Cofund on Biotechnologies

An orthogonal, organism-independent expression platform based on extracytoplasmic function (ECF) sigma factors

Consortium:

Summary

Orthogonality is a key feature of classical engineering approaches but a major challenge to Synthetic Biology (SynBio) due to the high degree of context dependence and interconnectivity in biological systems. ECFexpress will develop a SynBio design framework based on Extracytoplasmic Function sigma factors (ECFs) to implement highly orthogonal regulatory switches and circuits. ECFs represent ideal building blocks for SynBio applications, because they are modular, inherently orthogonal, universal, and scalable. Initially, we will evaluate the organism-independent potential of ECFs by plementing orthogonal ECF-based regulation in four phylogenetically highly diverse bacteria, including two biotechnological workhorses. This will allow extracting and establishing universal design rules for choosing and implementing orthogonal ECF-based natural switches in any bacterium. Subsequently, we will design and engineer novel synthetic ECF switches with increased orthogonality. This goal will be achieved by applying design strategies based on combinatorial synthesis and structure-guided mutational approaches. Finally, we will use rational forward design to build and evaluate complex synthetic circuits based on ECF switches. This combined theoretical and experimental evaluation of novel ECF-based circuits will allow us to benchmark their orthogonality and to explore the ECF circuit design space. ECFexpress strictly adheres to engineering principles by (i) applying defined standards in assembly, measurements and data management, (ii) establishing orthogonal parts and switches for biological circuit design, and (iii) developing a bioinformatics platform for data management, mathematical modelling and forward design. Taken together, ECFexpress aims at a foundational advance by implementing true orthogonality to bacterial cells. It will directly be applicable in and beneficial for projects in basic research and the knowledge-based bio-economy (KBBE).

Orthogonal biosystems based on phosphonate XNAs

Consortium:

Summary

Although RNA and DNA are the only genetic polymers in nature, synthetic nucleic acids (XNAs) can be suitable genetic materials. If not toxic and if unable to interact with the cellular machinery, such XNAs can be further developed into orthogonal genetic materials in vivo – rewriting the topology of information transfer in biology, redesigning the Central Dogma.

An XNA episome, maintained independent from and unable to interact with the cell’s genetic information storage, would give us insights into how information is stored and propagated in biology as well as establishing a minimal system from which complex functions based on XNA could be systematically developed. If XNA precursors cannot be made in the cell, then an XNA episomecan be immediately applied to develop safer, genetically-contained, engineered organisms.
Redesign of the Central Dogma pose a number of challenges that can be systematically overcome. It requires an XNA that is bio-orthogonal, a replication system for maintaining the information, and a route of communication with the cell. Recent advances highlight that this is a feasible goal.
Using oxymethylphosphonates as a bio-orthogonal XNA and an orthogonal DNA replication system, we propose to develop all key components required to establish and maintain an XNA episome in vivo. This will include engineered polymerases to transfer information to XNA in vitro (DNA>XNA and XNA>DNA) as well as an XNA replicase (XNA>XNA) dedicated to replicating the linear XNA plasmid being developed. Functional XNAzymes, will link genetic information stored in XNA to cell survival, ensuring episome maintenance in the semi-synthetic cell and establishing the episome as a platform for further creation and evolution of XNA function and circuits.

In addition to forging the first orthogonal genetic element, the methodologies and molecules generated in the project will in themselves be of great scientific interest and enable new avenues of research.

A model-driven approach to minimal cell engineering for medical therapy

Consortium:

Summary

Synthetic biology promises to enable researchers to design therapeutically and industrially valuable organisms. Achieving this promise requires new techniques for designing, synthesizing, and transplanting entire genomes. Here we propose to develop the first model-driven approach to synthetic biology, and use this approach to construct a bacterial chassis capable of synthesizing and delivering human lung therapies in situ. Specifically, we propose to develop a whole-cell model of the human lung pathogen M.pneumoniae, and use this model to design and construct a reduced, non-pathogenic chassis capable of delivering human lung therapies and/or vaccinations. This project will involve intimate integration of predictive modeling, genomic engineering, and systems and synthetic biology. Model predictions will provide direct input into genomic engineering, and the newly created strains will be characterized to refine the computational model. The project will produce the most accurate computational model of any organism to date, as well as produce the most reduced cell to date. In the future we anticipate this reduced chassis could be extended to synthesize and deliver small molecule and/or protein therapies to diseased lungs in situ.

Synthetic photoswitchable modulators to harness endogenous proteins in vivo: remote control of pentameric ligand-gated ion channels with light

Consortium:

Summary

The objective of the MODULIGHTOR proposal is to develop synthetic photochromic ligands (PCLs) to control neurotransmitter-activated receptors with light, and to test them in neuronal preparations and freely behaving animals. We will target ionotropic gamma-aminobutyric acid (GABA), glycine and nicotinic acetylcholine receptors, whose structure and pharmacology is well characterized. These receptors have a particularly important role in the functioning of mammalian nervous system including inhibitory neurotransmission in the brain and spinal cord (GABA, glycine) and muscle excitation (acetylcholine). The PCLs generated in this project will thus allow to spatiotemporally control inhibitory neuronal circuits and muscles in vivo.

A multidisciplinary approach will be applied to design, synthesize and characterize the PCLs in vitro and in vivo by combining the efforts of computational and synthetic chemists, electrophysiologists and bioengineers. Synthesis of compound libraries will be guided by molecular dynamics simulations of target ligands docked in their corresponding receptor and tethered to photochromic groups. After characterizing their solubility and photophysical properties,
the compounds will be subject to electrophysiological assays in cell lines, neuronal cultures and brain slices. Successful PCLs will be tested in vivo in tadpoles using behavioural assays and microscopy imaging of receptor activity.

The PCLs proposed in this project will enable remotely controlling endogenous proteins with pharmacologic and spatiotemporal selectivity. This approach can be applied to specific signalling pathways with known pharmacology or to highly specialized cells like neurons. MODULIGHTOR will thus enable synthetic biology to gain control over high-order biological structures like physiologically intact neuronal circuits and specific aspects of the behaviour of organisms, leading to unexplored technological and therapeutic benefits.

Control of Engineered Metabolism by Flowering and Temperature Triggered Plant Regulatory Networks

Consortium:

Summary

A major goal of plant synthetic biology is to create smart plants that are able to respond to key cues and display a variety of agronomically valuable traits such as enhanced stress resilience or the biosynthesis of high value compounds. The objectives of the SMARTPLANTS consortium are to develop parallel regulatory networks (PaRNets) that are based on cues that plants normally encounter in their growth cycle, namely flowering and temperature changes, and translate these into metabolic engineering-based outputs to produce high value or stress-protecting compounds. Flowering is accompanied by a dramatic metabolic switch leading to the massive transfer of resources from the leaves to the seeds or the fruits. However there is still significant biomass remaining in the leaves and stems. By developing a PaRNet that uses flowering as a trigger, we will capture part of this biomass to convert it to a high value compound, the diterpene cis-abienol. Our flowering PaRNet will be based on the florigen signal encoded by the conserved FT gene. Similarly we will develop a PaRNet based on temperature fluctuations to induce upon higher temperatures the production of isoprene, a compound conferring heat-stress protection. One key aspect in the design of these networks is signal propagation, which will be mediated by mobile orthogonal Transcription Activator-Like Effectors (TALEs) activating synthetic promoters. Mobility will be engineered by fusing the TALEs to Intercellular Trafficking Motifs or viral movement proteins, or by using deconstructed viral vectors. Both regulatory network and metabolic engineering optimisation will be assisted by modelling in iterative rounds. Finally, to initiate and promote a community effort in the development of artificial plant regulatory networks we will organize an international symposium on this topic. SMARTPLANTS is a true interdisciplinary project, which will be carried out by a team of highly experienced scientists with complementary skills.

Design and Synthesis of a Bio-orthogonal Genetic System Based on Threose Nucleic acids In Vivo

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Summary

The development of synthetic biopolymers as an effective engineering tool for in vivo applications has been limited by our ability to demonstrate an intra-cellular system that replicates and transfers encoded chemical information. The maintenance and interpretation of cellular chemical information is central to biology and best known as the Central Dogma. Through the re-design of information storage systems (genetic material) and the enzymes involved in accessing and maintaining biological information (polymerases, ribosomes), synthetic biology has the potential to re-write the Central Dogma and extend our understanding of life's most basic processes. Our proposed synthesis of an all-TNA episome based on the bacteriophage Phi29 would revolutionize synthetic biology by providing an in vivo, stable replicon for storing sequence-defined genetic information completely isolated from the cellular genome and able to replicate independently and therefore function as a safe and effective tool for synthetic biology.

A Unified Nucleic Acid Computation System for Organisms

Consortium:

Summary

We will develop a Unified Nucleic-Acid Computation System (UNACS) for cells that has genetic encodability, the ability to probe endogenous transcripts, programmability, orthogonality, composability, uniform thermodynamics and kinetics, low background and high signal. UNACS will provide a programmable and general purpose means of regulating metabolism in a wide variety of cell types.
UNACS utilizes RNA programming via riboregulators, engineered versions of T7 RNA polymerase and the ribonucleoprotein dCas9 (a catalytically inactive version of the Cas9 nuclease derived from a CRISPR type II system) as a general purpose ‘universal join’ between RNA programming and protein functions such as repression and transcription. These components will be combined to enable RNA-based sensing, logic computation, and sequence-programmable (re)-wiring of genes into circuits. Synthetic triplex-forming oligonucleotides ((L)-aTNAs) will be developed that can be utilized as exogenous triggers for UNACS gates.
We will demonstrate the capabilities of our approach by developing checkpoint circuits in E.coli thatcan be used to enhance the performance of engineered metabolic pathways such as that for the isoprenoid lycopene. The checkpoint circuits monitor ribosomal abundance, cell density or the presence of metabolic key enzymes. When conditions are good, cellular production resources are re-distributed by temporarily halting growth and switching on the production of the target metabolite.
The UNACS scheme will be also adjusted to enable RNA sensing and regulation in eukaryotic cells, demonstrating the generality of our approach. To this end, riboregulators and T7 RNA polymerase will be modified to function in the eukaryotic context, and exogenous (L)-aTNAs will be used to regulate the translation of proteins.