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Design, Construction and Characterization of Dynamic Genetic Circuits in Bacteria

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Design, Construction and Characterization of Dynamic Genetic Circuits in Bacteria Design, Construction and Characterization of Dynamic Genetic Circuits in Bacteria A Thesis Presented by BORIS KIROV Submitted to the Ecole doctorale des Génomes Aux Organismes of the University of Evry Val-d’Essonne in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY JANUARY 2014 Acknowledgements I would like to thank all the people that helped me fulfill this research. I express my sincere gratitudes to: • all the members of the jury who agreed to help me make the final step of a long journey • my supervisor Alfonso Jaramillo for taking me on this journey • Jeff Hasty and his group for the transfer of all aspects of microfluidics technology and their constant support • Mohammed Atari, Claudiu Giuraniuc, Fabio Cancare' and Jangir Selimkhanov for the help with the development of the image processing scripts • Octavio Mondragón-Palomino, Fabrice Monti and Ivan Razinkov for teaching me microfluidics fabrication • all my colleagues, or better all my friends from iSSB for the great environment and for the work we did and for the fun we had together Finally, I would like to thank all my close friends for the constant support and understanding, and patience and love. I could have never reached that far without Kiril, Charles, Jirair, father Emilian and my loving sister Vesela. 1 Abstract Engineering of synthetic genetic devices capable of controlling different aspects of the cellular physiology in a predefined manner and with precise timing is regarded as crucial for modern bioengineering and synthetic biology. The task to design and construct parts for synthetic biology is not simple and needs to meet a number of requirements. The parts utilized for the construction of genetic circuits should be modular, well-characterized, well-behaved and robust to changes in the environment. They should be insulated from cross-talk with the environment and be resilient to mutations. Finally, they should also be properly modeled based on parameters derived from single-cell level experiments. In my thesis I researched in detail the general requirements for the engineering of individual parts like promoters, ribosome binding site, transcription factors and of some important type of devices. Furthermore, I established a complete platform for the single-cell level characterization of engineered genetic devices. All the required hardware and know-how for the fabrication of microfluidics devices capable of sustained bacterial growth was acquired. The whole process from the design of microfluidics devices with aimed functionality to their fabrication and utilization for microbial experiments was successfully developed. An efficient image processing tool for distributed computational analysis of the data acquired during the microscopy experiments was also developed. The experimental results proved that the engineered genetic devices were behaving according to theoretical expectations. Furthermore, the established experimental procedures, fabrication process and automated data analysis showed to be well- adapted to the task of single-cell characterization of engineered bacteria and efficient. 2 Table of contents ACKNOWLEDGEMENTS ................................................................................................................................... 1 ABSTRACT .......................................................................................................................................................... 2 TABLE OF CONTENTS ...................................................................................................................................... 3 INDEX OF ILLUSTRATIONS ............................................................................................................................ 5 INDEX OF TABLES ............................................................................................................................................ 6 I. OVERVIEW ...................................................................................................................................................... 7 II. ENGINEERING OF GENETIC PARTS ...................................................................................................... 10 II.1. INTRODUCTION ........................................................................................................................................................................ 10 II.2. PROMOTER ENGINEERING .................................................................................................................................................... 13 II.3. SYNTHETIC PROMOTERS ....................................................................................................................................................... 20 II.4. RIBOSOME BINDING SITE ...................................................................................................................................................... 23 II.5. TRANSCRIPTION FACTORS .................................................................................................................................................... 24 II.6. FLUORESCENCE REPORTER PROTEINS .............................................................................................................................. 26 II.7. PROTEIN DEGRADATION TAGS ............................................................................................................................................. 27 II.8. TRANSCRIPTIONAL TERMINATORS ..................................................................................................................................... 28 II.9. EXPRESSION VECTORS ........................................................................................................................................................... 29 II.10. CONSTRUCTION OF GENETIC CIRCUITS ........................................................................................................................... 32 II.11. GENETIC PARTS ENGINEERED FOR THIS RESEARCH .................................................................................................... 32 II.11.1. Synthetic promoters (APPENDIX A) ............................................................................................................... 32 II.11.2. XOR gate promoters ............................................................................................................................................... 34 II.12. CONCLUSION ......................................................................................................................................................................... 38 II.13. REFERENCES ......................................................................................................................................................................... 38 III. ENGINEERING OF GENETIC OSCILLATORS ....................................................................................... 43 III.1. INTRODUCTION ...................................................................................................................................................................... 43 III.2. MATHEMATICAL MODELING ............................................................................................................................................... 45 III.3. SYNTHETIC GENETIC OSCILLATORS .................................................................................................................................. 63 III.4. GENETIC OSCILLATORS ENGINEERED FOR THIS RESEARCH ........................................................................................ 72 III.4.1. Goodwin-type oscillators ....................................................................................................................................... 72 III.4.2. Double genetic oscillators..................................................................................................................................... 79 III.4.3. Oscillatory copy number plasmid ...................................................................................................................... 83 III.4.4. Phage-communication-based oscillator ........................................................................................................ 85 III.5. CONCLUSION ........................................................................................................................................................................... 88 III.6. REFERENCES .......................................................................................................................................................................... 89 IV. ENGINEERING OF MICROFLUIDICS DEVICES .................................................................................... 94 IV.1. INTRODUCTION ...................................................................................................................................................................... 94 IV.2. MICROFLUIDICS DEVICES DESIGN ...................................................................................................................................... 98 IV.2.1. Growth chambers ...................................................................................................................................................... 98 IV.2.2. Channel system........................................................................................................................................................
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