Bottom-up synthetic biology aims to engineer artificial cells capable of responsive behaviors by using a minimal set of molecular components. An important challenge toward this goal is the development of programmable biomaterials that can provide active spatial organization in cell-sized compartments. Here, we demonstrate the dynamic self-assembly of nucleic acid (NA) nanotubes inside water-in-oil droplets. We develop methods to encapsulate and assemble different types of DNA nanotubes from programmable DNA monomers, and demonstrate temporal control of assembly via designed pathways of RNA production and degradation. We examine the dynamic response of encapsulated nanotube assembly and disassembly with the support of statistical analysis of droplet images. Our study provides a toolkit of methods and components to build increasingly complex and functional NA materials to mimic life-like functions in synthetic cells

Dynamic self-assembly of compartmentalized DNA nanotubes

A major goal in Engineering Biology and Materials Science is the development of active, autonomous scaffolds that mimic those present in biological cells. Here the authors report a toolkit for programming the dynamic behaviour of nucleic acid scaffolds in minimal cell-like compartments

Circulating Neoplastic-Immune Hybrid Cells Predict Metastatic Progression in Uveal Melanoma

Background: Uveal melanoma is an aggressive cancer with high metastatic risk. Recently, we identified a circulating cancer cell population that co-expresses neoplastic and leukocyte antigens, termed circulating hybrid cells (CHCs). In other cancers, CHCs are more numerous and better predict oncologic outcomes compared to circulating tumor cells (CTCs). We sought to investigate the potential of CHCs as a prognostic biomarker in uveal melanoma. Methods: We isolated peripheral blood monocular cells from uveal melanoma patients at the time of primary treatment and used antibodies against leukocyte and melanoma markers to identify and enumerate CHCs and CTCs by immunocytochemistry. Results: Using a multi-marker approach to capture the heterogeneous disseminated tumor cell population, detection of CHCs was highly sensitive in uveal melanoma patients regardless of disease stage. CHCs were detected in 100% of stage I-III uveal melanoma patients (entire cohort, n = 68), whereas CTCs were detected in 58.8% of patients. CHCs were detected at levels statically higher than CTCs across all stages (p = 0.05). Moreover, CHC levels, but not CTCs, predicted 3 year progression-free survival (p p < 0.04). Conclusion: CHCs are a novel and promising prognostic biomarker in uveal melanoma