Implicit Neural Multiple Description for DNA-based data storage

DNA exhibits remarkable potential as a data storage solution due to its impressive storage density and long-term stability, stemming from its inherent biomolecular structure. However, developing this novel medium comes with its own set of challenges, particularly in addressing errors arising from storage and biological manipulations. These challenges are further conditioned by the structural constraints of DNA sequences and cost considerations. In response to these limitations, we have pioneered a novel compression scheme and a cutting-edge Multiple Description Coding (MDC) technique utilizing neural networks for DNA data storage. Our MDC method introduces an innovative approach to encoding data into DNA, specifically designed to withstand errors effectively. Notably, our new compression scheme overperforms classic image compression methods for DNA-data storage. Furthermore, our approach exhibits superiority over conventional MDC methods reliant on auto-encoders. Its distinctive strengths lie in its ability to bypass the need for extensive model training and its enhanced adaptability for fine-tuning redundancy levels. Experimental results demonstrate that our solution competes favorably with the latest DNA data storage methods in the field, offering superior compression rates and robust noise resilience.Comment: Xavier Pic and Trung Hieu Le are both equal contributors and primary author

Proceedings of Abstracts Engineering and Computer Science Research Conference 2019

© 2019 The Author(s). This is an open-access work distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. For further details please see https://creativecommons.org/licenses/by/4.0/. Note: Keynote: Fluorescence visualisation to evaluate effectiveness of personal protective equipment for infection control is © 2019 Crown copyright and so is licensed under the Open Government Licence v3.0. Under this licence users are permitted to copy, publish, distribute and transmit the Information; adapt the Information; exploit the Information commercially and non-commercially for example, by combining it with other Information, or by including it in your own product or application. Where you do any of the above you must acknowledge the source of the Information in your product or application by including or linking to any attribution statement specified by the Information Provider(s) and, where possible, provide a link to this licence: http://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/This book is the record of abstracts submitted and accepted for presentation at the Inaugural Engineering and Computer Science Research Conference held 17th April 2019 at the University of Hertfordshire, Hatfield, UK. This conference is a local event aiming at bringing together the research students, staff and eminent external guests to celebrate Engineering and Computer Science Research at the University of Hertfordshire. The ECS Research Conference aims to showcase the broad landscape of research taking place in the School of Engineering and Computer Science. The 2019 conference was articulated around three topical cross-disciplinary themes: Make and Preserve the Future; Connect the People and Cities; and Protect and Care

Design principles of cell-free replicators

Der heilige Gral der synthetischen Biologie ist die Erschaffung einer minimalen Zelle, welche sowohl zu autonomer Selbstreplikation als auch zu natürlicher Evolution befähigt ist. Bereits heute ist es möglich das zentrale Dogma der Molekularbiologie, also die Implementierung des genetischen Codes mittels Transkription-Translation, in vitro zu rekonstruieren. Doch die Kopplung dieses Prozesses mit einem vollständigen DNA-Selbstreplikationssystem war bisher nur auf ein paar Kilobasen (kbp) beschränkt, weit entfernt von den vorgeschlagenen 113 kbp die für eine minimale Zelle nötig wären. In dieser Arbeit wird die Entwicklung einer Plattform für die transkriptions-translations-gekoppelte DNA-Replikation vorgestellt, genannt PURErep, welche in der Lage ist Genome mit der vorhergesagten Größe einer Minimalzelle zu replizieren. Als wichtiger Schritt in Richtung natürlicher Evolution kann sich der hier beschriebene Selbstreplikator pREP über mehrere Generationen fortpflanzen, sowohl in vitro als auch in vivo. PURErep ist modular aufgebaut und frei verfügbar, sodass es mit beliebigen Funktionen erweitert werden kann. Neben der DNA gibt es weitere Komponenten, die zum Selbsterhalt einer Zelle vermehrt werden müssen. Es konnte gezeigt werden, dass PURErep die simultane Co-Expression mehrerer seiner Proteinkomponenten ermöglicht. Diese Faktoren waren in der Lage sich aktiv an der Selbst-Regeneration des Systems beteiligen, was einen wichtigen Schritt in Richtung biochemischer Autonomie darstellt. Weiterhin wurden Möglichkeiten zur Selbstreplikation des komplexen Ribosoms erforscht, einem wesentlichen Bestandteil des Translationsapparates. Die de novo Synthese und Assemblierung solcher Ribosomen wird eine entscheidende Rolle für zukünftige Entwicklungen spielen. Ein weiteres Merkmal von Zellen stellt ihre Hülle dar, die Zellmembran. Eine von Grund auf neu geschaffene Minimalzelle müsste in der Lage sein, eine ähnliche Hülle selbst zu produzieren. Es wurde ein effizientes Konzept zur Selbst-Verkapselung des pREP Replikators entwickelt, welches vollkommen ohne zusätzlichen Energiebedarf auskommt. Es konnte gezeigt werden, dass diese sogenannten DNA-Nanoflowers Kernstrukturen bildeten und sich über Generation hinweg vermehren können. Insgesamt dienen die in dieser Arbeit dargelegten Entwürfe der Weiterentwicklung unabhängiger Selbstreplikatoren, welche vielleicht in der Lage sein werden eines Tages natürliche Zellen zu imitieren.The holy grail of bottom-up synthetic biology is the creation of a minimal cell capable of autonomous self-replication and open-ended Darwinian evolution. Reconstituting molecular biology’s central dogma, the implementation of genetic information via transcription-translation, is already feasible in vitro. Yet coupling this process to a DNA self-replication system has so far been limited to only a few kilobases (kbp), a far cry from the proposed 113 kbp proposed for a minimal cell. This work presents the development of a transcription-translation coupled DNA replication platform, called PURErep, which is capable of replicating DNA genomes approaching the proposed size of a minimal cell. As an important step towards Darwinian evolution, the herein described self-replicator pREP can propagate over several generations, both in vitro and in vivo. PURErep is modular and freely available, so that it can be extended with further functions as desired. In addition to DNA, there are other components that need to be replicated for the self-preservation of a cell. It could be shown that PURErep enables the simultaneous co-expression for several of its protein components. These factors were able to actively participate in the self-regeneration of the system, representing an important hallmark of biochemical autonomy. Furthermore, the self-reproduction of the complex ribosome was investigated, an essential component of the translational apparatus. The de novo synthesis and assembly of such ribosomes will be a crucial step towards future developments. Another feature of cells is their envelope, the cell membrane. A minimal cell created from scratch should be able to produce a similar compartment by itself. An efficient concept for the self-compartmentalization of the pREP replicator has been developed, which requires no additional energy and is entirely based on self-organization. It could be shown that these so-called DNA nanoflowers formed nuclear structures and could reproduce over generations. Overall, the designs laid out in this work serve to further develop independent self-replicators, which may one day be able to mimic a natural cell

Novel amphiphilic block copolymers and their self-assembled injectable hydrogels for gene delivery

This work describes the development and investigation of a family of novel smart copolymers as non-viral gene delivery vectors. The copolymers have five blocks, and thus named pentablock, with a central block of a hydrophobic polymer, surrounded by two blocks of a hydrophilic polymer, and capped at each terminal end with cationic polymer blocks, arranged in an architecture to provide temperature and pH sensitivity to the copolymers. They are derived from commercially available triblock Pluronic copolymers. The cationic copolymers can efficiently condense negatively charged plasmid DNA in nanostructures with efficient cellular uptake. The amphiphilic nature of copolymers causes them to exist as micelles in aqueous solutions that help them traverse cellular membranes with minimal cell membrane damage. Intra-cellular trafficking of copolymer/DNA complexes revealed that they are up-taken by the cells predominately via endocytosis and are able to deliver the ferried gene into the nuclei. The copolymers efficiently protect the condensed DNA against degradation by nucleases while their protonation capability at low pH assists them in escape from endosomal vesicles into the cytoplasm. The efficiency of the copolymers to deliver condensed DNA into the cells in vitro was comparable to the commercially available polymeric transfection vectors, and they were also found to be significantly less cytotoxic. Adding non-ionic Pluronic copolymers to the formulation of pentablock copolymer/DNA complexes sterically shielded their surface charge and protected them against aggregation with serum proteins. These stabilized formulations were able to retain their ability to transfect cells even in complete growth media supplemented with serum proteins, warranting efficient transfection efficiency in an in vivo application. The amphiphilic nature of copolymers further permits copolymer/DNA complexes to form thermo-reversible hydrogels at physiological temperatures. At concentrations above 15 wt%, copolymer/DNA complexes existed as solutions at room temperature and formed elastic hydrogels at 37°C that dissolved over seven days in excess buffers to release colloidally stable polyplexes. The system thus permits an injectable aqueous pharmaceutical preparation at room temperature that can be injected subcutaneously in tissues/cavities to form a localized depot in situ, which provides a long-term sustained release of therapeutic genes well protected inside the copolymer/DNA complexes

Engineering morphogenesis of Marchantia polymorpha gemmae

Morphogenesis is an apparent yet complex process: emergence of a plant shape is the result of an intricate interplay between genetic regulation, cell physiology and mechanical processes at the tissue scale. Morphogenesis spans three levels of biological organisation, forming a nested complex system. Genes, which form the lowest level of the system, are arranged into networks that control properties of cells. Cells, which form the middle level of the system, are arranged into geometric networks, and their mechanical and chemical interactions give rise to the morphology of a whole organism. Therefore, the study of plant morphogenesis relies on understanding how genetically-driven cellular interactions influence the formation of a plant shape. Clonal propagules of Marchantia polymorpha (Marchantia), also known as gemmae, are an attractive system for studying these interactions. Gemmae are small, have a simple disk-like shape and are resilient to environmental conditions. As such, they are well-suited for fluorescent microscopy and the collection of gene expression data. These features create an opportunity to study the processes of gemma morphogenesis at tissue, cellular and genetic levels through fluorescent microscopy in a single assay. In order to enable the engineering of morphogenesis in Marchantia gemmae, tools and frameworks for obtaining, storing and analysing the observations from the three levels of biological organisation should be put into place. The work presented in this thesis focuses on the development of such tools and their application in studying the role of phytohormone auxin in Marchantia development. I introduced novel sample preparation and time-lapse imaging assays for Marchantia gemmae along with image- processing methods for the estimation of tissue expansion rates with sub-cellular resolution. These methods allowed me to hypothesise a mechanism behind the regulation of cell proliferation in Marchantia and the role auxin plays in controlling this process. Together with Bernardo Pollak, I developed MarpoDB, a gene-centric representation of the Marchantia genome that enables the search and preparation of Marchantia genetic parts for assembly into synthetic DNA constructs. I used MarpoDB to extract parts and build fluorescent reporters, providing proxies for auxin biosynthesis and signalling. The reporters were then introduced into the Marchantia genome, and the gemmae of the transgenic lines were imaged to estimate the average patterns of the reporters’ expression. The collected patterns were then overlaid on the patterns of relative tissue expansion to validate the proposed role of auxin as an inhibitor of cell proliferation and the mechanism behind its transport in Marchantia.BBSR

Novel strategies for targeting botulinum neurotoxin-based therapeutics to neuronal populations involved in pain sensation

Botulinum neurotoxin serotype A (BoNT/A) is a bacterial di-chain protein containing a protease light-chain which cleaves synaptosomal-associated protein of Mr = 25 k (SNAP-25), a protein essential for neuronal exocytosis; this produces prolonged, but ultimately reversible, abolishment of neurotransmitter release. The retargeting of a BoNT/A-derivative to sensory neurons involved in heightened pain sensation is a potential therapeutic strategy for the treatment chronic pain. The development of such a therapeutic is, however, a complex multi-step process dependent on the identification of suitable cell-surface target receptors, functional expression of appropriate targeting ligands, and the employment of an effective means of conjugating the latter to a binding domain-deficient BoNT/A core-therapeutic. An extensive literature research was conducted which identified both transient receptor potential vallinoid 1 (TRPV1) and tyrosine kinase A (TrkA) as promising target candidates and, furthermore, a spider-venom peptide (double-knot toxin) and nerve-growth factor (NGF) as respective means by which to target them. Two published approaches for the conjugation of targeting ligands to the core-therapeutic were initially assessed: conventional protein fusion and “protein stapling” technology. Comprehensive investigation of these strategies highlighted relative advantages and deficiencies; hence, an additional novel method was developed, based on the binding of S. aureus protein A to immunoglobulin G (IgG) antibodies. This latter strategy was optimized to provide a means to couple the core-therapeutic to either antibodies raised against TrkA, or a recombinantly expressed fusion of NGF and fragment crystallizable (Fc) of rabbit IgG. These approaches both culminated in detectable delivery of the protease into target cells, demonstrating the versatility of this innovative technology. However, NGF produced considerably more efficient protease delivery than its IgG counterpart, thereby, highlighting that for effective toxin retargeting the biological activity of the employed ligand is paramount. Consequently, NGF-mediated targeting via TrkA represents a potentially viable strategy for the intracellular delivery of engineered BoNT/A-based pain therapeutics

6S RNAs in Bacillus subtilis. Investigation of biological function and molecular mechanism

6S-1 RNA (bsrA) is a 190 nucleotides small non-coding RNA found in Bacillus subtilis, which binds to the housekeeping sigma A (σA) RNA polymerase holoenzyme (RNAP). 6S-1 RNA levels peak in stationary phase where the RNA supports adaptation to nutrient scarcity (or other stresses) and prepares cells for an instant outgrowth from stationary phase upon nutrient re-supply. In addition to inhibiting cellular transcription by binding to σA-RNAP, 6S-1 RNA also serves as a template for this enzyme to direct the synthesis of small product RNAs (pRNAs) in an RNA-dependent RNA polymerization reaction. These pRNAs, if long enough (~ 14-mer), are able to rearrange the structure of 6S-1 RNA such that the polymerase is released from the 6S-1 RNA and transcription of DNA promoters can be resumed. The kinetics of the process and key nucleotides involved in release and rearrangement of 6S-1 are unknown. Towards a deeper understanding of this process, we introduce substitutions/deletions along the secondary structure of 6S-1 RNA and analyse the impact thereof on the kinetics of 6S-1 rearrangement. In our study, we found that mutations C44/45 in the 5’-Central Bulge (CB) weaken 6S-1 RNA:σA-RNAP ground state binding two- to threefold while stabilizing the Central Bulge Collapse Helix (CBC) and shifting the pRNA length pattern to shorter pRNAs. A 6S-1 RNA variant with a weakened helix P2 and CBC stabilized by the the C44/45 mutation was more effectively rearranged and released from the enzyme. Our mutational analysis also revealed that formation of a second short hairpin in 3’-CB is detrimental to 6S-1 RNA release. From our results we infer that formation of the CBC subtly supports pRNA-induced 6S-1 RNA rearrangement and release. Additionally, truncated variants of 6S-1 RNA, solely consisting of the CB flanked by two short helical arms, can still traverse the functional 6S RNA cycle in vitro, despite decreased σA-RNAP affinity. This indicates that the ‘- 35-like’ region is not strictly essential for 6S-1 RNA function, at least in B. subtilis. We also showed that pRNA-isosequential locked nucleic acids (pLNAs) as short as 6 nt were able to induce 6S-1 RNA rearrangement and disrupt/prevent complex formation with σA-RNAP. Additionally, we analyzed the spatial dimensions of free 6S-1 RNA versus 6S-1:pLNA complexes by atomic force microscopy, revealing that 6S-1:pRNA hybrid structures, on average, adopt a more bent/constrained structure than 6S-1 RNA alone. The pLNA studies help us interpret the 6S-1 rearrangement to be a progressive process with intermediate states, ultimately leading to a maximally constrained and bent structure. Finally, we observed that the pRNA-mediated rearrangement of 6S-1 RNA and its release from σA-RNAP largely accelerates at NTP concentrations > 40 μM, which supports the role of 6S-1 RNA during nutrient re-supply. In gel assays, the regulatory 6S-1 and 6S-2 RNAs of Bacillus subtilis bind to the housekeeping RNA polymerase holoenzyme (σA-RNAP) with submicromolar affinity. We observed copurification of endogenous 6S RNAs from a published B. subtilis strain expressing a His-tagged RNAP. Such 6S RNA contaminations in σA-RNAP preparations reduce the fraction of enzymes that are accessible for binding to DNA promoters. In addition, this leads to background RNA synthesis by σA-RNAP utilizing copurified 6S RNA as template for the synthesis of short abortive transcripts termed product RNAs (pRNAs). To avoid this problem, we constructed a B. subtilis strain expressing His-tagged RNAP but carrying deletions of the two 6S RNA genes. The His-tagged, 6S RNA-free σA-RNAP holoenzyme can be prepared with sufficient purity and activity by a single affinity step. We also reported expression and separate purification of B. subtilis σA that can be added to the His-tagged RNAP to maximize the amount of holoenzyme and, by inference, in vitro transcription activity. In order to test our hypothesis that the regulatory role of 6S RNAs may be particularly important under natural, constantly changing environmental conditions, we constructed 6S RNA deletion mutants of the undomesticated B. subtilis wild-type strain NCIB 3610. We observed a strong phenotype under stress conditions in which the Δ6S-2 RNA strain exhibited retarded swarming activity and earlier spore formation. In contrast, the Δ6S-1&2 double knockout strain exhibited lesser spore formation than the wild-type. Additionally, we could show that 6S mutants grown under nutrient rich conditions revealed no strong phenotype. Our data suggests that both 6S RNAs contributes to the fitness of B. subtilis under the unsteady and temporarily harsh conditions encountered in natural habitats

Light-activated ATP synthesis in droplet networks

The fabrication of synthetic tissues that mimic the complex collective functionality of living tissues is the central objective of synthetic biology. A key step in this endeavour is the construction of an energy production system. Energised synthetic tissues could produce therapeutic oligonucleotides and proteins on demand, or transport substrates to modulate the behaviour of interfaced living tissues. This thesis describes the assembly of synthetic tissues with an encapsulated light- activated adenosine triphosphate (ATP) production system. The synthetic tissues comprised droplet networks, assemblies of aqueous droplets in a lipid-containing oil separated and stabilised by droplet interface bilayers (DIBs). Two membrane proteins were reconstituted into proteoliposomes to create a synthetic organelle able to generate ATP on demand: a modified light-activated proton pump, proteorhodopsin-mCherry and E. coli F1FO ATP synthase. An ATP synthesis rate of 53.2 nmol ATP.min-1.mgF1FO-1 was achieved in bulk studies. Transfer of the energy generating proteoliposomes to pL-sized droplets created a system capable of light-activated transcription of a ribonucleic acid (RNA) aptamer, which was detected with a novel hydrophilic analogue of the cognate fluorophore, DFHBI. To facilitate small molecule communication between compartments of the synthetic tissue, DIBs were permeabilised with a pore forming protein, alpha-hemolysin (aHL). ATP synthesised under control by light could then be transferred to and used in a neighbouring compartment