Rapid cell-free forward engineering of novel genetic ring oscillators

Abstract

While complex dynamic biological networks control gene expression in all living organisms, the forward engineering of comparable synthetic networks remains challenging. The current paradigm of characterizing synthetic networks in cells results in lengthy design-build-test cycles, minimal data collection, and poor quantitative characterization. Cell-free systems are appealing alternative environments, but it remains questionable whether biological networks behave similarly in cell-free systems and in cells. We characterized in a cell-free system the ‘repressilator’, a three-node synthetic oscillator. We then engineered novel three, four, and five-gene ring architectures, from characterization of circuit components to rapid analysis of complete networks. When implemented in cells, our novel 3-node networks produced population-wide oscillations and 95% of 5-node oscillator cells oscillated for up to 72 hr. Oscillation periods in cells matched the cell-free system results for all networks tested. An alternate forward engineering paradigm using cell-free systems can thus accurately capture cellular behavior.DOI: http://dx.doi.org/10.7554/eLife.09771.001

In Vitro Reconstruction of Nonribosomal Peptide Biosynthesis Directly from DNA Using Cell-Free Protein Synthesis

“…Indeed, our discovery-centered cell-free approach sets the stage for high-throughput experimentation in a cell-free environment, where design–build–test iterations can be performed without the need to reengineer organisms, DNA for pathway enzymes is directly input with plasmid refactoring, and substrates and cofactors needed for secondary metabolism can be controlled and maintained at defined concentrations. 37 …”

Control theory meets synthetic biology

“…Synthetic negative feedback loops, together with many other synthetic biology parts, such as the toggle switch and the repressilator, have been successfully reconstructed in cell-free systems [92,93]. Because experimental results have suggested that circuit performance in a cell-free system highly resembles its in-cell counterpart [91], cell-free systems can potentially serve as a rapid controller prototyping platform, similar to a wind tunnel for fluid dynamics, to investigate more sophisticated in vivo control strategies.…”

“…Finally, cell-free systems, where circuits are studied in cell extracts in vitro, provide appealing testing platforms to accelerate synthetic circuits prototyping, and to deepen our understanding of natural systems [90,91] by removing issues such as cellular context dependence, noise and cell heterogeneity, and cell growth. Synthetic negative feedback loops, together with many other synthetic biology parts, such as the toggle switch and the repressilator, have been successfully reconstructed in cell-free systems [92,93].…”

Using synthetic biology to explore principles of development

“…More recent work has addressed some of these limitations by improving the system in various ways (e.g. Stricker et al, 2008;Hussain et al, 2014;Niederholtmeyer et al, 2015). Some of these have been particularly useful in highlighting features that can make great differences to the precision of systems, some of which might be counter-intuitive.…”