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Towards the Construction of Escherichia Coli Cell-Free Protein Synthesis System Platform

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Towards the Construction of Escherichia Coli Cell-Free Protein Synthesis System Platform Towards the construction of Escherichia coli cell-free protein synthesis system platform Sara Alexandra Peça de Sousa Rosa Thesis to obtain the Master of Science Degree in Biotechnology Supervisor(s): Professor Luís Joaquim Pina da Fonseca Examination Committee Chairperson: Professor Arsénio do Carmo Sales Mendes Fialho Supervisor: Professor Luís Joaquim Pina da Fonseca Member of the Committee: Professor Gabriel António Amaro Monteiro July 2015 ii Acknowledgments I would like to express my gratitude to professor Lu´ıs Fonseca, for giving me the opportunity to work in a subject that I fell in love with. I would like to thank to all my colleges of 7th and 8th floor, for all the help, advice, and support. Without all of you I would not be able to finish my work. I would also like to thank to Doctor Ana Azevedo and Sofia Duarte, for all the help and tips. I would like to give a special thanks to Ana, Andreia, Catia,´ Elsa, Pedro, Rita and Ricardo, for help and support during the best and worst times. I also want to thank to Claudia,´ for the time spent inside IST walls. Most of all, I would like to thank my mom and dad for all the support. I also want to thank to my grandmother, for all the concern, and to my nieces, Beatriz and Margarida. Finally, I want to thank Davide, for always being there. My words will be never enough to express my gratitude towards you. Thank you all. iii iv Resumo Sistemas produtores de prote´ınas livres de celulas´ sao˜ descritos como a expressao˜ in vitro de prote´ınas recombinantes sem o recurso a celulas´ vivas. Esta abordagem usa lisados celulares que contem´ varios´ componentes qu´ımicos e biologicos´ necessarios´ para a transcric¸ao,˜ traduc¸ao,˜ enrolamento de prote´ınas, e metabolismo energetico;´ tudo o que e´ necessario´ para sintetizar directamente prote´ınas. Contudo, existem problemas associados ao uso destes sistemas: capacidade de sintetizar com fiabil- idade uma prote´ına activa numa plataforma universal, falta de uma plataforma rentavel´ e escalavel,´ e incapacidade de realizar padroes˜ de glicosilac¸ao.˜ O objectivo principal deste trabalho e´ construir uma plataforma robusta e rentavel´ para s´ıntese de prote´ınas in vitro num sistema de transcric¸ao˜ e traduc¸ao˜ copulado. Neste trabalho, um modelo de DNA foi desenhado e purificado. Este foi purificado por lise alcalina e cromatografia por interac¸ao˜ hidrofobica.´ Este metodo´ permite produzir em maior quanti- dade e com um menor custo. RNA de transferenciaˆ foi purificado extraindo os acidos´ nucleicos por extrac¸ao˜ com fenol, separando o DNA com acetato de sodio´ e removendo os restante contaminantes por cromatografia de troca anionica.´ Aquando da produc¸ao˜ do lisado S30, nao˜ foi poss´ıvel atingir a concentrac¸ao˜ proteica necessaria´ para ter um lisado activo. Apesar de o objectivo principal, construir uma plataforma de produc¸ao˜ de prote´ınas livre de celulas´ reprodut´ıvel nao˜ foi conseguido, foram dados alguns passos importantes para atingir esse objectivo. No futuro, uma das prioridades e´ melhorar o metodo´ de produc¸ao˜ de lisados para se obter um maior rendimento a partir do sistema livre de celulas.´ Palavras-chave: Sistemas livres de celulas,´ Escherichia coli, lisado S30, Purificac¸ao˜ de tRNA, Purificac¸ao˜ de plasm´ıdeo v vi Abstract Cell-free protein systems are described as the in vitro expression of recombinant proteins without the use of living cells. This approach uses a cell lysate containing a wide array of biological and chemical components for transcription, translation, protein folding, and energy metabolism; all required to directly synthesise the target protein. However, there are some problems when using these systems: the ability to reliably synthesize any biologically active protein in a universal platform, the lack of a cost-effective and scalable platform, and the inability to carry out humanized glycosylation patterns. The main aim of this work is to construct a robust and cost effective platform for in vitro protein synthesis in a coupled transcription–translation system. In this work, a DNA template was design and purified. The DNA template was purified using an alkaline lysis and hydrophobic interaction chromatography. This method allows us to produce in higher quantity and lower cost. The tRNA purification was achieved by extracting the nucleic acids by phenol extraction, separate the DNA with sodium acetate, and remove the remaining contaminants by anion exchange chromatography. When producing the S30 lysate, it was not possible to achieve the protein concentration necessary to have a highly active lysate. Although the main goal of creating a reproducible E. coli cell-free protein synthesis system platform was not achieved, some major steps were taken towards this goal. In the future, one of the priorities is to improve the lysate production method so that we get a better yield from the cell-free system. Keywords: Cell-free systems, Escherichia coli, S30 lysate, tRNA purification, Plasmid purifica- tion, Cytomimic system vii viii Contents Acknowledgments........................................... iii Resumo.................................................v Abstract................................................. vii List of Tables.............................................. xiii List of Figures............................................. xviii Nomenclature.............................................. xix 1 Introduction 1 1.1 State-of-the-art..........................................2 1.1.1 Cell-free Protein Synthesis...............................2 1.1.2 cell lysates........................................5 1.1.3 Cell free protein synthesis system templates.....................9 1.1.4 Other components and Energy systems........................ 12 1.1.5 Configurations of Cell-free systems........................... 17 1.1.6 Folding and post-translational modifications...................... 20 1.1.7 History and applications................................. 22 1.1.8 Applications........................................ 23 1.1.9 Synthetic biology frontiers............................... 28 2 Objectives 31 3 Materials and Methods 33 3.1 Template preparation....................................... 33 3.1.1 pEXP5-NT/GFP construction.............................. 34 3.2 Agarose gel electrophoresis................................... 36 3.3 Cell transformation........................................ 36 3.3.1 Preparation of chemically competent cells....................... 36 3.3.2 Transformation of chemically competent cells..................... 37 3.3.3 Cell banks preparation.................................. 37 3.4 Plasmid production and purification............................... 37 3.4.1 Cell production...................................... 37 3.4.2 Alkaline lysis....................................... 38 ix 3.4.3 Plasmid purification................................... 38 3.4.4 HIC purification...................................... 38 3.4.5 SEC purification..................................... 39 3.4.6 Dialysis.......................................... 39 3.4.7 Plasmid concentration.................................. 39 3.5 tRNA purification......................................... 39 3.5.1 Cell growth........................................ 39 3.5.2 Phenol extraction..................................... 40 3.5.3 Contaminant removal.................................. 40 3.5.4 Anion-exchange chromatography............................ 40 3.5.5 RNA electrophoresis................................... 41 3.5.6 tRNA concentration................................... 41 3.6 GFP production and purification................................. 42 3.6.1 Cell production...................................... 42 3.6.2 Cell lysis......................................... 42 3.6.3 HIC purification...................................... 42 3.6.4 IMAC purification..................................... 43 3.7 S30 Lysate preparation..................................... 43 3.7.1 Cell production...................................... 43 3.7.2 Biomass processing................................... 44 3.7.3 Cell lysis efficiency determination............................ 44 3.8 Cell free protein synthesis.................................... 45 3.8.1 Expressway cell-free kit................................. 45 3.8.2 Cytomim system..................................... 45 3.8.3 Optimisation....................................... 46 3.9 Protein analysis.......................................... 46 3.9.1 Protein concentration.................................. 46 3.9.2 SDS-PAGE........................................ 47 3.9.3 Western blotting..................................... 47 3.9.4 Fluorimetry........................................ 48 4 Results and Discussion 49 4.1 Template preparation....................................... 49 4.1.1 pEXP5-NT/GFP..................................... 49 4.2 Plasmid purification....................................... 51 4.2.1 Hydrophobic interaction chromatography....................... 52 4.2.2 Plasmid desalting.................................... 54 4.3 tRNA purification......................................... 57 4.4 GFP purification......................................... 61 x 4.5 S30 lysate preparation...................................... 63 4.6 Cell-free protein synthesis.................................... 67 5 Conclusions 73 Bibliography 89 xi xii List of Tables 3.1 Digestion tests. Four different tests were performed. In all tests, 1000 ng of plasmid and 0.5 of each enzyme (PvuII and SacII) were used. In the
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