SynBioFromLeukipposInstitute

Biological regulatory systems are complex due to their role in living organisms in modulating precise responses to changes in internal and external conditions. In this respect, mathematical models have become essential tools to address their complexity for a better understanding of their mechanisms. The vision here, based on integrating experimental and theoretical techniques, provides a systematic means to quantitatively study the characteristics of the interactions that occur in living organisms. The outcome of such an endeavour should provide insights in terms of predictions and quantifications for further investigations in systems and synthetic biology. In this thesis, we establish an integrated modelling framework that can ensure the interaction of experimental biology with the development of quantitative mathematical descriptions of biological systems. To this end, we develop a framework to simulate and analyse biological regulatory systems by integrating different layers of regulatory information. The work herein presents a biological model development workflow in terms of a step by step approach, highlighting challenges and “real life” problems associated with each stage of model development. In the first part, we have focused on applying systems and synthetic biology modelling tools to the phosphate system at the cellular and genetic levels in Escheria coli. Then, we have analysed the interaction mechanisms and the dynamic behaviour of the phosphate starvation response deactivation and evaluated the role of phosphatase activity. We have investigated how the properties of these signalling systems depend on the network structure. Moreover, we have constructed detailed transcriptional regulatory network models and models for promoter design. In the second part, we have designed a multi-level dynamical set up by providing a novel closed loop whole body model of glucose homeostasis coupled with molecular signalling. We have then developed a system embracing the intracellular metabolic level, the cellular level involving the dynamics of the cells, the organ level, and the processes within the whole body. The output of each model directly has been fed with the variables and the parameters of the next aggregated model. This allowed us to observe the metabolic changes that occur at all levels and monitor inter-level communications for Type 2 Diabetes disease.

Living therapeutics have been engineered to diagnose diseases and produce and deliver therapeutics in situ. These therapeutics can be equipped with devices for sensing inputs, controlling gene expression, building memory, and producing and delivering an active compound.
Ingenious devices responding to stress, temperature, quorum-sensing signals, and other small molecules have been built to control the production and delivery of therapeutic molecules.
To deal with biosafety, some living therapeutics carry biocontainment devices based on cell auxotrophy, temperature-sensitive regulators, and toxin/antitoxin counteraction.
Recent advances in synthetic biology greatly expanded the toolbox for engineering living therapeutics; however, new parts are still needed to help synthetic biologists engineer more diverse and fully functional living therapeutics.
Microbes can be engineered to act like living therapeutics designed to perform specific actions in the human body. From fighting and preventing infections to eliminating tumors and treating metabolic disorders, engineered living systems are the next generation of therapeutics. In recent years, synthetic biologists have greatly expanded the genetic toolbox for microbial living therapeutics, adding sensors, regulators, memory circuits, delivery devices, and kill switches. These advances have paved the way for successful engineering of fully functional living therapeutics, with sensing, production, and biocontainment devices. However, some important tools are still missing from the box. In this review, we cover the most recent biological parts and approaches developed and describe the missing tools needed to build robust living therapeutics.

CRISPR-based genome editing provides a simple and scalable toolbox for a variety of therapeutic and biotechnology applications. Whilst the fundamental properties of CRISPR proved easily transferable from the native prokaryotic hosts to eukaryotic and multicellular organisms, the tight control of the CRISPR-editing activity remains a major challenge. Here we summarise recent developments of CRISPR and riboswitch technologies and recommend novel functionalised synthetic-gRNA (sgRNA) designs to achieve inducible and spatiotemporal regulation of CRISPR-based genetic editors in response to cellular or extracellular stimuli. We believe that future advances of these tools will have major implications for both basic and applied research, spanning from fundamental genetic studies and synthetic biology to genetic editing and gene therapy.

CRISPR-Cas systems provide microbes with adaptive immunity to infectious nucleic acids and are widely employed as genome editing tools. These tools utilize RNA-guided Cas proteins whose large size (950–1400 amino acids) has been considered essential to their specific DNA- or RNA-targeting activities. Here we present a set of CRISPR-Cas systems from uncultivated archaea that contain Cas14, a family of exceptionally compact RNA-guided nucleases (400–700 amino acids). Despite their small size, Cas14 proteins are capable of targeted single-stranded DNA (ssDNA) cleavage without restrictive sequence requirements. Moreover, target recognition by Cas14 triggers non-specific cutting of ssDNA molecules, an activity that enables high-fidelity SNP genotyping (Cas14-DETECTR). Metagenomic data show that multiple CRISPR-Cas14 systems evolved independently and suggest a potential evolutionary origin of single-effector CRISPR-based adaptive immunity.

RetroRules is a database of reaction rules for metabolic engineering (https://retrorules.org). Reaction rules are generic descriptions of chemical reactions that can be used in retrosynthesis workflows in order to enumerate all possible biosynthetic routes connecting a target molecule to its precursors. The use of such rules is becoming increasingly important in the context of synthetic biology applied to de novo pathway discovery and in systems biology to discover underground metabolism due to enzyme promiscuity. Here, we provide for the first time a complete set containing >400 000 stereochemistry-aware reaction rules extracted from public databases and expressed in the community-standard SMARTS (SMIRKS) format, augmented by a rule representation at different levels of specificity (the atomic environment around the reaction center). Such numerous representations of reactions expand natural chemical diversity by predicting de novo reactions of promiscuous enzymes.

The rate at which scientists can read DNA sequences has risen dramatically in recent years. But the speed at which they can write DNA hasn't kept pace. That may soon change. Researchers from a French biotech company reported last week that by using close relatives of the DNA-writing enzymes at work in living cells, they can build DNA strands as long as 150 "letters," or nucleotide bases. That's up from 50 nucleotides just a few months ago, and nearly on par with the standard chemical approach to writing DNA that has been around for decades. With numerous companies and academic groups aiming to further improve the technology, experts expect the new enzymatic approach to writing DNA to continue to improve steadily. That's likely to drop prices for made-to-order DNA strands and turbocharge research in synthetic biology and efforts to archive data in DNA.

Researchers at Stanford University have reworked CRISPR-Cas9 gene-editing technology to manipulate the genome in three-dimensional space, allowing them to ferry genetic snippets to different locations in a cell's nucleus.

Mutations in the gene encoding dystrophin, a protein that maintains muscle integrity and function, cause Duchenne muscular dystrophy (DMD). The deltaE50-MD dog model of DMD harbors a mutation corresponding to a mutational “hotspot” in the human DMD gene. We used adeno-associated viruses to deliver CRISPR gene editing components to four dogs and examined dystrophin protein expression 6 weeks after intramuscular delivery (n = 2) or 8 weeks after systemic delivery (n = 2). After systemic delivery in skeletal muscle, dystrophin was restored to levels ranging from 3 to 90% of normal, depending on muscle type. In cardiac muscle, dystrophin levels in the dog receiving the highest dose reached 92% of normal. The treated dogs also showed improved muscle histology. These large-animal data support the concept that, with further development, gene editing approaches may prove clinically useful for the treatment of DMD.

Synthetic Biology is characterized by a forward engineering approach to the design of biological systems implementing desired functionalities. The Synthetic Biology design cycle benefits from the understanding and the proper representation of the underling biological complexity, allowing predicting the behavior of the target system. Considering the intrinsic nature of the systems to be designed with a Systems Biology perspective is a key requirement to support the Synthetic Biology design cycle. In particular, good models for synthetic biological systems must express hierarchy, encapsulation, selective communication, spatiality, quantitative mechanisms, and stochasticity. Computational models in general not only properly handle such modeling requirements. They can also manage heterogeneous information in compositional processes, support formal analysis and simulation, and can further be exploited for knowledge interchange among the scientific community. In particular, the nets-within-nets formalism expresses all of these features providing high flexibility in the modeling task. The formalism is well suited to represent heterogeneous systems and in general provide an extraordinary expressivity. This is achieved thanks its capability of tuning the abstraction level in each part of the model.

Microbial electrochemical systems are able to harvest electricity or synthesize valuable chemicals from organic matters while simultaneously cleaning environmentally hazardous wastes. The sluggish extracellular electron transfer (EET) between “non- or poor-conductive” microbes and electrode involves both bio- and electrocatalytic processes but is one of the main impediments to fast microbial electrode kinetics. To boost EET, researches have been focused on engineering electrochemically active microbes, constructing a unique nanostructured electrode endowed with a large amount loading of microbes and enhancing biotic–abiotic interactions for rapid electrode kinetics. After surveys of fundamentals of microbial electrocatalysis, particularly the diverse EET mechanisms with discussions on scientific insights, this review summarizes and discusses the recent advances in bioengineering highly active biocatalytic microbes and nanoengineering unique electrode nanostructures for significantly improved microbial EET processes. In particular, this review associated with our researches analyzes in more detail the EET pathways, which contain direct and mediated electron transfer. The confusion between the energy efficiency and electron transfer rate is clarified and the approaches to elevate the EET rate are further discussed. These discussions shed both theoretical and practical lights on further research and development of more high-performance microbial catalysts by using synthetic biology coupled with nanoengineering approach for high energy conversion efficiency while achieving high power density for practical applications. The challenges and perspectives are presented. It is believed that a next wave of research of microbial electrochemical systems will produce a new generation of sustainable green energy technologies and demonstrate great promise in their broad applications and industrializations.