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Automated Biopart Characterisation for Synthetic Biology

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Automated Biopart Characterisation for Synthetic Biology Automated BioPart Characterisation for Synthetic Biology Christopher David Hirst Imperial College London Department of Bioengineering Centre for Synthetic Biology and Innovation Submitted for Degree of Doctor of Philosophy February 2014 Page | 0 Declaration All work and data contained within this thesis is my own except where referenced. ‘The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work’ Page | 1 Acknowledgments I would like to thank my primary supervisor Richard Kitney for the many helpful discussions and support during this project, allowing me to grow as a researcher. His tireless belief in the future for engineering biology has been an inspiration. I would particularly like to thank him for the many opportunities including conferences, events and experiences that I have benefited so much from but most importantly the opportunity to work on this project. I also greatly appreciate the guidance of my other supervisors Geoff Baldwin and Paul Freemont for many useful discussions about the technology in development and overall direction of the project. For my supervisors collectively I must also express my thanks for selecting me to compete as part of the Imperial iGEM team in 2008 and supervise the team in 2010, those experiences have taught me much about myself, the communication of science and re-enforced my decision to become a researcher. I appreciate the many useful discussions with Dr. Tom Ellis regarding the initial design for the datasheets. Special thanks are also reserved for Kirsten Jensen for her many years of imparting knowledge and useful discussions and to Dr. Matthieu Bultelle for continually challenging my assumptions and always requiring more (either data or biological knowledge). To my colleagues in the CSynBI and the Department of Bioengineering, I thank you for all the interesting debates and scientific discussions as well as helping me to cope with the many ups and downs we all share as scientists as part of our projects. My appreciation in particular goes out to the members of the CSynBI who used the automated platform while it was being set up and were tolerant of its occasional bugs and errors and always pushed what was required from the platform. Those occasional failed runs were vital in the identification of the small bugs that would otherwise have remained a mystery. I would like to thank the EPSRC for providing the scholarship funding to carry out this research. Additionally I would like to thank the SynBioStandards Network for the travel grant that allowed me to attend the SB5.0 conference and meet with the members of the BioFAB in Stanford. To my family and friends collectively, the deepest appreciation goes out to you for being a constant source of support and inspiration during the length of this project. Page | 2 1 Abstract Synthetic Biology is an approach to the development of biological systems based on engineering principles. By using concepts from other engineering disciplines such as abstraction it is possible to break down complicated biological functions into components termed ‘BioParts’. BioParts can be assembled collectively into modules and systems to carry out advanced functions, designed from the bottom up. A key part of this approach is the standardisation of BioParts and practices to aid design, testing and implementation. An automated characterisation methodology focused primarily on promoter BioParts has been developed which is potentially scalable to other BioPart families. The standardised workflow is optimised to enable BioPart characterisation under highly reproducible growth conditions, reliably producing high quality data. It has been designed around automation equipment which should ensure accurate reproduction of experiments at other sites. The automated characterisation workflow has been demonstrated to produce high quality data for both constitutive and inducible promoters. The entire Anderson promoter collection has been characterised and high details results for all library members are available for the first time. A pair of inducible promoter BioPart were characterised to obtain a deep data level regarding their activity in response to inducer over time. To allow the characterisation of more inducible BioParts in a shorter period of time, promoter engineering was also used to generate novel promoters which are induced by xylose. The development of the automated workflow should be a step towards the standardisation of characterisation protocols and production of large numbers of BioParts with associated high quality, reproducible characterisations. Standardisation will further aid the comparison of the data sets produced, potentially shining light on unknown interactions between BioParts and their environment and improving the ability of Synthetic Biologists to design novel biological systems from the ground up. Page | 3 Contents Declaration ................................................................................................................................... 1 Acknowledgments ........................................................................................................................ 2 1 Abstract ............................................................................................................................... 3 Contents ........................................................................................................................................ 4 List of tables .................................................................................................................................. 8 List of figures................................................................................................................................. 9 2 Introduction ...................................................................................................................... 11 2.1 Lay Summary ................................................................................................................. 11 2.2 Synthetic Biology ........................................................................................................... 12 2.3 Rational design of biology ............................................................................................. 13 2.4 Applications ................................................................................................................... 15 3 Specific Problem ................................................................................................................ 18 3.1 Characterisation ............................................................................................................ 18 3.1.1 Data and Metrology .............................................................................................. 19 3.1.2 Chassis and Characterisation conditions............................................................... 21 3.1.3 Genetic and host context ...................................................................................... 23 3.2 Standards and documentation ..................................................................................... 25 3.3 Promoter BioParts and Promoter Engineering ............................................................. 27 3.4 Automation in Biology .................................................................................................. 28 3.5 The approach at CSynBI: SynBIS .................................................................................... 29 3.6 Project Aims .................................................................................................................. 29 4 General Methods .............................................................................................................. 30 4.1 Bacterial strains and media ........................................................................................... 30 4.2 Cloning and DNA manipulation ..................................................................................... 31 4.2.1 Plasmids and DNA preparations ........................................................................... 31 4.2.2 General Cloning and PCR....................................................................................... 32 4.2.2.1 Cloning of Minimal Vectors ............................................................................. 33 4.2.2.2 Cloning of FNR plasmids .................................................................................. 35 4.2.2.3 Cloning of SVa plasmids ................................................................................... 35 4.2.2.4 Cloning of SVb plasmid .................................................................................... 39 4.2.2.5 Cloning of SVc plasmids ................................................................................... 39 4.2.2.6 Cloning of Synthetic Promoters ....................................................................... 42 4.3 Manual characterisation experiments .......................................................................... 43 Page | 4 4.3.1 Ideal growth condition experiments ....................................................................
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