Design and Functional Assembly of Synthetic Biological Parts and Devices Baojun Wang A thesis submitted to the Imperial College London for the degree of Doctor of Philosophy Department of Bioengineering Imperial College London Nov 2010 Declaration of Originality I certify that the work presented in this thesis is my own and original. The work or any part of the work has not been previously submitted anywhere for any degree or diploma. I confirm that any ideas or work of others have been clearly referenced in the thesis and all main sources of help have been acknowledged. Baojun Wang 2 Abstract Programming living cells with synthetic gene circuits to perform desired tasks has been a major theme in the emerging field of synthetic biology. However, gene circuit engineering currently lacks the same predictability and reliability as seen in other mature engineering disciplines. This thesis focuses on the design and engineering of novel modular and orthogonal biological devices, and the predictable functional assembly of modular biological elements (BioParts) into customisable larger biological devices. The thesis introduces the design methodology for engineering modular and orthogonal biological devices. A set of modular biological devices with digital logic functions, including the AND, NOT and combinatorial NAND gates, were constructed and quantitatively characterised. In particular, a novel genetic AND gate was engineered in Escherichia coli by redesigning the natural HrpR/HrpS hetero- regulation motif in the hrp system of Pseudomonas syringae. The AND gate is orthogonal to E. coli chassis, and employs the alternative σ54-dependent gene transcription to achieve tight transcriptional control. Results obtained show that context has a large impact on part and device behaviour, established through the systematic characterisation of a series of biological parts and devices in various biophysical and genetic contexts. A new, effective strategy is presented for the assembly of BioParts into functional customised systems using engineered ‘in- context’ characterised modules aided by modelling, which can significantly increase the predictability of circuit construction by characterising the component parts and modules in the same biophysical and genetic contexts as anticipated in their final systems. Finally, the thesis presents the design and construction of an application- oriented integrated system – the cell density-dependent microbe-based biosensor. The in vivo biosensor was programmed to be able to integrate its own cell density signal through an engineered cell-cell communication module and a second environmental signal through an environment-responsive promoter in the logic AND manner, with GFP as the output readout. 3 Contents Declaration of Originality ....................................................................................................... 2 Abstract .................................................................................................................................... 3 Acknowledgements .................................................................................................................. 4 Contents .................................................................................................................................... 5 List of Figures .......................................................................................................................... 9 List of Tables .......................................................................................................................... 12 Chapter 1 Introduction ......................................................................................................... 13 1.1 Thesis Statement and Summary of Contributions ......................................................... 14 1.1.1 Thesis statement ..................................................................................................... 14 1.1.2 Summary of contributions ..................................................................................... 14 1.1.3 Thesis outline ......................................................................................................... 16 1.2 Synthetic Biology and the State-of-the-Art ................................................................... 17 1.2.1 Synthetic biology – an emerging discipline ........................................................... 17 1.2.2 The state-of-the-art of synthetic biology ................................................................ 20 1.3 Current Design Principles and Challenges of Engineering Gene Circuits .................... 29 1.3.1 Current design principles for engineering synthetic gene circuits ......................... 29 1.3.2 Challenges for engineering gene circuits ............................................................... 31 1.4 Objectives of This Study ............................................................................................... 35 Chapter 2 Materials and Methods ....................................................................................... 36 2.1 Materials ........................................................................................................................ 37 2.1.1 Media ..................................................................................................................... 37 2.1.2 Antibiotics .............................................................................................................. 37 2.1.3 Kits ......................................................................................................................... 38 2.1.4 Dyes, chemicals and enzymes ............................................................................... 38 2.1.5 Bacterial strains...................................................................................................... 39 2.2 DNA Methods ............................................................................................................... 39 2.2.1 DNA purification ................................................................................................... 40 2.2.2 Polymerase Chain Reaction (PCR) ........................................................................ 40 2.2.3 Agarose gel electrophoresis ................................................................................... 41 5 2.2.4 Gel extraction and purification of DNA ................................................................ 42 2.2.5 Restriction digest ................................................................................................... 42 2.2.6 Ligation .................................................................................................................. 42 2.2.7 Assembly strategy for DNA constructs ................................................................. 43 2.2.8 DNA sequencing and synthesis ............................................................................. 45 2.2.9 Competent cell preparation .................................................................................... 46 2.2.10 Heat shock transformation ................................................................................... 46 2.3 In Vivo Assay Methods ................................................................................................. 46 2.3.1 Growth of bacteria cells ......................................................................................... 46 2.3.2 Fluorescence assay for gene expression in living cells .......................................... 47 2.4 Modelling and Data Analysis Methods ......................................................................... 51 2.4.1 Deterministic approach using ODE-based rate equations ...................................... 51 2.4.2 Basic biochemical kinetics ..................................................................................... 52 2.4.3 Modelling a constitutive single gene expression ................................................... 53 Chapter 3 The Design and Engineering of Modular Biological Devices .......................... 56 3.1 Approaches to Design Modular and Orthogonal Biological Devices ........................... 57 3.2 The Two Paradigms of Bacterial Gene Transcription and the hrp Regulatory System in Pseudomonas syringae ........................................................................................................ 61 3.2.1 The two mechanistic paradigms of bacterial gene transcription ............................ 61 3.2.2 The hrp gene regulation system in Pseudomonas syringae ................................... 63 3.3 Designing a Set of Modular Logic Devices and Functional Assembly of BioParts ..... 68 3.3.1 The design of a modular logic AND gate .............................................................. 68 3.3.2 The design of a set of modular NOT gates and the composite NAND gate .......... 70 3.3.3 Functional assembly of BioParts using engineered in-context characterised modules ........................................................................................................................... 71 3.4 Deriving Transfer Functions of the Designed Logic Devices ....................................... 74 3.4.1 Deriving the transfer function of environment-responsive promoters ................... 74 3.4.2 Deriving the transfer function of the AND gate .................................................... 75 3.4.3 Deriving the transfer function of the NOT gate ....................................................