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Development of a Combined in Vitro Transcription and Translation System for Rapid Cell-Free Screening of Metagenomic DNA

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Development of a Combined in Vitro Transcription and Translation System for Rapid Cell-Free Screening of Metagenomic DNA Development of a combined in vitro transcription and translation system for rapid cell-free screening of metagenomic DNA Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) at the Department of Biology Subdivision of the Faculty of Mathematics, Informatics and Natural Sciences of the University of Hamburg by Mareike Janus born on 06.02.1989 in Hamburg Genehmigt vom Fachbereich Biologie der Universität Hamburg auf Antrag von Prof. Dr. J. Kehr Weiterer Gutachter der Dissertation: Prof. Dr. S. J. Wittke Tag der Disputation: 19.06.2020 “When life gives you lemons, make lemonade.” Table of Contents List of figures ................................................................................................................................. I List of tables ................................................................................................................................. II Abstract ...................................................................................................................................... VI Introduction ....................................................................................................................... 1 1.1 Metagenomics ..................................................................................................................... 1 1.1.1 Functional screening for new enzymes in metagenomics ..................................... 3 1.1.2 Challenges and limitations ..................................................................................... 4 1.2 Cell-free protein synthesis ................................................................................................... 6 1.2.1 DNA-dependant RNA polymerases for in vitro transcription ................................. 7 1.2.2 In vitro transcription and translation ..................................................................... 9 1.2.3 Existing systems .................................................................................................... 11 1.2.4 Advantages and disadvantages ............................................................................ 12 1.3 Cell-free protein expression for function-based metagenomics ....................................... 13 1.4 In vitro compartmentalization ........................................................................................... 15 Aim of this work ............................................................................................................... 17 Material and Methods ...................................................................................................... 19 3.1 Bacterial strains ................................................................................................................. 19 3.2 Vectors and constructs ...................................................................................................... 20 3.3 Primers ............................................................................................................................... 21 3.4 Culture media and supplements ........................................................................................ 22 3.5 Culture conditions .............................................................................................................. 23 3.5.1 Cultivation of bacteria .......................................................................................... 23 3.5.2 Strain maintenance .............................................................................................. 24 3.5.3 Measurement of optical density (OD) .................................................................. 24 3.5.4 Cell harvesting ...................................................................................................... 24 3.6 Preparation of cell extracts for in vitro translation ........................................................... 25 3.7 Standard techniques for working with DNA ...................................................................... 26 3.7.1 Isolation of DNA .................................................................................................... 26 3.7.2 Purification and concentration of DNA ................................................................ 26 3.7.3 Agarose gel electrophoresis for DNA ................................................................... 27 3.7.4 PCR ........................................................................................................................ 27 3.7.5 Cloning of the T. thermophilus RNAP subunits ..................................................... 28 3.7.6 Cloning of the RNAPE ............................................................................................ 30 3.7.7 Cloning of templates for in vitro protein expression............................................ 30 3.7.8 Heat shock transformation of E. coli .................................................................... 31 3.7.9 Blue-white screening ............................................................................................ 31 3.7.10 Sequencing ........................................................................................................... 31 3.8 Standard techniques for working with proteins ................................................................ 32 3.8.1 Heterologous expression in E. coli ........................................................................ 32 3.8.2 Purification ........................................................................................................... 32 3.8.3 Buffer exchange .................................................................................................... 34 3.8.4 In vitro reconstitution of the RNAP of T. thermophilus ........................................ 34 3.8.5 Protein quantification (Bradford, 1976) ............................................................... 35 3.8.6 SDS - polyacrylamide gel electrophoresis (SDS-PAGE; (Laemmli, 1970)) ............. 35 3.8.7 Semi-native gels .................................................................................................... 36 3.8.8 Coomassie staining of proteins and estimation of molecular weight .................. 37 3.8.9 Transfer of proteins on nitrocellulose membranes (Western Blot) ..................... 37 3.8.10 Immunodetection of six-fold histidines ................................................................ 38 3.9 Techniques for working with RNA ..................................................................................... 39 3.9.1 Transcriptional activity of RNAPs ......................................................................... 39 3.9.2 Cleaning & concentration of RNA ......................................................................... 39 3.9.3 DNase I treatment of RNA samples ...................................................................... 40 3.9.4 Measurement of RNA concentration ................................................................... 40 3.9.5 Agarose gel electrophoresis for RNA .................................................................... 40 3.10 In vitro protein expression ................................................................................................. 41 3.10.1 Coupled ivTT ......................................................................................................... 41 3.10.2 Linked ivTT ............................................................................................................ 42 3.11 Screening methods for activity of in vitro expressed enzymes ......................................... 43 3.11.1 Para-nitrophenol (pNP) ester assay ..................................................................... 43 3.11.2 Methylumbelliferyl (MU) ester assay with immobilized enzymes ....................... 43 3.11.3 ß-Lactamase ....................................................................................................... 44 3.12 In vitro compartmentalization ........................................................................................... 45 3.13 Computational analysis ...................................................................................................... 46 3.13.1 Programs............................................................................................................... 46 3.13.2 Databases ............................................................................................................. 46 Results.............................................................................................................................. 47 4.1 Recombinant RNAPs .......................................................................................................... 47 4.1.1 Recombinant RNAP from T. thermophilus............................................................ 47 4.1.2 Recombinant viral RNAP from the elephant metagenome .................................. 50 4.2 Cell-free protein synthesis ................................................................................................. 58 4.2.1 Coupled vs linked cell-free expression ................................................................. 58 4.2.2 Comparison of different bacterial translation extracts ........................................ 59 4.2.3 In vitro expression of metagenomic-derived genes ............................................. 63 4.2.4 Photometric detection of in vitro expressed enzymes ......................................... 70 4.3 In vitro protein expression
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