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 hours. 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.
Article and author information
Author details
Reviewing Editor
- Friedrich Simmel, Technische Universität München, Germany
Version history
- Received: June 29, 2015
- Accepted: October 1, 2015
- Accepted Manuscript published: October 2, 2015 (version 1)
- Accepted Manuscript updated: October 5, 2015 (version 2)
- Version of Record published: December 9, 2015 (version 3)
Copyright
© 2015, Niederholtmeyer et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 7,354
- views
-
- 1,667
- downloads
-
- 203
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Biochemistry and Chemical Biology
Prions replicate via the autocatalytic conversion of cellular prion protein (PrPC) into fibrillar assemblies of misfolded PrP. While this process has been extensively studied in vivo and in vitro, non-physiological reaction conditions of fibril formation in vitro have precluded the identification and mechanistic analysis of cellular proteins, which may alter PrP self-assembly and prion replication. Here, we have developed a fibril formation assay for recombinant murine and human PrP (23-231) under near-native conditions (NAA) to study the effect of cellular proteins, which may be risk factors or potential therapeutic targets in prion disease. Genetic screening suggests that variants that increase syntaxin-6 expression in the brain (gene: STX6) are risk factors for sporadic Creutzfeldt-Jakob disease (CJD). Analysis of the protein in NAA revealed, counterintuitively, that syntaxin-6 is a potent inhibitor of PrP fibril formation. It significantly delayed the lag phase of fibril formation at highly sub-stoichiometric molar ratios. However, when assessing toxicity of different aggregation time points to primary neurons, syntaxin-6 prolonged the presence of neurotoxic PrP species. Electron microscopy and super-resolution fluorescence microscopy revealed that, instead of highly ordered fibrils, in the presence of syntaxin-6 PrP formed less-ordered aggregates containing syntaxin-6. These data strongly suggest that the protein can directly alter the initial phase of PrP self-assembly and, uniquely, can act as an 'anti-chaperone', which promotes toxic aggregation intermediates by inhibiting fibril formation.
-
- Biochemistry and Chemical Biology
- Cell Biology
Numerous lipids are heterogeneously distributed among organelles. Most lipid trafficking between organelles is achieved by a group of lipid transfer proteins (LTPs) that carry lipids using their hydrophobic cavities. The human genome encodes many intracellular LTPs responsible for lipid trafficking and the function of many LTPs in defining cellular lipid levels and distributions is unclear. Here, we created a gene knockout library targeting 90 intracellular LTPs and performed whole-cell lipidomics analysis. This analysis confirmed known lipid disturbances and identified new ones caused by the loss of LTPs. Among these, we found major sphingolipid imbalances in ORP9 and ORP11 knockout cells, two proteins of previously unknown function in sphingolipid metabolism. ORP9 and ORP11 form a heterodimer to localize at the ER-trans-Golgi membrane contact sites, where the dimer exchanges phosphatidylserine (PS) for phosphatidylinositol-4-phosphate (PI(4)P) between the two organelles. Consequently, loss of either protein causes phospholipid imbalances in the Golgi apparatus that result in lowered sphingomyelin synthesis at this organelle. Overall, our LTP knockout library toolbox identifies various proteins in control of cellular lipid levels, including the ORP9-ORP11 heterodimer, which exchanges PS and PI(4)P at the ER-Golgi membrane contact site as a critical step in sphingomyelin synthesis in the Golgi apparatus.