Alexander Templar EngD Thesis Industrially Robust Synthetic Biology Standards for the Polymerase Chain Reaction A thesis submitted to the University College London for the degree of ENGINEERING DOCTORATE by Alexander Templar BSc MRes 2017 Advanced Centre of Biochemical Engineering Department of Biochemical Engineering University College London Torrington Place London WC1E 7JE Alexander Templar EngD Thesis Table of Contents Abstract ................................................................................................................................... 11 Thesis Declaration .................................................................................................................. 12 Acknowledgements ................................................................................................................. 13 Nomenclature.......................................................................................................................... 14 List of Figures ......................................................................................................................... 18 List of Tables .......................................................................................................................... 21 1 General Introduction ....................................................................................................... 1 1.1 Introduction to synthetic biology and standardisation........................................ 1 1.2 The quantitative polymerase chain reaction ....................................................... 1 1.2.1 PCR nomenclature ...................................................................................... 2 1.2.2 PCR data capture and analysis .................................................................... 3 1.2.3 End-point PCR ............................................................................................ 3 1.2.4 qPCR amplification curve ........................................................................... 3 1.2.5 Efficiency of qPCR amplification ............................................................... 6 1.2.6 Quantification cycles (Cq) in qPCR ............................................................ 7 1.2.7 Relative quantification from qPCR ............................................................. 7 1.2.8 Absolute quantification qPCR .................................................................... 7 1.2.9 Limitations of absolute quantification ........................................................ 8 i Alexander Templar EngD Thesis 1.3 Diversity of qPCR data analysis ......................................................................... 9 1.3.1 Sigmoidal curve fitting and Linear Regression of Efficiency (LRE)-qPCR 9 1.3.2 Optical Calibration Factor (OCF) and LRE-qPCR ................................... 11 1.3.3 Cy0 ............................................................................................................ 11 1.3.4 Methods for comparing competing analysis methods ............................... 13 1.3.5 qPCR instrumentation ............................................................................... 14 1.3.6 Rapid-cycle qPCR platforms..................................................................... 14 1.4 Introduction to Bioprocess Engineering ........................................................... 16 1.5 Bioprocess platforms ........................................................................................ 17 1.5.1 Pichia pastoris .......................................................................................... 17 1.5.2 Chinese Hamster Ovary (CHO) ................................................................ 18 1.5.3 Escherichia coli ......................................................................................... 18 1.6 Bioprocess monitoring ..................................................................................... 19 1.6.1 Scope for improvement in Bioprocess monitoring ................................... 20 1.6.2 Biomass and Growth ................................................................................. 21 1.6.3 Gasses ........................................................................................................ 21 1.6.4 Metabolites ................................................................................................ 21 1.6.5 Proteins and bio-products .......................................................................... 22 1.6.6 Gene expression ........................................................................................ 22 ii Alexander Templar EngD Thesis 1.7 Quantitative PCR as a bioprocess monitoring tool .......................................... 23 1.7.1 Sample preparation for bioprocess monitoring by qPCR ......................... 23 1.7.2 Target selection for qPCR bioprocess monitoring ... 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Bookmark not defined. 1.7.3 Standardisation of bioprocess monitoring ................................................. 24 1.8 Aims and objectives ......................................................................................... 24 2 Materials and Methods .................................................................................................. 26 2.1 Cell cultivation ................................................................................................. 26 2.1.1 P. pastoris cultivation ................................................................................ 26 2.1.2 CHO cultivation ........................................................................................ 27 2.1.3 E. coli cultivation ...................................................................................... 27 2.2 Sample preparation ........................................................................................... 28 2.2.1 Nucleic acid purification ........................................................................... 28 2.2.2 Sonication .................................................................................................. 28 2.2.3 Heat lysis ................................................................................................... 28 2.3 Oligonucleotide design ..................................................................................... 29 2.3.1 Primers ...................................................................................................... 29 2.3.2 Cal 1 sequence........................................................................................... 29 2.3.3 Plasmid design .......................................................................................... 29 2.4 PCR conditions ................................................................................................. 29 iii Alexander Templar EngD Thesis 2.4.1 e-pPCR ...................................................................................................... 29 2.4.2 qPCR ......................................................................................................... 30 2.4.3 PCR efficiency estimation......................................................................... 30 2.5 Nucleic acid quantitation .................................................................................. 30 2.5.1 Spectrophotometry .................................................................................... 30 2.5.2 Densitometry ............................................................................................. 31 2.5.3 Standard curve ........................................................................................... 31 2.5.4 LRE ........................................................................................................... 31 2.5.5 Cy0 ............................................................................................................ 32 2.6 Flow Cytometry ................................................................................................ 32 2.7 HPLC ................................................................................................................ 32 3 Assay optimisation through primer screening and validation ................................... 33 3.1 Introduction ...................................................................................................... 33 3.2 Results .............................................................................................................. 35 3.2.1 Design strategy .......................................................................................... 35 3.2.2 Transketolase (Tkt) ................................................................................... 36 3.2.3 Glyceraldehyde 3-phosphate dehydrogenase (GapDH) ............................ 39 3.2.4 Biotin retention locus (BirA) .................................................................... 41 3.2.5 Fragment antigen binding (Fab) antibody fragment ................................. 43 3.2.6 Green fluorescence protein (GFP) ............................................................ 45 iv Alexander Templar EngD Thesis 3.2.7 Mycoplasma consensus sequence ............................................................. 47 3.2.8 Direct comparison of Standard Curve-derived copy number and LRE- qPCR derived copy number .................................................................................... 49 3.3 Discussion ........................................................................................................ 51 3.4 Conclusions .....................................................................................................