Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge- and column-based protocols require specialised equipment, often use toxic reagents, and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA, RNA and total nucleic acid (TNA) from a range of bacterial, animal, plant, environmental and synthetic sources. We also provide a bead-based protocol for bisulfite conversion and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility, and extreme cost-effectiveness of using magnetic beads. These open-source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement.

Biosynthesis of vitamins is fundamental to malaria parasites. Plasmodia synthesize the active form of vitamin B(6) (pyridoxal 5'-phosphate, PLP) using a PLP synthase complex. The EM analysis shown here reveals a random association pattern of up to 12 Pdx2 glutaminase subunits to the dodecameric Pdx1 core complex. Interestingly, Plasmodium falciparum PLP synthase organizes in fibers. The crystal structure shows differences in complex formation to bacterial orthologs as interface variations. Alternative positioning of an α helix distinguishes an open conformation from a closed state when the enzyme binds substrate. The pentose substrate is covalently attached through its C1 and forms a Schiff base with Lys84. Ammonia transfer between Pdx2 glutaminase and Pdx1 active sites is regulated by a transient tunnel. The mutagenesis analysis allows defining the requirement for conservation of critical methionines, whereas there is also plasticity in ammonia tunnel construction as seen from comparison across different species.

Acinetobacter baumannii is a Gram-negative pathogen that causes a multitude of nosocomial infections. The Acinetobacter trimeric autotransporter adhesin (Ata) belongs to the superfamily of trimeric autotransporter adhesins which are important virulence factors in many Gram-negative species. Phylogenetic profiling revealed that ata is present in 78% of all sequenced A. baumannii isolates but only in 2% of the closely related species A. calcoaceticus and A. pittii. Employing a markerless ata deletion mutant of A. baumannii ATCC 19606 we show that adhesion to and invasion into human endothelial and epithelial cells depend on Ata. Infection of primary human umbilical cord vein endothelial cells (HUVECs) with A. baumannii led to the secretion of interleukin (IL)-6 and IL-8 in a time- and Ata-dependent manner. Furthermore, infection of HUVECs by WT A. baumannii was associated with higher rates of apoptosis via activation of caspases-3 and caspase-7, but not necrosis, in comparison to ∆ata. Ata deletion mutants were furthermore attenuated in their ability to kill larvae of Galleria mellonella and to survive in larvae when injected at sublethal doses. This indicates that Ata is an important multifunctional virulence factor in A. baumannii that mediates adhesion and invasion, induces apoptosis and contributes to pathogenicity in vivo.

19Current molecular biology laboratories rely heavily on the purification and manipulation of 20 nucleic acids. Yet, commonly used centrifuge-and column-based protocols require 21 specialised equipment, often use toxic reagents and are not economically scalable or practical 22 to use in a high-throughput manner. Although it has been known for some time that magnetic 23 beads can provide an elegant answer to these issues, the development of open-source 24 protocols based on beads has been limited. In this article, we provide step-by-step 25 instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols 26 for their use in the high-throughput purification of plasmids, genomic DNA and total RNA from 27 different sources, as well as environmental TNA and PCR amplicons. We also provide a bead-28 based protocol for bisulfite conversion, and size selection of DNA and RNA fragments. 29Comparison to other methods highlights the capability, versatility and extreme cost-30 effectiveness of using magnetic beads. These open source protocols and the associated 31 webpage (https://bomb.bio) can serve as a platform for further protocol customisation and 32 community engagement. 33 3 Abbreviations 34 BOMB: Bio-On-Magnetic-Beads 35 SPRI: Solid-Phase Reversible Immobilisation 36 MNP: magnetic nanoparticle 37 38The authors would like to thank all members of the Jurkowski and Hore laboratories for 456helping to optimise and test the BOMB protocols. We are also indebted to Ken Wyber (Otago 457Polytechnic) for help with laser cutting magnetic plates. We are grateful to Dr. Renata 458Jurkowska for critical reading of the manuscript. We would like to thank the wider research 459 community for offering unpublished information and resources concerning magnetic bead 460 22 preparation and utility, in particular, Dr Ethan Ford, Dr James Hadfield, Dr Brant Faircloth, Dr 461 Nadin Rohland and associated authors. 462 Author's contribution 463 The idea was conceived by TPJ and TH. Protocol setup and optimisation was led by PO, PS, 464 DB, TPJ and TH, with contributions from SH, JF, VM, LS, VJS, G-JJ and FvM. Laser cutting 465 designs were contributed by SRH. The electron microscope analysis was done by KH. The 466 website and its content were created by TM, PS, PO, TPJ and TH. The manuscript was written 467 by TPJ, TH, PS and PO. All authors contributed to the editing of the manuscript and approved 468 its final version. 469