Biocomputing in plants, from proof of concept to application.

In response to the challenges of climate change and the transition toward sustainability, synthetic biology offers innovative solutions. Most current plant synthetic biology applications rely on the constitutive expression of enzymes and regulators. To engineer plant phenotypes tuneable to environmental conditions and plant cellular states, the integration of multiple signals in synthetic circuits is required. While most circuits are developed in model organisms, numerous tools were recently developed to implement biocomputation in plant synthetic circuits. I presented in this review the tools and design methods for logic circuit implementation in plants. I highlighted recent and potential applications of those circuits to understand and engineer plant interaction with the environment, development, and metabolic pathways.

Engineering Plant Cell Fates and Functions for Agriculture and Industry.

Many plant species are grown to enable access to specific organs or tissues, such as seeds, fruits, or stems. In some cases, a value is associated with a molecule that accumulates in a single type of cell. Domestication and subsequent breeding have often increased the yields of these target products by increasing the size, number, and quality of harvested organs and tissues but also via changes to overall plant growth architecture to suit large-scale cultivation. Many of the mutations that underlie these changes have been identified in key regulators of cellular identity and function. As key determinants of yield, these regulators are key targets for synthetic biology approaches to engineer new forms and functions. However, our understanding of many plant developmental programs and cell-type specific functions is still incomplete. In this Perspective, we discuss how advances in cellular genomics together with synthetic biology tools such as biosensors and DNA-recording devices are advancing our understanding of cell-specific programs and cell fates. We then discuss advances and emerging opportunities for cell-type-specific engineering to optimize plant morphology, responses to the environment, and the production of valuable compounds.

An integrase toolbox to record gene-expression during plant development.

There are many open questions about the mechanisms that coordinate the dynamic, multicellular behaviors required for organogenesis. Synthetic circuits that can record in vivo signaling networks have been critical in elucidating animal development. Here, we report on the transfer of this technology to plants using orthogonal serine integrases to mediate site-specific and irreversible DNA recombination visualized by switching between fluorescent reporters. When combined with promoters expressed during lateral root initiation, integrases amplify reporter signal and permanently mark all descendants. In addition, we present a suite of methods to tune the threshold for integrase switching, including: RNA/protein degradation tags, a nuclear localization signal, and a split-intein system. These tools improve the robustness of integrase-mediated switching with different promoters and the stability of switching behavior over multiple generations. Although each promoter requires tuning for optimal performance, this integrase toolbox can be used to build history-dependent circuits to decode the order of expression during organogenesis in many contexts.

Computational Methods for the Design of Recombinase Logic Circuits with Adaptable Circuit Specifications.

Synthetic biology aims at engineering new biological systems and functions that can be used to provide new technological solutions to worldwide challenges. Detection and processing of multiple signals are crucial for many synthetic biology applications. A variety of logic circuits operating in living cells have been implemented. One particular class of logic circuits uses site-specific recombinases mediating specific DNA inversion or excision. Recombinase logic offers many interesting features, including single-layer architectures, memory, low metabolic footprint, and portability in many species. Here, we present two automated design strategies for both Boolean and history-dependent recombinase-based logic circuits. One approach is based on the distribution of computation within multicellular consortia, and the other is a single-cell design. Both are complementary and adapted for non-expert users via a web design interface, called CALIN and RECOMBINATOR, for multicellular and single-cell design strategies, respectively. In this book chapter, we are guiding the reader step by step through recombinase logic circuit design, from selecting the design strategy fitting to their final system of interest to obtaining the final design using one of our design web interfaces.

A single-cell view of the transcriptome during lateral root initiation in Arabidopsis thaliana.

Root architecture is a major determinant of plant fitness and is under constant modification in response to favorable and unfavorable environmental stimuli. Beyond impacts on the primary root, the environment can alter the position, spacing, density, and length of secondary or lateral roots. Lateral root development is among the best-studied examples of plant organogenesis, yet there are still many unanswered questions about its earliest steps. Among the challenges faced in capturing these first molecular events is the fact that this process occurs in a small number of cells with unpredictable timing. Single-cell sequencing methods afford the opportunity to isolate the specific transcriptional changes occurring in cells undergoing this fate transition. Using this approach, we successfully captured the transcriptomes of initiating lateral root primordia in Arabidopsis thaliana and discovered many upregulated genes associated with this process. We developed a method to selectively repress target gene transcription in the xylem pole pericycle cells where lateral roots originate and demonstrated that the expression of several of these targets is required for normal root development. We also discovered subpopulations of cells in the pericycle and endodermal cell files that respond to lateral root initiation, highlighting the coordination across cell files required for this fate transition.

Decoding and recoding plant development.

The development of multicellular organisms has been studied for centuries, yet many critical events and mechanisms of regulation remain challenging to observe directly. Early research focused on detailed observational and comparative studies. Molecular biology has generated insights into regulatory mechanisms, but only for a limited number of species. Now, synthetic biology is bringing these two approaches together, and by adding the possibility of sculpting novel morphologies, opening another path to understanding biology. Here, we review a variety of recently invented techniques that use CRISPR/Cas9 and phage integrases to trace the differentiation of cells over various timescales, as well as to decode the molecular states of cells in high spatiotemporal resolution. Most of these tools have been implemented in animals. The time is ripe for plant biologists to adopt and expand these approaches. Here, we describe how these tools could be used to monitor development in diverse plant species, as well as how they could guide efforts to recode programs of interest.

Computational Methods for the Design of Recombinase Logic Circuits.

Synthetic biology aims at engineering new biological systems and functions that can be used to provide new technological solutions to worldwide challenges. Detection and processing of multiple signals are crucial for many synthetic biology applications. A variety of logic circuits operating in living cells have been implemented. One particular class of logic circuits uses site-specific recombinases mediating specific DNA inversion or excision. Recombinase logic offers many interesting features, including single-layer architectures, memory, low metabolic footprint, and portability in many species. Here, we present two automated design strategies for recombinase-based logic circuits, one based on the distribution of computation within a multicellular consortia and the other one being a single-cell design. The two design strategies are complementary and are both adapted for none expert as a design web-interface exits for each strategy, the CALIN and RECOMBINATOR web-interface for respectively the multicellular and single-cell design strategy. In this book chapter, we are guiding the reader step by step through recombinase-logic circuit design from selecting the design strategy fitting to his/her final system of interest to obtaining the final design using one of our design web-interface.

Rational programming of history-dependent logic in cellular populations.

Genetic programs operating in a history-dependent fashion are ubiquitous in nature and govern sophisticated processes such as development and differentiation. The ability to systematically and predictably encode such programs would advance the engineering of synthetic organisms and ecosystems with rich signal processing abilities. Here we implement robust, scalable history-dependent programs by distributing the computational labor across a cellular population. Our design is based on standardized recombinase-driven DNA scaffolds expressing different genes according to the order of occurrence of inputs. These multicellular computing systems are highly modular, do not require cell-cell communication channels, and any program can be built by differential composition of strains containing well-characterized logic scaffolds. We developed automated workflows that researchers can use to streamline program design and optimization. We anticipate that the history-dependent programs presented here will support many applications using cellular populations for material engineering, biomanufacturing and healthcare.

Engineering Synthetic Signaling in Plants.

Synthetic signaling is a branch of synthetic biology that aims to understand native genetic regulatory mechanisms and to use these insights to engineer interventions and devices that achieve specified design parameters. Applying synthetic signaling approaches to plants offers the promise of mitigating the worst effects of climate change and providing a means to engineer crops for entirely novel environments, such as those in space travel. The ability to engineer new traits using synthetic signaling methods will require standardized libraries of biological parts and methods to assemble them; the decoupling of complex processes into simpler subsystems; and mathematical models that can accelerate the design-build-test-learn cycle. The field of plant synthetic signaling is relatively new, but it is poised for rapid advancement. Translation from the laboratory to the field is likely to be slowed, however, by the lack of constructive dialogue between researchers and other stakeholders.

Hierarchical composition of reliable recombinase logic devices.

A major goal of synthetic biology is to reprogram living organisms to solve pressing challenges in manufacturing, environmental remediation, and healthcare. Recombinase devices can efficiently encode complex logic in many species, yet current designs are performed on a case-by-case basis, limiting their scalability and requiring time-consuming optimization. Here we provide a systematic framework for engineering reliable recombinase logic devices by hierarchical composition of well-characterized, optimized recombinase switches. We apply this framework to build a recombinase logic device family supporting up to 4-input Boolean logic within a multicellular system. This work enables straightforward implementation of multicellular recombinase logic and will support the predictable engineering of several classes of recombinase devices to reliably control cellular behavior.

An Automated Design Framework for Multicellular Recombinase Logic.

Tools to systematically reprogram cellular behavior are crucial to address pressing challenges in manufacturing, environment, or healthcare. Recombinases can very efficiently encode Boolean and history-dependent logic in many species, yet current designs are performed on a case-by-case basis, limiting their scalability and requiring time-consuming optimization. Here we present an automated workflow for designing recombinase logic devices executing Boolean functions. Our theoretical framework uses a reduced library of computational devices distributed into different cellular subpopulations, which are then composed in various manners to implement all desired logic functions at the multicellular level. Our design platform called CALIN (Composable Asynchronous Logic using Integrase Networks) is broadly accessible via a web server, taking truth tables as inputs and providing corresponding DNA designs and sequences as outputs (available at http://synbio.cbs.cnrs.fr/calin ). We anticipate that this automated design workflow will streamline the implementation of Boolean functions in many organisms and for various applications.

A part toolbox to tune genetic expression in Bacillus subtilis.

Libraries of well-characterised components regulating gene expression levels are essential to many synthetic biology applications. While widely available for the Gram-negative model bacterium Escherichia coli, such libraries are lacking for the Gram-positive model Bacillus subtilis, a key organism for basic research and biotechnological applications. Here, we engineered a genetic toolbox comprising libraries of promoters, Ribosome Binding Sites (RBS), and protein degradation tags to precisely tune gene expression in B. subtilis We first designed a modular Expression Operating Unit (EOU) facilitating parts assembly and modifications and providing a standard genetic context for gene circuits implementation. We then selected native, constitutive promoters of B. subtilis and efficient RBS sequences from which we engineered three promoters and three RBS sequence libraries exhibiting ∼14 000-fold dynamic range in gene expression levels. We also designed a collection of SsrA proteolysis tags of variable strength. Finally, by using fluorescence fluctuation methods coupled with two-photon microscopy, we quantified the absolute concentration of GFP in a subset of strains from the library. Our complete promoters and RBS sequences library comprising over 135 constructs enables tuning of GFP concentration over five orders of magnitude, from 0.05 to 700 µM. This toolbox of regulatory components will support many research and engineering applications in B. subtilis.

Analytic framework for a stochastic binary biological switch.

We propose and solve analytically a stochastic model for the dynamics of a binary biological switch, defined as a DNA unit with two mutually exclusive configurations, each one triggering the expression of a different gene. Such a device has the potential to be used as a memory unit for biological computing systems designed to operate in noisy environments. We discuss a recent implementation of this switch in living cells, the recombinase addressable data (RAD) module. In order to understand the behavior of a RAD module we compute the exact time-dependent joint distribution of the two expressed genes starting in one state and evolving to another asymptotic state. We consider two operating regimes of the RAD module, a fast and a slow stochastic switching regime. The fast regime is aggregative and produces unimodal distributions, whereas the slow regime is separative and produces bimodal distributions. Both regimes can serve to prepare pure memory states when all cells are expressing the same gene. The slow regime can also separate mixed states by producing two subpopulations, each one expressing a different gene. Compared to the genetic toggle switch based on positive feedback, the RAD module ensures more rapid memory operations for the same quality of the separation between binary states. Our model provides a simplified phenomenological framework for studying RAD memory devices and our analytic solution can be further used to clarify theoretical concepts in biocomputation and for optimal design in synthetic biology.