Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways

Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.European Research Council ERC2014-ADG669898 TARLOOPMinisterio de Economía y Competitividad BFU2016-75058-PJunta de Andalucía BIO123

Modified polymer surfaces with antimicrobial properties

Biofilms are a multispecies community of bacterial cells that can establish over time on biotic and abiotic surfaces. Biofilm growth and maturation on industrial polymer surfaces poses a serious challenge to public health, industrial manufacturing, oil and gas industries, food production and healthcare. As a result, there has been increasing interest in the development of easily synthesised low-cost antimicrobial polymer surfaces and coatings to inhibit, and/or perturb bacterial attachment and biofilm viability. This thesis was focused on modifying the surface of low cost commercial polymers, with high-density polyethylene (HDPE) being used as the model substrate. Surface modifications were performed using a range of approaches. These included different surface oxidative treatment methods in order to evaluate their intrinsic antimicrobial performance, and as an approach for improving the adhesion for other antimicrobial coatings being investigated. Tin-dioxide thin-films and low/medium molecular weight chitosan-zinc oxide nanocomposite coatings were also investigated and examined as possible antimicrobial surface coating technologies for polymer materials, primarily for HDPE. A key aim of this work was to evaluate microbial adhesion and biofilm viability at the initial stages of biofilm maturation (early-stage biofilm), with successful antimicrobial approaches then being further evaluated against a more mature biofilm system. This study confirms that the sulfuric-chromic acid surface oxidisation of HDPE and polypropylene surfaces showed marked reductions in bacterial adhesion and viability, with a decrease in attachment seen for both Gram-negative and positive bacterial species with increasing surface oxidisation treatment time periods from 0 to 60 minutes. In particular, extended sulfuric-chromic acid oxidisation treatment times of 30 and 60 minutes significantly compromised microbial viability, such that there was no agar growth of early-stage biofilm of Gram-negative organisms recovered from these surfaces when cultured, even after 3 months of aging this polymer material under standard laboratory conditions. The mechanism for this activity was ascribed to the formation of chromium esters which are known reactive intermediates in the sulfuric-chromic acid oxidisation of HDPE, and their surface presence was further confirmed using TOF-SIMS. Tin-dioxide nanoparticle coatings showed a reduction in the earlystage biofilm formation of Pseudomonas aeruginosa, irrespective of nanoparticle size of the tin-dioxide coating used, which was also observed when coatings were deployed on polypropylene and polycarbonate materials. A range of chitosan-zinc oxide composite thin-films were examined which exhibited minimal viability reduction against Escherichia coli early-stage biofilms. Therefore, this study could not affirm a significant antimicrobial performance for the chitosan-ZnO nanocomposite coatings on oxidised HDPE materials which was attributed to the loss of a key antimicrobial active functional group in chitosan, the primary amines. However, optimisation of the chitosan-ZnO formulations revealed some key trends, where increased concentrations of low molecular weight chitosan in the presence of zinc-oxide in 1% acetic acid, resulted in a 2.8-log reduction of Escherichia coli due to the presence of primary amine substituents, confirmed by a ninhydrin assay. Experiments performed against mature biofilms prepared in a 24-hour aqueous environment found no antimicrobial performance on all substrates and coatings tested.Open Acces

Proceedings of Abstracts, School of Physics, Engineering and Computer Science Research Conference 2022

© 2022 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/. Plenary by Prof. Timothy Foat, ‘Indoor dispersion at Dstl and its recent application to COVID-19 transmission’ is © Crown copyright (2022), Dstl. This material is licensed under the terms of the Open Government Licence except where otherwise stated. To view this licence, visit http://www.nationalarchives.gov.uk/doc/open-government-licence/version/3 or write to the Information Policy Team, The National Archives, Kew, London TW9 4DU, or email: [email protected] present proceedings record the abstracts submitted and accepted for presentation at SPECS 2022, the second edition of the School of Physics, Engineering and Computer Science Research Conference that took place online, the 12th April 2022

Development and integration of chemical imaging methods for applications in biomedical and pharmaceutical research

Imaging of biomolecules in biological substrates by mass spectrometry or spectroscopic imaging techniques plays a major role in biomedical, clinical, and pharmaceutical research. The work presented in this thesis investigates the capabilities of three imaging techniques, liquid extraction surface analysis (LESA) mass spectrometry imaging (MSI), matrix assisted laser desorption ionisation (MALDI) MSI and stimulated Raman scattering (SRS) microscopy. A method for combined LESA and MALDI analysis was developed and results provided high resolution imaging of multiple analyte classes (proteins, lipids and small molecule drugs) in thin tissue sections. SRS microscopy was used for the quantitative imaging of MALDI sampling effects and sample preparation, providing insight into fundamental processes of MALDI MS. Multimodal SRS, LESA and MALDI imaging was executed on a single tissue sample revealing the complementarity between the three approaches. Specific challenges for LESA were further explored, namely quantification, improved spatial resolution and alternative biological substrates. A quantitative LESA method based on the production of mimetic tissue models containing stable isotope-labelled proteins was developed. An alternative platform, the Flow-Probe™, with the potential to achieve higher spatial resolution was assessed. Finally, a LESA method for the direct analysis of proteins from live bacterial colonies was developed

Three-dimensional imaging of bacterial microcolonies

Previous research into microbial colonies and biofilms shows a significant gap in our current understanding of how bacterial structures develop. Despite the huge body of research undertaken into the formation, genetic makeup, composition, and optimal growth conditions of colonies, no study has been successful in identifying all individual bacteria in a colony in three-dimensions as a function of time. This lack of bacterial cell lineage in such a simple class of organisms is conspicuous in the light of what is known about other organisms, such as Caenorhabditis elegans [1]. In this thesis I show that using laser scanning confocal microscopy in conjunction with developments in sample preparation and post acquisition image analysis, it is possible to fully reconstruct all individual bacteria within an Escherichia coli (E. coli ) microcolony grown in viscoelastic media. Additionally, I show that by further pushing the resolution of confocal microscopes, commercial systems are capable of extracting three-dimensional information on protein structures inside bacteria at early stages of growth. This thesis is in three parts. The first part shows that by pushing the resolution of a commercial laser scanning confocal microscope system it is possible to achieve single cell resolution of a bacterial colony growing in three dimensions in a viscoelastic medium (agarose) from a seed bacterium. The growth of individual bacteria is examined as the concentration of agarose in the media is altered. Results show there is a nonlinear dependence between the rate of growth of a bacterium and the concentration of the agarose in the media with a peak in growth rate at 3% (weight) concentrations of agarose in M9 media. The second part of this work presents a study of how an initially two-dimensional colony growing between a glass slide and agarose gel suddenly invades the third spatial dimension by buckling. The results show that the cells within the centre of the colony flex and buckle, due to confinement by their neighbours, creating additional layers. Indeed, flexing is not limited to the buckling event but occurs throughout the early growth cycle of a colony. The final part of this thesis shows that by further pushing the resolution of confocal microscopes, commercial systems are capable of extracting three-dimensional information about the temporal evolution of the spatial distribution of the FtsZ septation ring within the cell. As the bacterial colony grows from a seed bacterium to a microcolony, the error in placing the division accurately at the cell centre is seen to increase as the number of bacteria within the colony increases and spatial confinement occurs

Electrically Connecting Bacteria to Nanoparticles for Biotechnological Applications

Combining abiotic photosensitisers such as semiconductor fluorescence emitting nanoparticles – quantum dots (QDs), with non-photosynthetic bacteria ‘in vivo’ presents an intriguing concept into the design of artificial photosynthetic organisms and solar-driven fuel production. Shewanella oneidensis MR-1 (MR-1) is a versatile bacterium concerning respiration, metabolism and biocatalysis, and is a very promising organism for artificial photosynthesis. The bacteria’s synthetic and catalytic abilities, together with their longevity, provide a promising system for bacterial biohydrogen production. MR-1’s hydrogenases are present in the periplasmatic space, and it follows QDs or their electrons will need to enter the periplasm via the Mtr pathway that is responsible for the extracellular electrontransfer ability of MR-1. Firstly, various QDs were tested for their nanotoxicology and further for interaction with MR-1 by fluorescence and electron microscopy. CdTe/CdS/TGA, CdTe/CdS/Cysteamine, commercial negatively charged CdTe and CuIn2S/ZnS/PMAL QD were examined, and it was found that the latter two showed no toxicity for MR-1 as evaluated by a colony-forming units method and a fluorescence viability assay. Only commercial negatively charged CdTe QDs showed good interaction with MR-1. Detailed investigation of the above interaction by transmission electron microscopy showed QDs were placed both inside the cell and close to the membrane. Subsequently, the photoreduction power of QDs was evaluated by the methyl viologen assays with different sacrificial electron donors. It was indeed found that QDs have reduction potential sufficiently low to perform MV photoreduction. As assessed by gas chromatography, CdTe/CdS/TGA and negatively charged CdTe QDs supported hydrogen evolution in Shewanella putrefaciens CN-32. The above results establish a proof of concept for photosynthetic production of biohydrogen by CN-32. Further research should be invested in the use of biocompatible sacrificial electron donors and the development of appropriate bacteria mutants that would help to understand the assisted by QDs hydrogen evolution in this bacterium

Single-Cell Analysis of Microbial Production Strains in Microfluidic Bioreactors

Grünberger A. Single-Cell Analysis of Microbial Production Strains in Microfluidic Bioreactors. Schriften des Forschungszentrums Jülich. Reihe Schlüsseltechnologien / Key Technologies. Vol 114. Jülich: RWTH Aachen; 2015