Design and Evaluation of Synthetic RNA-Based Incoherent Feed-Forward Loop Circuits.

RNA-based regulators are promising tools for building synthetic biological systems that provide a powerful platform for achieving a complex regulation of transcription and translation. Recently, de novo-designed synthetic RNA regulators, such as the small transcriptional activating RNA (STAR), toehold switch (THS), and three-way junction (3WJ) repressor, have been utilized to construct RNA-based synthetic gene circuits in living cells. In this work, we utilized these regulators to construct type 1 incoherent feed-forward loop (IFFL) circuits in vivo and explored their dynamic behaviors. A combination of a STAR and 3WJ repressor was used to construct an RNA-only IFFL circuit. However, due to the fast kinetics of RNA-RNA interactions, there was no significant timescale difference between the direct activation and the indirect inhibition, that no pulse was observed in the experiments. These findings were confirmed with mechanistic modeling and simulation results for a wider range of conditions. To increase delay in the inhibition pathway, we introduced a protein synthesis process to the circuit and designed an RNA-protein hybrid IFFL circuit using THS and TetR protein. Simulation results indicated that pulse generation could be achieved with this RNA-protein hybrid model, and this was further verified with experimental realization in E. coli. Our findings demonstrate that while RNA-based regulators excel in speed as compared to protein-based regulators, the fast reaction kinetics of RNA-based regulators could also undermine the functionality of a circuit (e.g., lack of significant timescale difference). The agreement between experiments and simulations suggests that the mechanistic modeling can help debug issues and validate the hypothesis in designing a new circuit. Moreover, the applicability of the kinetic parameters extracted from the RNA-only circuit to the RNA-protein hybrid circuit also indicates the modularity of RNA-based regulators when used in a different context. We anticipate the findings of this work to guide the future design of gene circuits that rely heavily on the dynamics of RNA-based regulators, in terms of both modeling and experimental realization.

Mercury proxy for hydrothermal and submarine volcanic activities in the sediment cores of Central Indian Ridge.

Hydrothermal vent is the one of the main natural Hg sources to the deep ocean. Thus, we investigated which Hg speciation in the sediment core can be the past records for geothermal activities in mid-ocean ridges of the Central Indian Ocean. The result showed that the hydrothermal Hg in the core sediments was mainly associated with Fe-Mn oxides with the elevated concentrations of other hydrothermal-derived trace metals [Co + Zn + Cu]. In addition, the [Sm]/[Nd] and [Rb]/[Sr] ratios and ɛNdCHUR and 87Sr/86Sr isotopic values supported that the extremely high Hg concentrations were possibly originated from the hydrothermal vent. However, the Hg emitted from submarine volcano was mainly associated with sulfides-organic matters because the volcanos did not release Fe and Mn. Thus, our results showed that the sedimentary Hg is an independent toll for reconstruction of paleodynamics of hydrothermal and/or volcanic activities in deep sea basin of the Central Indian Ocean.

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.