Jajesniak pawel redacted phd thesis

EXPANDING MOLECULAR TOOLBOX FOR SYNTHETIC BIOLOGY Thesis submitted for the degree of Doctor of Philosophy Pawel Jajesniak Department of Chemical and Biological Engineering University of Sheffield September 2017 DECLARATION This thesis and the work presented in it are my own and has been generated by me as the result of my own original research. Where other sources of information have been used, they have been duly acknowledged. No part of this thesis has previously been submitted for a degree or any other qualification at this University or any other institution. Parts of this work have been, or will be, presented elsewhere - e., in the form of scientific publications.

This dissertation conforms to the rules of Alternative Format Thesis, as described by the Code Of Practice For Research Degree Programmes 2017–18 of the University of Sheffield. In accordance with the underlined guidelines, this thesis contains sections that are written in the form of scientific publications, some of which have already been published in peer-reviewed journals and other outlets for academic research. Whenever such materials are used, their sources are clearly acknowledged. | ii | ASSOCIATED PUBLICATIONS (1) Jajesniak P, Wong TS.

(2015) Tapping into biodiversity: From metagenomics to industrial enzymes. In: Biodiversity and Conservation (Vol. USA: Studium Press LLC. (2) Jajesniak P, Wong TS.

(2015) From genetic circuits to industrial-scale biomanufacturing: bacterial promoters as a cornerstone of biotechnology. (3) Jajesniak P, Wong TS. (2015) QuickStep-Cloning: a sequence-independent, ligation-free method for rapid construction of recombinant plasmids. Journal of Biological Engineering, 9, 15.

(4) Jajesniak P, Wong TS. (2017) Rapid Construction of Recombinant Plasmids by QuickStep-Cloning. In: Synthetic DNA. Methods in Molecular Biology (Vol.

USA: Humana Press, New York, NY; 205-214. | iii | ACKNOWLEDGEMENTS A pursuit of PhD degree, while seemingly a solitary endeavour, is never conducted in isolation. Its successful completion depends heavily upon the goodwill and all- encompassing support – academic, technical, financial, emotional - of many people whose role and generosity simply cannot be overstated. I would like to sincerely thank my supervisor, Dr Tuck Seng Wong, for his invaluable guidance during the course of my degree, in particular, for always keeping his door open and being willing to help.

I am grateful to the Department of Chemical and Biological Engineering, the funder of my project, for providing me with an opportunity of pursuing the PhD degree at the University of Sheffield. I would like to express my gratitude to Dr Kang Lan Tee for her technical support, fruitful scientific discussions and invaluable help in refining my experimental techniques. I wish to give special thanks to my friends, and fellow PhD students - Hossam, Yomi, Miriam, Zaki, Abdul, Inas and José - for making the past four years, including even the most daunting laboratory sessions, truly enjoyable. Last but certainly not least, I am extremely grateful to my whole family for their unceasing and unconditional support.

| iv | SUMMARY Synthetic biology can broadly be defined as a construction of new biological systems and modification of the existing ones. In recent years, synthetic biology has presented itself as a potential solution to many global challenges - e., climate change and scarcity of natural resources. The progress of synthetic biology is, however, largely reliant on the availability of a large repertoire of functional genetic parts. As a result, there exists an urgent need for enhancing and expediting the process of tapping into genetic diversity - both natural and laboratory-induced.

Analysing the current landscape of synthetic biology and various approaches of accessing biological diversity, two high-impact areas, namely the basic molecular cloning and identification of genes encoding for complex microbial phenotypes, have been identified as particularly lacking and, as such, constitute the focal point of my PhD project. The culmination of the work presented in this dissertation are two molecular methods, QuickStep-Cloning and Multi-Genius, that aim to accelerate the development of synthetic biology. QuickStep-Cloning is a new molecular cloning technique that builds upon recent advances in megaprimer-based cloning to allow for seamless integration of a DNA fragment of interest into a plasmid in less than 6 hours – the result that could not be reproduced using state-of-the-art methods. The new improved version of the method, QuickStepS-Cloning, utilises phosphorothioate oligonucleotides to not only simplify the overall procedure but also significantly increase its cloning efficiency.

It also shown that |v| incorporating random mutagenesis into the method allows for streamlining directed evolution experiments. Whereas the potential applications of QuickStep-Cloning revolve around artificially- induced diversity, Multi-Genius builds upon the concept of genomic libraries to tap into naturally-existing diversity and expedite identification of genes encoding for useful phenotypes. The usefulness of the method has been proven by isolating thermotolerant variants of Escherichia coli DH5α and indentifying the gene responsible for the observed phenotype. | vi | TABLE OF CONTENTS DECLARATION.

ii ASSOCIATED PUBLICATIONS. v TABLE OF CONTENTS. vii LIST OF FIGURES. xii LIST OF TABLES.

xx Chapter 1 General introduction .1 Brief introduction to synthetic biology .2 Traditional approaches to accessing and investigating biodiversity .3 What is metagenomics? .4 Environmental sample extraction .5 Sample enrichment and DNA extraction.6 Function-based metagenomics .7 Sequence-based metagenomics .9 Future prospects and conclusions .4 Diversity of non-coding DNA sequences .4 Protein expression and promoter selection .5 Synthetic biology and metabolic engineering .6 Databases and bioinformatics tools .7 Standardization and quantification of promoter strength .8 Complementary technologies and future prospects .5 Genetic diversity and interactions between multiple genes .6 Scope and objectives. 88 Chapter 2 QuickStep-Cloning .3 Results and Discussion .1 QuickStep-Cloning: Principle and molecular mechanism .2 Primer design for QuickStep-Cloning.3 Demonstration of QuickStep-Cloning.4 Optimizing QuickStep-Cloning .5 Comparison to restriction-free (RF) cloning .6 General applicability of QuickStep-Cloning .7 Comparison to other cloning methods.4 Restriction-free (RF) cloning .5 DNA gel electrophoresis.6 Transformation and clone analysis .7 Estimated cloning times reported in Table 2.122 Chapter 3 QuickStepS-Cloning .2 Results and Discussion .4 Application to directed evolution .4 Restriction-free (RF) cloning .6 DNA gel electrophoresis.7 Chemical transformation and clone analysis .8 Error-prone PCR .10 Transformation of rfp library .11 Protein expression purification .148 Chapter 4 Multi-Genius: method development .2 Genomic DNA extraction .5 Vector design and construction.3 Genomic DNA extraction .4 Genomic DNA amplification .8 DNA gel electrophoresis. 173 Chapter 5 Multi-Genius: validation and application .1 Thermotolerance and halotolerance of E.2 Primers and genomic DNA .3 Initial expression studies .4 Further expression studies .5 Investigation of halotolerance of E.6 Investigation of thermotolerance of E.7 Multi-Genius - application .204 Chapter 6 Conclusions and future work .213 | xi | LIST OF FIGURES Figure 1.1 Factors affecting microbial growth.2 General scheme of the metagenomic approach to finding 39 new functional biomolecules.4 Important characteristics of a biocatalyst; adapted from 55 (Lorenz and Eck, 2005).5 Typical steps involved in a directed protein evolution 56 experiment.6 Outline of prokaryotic promoter structure - simplified 62 structure and consensus sequence of E.7 Main factors affecting protein expression levels during 67 heterologous protein production.8 Proposed flowchart for a quick selection of an appropriate 71 inducible promoter (based on the list of 13 most common inducible E. coli expression systems listed in Table 1.1 Overview of QuickStep-Cloning 96 Figure 2.2 Outline of primer design for QuickStep-Cloning.3 Plasmid map of pEGFP vector used in egfp cloning 100 experiment (created with SnapGene).4 Outline of egfp gene cloning experiment.6 Cell pellets from cell cultures grown as part of egfp cloning 102 experiment.7 Investigation and optimization of asymmetric PCR stage of 103 QuickStep-Cloning.8 Yield of whole plasmid amplification for different primer 104 ratios used during asymmetric PCRs Figure 2.9 Optimization of megaprimer PCR stage of QuickStep- 105 Cloning.10 Plasmid map of pBbA8k-RFP vector used in rfp cloning 107 experiment (created with SnapGene).12 Cell pellets from cell cultures grown as part of rfp cloning 109 experiment.13 Cell pellets from cell cultures grown as part of rfp cloning 110 experiment.1 Chemical structure of (A) DNA, containing standard 129 phosphodiester bond between two neighbouring nucleosides, and (B) DNA containing phosphorothioate bond.2 Comparison of the megaprimer synthesis stage between (A) 130 original QuickStep-Cloning and (B) improved version of the method, utilising phosphorothioate oligonucleotides.3 Optimisation of QuickStepS-Cloning.4 Yield of RF cloning, QuickStep-Cloning and QuickStepS- 135 Cloning (25 cycles of megaprimer PCR) ), as determined for rfp cloning experiment.5 Outline of P450 BM3 reductase gene cloning experiment.6 Yield of RF cloning and QuickStepS-Cloning, as determined 136 for P450 BM3 reductase gene cloning experiment.7 Comparison of the wild-type RFP and three isolated mutants 139 Figure 3.8 Absorbance spectra of the wild-type RFP and three isolated 139 mutants.1 Overview of Multi-Genius.2 Design of expression vector for Multi-Genius 151 Figure 4.3 Product of genomic DNA amplification.4 Yield of genomic DNA amplification carried out at different 155 concentrations of MnCl2.5 Amplified genomic DNA (1a) before and (1b) after 156 purification with QIAquick PCR Purification Kit.6 Optimisation of DNA fragmentation.7 Outline of pD441-SR, used as a first building block for the 161 assembly of Multi-Genius expression vector.8 530 bp DNA fragment containing the cloning site under a 162 control of rhamnose-inducible promoter, used as the second building block for the assembly of Multi-Genius expression vector.9 Outline of the Multi-Genius expression vector, containing 162 two cloning sites under the control of T5 and rhamnose- inducible promoter.10 Outline of the cloning strategy for Multi-Genius.11 The result of applying optimised cloning protocol for 166 incorporating two genomic libraries into Multi-Genius vector via blunt-end ligation; the integration of the first fragment.12 Plasmids extracted from four different population cells 167 resulting from applying the optimised cloning protocol to integrate: (1) EGFP into cloning site 1, (2) RFP into cloning site 1, (3) EGFP into cloning site 1, and (4) RFP into cloning site 2.13 The result of applying optimised cloning protocol for 168 incorporating two genomic libraries into Multi-Genius vector via blunt-end ligation; the integration of the second fragment.1 Cell pellets of CS1:EGFP/CS2:RFP-transformed cells after 176 incubation for 12 h (at 37°C and 500 rpm) in LB media supplemented with: (1) kanamycin only, (2) Kan + 2% glucose, (3) Kan + 1mM IPTG, (4) Kan + 0.2% L- rhamnose, and (5) Kan + 1 mM IPTG + 0.2 Cell pellets of CS1:RFP/CS2:EGFP-transformed cells after 177 incubation for 12 h at (37°C and 500 rpm) in LB media supplemented with: (1) kanamycin only, (2) Kan + 2% glucose, (3) Kan + 1 mM IPTG, (4) Kan + 0.2% L- rhamnose, and (5) Kan + 1 mM IPTG + 0.3 Fluorescence of CS1:EGFP/CS2:RFP-transformed cells after 179 12 h incubation with different concentration of IPTG.4 Fluorescence of CS1:EGFP/CS2:RFP-transformed cells after 180 12 h incubation with different concentration of L-rhamnose.5 Fluorescence of CS1:RFP/CS2:EGFP-transformed cells after 181 12 h incubation with different concentration of IPTG.6 Fluorescence of CS1:RFP/CS2:EGFP-transformed cells after 182 12 h incubation with different concentration of L-rhamnose.7 Natural thermotolerance of E.8 Natural halotolerance of E.

184 | xvi | LIST OF TABLES Table 1.1 Examples of important biocatalysts identified through 38 metagenomic analysis (Lorenz and Eck, 2005; Mocali and Benedetti, 2010; Riesenfeld et al., 2004; Xing et al.2 Comparison of the three main second-generation sequencing 49 platforms (Liu et al., 2012; van Dijk et al.3 Comparison between two main strategies of promoter 65 engineering - saturation mutagenesis of spacer regions and error-prone PCR (based on the information provided in (Alper et al., 2005; Hammer et al., 2006; Jensen and Hammer, 1998)).4 Most commonly-used inducible E. coli expression systems 69 and their important properties (adapted from (Terpe, 2006) and (Weickert et al., 1996)); inducer prices are based on the information provided by Sigma-Aldrich Corporation, http://sigmaaldrich.5 Novel expression systems developed for Escherichia coli, 73 Bacillus spp. and Pseudomonas spp. over the last decade.1 Results of egfp cloning experiment.2 Results of rfp cloning experiment.3 A comparison of QuickStep-Cloning to other recently 111 reported megaprimer-based cloning methods.1 Comparison of QuickStepS-Cloning, using 25 or 30 cycles 134 of megaprimer PCR, with RF cloning and QuickStep- Cloning, as determined by rfp cloning experiment.2 Results of P450 BM3 reductase gene cloning experiment.3 Comparison of the library size when performing an 138 experiment involving rfp mutagenesis via QuickStepS- Cloning and MEGAWHOP.