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.
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
The β-hydroxyacid dehydrogenase from Thermocrinus albus (Ta-βHAD), which catalyzes the NADP +-dependent oxidation of βhydroxyacids, was engineered to accept imines as substrates. The catalytic activity of the proton-donor variant K189D was further increased by the introduction of two nonpolar flanking residues (N192 L, N193 L). Engineering the putative alternative proton donor (D258S) and the gate-keeping residue (F250 A) led to a switched substrate specificity as compared to the single and triple variants. The two most active Ta-βHAD variants were applied to biocatalytic asymmetric reductions of imines at elevated temperatures and enabled enhanced product formation at a reaction temperature of 50°C.
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