Polydopamine (PDA) is a simple and versatile conformal coating material that has been proposed for a variety of uses; however in practice its performance is often hindered by poor mechanical properties and high roughness. Here, we show that blue-diode laser annealing dramatically improves mechanical performance and reduces roughness of PDA coatings. Laser-annealed PDA (LAPDA) was shown to be >100-fold more scratch resistant than pristine PDA and even better than hard inorganic substrates, which we attribute to partial graphitization and covalent coupling between PDA subunits during annealing. Moreover, laser annealing provides these benefits while preserving other attractive properties of PDA, as demonstrated by the superior biofouling resistance of antifouling polymer-grafted LAPDA compared to PDA modified with the same polymer. Our work suggests that laser annealing may allow the use of PDA in mechanically demanding applications previously considered inaccessible, without sacrificing the functional versatility that is so characteristic of PDA.
Synthetic genetic circuits incorporating regulatory components based on RNA interference (RNAi) have been used in a variety of systems. A comprehensive understanding of the parameters that determine the relationship between microRNA (miRNA) and target expression levels is lacking. We describe a quantitative framework supporting the forward engineering of gene circuits that incorporate RNAi-based regulatory components in mammalian cells. We developed a model that captures the quantitative relationship between miRNA and target gene expression levels as a function of parameters, including mRNA half-life and miRNA target-site number. We extended the model to synthetic circuits that incorporate protein-responsive miRNA switches and designed an optimized miRNA-based protein concentration detector circuit that noninvasively measures small changes in the nuclear concentration of β-catenin owing to induction of the Wnt signaling pathway. Our results highlight the importance of methods for guiding the quantitative design of genetic circuits to achieve robust, reliable and predictable behaviors in mammalian cells.
BackgroundHomeostasis within mammalian cells is achieved through complex molecular networks that can respond to changes within the cell or the environment and regulate the expression of the appropriate genes in response. The development of biological components that can respond to changes in the cellular environment and interface with endogenous molecules would enable more sophisticated genetic circuits and greatly advance our cellular engineering capabilities.ResultsHere we describe a platform that combines a ligand-responsive ribozyme switch and synthetic miRNA regulators to create an OFF genetic control device based on RNA interference (RNAi). We developed a mathematical model to highlight important design parameters in programming the quantitative performance of RNAi-based OFF control devices. By modifying the ribozyme switch integrated into the system, we demonstrated RNAi-based OFF control devices that respond to small molecule and protein ligands, including the oncogenic protein E2F1. We utilized the OFF control device platform to build a negative feedback control system that acts as a proportional controller and maintains target intracellular protein levels in response to increases in transcription rate.ConclusionsOur work describes a novel genetic device that increases the level of silencing from a miRNA in the presence of a ligand of interest, effectively creating an RNAi-based OFF control device. The OFF switch platform has the flexibility to be used to respond to both small molecule and protein ligands. Finally, the RNAi-based OFF switch can be used to implement a negative feedback control system, which maintains target protein levels around a set point level. The described RNAi-based OFF control device presents a powerful tool that will enable researchers to engineer homeostasis in mammalian cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13036-015-0002-3) contains supplementary material, which is available to authorized users.
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