Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts.

Cell-free gene expression systems have emerged as a promising platform for field-deployed biosensing and diagnostics. When combined with programmable toehold switch-based RNA sensors, these systems can be used to detect arbitrary RNAs and freeze-dried for room temperature transport to the point-of-need. These sensors, however, have been mainly implemented using reconstituted PURE cell-free protein expression systems that are difficult to source in the Global South due to their high commercial cost and cold-chain shipping requirements. Based on preliminary demonstrations of toehold sensors working on lysates, we describe the fast prototyping of RNA toehold switch-based sensors that can be produced locally and reduce the cost of sensors by two orders of magnitude. We demonstrate that these in-house cell lysates provide sensor performance comparable to commercial PURE cell-free systems. We further optimize these lysates with a CRISPRi strategy to enhance the stability of linear DNAs by knocking-down genes responsible for linear DNA degradation. This enables the direct use of PCR products for fast screening of new designs. As a proof-of-concept, we develop novel toehold sensors for the plant pathogen Potato Virus Y (PVY), which dramatically reduces the yield of this important staple crop. The local implementation of low-cost cell-free toehold sensors could enable biosensing capacity at the regional level and lead to more decentralized models for global surveillance of infectious disease.

Artificial Symmetry-Breaking for Morphogenetic Engineering Bacterial Colonies.

Morphogenetic engineering is an emerging field that explores the design and implementation of self-organized patterns, morphologies, and architectures in systems composed of multiple agents such as cells and swarm robots. Synthetic biology, on the other hand, aims to develop tools and formalisms that increase reproducibility, tractability, and efficiency in the engineering of biological systems. We seek to apply synthetic biology approaches to the engineering of morphologies in multicellular systems. Here, we describe the engineering of two mechanisms, symmetry-breaking and domain-specific cell regulation, as elementary functions for the prototyping of morphogenetic instructions in bacterial colonies. The former represents an artificial patterning mechanism based on plasmid segregation while the latter plays the role of artificial cell differentiation by spatial colocalization of ubiquitous and segregated components. This separation of patterning from actuation facilitates the design-build-test-improve engineering cycle. We created computational modules for CellModeller representing these basic functions and used it to guide the design process and explore the design space in silico. We applied these tools to encode spatially structured functions such as metabolic complementation, RNAPT7 gene expression, and CRISPRi/Cas9 regulation. Finally, as a proof of concept, we used CRISPRi/Cas technology to regulate cell growth by controlling methionine synthesis. These mechanisms start from single cells enabling the study of morphogenetic principles and the engineering of novel population scale structures from the bottom up.

Synthetic Biology: opportunities for Chilean bioindustry and education.

In an age of pressing challenges for sustainable production of energy and food, the new field of Synthetic Biology has emerged as a promising approach to engineer biological systems. Synthetic Biology is formulating the design principles to engineer affordable, scalable, predictable and robust functions in biological systems. In addition to efficient transfer of evolved traits from one organism to another, Synthetic Biology offers a new and radical approach to bottom-up engineering of sensors, actuators, dynamical controllers and the biological chassis they are embedded in. Because it abstracts much of the mechanistic details underlying biological component behavior, Synthetic Biology methods and resources can be readily used by interdisciplinary teams to tackle complex problems. In addition, the advent of robust new methods for the assembly of large genetic circuits enables teaching Biology and Bioengineering in a learning-by-making fashion for diverse backgrounds at the graduate, undergraduate and high school levels. Synthetic Biology offers unique opportunities to empower interdisciplinary training, research and industrial development in Chile for a technology that promises a significant role in this century's economy.

Synthetic Biology: opportunities for Chilean bioindustry and education

In an age of pressing challenges for sustainable production of energy and food, the new field of Synthetic Biology has emerged as a promising approach to engineer biological systems. Synthetic Biology is formulating the design principles to engineer affordable, scalable, predictable and robust functions in biological systems. In addition to efficient transfer of evolved traits from one organism to another, Synthetic Biology offers a new and radical approach to bottom-up engineering of sensors, actuators, dynamical controllers and the biological chassis they are embedded in. Because it abstracts much of the mechanistic details underlying biological component behavior, Synthetic Biology methods and resources can be readily used by interdisciplinary teams to tackle complex problems. In addition, the advent of robust new methods for the assembly of large genetic circuits enables teaching Biology and Bioengineering in a learning-by-making fashion for diverse backgrounds at the graduate, undergraduate and high school levels. Synthetic Biology offers unique opportunities to empower interdisciplinary training, research and industrial development in Chile for a technology that promises a significant role in this century's economy.

A molecular framework for the inhibition of Arabidopsis root growth in response to boron toxicity.

Boron is an essential micronutrient for plants and is taken up in the form of boric acid (BA). Despite this, a high BA concentration is toxic for the plants, inhibiting root growth and is thus a significant problem in semi-arid areas in the world. In this work, we report the molecular basis for the inhibition of root growth caused by boron. We show that application of BA reduces the size of root meristems, correlating with the inhibition of root growth. The decrease in meristem size is caused by a reduction of cell division. Mitotic cell number significantly decreases and the expression level of key core cell cycle regulators is modulated. The modulation of the cell cycle does not appear to act through cytokinin and auxin signalling. A global expression analysis reveals that boron toxicity induces the expression of genes related with abscisic acid (ABA) signalling, ABA response and cell wall modifications, and represses genes that code for water transporters. These results suggest that boron toxicity produces a reduction of water and BA uptake, triggering a hydric stress response that produces root growth inhibition.