Modelling tools and methodologies for rapid protocell prototyping

The field of unconventional computing considers the possibility of implementing computational devices using novel paradigms and materials to produce computers which may be more efficient, adaptable and robust than their silicon based counterparts. The integration of computation into the realms of chemistry and biology will allow the embedding of engineered logic into living systems and could produce truly ubiquitous computing devices. Recently, advances in synthetic biology have resulted in the modification of microorganism genomes to create computational behaviour in living cells, so called “cellular computing”. The cellular computing paradigm offers the possibility of intelligent bacterial agents which may respond and communicate with one another according to chemical signals received from the environment. However, the high levels of complexity when altering an organism which has been well adapted to certain environments over millions of years of evolution suggests an alternative approach in which chemical computational devices can be constructed completely from the bottom up, allowing the designer exquisite control and knowledge about the system being created. This thesis presents the development of a simulation and modelling framework to aid the study and design of bottom-up chemical computers, involving the encapsulation of computational re-actions within vesicles. The new “vesicle computing” paradigm is investigated using a sophisticated multi-scale simulation framework, developed from mesoscale, macroscale and executable biology techniques

Synthetic logic circuits encoded on toehold strand-displacement switchable CRISPR guide RNAs

The field of biological computing offers the potential to construct devices using complex and dynamic regulation for applications ranging from theranostics to the production of high value chemicals in bioreactors. However, building these complex regulatory systems depends on the creation of effective logic gates, which form the basis of digital systems. The low metabolic load, small genetic footprint and the low latency of expression that can be achieved with a small RNA-based regulatory system in contrast to protein regulators, emphasises the potential within RNA regulation. From an engineering perspective, the great advantage of an RNA approach is the highly predictable nature of RNA folding and the availability of established in silico tools. This thesis describes a novel de novo mechanism for NAND gate implementation in vivo using two RNAs, both of which must be expressed for the repression of an output gene. To construct this regulatory system a guide RNA of the CRISPR-Cas9 system is modified through the addition of a cis-repressing element, which complements part of the guide RNA and represses its activity. The activity of the cis-repressed guide RNA (crgRNA) can be rescued by the expression of an antisense RNA, which complements the cis-repressing element. This allows the guide RNA to return to an active conformation and repress the target promoter through CRISPRi (in strains expressing dCas9). This represents a NAND gate, as the output is repressed (OFF) only when both input RNAs are expressed. The design and optimisation of this system was performed using modelling of system energy states and dynamics and machine learning optimisation in a process which was automated into a single pipeline for future users. This system was characterised over a range of crgRNA and dCas9 expression levels and temperatures, and in different growth phases. Eight designs were tested and the optimal variant, for which output gene expression most closely approximated the OFF (repressed) and ON (un-repressed) states required for a logic gate, was chosen. The resulting NAND gate has a 10-fold repression of the output promoter when both RNAs were present; in contrast, only 1.2 fold repression was obtained when only the crgRNA was expressed. Consequently, multiple versions of the optimal variant were synthesised, each with different sequences but the same design principles. These performed similarly when applied to the repression of different reporter genes. Finally, an in silico approach was used to maximise orthogonality of different versions of the optimal variant which was then demonstrated in vivo. This novel NAND gate design offers the ability to build large libraries of logic gates with small genetic footprints (304 bp) and the potential to be combined to produce complex regulatory networks

Development of methods for combinational approaches to cis-regulatory module interactions

The complexity and size of the higher animal genome and relative scarcity of DNA-binding factors with which to regulate it imply a complex and pleiotropic regulatory system. Cisregulatory modules (CRMs) are vitally important regulators of gene expression in higher animal cells, integrating external and internal information to determine an appropriate response in terms of gene expression by means of direct and indirect interactions with the transcriptional machinery. The interaction space available within systems of multiple CRMs, each containing several sites where one or more factors could be bound is huge. Current methods of investigation involve the removal of individual sites or factors and measuring the resulting effect on gene expression. The effects of investigations of this type may be masked by the functional redundancy present in some of these regulatory systems as a result of their evolutionary development. The investigation of CRM function is limited by a lack of technology to generate and analyse combinatorial mutation libraries of CRMs, where putative transcription factor binding sites are mutated in various combinations to achieve a holistic view of how the factors binding to those sites cooperate to bring about CRM function. The principle work of this thesis is the generation of such a library. This thesis presents the development of microstereolithography as a method for making microfluidic devices, both directly and indirectly. A microfluidic device was fabricated that was used to generate oligonucleotide mixtures necessary to synthesise combinatorial mutants of a CRM sequence from the muscle regulatory factor MyoD. In addition, this thesis presents the development of the optimisation algorithms and assembly processes necessary for successful sequence assembly. Furthermore, it was found that the CRM, in combination with other CRMs, is able to synergistically regulate gene expression in a position and orientation independent manner in three separate contexts. Finally, by testing a small portion of the available combinatorial mutant library it was shown that mutation of individual binding sites within of the CRM is not sufficient to show a significant change in the level of reporter gene expression

From CoA ester supply to a yeast communication toolkit in Saccharomyces cerevisiae

Saccharomyces cerevisiae is the most widely used eukaryotic chassis in synthetic biology, as hu-manity and yeast share a long and fruitful history. For synthetic biology applications, S. cerevisiae was extensively used for metabolic engineering as well as for the construction of artificial net-works. To contribute to the metabolic engineering achievements conducted in S. cerevisiae, we extended its metabolic capacities by providing non-native short-chain acyl-coenzyme A esters as metabolic precursors. In order to advance the construction of artificial networks to multicel-lular systems we provided a comprehensive yeast communication toolkit (YCTK), and demon-strated its usability for the rapid assembly of synthetic cell-cell communication systems. Engineered production of short-chain acyl-coenzyme A esters in Saccharomyces cerevisiae Globally, S. cerevisiae is one of the most commonly used chassis organisms in modern biotech-nology and constitutes a high economic value to the growing bioecomomy. With the objective to produce novel natural products in S. cerevisiae a bottleneck of the chassis was uncovered. Short-chain acyl-coenzyme A esters serve as intermediate compounds in fatty acid biosynthesis, and are building blocks for the production of polyketides, biopolymers, and other value-added chemicals. However, S. cerevisiae’s limited repertoire of short-chain acyl-CoAs effectively pre-vents its application as a production host for a plethora of natural products. To address and re-solve this limitation, we introduced metabolic pathways to five different acyl-CoA esters into S. cerevisiae. We engineered plasmid-based yeast strains that provide propionyl-CoA, methylmalonyl-CoA, n-butyryl-CoA, isovaleryl-CoA, and n-hexanoyl-CoA. For the production of propionyl-CoA and methylmalonyl-CoA, we reestablished a published feeding-dependent pro-duction route using the PrpE and Pcc enzymes to serve as benchmark for our feeding-independent production pathways that provided in our study comparable product concentra-tions. To ensure efficient extraction of the produced metabolites we established a yeast-specific metabolite extraction protocol to determine the intracellular acyl-CoA concentrations in the engineered strains. For the production of isovaleryl-CoA, we tested two different pathways but only obtained product formation from the alternative isovaleryl-CoA biosynthetic (AIB) pathway originating from Myxococcus xanthus and obtained 5.5±1.2 µM isovaleryl-CoA. To our knowledge, this is the first reported functional heterologous expression of this pathway in S. cerevisiae. For the production of n-butyryl-CoA and n-hexanoyl-CoA, we adapted the butanol production pathway for our purposes and measured approximately 6 µM intracellular concen-tration of butyryl-CoA and hexanoyl-CoA. For the feeding-dependent pathway towards propio-nyl-CoA we obtained intracellular concentrations of 5.3 ± 2.4 µM while the feeding independ-ent 3-hydroxypropionate (3HP) pathway produced 8.5 ± 3.7 µM. The extension of both propio-nyl-CoA pathways to produce methylmalonyl-CoA resulted only into production of 0.5 ± 0.1 µM and 0.3 ± 0.3 µM methylmalonyl-CoA. Not only but particularly for the production of methylmalonyl-CoA further optimization is required. To allow rapid pathway prototyping, op-timization and testing of alternative enzymes, we established a short-chain acyl-CoA Golden Gate collection. This collection enables together with the well-known Dueber yeast toolkit YTK collection the examination of different enzymes variants and to investigate optimized expres-sion of the corresponding genes. We conclude that the acyl-CoAs produced here, that are common building blocks of secondary metabolites, prepared the ground for prospective engineered production of a variety of natural products in S. cerevisiae. These acyl-CoA producing strains together with the short-chain acyl-CoA collection lay the foundation to further explore S. cerevisiae as a heterologous production host for high-value secondary metabolite production. Yeast communication toolkit The construction of multicellular networks was a proposed aim already early on in synthetic biology. Today, they still hold many promises like the division of labor or the performance of more complex tasks. Most of the systems so far were implemented in bacterial chassis and only a few examples exist for the eukaryotic chassis S. cerevisiae. Especially for gram-negative bacterial chassis, the quorum sensing system provides a large diversity of ready to use communication systems. Also, yeast species evolved a communication system using peptide-based pheromones to interact with the opposite mating type. Here, we employed the natural diversity of the pep-tide α-factor pheromones, the corresponding GPCR receptors, as well as of barrier proteases, that function similarly to quorum quenching enzymes. With the establishment of the Golden Gate yeast communication toolkit (YCTK) we provide a standardized collection of parts that al-low the rapid construction of multicellular networks in the model organism S. cerevisiae. The feasible designs are limitless as well as the number of envisioned applications. The YCTK collec-tion consists of responder (pheromone-responsive promoters), sender (mfα1 genes – α-factors), receiver (Ste2 receptors) and barrier (Bar1 proteases) parts. We characterized the dynamics of the pheromone-inducible promoters in the different mating-type strain backgrounds and de-termined the dose-response to the α-factor as well as their temporal response. The different promoters exhibited a range of different dynamics and properties that enable the implementa-tion of different prospective network design motives. The characterization results of the Ste2 receptors indicated that our collection is comprised of receptors with high α-factor promiscuity and of receptors with high substrate specificity for their cognate α-factor. Further we found that different Ste2 receptors exhibit different sensitivities towards the cognate as well as to non-cognate α-factors. The promiscuity of the Ste2 receptors did not correlate with the α-factor se-quences. Our likelihood analysis of the Ste2 receptors indicated that the ones closer related to S. cerevisiae tend to be stimulated by the α-factors of related species. Our likelihood analysis of the Ste2 receptors coincided with the phylogenetic relationships of the species. Interesting is also the finding that α-factors of species for which the receptor exhibited high α-factor promis-cuity stimulated only a few receptors. Even though only five of the selected barrier proteases were functionally expressed the characterization of the protease promiscuity was to our knowledge the most comprehensive study of its kind so far. Similar to the receptors we identi-fied promiscuous and substrate specific barrier proteases. The proposed model of a coevolution between the receptor and barrier proteases to recognize similar sequence motives of the α-factor was partly validated, however, the model is not universally applicable according to our results. The extended knowledge of the pheromone-inducible promoters, the crosstalk be-tween α-factors, receptors and barrier proteases, and an initial tunability test enabled proof of principle construction of multicellular systems using the YTCK collection. We engineered mul-ticellular logic gate-like population networks that allow the receiver cells to conditionally re-spond to the population composition. While the α-factor signaling motif is functional and was used to successfully establish OR and AND gate-like systems, signal disruption by a barrier pro-tease of a self-stimulating or a signaling motif requires further optimization. Overall, the reali-zation of multicellular networks using the YCTK was proven to be successful. To summarize, with the YCTK we provide a set of comprehensively characterized sender, re-ceiver, and barrier parts to facilitate the implementation of cell-cell and thus multicellular communication networks in S. cerevisiae

Error control in bacterial quorum communications

Quorum sensing (QS) is used to describe the communication between bacterial cells, whereby a coordinated population response is controlled through the synthesis, accumulation and subsequent sensing of specific diffusible chemical signals called autoinducers, enabling a cluster of bacteria to regulate gene expression and behavior collectively and synchronously, and assess their own population. As a promising method of molecular communication (MC), bacterial populations can be programmed as bio-transceivers to establish information transmission using molecules. In this work, to investigate the key features for MC, a bacterial QS system is introduced, which contains two clusters of bacteria, specifically Vibrio fischeri, as the transmitter node and receiver node, and the diffusive channel. The transmitted information is represented by the concentration of autoinducers with on-off keying (OOK) modulation. In addition, to achieve better reliability and energy efficiency, different error control techniques, including forward error correction (FEC) and Automatic Repeat reQuest (ARQ) are taken into consideration. For FEC, this work presents a comparison of the performance of traditional Hamming codes, Minimum Energy Codes (MEC) and Luby Transform (LT) codes over the channel. In addition, it applied several ARQ protocols, namely Stop-N-Wait (SW-ARQ), Go-Back-N (GBN-ARQ), and Selective-Repeat (SR-ARQ) combined with error detection codes to achieve better reliability. Results show that both the FEC and ARQ techniques can enhance the channel reliability, and that ARQ can resolve the issue of out-of-sequence and duplicate packet delivery. Moreover, this work further addresses the question of optimal frame size for data communication in this channel capacity and energy constrained bacterial quorum communication system. A novel energy model which is constructed using the experimental validated synthetic logic gates has been proposed to help with the optimization process. The optimal fixed frame length is determined for a set of channel parameters by maximizing the throughput and energy efficiency matrix

Physics of Ionic Conduction in Narrow Biological and Artificial Channels

The book reprints a set of important scientific papers applying physics and mathematics to address the problem of selective ionic conduction in narrow water-filled channels and pores. It is a long-standing problem, and an extremely important one. Life in all its forms depends on ion channels and, furthermore, the technological applications of artificial ion channels are already widespread and growing rapidly. They include desalination, DNA sequencing, energy harvesting, molecular sensors, fuel cells, batteries, personalised medicine, and drug design. Further applications are to be anticipated.The book will be helpful to researchers and technologists already working in the area, or planning to enter it. It gives detailed descriptions of a diversity of modern approaches, and shows how they can be particularly effective and mutually reinforcing when used together. It not only provides a snapshot of current cutting-edge scientific activity in the area, but also offers indications of how the subject is likely to evolve in the future

Towards light based dynamic control of synthetic biological systems

For the field of synthetic biology, the adaptation of principles, from the well established traditional engineering disciplines, like mechanical and electrical engineering, in order to realise complex synthetic biological circuits, is an intriguing prospect. These principles can enable a forward engineering, rational design and implementation approach, where a system's properties can be predicted or designed in silico followed by the manufacturing of the in vivo system, that can be tested, used or redesigned in the most efficient possible way. Achieving control over these circuits, is one of the important topics of the field, for these applications to become robust and render useful functions applicable to energy, medicine, pharmaceuticals and agriculture industries. In this work, I attempt to explore light, as a promising control 'dial' for synthetic circuitry. Light is fast, economic compared to chemicals, it can be interfaced with electronics, it is reversible in its effect and can be applied at a fine spatio-temporal resolution. These characteristics, are absent from the classically used chemical inducers, meaning that light, can open new possibilities for the user to control synthetic systems, or even facilitate the cell to cell communication, within population based networks. This work, is a contribution towards harnessing the advantages of light, for achieving control over synthetic circuits. More specifically, I start with the detailed theoretical and experimental study of the Cph8 two component system, a synthetic chimeric receptor which is responsive to red light. This is done, in order to develop a sufficient theoretical understanding of it, through detailed mechanistic modelling, in order to connect the specific system with the toggle switch and the dual feedback oscillator, in an optimal way and achieve control of these devices through light. The developed model, was able to highlight the main aspects and mechanisms inherent to its structure, describe most of the observations from the experimental system, to also make quantitative predictions. The second part of this work, was the development of novel promoters, that can be regulated by a commonly used transcription factor, such as LacI, but also, light responsive regulators like OmpR and CcaR. This yielded a direct way to integrate light and chemical inputs, into a single output, while the dual regulation, allowed to connect and modulate the toggle switch without the need of additional transcription factors. The latter, a light tuneable toggle switch, showed indications that it can function as a memory controller that can be reset by light. Finally, I show the design and modelling of a light tuneable dual feedback oscillator, where light of one wavelength can be used to tune the amplitude, while another wavelength can tune the period. The developed models and synthetic circuits are expected to contribute towards implementing finely tuned and controlled synthetic circuits through light.Open Acces

Physics of Ionic Conduction in Narrow Biological and Artificial Channels

This is a book about ion channels. It has been written mostly by physical scientists and mathematicians, even though the most widespread and important manifestation of ion channels is in biology, where they are essential to life in all its forms. How do non-biologists get involved in such investigations? Everyone will have their own particular story but, for ourselves, it was the heady combination of scientific curiosity, a wish to contribute to the fundamental understanding of natural phenomena that clearly have crucially important applications, and the realisation that some of our physics knowledge and expertise might be relevant