Hong, Kelly

English

Many soil-dwelling microbes have the natural capacity to produce toxic compounds that inhibit growth of competing bacteria; most traditional antibiotics have been derived 
from small molecules made by such soil-based microorganisms, of which only a small fraction can be grown in the laboratory. Since techniques that require culturing of these microbes in the lab have been the starting point for studying them in the past, our 
knowledge of the uncultured majority remains limited. Functional metagenomics is a method that circumvents the need for culturing, and thus has the potential to revel a yet 
untapped reservoir of antibacterial compounds. Here we present a potential application of functional metagenomics using genes isolated from soil microbes that employs high 
throughput sequencing to identify microbial genes encoding novel compounds that inhibit bacterial growth

Massey Research Online

Copyright is owned by the Author of the thesis.  Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only.  The thesis may not be reproduced elsewhere without the permission of the Author.  ?? i  Bioprospecting soil metagenomes for potential new antibiotics  A thesis presented in partial fulfilment of the requirements for the degree of  Master of Science In Genetics  At Massey University, Albany, New Zealand  Kelly Hong 2017  ?? ii 0.1? Abstract Many soil-dwelling microbes have the natural capacity to produce toxic compounds that inhibit growth of competing bacteria; most traditional antibiotics have been derived from small molecules made by such soil-based microorganisms, of which only a small fraction can be grown in the laboratory. Since techniques that require culturing of these microbes in the lab have been the starting point for studying them in the past, our knowledge of the uncultured majority remains limited. Functional metagenomics is a method that circumvents the need for culturing, and thus has the potential to revel a yet untapped reservoir of antibacterial compounds. Here we present a potential application of functional metagenomics using genes isolated from soil microbes that employs high throughput sequencing to identify microbial genes encoding novel compounds that inhibit bacterial growth. ?? iii 0.2? Acknowledgements  First and foremost, I would like to thank my supervisor, Nikki Freed, for providing me an environment that was always open to new ideas, and encouraging me to take every opportunity to learn new tools that enabled me to grow as a researcher. I would also like to acknowledge my co-supervisor, Olin Silander, for his constant constructive criticisms that challenged me and made every day a bit more interesting. Together, I could not have wished for better support, and I feel incredibly grateful for their mentorship. To the friends I have made; especially Danielle, Joanna, and Richard – I now consider us a tiny tribe of science warriors. Thank you to Wayne Patrick of Otago University for providing us with the plasmid. And last but not least, mum & dad – thank you for your unconditional love and support.   ?? iv0.3? Table of Contents 0.1 Abstract ....................................................................................................................... ii 0.2 Acknowledgements ................................................................................................... iii 0.3 Table of Contents ....................................................................................................... iv 0.4 List of Figures ............................................................................................................ vi 0.6 List of Tables ............................................................................................................. vii 0.7 Abbreviations .......................................................................................................... viii 1 Introduction ............................................................................................................. 1 1.1 Bioprospecting soil for novel antibiotics .................................................................. 2 1.2 Functional metagenomics: A cultivation-independent approach .......................... 4 1.2.1 Soil as a sample for functional metagenomics ......................................................... 4 1.3 Steps in generating a metagenomic library .............................................................. 5 1.3.1 Extracting DNA from soil ........................................................................................ 5 1.3.2 Fragmenting DNA .................................................................................................... 8 1.3.3 Selection of insert size and choice of expression vector .......................................... 9 1.3.4 Selecting a heterologous host organism ................................................................. 11 1.4 Screening metagenomic libraries ............................................................................ 12 1.5 Limitations and Challenges ..................................................................................... 15 1.7 Aims and Objectives of this study ........................................................................... 18 2 Materials and Methods ......................................................................................... 20 2.1 Proof-of-principle: testing the vector-host system with a toxin-encoding gene .. 20 2.1.1 Using PSTPO as a toxic gene to verify the pBAD system .................................... 20 2.1.2 Using sacB as a toxic gene to test the pBAD system ............................................. 24 2.2 Extracting genomic DNA from the soil .................................................................. 24 2.3 Preparing the vector for cloning ............................................................................. 25 2.4 Preparing electrocompetent E. coli TOP10 ............................................................ 25 ?? v 2.5 Construction of a mini library ................................................................................ 25 2.6 TSS transformation .................................................................................................. 26 2.7 Expression Screening ............................................................................................... 27 2.8 Sanger Sequencing ................................................................................................... 27 3 Results and Discussion .......................................................................................... 28 3.1 Proof-of-principle experiment with PSPTO failed to show cell death upon induction ................................................................................................................................. 28 3.2 SacB is toxic to E. coli and expression in pBAD is regulated in a dosage-dependent manner. ................................................................................................................ 31 3.3 Difficulties in extracting high yields of unfragmented DNA from soil ................ 34 3.4 Low ligation and transformation efficiencies hinder efforts in generating a large metagenomic library. ............................................................................................................ 36 3.5 Expression screening to identify any clones carrying toxic eDNA fragments .... 39 3.6 Analysis of sequences reveal cloning errors ........................................................... 43 4 Conclusion ............................................................................................................. 48 4.1 Improvements in DNA extraction methods to be pursued ................................... 48 4.2 Optimising ligation and transformation efficiencies ............................................. 49 4.3 A prospective large-scale library ............................................................................ 49 Bibliography .................................................................................................................. 52 Appendix ........................................................................................................................ 63    ?? vi0.4? List of Figures 1-1. Steps in constructing a metagenomic library. ............................................................ 7 1-2. Functional metagenomics for identifying toxic genes.. ........................................... 19 2-1. Inserting PSPTO in the correct orientation translates to a functional product. ....... 21 2-2. Inserting PSPTO in the reverse orientation translates to a non-functional product................................................................................................................................... 21 3-1. PSPTO inserted out-of-frame has no effect on the growth of E. coli TOP10 ......... 31 3-2. Expression of SacB inhibits growth of E. coli in the presence of sucrose.  ............ 32 3-3. Salt-free LB best supports expression of SacB in E. coli TOP10. .......................... 32 3-4. Soil samples collected from Massey University Auckland, NZ. ............................. 34 3-5. Extracting fragments of environmental DNA and cloning vectors with compatible ends ......................................................................................................................... 37 3-6. Growth assays with and without the inducer present. ............................................. 41 3-7. Five clones inhibited bacterial growth upon expression of the eDNA insert. ......... 42 3-8. All five inserts inhibiting growth were SacB. ......................................................... 43 3-10. T6A1 to T10A1 control inserts were all the same fragment of the TOPO cloning vector. ...................................................................................................................... 44 3-11. XbaI and XhoI restriction sites flank the spectinomycin marker in the TOPO vector. ...................................................................................................................... 45 3-12. Most clones contain an empty vector with no insert. ............................................ 46    ?? vii0.6? List of Tables 3-1. Salt-free LB best supports expression of SacB in E. coli TOP10. ............................. 32 3-2. Metagenomic DNA extractions from the PowerSoil Kit are similar in quantity and quality for the three different soil samples. ............................................................... 35 6-1. List of strains used in this study ................................................................................. 63 6-3. List of primers used in this study. ............................................................................... 64   ?? viii 0.7? Abbreviations eDNA: environmental DNA pBAD: pBAD/Myc-His B, the plasmid cloning vector used NGS: next generation sequencing PCR: polymerase chain reaction E. coli: Escherichia coli  TOPO: PCR8/GW/TOPO cloning vector (Invitrogen) TSS: transformation and storage solution LB: lysogeny broth SOC: super optimal broth with catabolite repression bp: base pairs kbp or kb: kilo base pairs OD600: optical density at 600nm. Indicator of bacterial growth. ORF: open reading frame ??

Bioprospecting soil metagenomes for potential new antibiotics : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Albany, New Zealand

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Bioprospecting soil metagenomes for potential new antibiotics : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Albany, New Zealand

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