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Developing Genetic Tools in Methanococcus Maripaludis Developing Genetic Tools in Methanococcus maripaludis Nathan Innard MSc by Research University of York Biology January 2018 Abstract Highly sophisticated genetic tools have been deployed in many eukaryotes and bacteria, yet those available in the third domain of life: the archaea, remain comparatively limited. The constraints this imposes must be overcome if the unique capabilities of this domain are to be tapped. One biological pathway found solely within members of the archaea is methanogenesis: the conversion of simple molecules into methane. The manipulation of methane production through genetic modification of methanogens is highly desirable, since in addition to its potential use as a biofuel, methane contributes significantly to the greenhouse effect. Among the methanogens, Methanococcus maripaludis (M. maripaludis) is one of the best developed as a model, however many of its available genetic tools are outdated. The CRISPR/Cas9 system is a relatively recently developed and highly versatile genetic technique, and at the outset of this work had not been deployed in any archaeal system. The aim of this project was therefore to provide a proof of principle that the CRISPR/Cas9 system could be deployed successfully in this organism. The experimental approach selected was a plasmid invader assay, in which the activity of the system could be demonstrated by Cas9 mediated plasmid destruction resulting in reduced M. maripaludis cell viability. A scheme by which this assay could be conducted using existing genetic tools in two different M. maripaludis strains was designed, and the full series of required plasmids was produced. Attempts to use these plasmids to produce the M. maripaludis strains required for the invader assay were unsuccessful, and it could therefore not be carried out. However, it is anticipated that the assay system designed and plasmids produced here should enable the rapid testing of this system were this work continued, which should enable the addition of the CRISPR/Cas9 system to the M. maripaludis genetic toolbox. 2 Table of Contents Abstract ............................................................................................................................... 2 Table of Contents ................................................................................................................ 3 List of Figures ..................................................................................................................... 9 List of Tables .................................................................................................................... 11 Acknowledgements ........................................................................................................... 12 Declaration ........................................................................................................................ 13 Chapter 1: General Introduction ....................................................................................... 14 1.1.1 Genetic Tools in the Archaea ........................................................................... 14 1.1.2 Methanogenesis and Methanogens .................................................................. 14 1.1.3 M. maripaludis ................................................................................................. 14 1.1.4 Existing Genetic Tools in M. maripaludis ....................................................... 15 1.1.4.1 Chromosomal modification ....................................................................... 15 1.1.4.2 Shuttle vectors ........................................................................................... 16 1.1.4.3 Reporter genes........................................................................................... 17 1.1.4.4 Gene expression ........................................................................................ 17 1.1.5 The CRISPR/Cas System ................................................................................. 18 1.1.5.1 CRISPR/Cas as a genetic tool ................................................................... 18 1.1.5.2 CRISPR/Cas in archaea ............................................................................ 19 1.1.5.3 Deploying the CRISPR/Cas9 system in M. maripaludis .......................... 19 1.1.6 The Plasmid Invader Assay .............................................................................. 20 1.1.7 Project Aims ..................................................................................................... 23 Chapter 2: Materials and Methods .................................................................................... 24 2.1.1 Molecular Work ............................................................................................... 24 2.1.1.1 Agarose gel electrophoresis ...................................................................... 24 2.1.1.2 Resuspending gBlocks .............................................................................. 24 2.1.1.3 Restriction digestion ................................................................................. 24 2.1.1.4 PCR purification ....................................................................................... 24 3 2.1.1.5 Gel extraction ............................................................................................ 25 2.1.1.6 Plasmid dephosphorylation ....................................................................... 25 2.1.1.7 Ligation ..................................................................................................... 25 2.1.1.8 Golden Gate reactions ............................................................................... 25 2.1.1.9 Transformation of ligation products/plasmids into E. coli ........................ 26 2.1.1.10 Colony PCR ............................................................................................ 26 2.1.1.11 Plasmid DNA isolation (from E. coli)..................................................... 27 2.1.1.12 Generating chemically competent cells (E. coli DH5α) ......................... 27 2.1.1.13 E. coli culturing ....................................................................................... 27 2.1.1.14 Long term plasmid storage ...................................................................... 28 2.1.1.15 Sequencing reactions............................................................................... 28 2.1.2 Aerobic Media and Antibiotics ........................................................................ 28 2.1.2.1 LB medium ............................................................................................... 28 2.1.2.2 LB agar plates ........................................................................................... 28 2.1.2.3 Ampicillin and kanamycin ........................................................................ 28 2.1.2.4 IPTG .......................................................................................................... 29 2.1.2.5 X-gal ......................................................................................................... 29 2.1.3 M. maripaludis ................................................................................................. 29 2.1.3.1 M. maripaludis culturing........................................................................... 29 2.1.3.2 McCas medium ......................................................................................... 29 2.1.3.3 N-free trace minerals (1000x) ................................................................... 30 2.1.3.4 Vitamin solution (100x) ............................................................................ 31 2.1.3.5 N-free general salts solution ..................................................................... 31 2.1.3.6 FeSO₄ solution .......................................................................................... 32 2.1.3.7 K₂HPO₄ solution ....................................................................................... 32 2.1.3.8 Resazurin solution ..................................................................................... 32 2.1.3.9 Transformation of plasmids into M. maripaludis ..................................... 32 2.1.3.10 M. maripaludis outgrowth and selection following transformation ........ 33 4 2.1.4 Anaerobic Antibiotics and Solutions ............................................................... 33 2.1.4.1 Reducing agent .......................................................................................... 33 2.1.4.2 Transformation buffer ............................................................................... 34 2.1.4.3 PEG solution ............................................................................................. 34 2.1.4.4 Na₂S solution ............................................................................................. 35 2.1.4.5 Neomycin .................................................................................................. 35 2.1.5 In Silico Methods ............................................................................................. 35 2.1.5.1 Codon optimisation ................................................................................... 35 2.1.5.2 gRNA design ............................................................................................. 35 2.1.5.3 Random sequence generation .................................................................... 36 2.1.5.4 Checking complexity IDT gblocks ..........................................................
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