A unified platform for experimental and computational biology

PhD ThesisIn natural sciences, the correct engineering of a system’s chemical, biological and physical properties may allow it to sustain life. Bioengineering cells is probably one of the most complex challenges of biological research; yet, the little we do know about the nature of life is sufficient to guide scientific research, and to explore the elements beyond the apparent simple proliferation of living cells. Although Mendel first characterised the concept of genetic heredity over 150 years ago, we only recently became able to perform tailored genetic modification of living organisms. The development of digital technologies, in particular, has positively influenced the quality and reproducibility of experimental results emerging from biological assays. However, the use of any equipment may require the need for a specific expertise in order to perform a given experimental procedure. Therefore, multidisciplinary research can bring benefits to all fields of science by helping the development of analytical methods that cross the boundaries of individual disciplines. This emerges as a systematic view of scientific problems, and relies on the adequation and integration of results from different research areas. Nevertheless, there is a complex interface between hard sciences that often creates a gap between experimental and theoretical models. In this thesis, we explored synthetic biology approaches and created a unified platform to fill this gap. We propose the first barcoding platform (Bac2code) that allows the identification and the tracking of bacterial strains. In order to facilitate communication between researchers, we developed a barcode system in DNA that physically links bacteria to their genetic description. We designed DNA barcodes as bioorthogonal elements, elaborated a universal cloning strategy to integrate these sequences in Gram-negative and Gram-positive bacteria, and demonstrated their stability over time. Through a generic protocol, any barcoded strain can later be identified via a single sequencing read. With the engineering of a synthetic circuit library, we built a biorepository of genetic constructs for our barcoding platform. These biological devices were optimised based on the closest achievable interface between experimental biology and viii computational results. Following their characterisation, and in the context of intercellular communication, we studied the behaviour of small cohorts of bioengineered cells at the microscale in microfluidics. We pushed the biological and physical boundaries of engineering techniques to the maximum, in order to observe physiological changes between bacteria separated by distances down to 20µm. However, we also showed that we reached a technological barrier, where even the use of nanoscale features was found insufficient to maintain cells isolated under high cellular density. Yet, microfluidics remains a remarkable technology, and we propose the expansion of barcoding methods to automated systems, which would allow serial barcode integration and documentation retrieval at any one time. Here, we developed and tested a barcoding method to ensure the cohesion of experimental and computational biology resources. We demonstrated its use by the in vitro assembly and the in vivo or in silico characterisation of a series of genetic circuits via different techniques. The research output of this thesis is realised as a step forward in interdisciplinary studies, and is now being adapted to reach a larger community of users as a startup companyEngineering and Physical Sciences Research Council and Newcastle University’s School of Computing Science

DNA replication initiation in Bacillus subtilis: Structural and functional characterization of the essential DnaA-DnaD interaction

© 2018 The Author(s). The homotetrameric DnaD protein is essential in low G+C content gram positive bacteria and is involved in replication initiation at oriC and re-start of collapsed replication forks. It interacts with the ubiquitously conserved bacterial master replication initiation protein DnaA at the oriC but structural and functional details of this interaction are lacking, thus contributing to our incomplete understanding of the molecular details that underpin replication initiation in bacteria. DnaD comprises N-terminal (DDBH1) and C-terminal (DDBH2) domains, with contradicting bacterial two-hybrid and yeast two-hybrid studies suggesting that either the former or the latter interact with DnaA, respectively. Using Nuclear Magnetic Resonance (NMR) we showed that both DDBH1 and DDBH2 interact with the N-terminal domain I of DnaA and studied the DDBH2 interaction in structural detail. We revealed two families of conformations for the DDBH2-DnaA domain I complex and showed that the DnaA-interaction patch of DnaD is distinct from the DNA-interaction patch, suggesting that DnaD can bind simultaneously DNA and DnaA. Using sensitive single-molecule FRET techniques we revealed that DnaD remodels DnaA-DNA filaments consistent with stretching and/or untwisting. Furthermore, the DNA binding activity of DnaD is redundant for this filament remodelling. This in turn suggests that DnaA and DnaD are working collaboratively in the oriC to locally melt the DNA duplex during replication initiation

Detailed Monte-Carlo characterization of a Faraday cup for proton therapy.

BACKGROUND Experiments with ultra-high dose rates in proton therapy are of increasing interest for potential treatment benefits. The Faraday Cup (FC) is an important detector for the dosimetry of such ultra-high dose rate beams. So far, there is no consensus on the optimal design of a FC, or on the influence of beam properties and magnetic fields on shielding of the FC from secondary charged particles. PURPOSE To perform detailed Monte Carlo simulations of a Faraday cup to identify and quantify all the charge contributions from primary protons and secondary particles that modify the efficiency of the FC response as a function of a magnetic field employed to improve the detector's reading. METHODS In this paper, a Monte Carlo (MC) approach was used to investigate the Paul Scherrer Institute (PSI) FC and quantify contributions of charged particles to its signal for beam energies of 70, 150, and 228 MeV and magnetic fields between 0 and 25 mT. Finally, we compared our MC simulations to measurements of the response of the PSI FC. RESULTS For maximum magnetic fields, the efficiency (signal of the FC normalized to charged delivered by protons) of the PSI FC varied between 99.97% and 100.22% for the lowest and highest beam energy. We have shown that this beam energy-dependence is mainly caused by contributions of secondary charged particles, which cannot be fully suppressed by the magnetic field. Additionally, it has been demonstrated that these contributions persist, making the FC efficiency beam energy dependent for fields up to 250 mT, posing inevitable limits on the accuracy of FC measurements if not corrected. In particular, we have identified a so far unreported loss of electrons via the outer surfaces of the absorber block and show the energy distributions of secondary electrons ejected from the vacuum window (VW) (up to several hundred keV), together with electrons ejected from the absorber block (up to several MeV). Even though, in general, simulations and measurements were well in agreement, the limitation of the current MC calculations to produce secondary electrons below 990 eV posed a limit in the efficiency simulations in the absence of a magnetic field as compared to the experimental data. CONCLUSION TOPAS-based MC simulations allowed to identify various and previously unreported contributions to the FC signal, which are likely to be present in other FC designs. Estimating the beam energy dependence of the PSI FC for additional beam energies could allow for the implementation of an energy-dependent correction factor to the signal. Dose estimates, based on accurate measurements of the number of delivered protons, provided a valid instrument to challenge the dose determined by reference ionization chambers, not only at ultra-high dose rates but also at conventional dose rates

Linking Engineered Cells to Their Digital Twins: A Version Control System for Strain Engineering

As DNA sequencing and synthesis become cheaper and more easily accessible, the scale and complexity of biological engineering projects is set to grow. Yet, although there is an accelerating convergence between biotechnology and digital technology, a deficit in software and laboratory techniques diminishes the ability to make biotechnology more agile, reproducible, and transparent while, at the same time, limiting the security and safety of synthetic biology constructs. To partially address some of these problems, this paper presents an approach for physically linking engineered cells to their digital footprint—we called it digital twinning. This enables the tracking of the entire engineering history of a cell line in a specialized version control system for collaborative strain engineering via simple barcoding protocols.J.T.L., C.W., J.K., and N.K. were supported by the UK Engineering and Physical Research Council under project “Synthetic Portabolomics: Leading the way at the crossroads of the Digital and the Bio Economies (EP/N031962/1)”. N.K. is funded by a Royal Academy of Engineering Chair in Emerging Technology award. V.d.L. was supported by project “BioRoboost (H2020-NMBP-BIO-CSA-2018, grant agreement N820699)”

S. M. L'Impératrice des Français Entourée des Dames de sa Cour : [estampe]

Référence bibliographique : De Vinck, 16671Appartient à l’ensemble documentaire : Est19Vinc

SirA Inhibits the essential DnaA : DnaD Interaction to block helicase recruitment during Bacillus subtilis sporulation

Bidirectional DNA replication from a chromosome origin requires the asymmetric loading of two helicases, one for each replisome. Our understanding of the molecular mechanisms underpinning helicase loading at bacterial chromosome origins is incomplete. Here we report both positive and negative mechanisms for directing helicase recruitment in the model organism Bacillus subtilis. Systematic characterization of the essential initiation protein DnaD revealed distinct protein interfaces required for homo-oligomerization, interaction with the master initiator protein DnaA, and interaction with the helicase co-loader protein DnaB. Informed by these properties of DnaD, we went on to find that the developmentally expressed repressor of DNA replication initiation, SirA, blocks the interaction between DnaD with DnaA, thereby inhibiting helicase recruitment to the origin during sporulation. These results advance our understanding of the mechanisms underpinning DNA replication initiation in B. subtilis, as well as guiding the search for essential cellular activities to target for antimicrobial drug design

The DNA replication initiation protein DnaD recognises a specific strand of the Bacillus subtilis chromosome origin

Genome replication is a fundamental biological activity shared by all organisms. Chromosomal replication proceeds bidirectionally from origins, requiring the loading of two helicases, one for each replisome. However, the molecular mechanisms underpinning helicase loading at bacterial chromosome origins (oriC) are unclear. Here we investigated the essential DNA replication initiation protein DnaD in the model organism Bacillus subtilis. A set of DnaD residues required for ssDNA binding was identified, and photo-crosslinking revealed that this ssDNA binding region interacts preferentially with one strand of oriC. Biochemical and genetic data support the model that DnaD recognizes a new single-stranded DNA (ssDNA) motif located in oriC, the DnaD Recognition Element (DRE). Considered with single particle cryo-electron microscopy (cryo-EM) imaging of DnaD, we propose that the location of the DRE within oriC orchestrates strand-specific recruitment of helicase during DNA replication initiation. These findings significantly advance our mechanistic understanding of bidirectional replication from a bacterial chromosome origin. [Abstract copyright: © The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.