School of Engineering and Science - Jacobs University

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Contents 1 Summary ....................................................................................................... 1 2 Introduction................................................................................................... 2 2.1 Biological control of plant disease ....................................................... 3 2.1.1 Advantages and limitations ............................................................ 3 2.1.2 Host, pathogen and antagonist – more than a “ménage à trois” 6 2.2 Iron and its implications for disease and disease control................ 11 2.2.1 The biology of iron......................................................................... 11 2.2.2 Iron in biological control of bacteria............................................ 12 2.3 Biological control of bacterial blight of soybean .............................. 13 2.4 Aim of this study .................................................................................. 18 3 Material ........................................................................................................ 19 3.1 Equipment............................................................................................. 19 3.2 Chemicals, antibiotics and enzymes .................................................. 19 3.3 Kits ........................................................................................................ 20 3.4 Media ..................................................................................................... 20 3.4.1 Complex medium for Escherichia coli......................................... 21 3.4.2 Complex medium for Pseudomonas syringae ............................ 21 3.4.3 Minimal media for Pseudomonas syringae ................................. 21 3.4.4 Siderophore detection medium .................................................... 23 3.5 Microorganisms.................................................................................... 24 3.6 Plasmids................................................................................................ 26 3.7 Oligonucleotides .................................................................................. 27 4 Methods ....................................................................................................... 28 4.1 Bacterial growth conditions ................................................................ 28 4.1.1 Escherichia coli ............................................................................. 28
Contents 4.1.2 Pseudomonas syringae................................................................. 28 4.1.3 Other bacteria ................................................................................ 29 4.1.4 Determination of cell density........................................................ 29 4.1.5 Storage of bacterial strains........................................................... 29 4.2 Molecular biology methods................................................................. 29 4.2.1 Plasmid DNA isolation................................................................... 29 4.2.1.1 Isolation of plasmid DNA by alkaline lysis ............................... 29 4.2.1.2 Plasmid DNA isolation by NucleoSpin plasmid kit .................. 30 4.2.2 Preparation of genomic DNA ........................................................ 30 4.2.3 DNA separation by gel electrophoresis ....................................... 31 4.2.4 Polymerase chain reaction (PCR) ................................................ 32 4.2.5 Cloning techniques........................................................................ 33 4.2.5.1 Digestion of plasmid DNA with endonucleases....................... 33 4.2.5.2 De-phosphorylation of digested DNA ....................................... 33 4.2.5.3 Converting overhanging sticky ends to blunt ends................. 34 4.2.5.4 Precipitation of DNA by ethanol ................................................ 34 4.2.5.5 Extraction of DNA fragments out of agarose gels ................... 34 4.2.5.6 QIAquick PCR and reaction purification kit.............................. 35 4.2.5.7 Estimation of DNA concentration.............................................. 35 4.2.5.8 Ligation of DNA........................................................................... 35 4.2.5.9 Ligation of PCR fragments by T/A cloning ............................... 36 4.2.5.10 Preparation of electrocompetent E. coli cells ........................ 36 4.2.5.11 Preparation of electrocompetent P. syringae cells................ 37 4.2.5.12 Electroporation of DNA ............................................................ 37 4.2.5.13 Conjugation by triparental mating........................................... 37 4.2.6 DNA-analysis by Southern blot technique .................................. 38 4.2.6.1 Generation of digoxigenin labelled probes .............................. 38
Page 1 and 2: Siderophore production of Pseudomon
Page 3 and 4: Acknowledgements This project was s
Page 5: Abbreviations Ach achromobactin-lik
Page 9 and 10: Contents 5.3 Influence of sideropho
Page 11 and 12: Introduction 2 Introduction Like hu
Page 13 and 14: Introduction In contrast to the rhi
Page 15 and 16: Introduction 2.1.2 Host, pathogen a
Page 17 and 18: Introduction The relationship betwe
Page 19 and 20: Introduction developement of resist
Page 21 and 22: Introduction Fe 2+ + H 2 O 2 → Fe
Page 23 and 24: Introduction 1997a; May et al., 199
Page 25 and 26: Introduction Pss22d demonstrated a
Page 27 and 28: Introduction 2.4 Aim of this study
Page 29 and 30: Material and Methods Tab. 4. Antibi
Page 31 and 32: Material and Methods Iron-free 5b m
Page 33 and 34: Material and Methods Solution3, car
Page 35 and 36: Material and Methods 3.6 Plasmids T
Page 37 and 38: Material and Methods PCR-Screening
Page 39 and 40: Material and Methods on ice for 10
Page 41 and 42: Material and Methods stained in eth
Page 43 and 44: Material and Methods 4.2.5.3 Conver
Page 45 and 46: Material and Methods 4.2.5.9 Ligati
Page 47 and 48: Material and Methods strains were s
Page 49 and 50: Material and Methods 9.5). For sign
Page 51 and 52: Material and Methods gentle shaking
Page 53 and 54: Material and Methods columns were w
Page 55 and 56: Results 5 Results 5.1 Analysis of s
Page 57 and 58:
Results The 13-kb open reading fram
Page 59 and 60:
Results If integration is due to a
Page 61 and 62:
Results Yersiniabactin is a siderop
Page 63 and 64:
Results Next, a large number of tra
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Results Nevertheless the site of tr
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Results Achr2_KpnI_rev binding site
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Results Interestingly the mutant Ps
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Results XAD4-purification yielded i
Page 73 and 74:
Results After changing the mobile p
Page 75 and 76:
Results P P A Fig. 21. Isoelectic f
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Results reduced in comparison to th
Page 79 and 80:
Results A-E represents single inocu
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Results Tab. 10. Development of pop
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Results Tab. 11. Distribution of th
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Discussion bacteria with redundande
Page 87 and 88:
Discussion 6.2 Possible regulation
Page 89 and 90:
Discussion stationary phase transit
Page 91 and 92:
Discussion and bacterium (Loper et
Page 93 and 94:
Literature Bultreys, A., Gheysen, I
Page 95 and 96:
Literature Galperin, M. Y. (2006).
Page 97 and 98:
Literature Mazzola, M. (2002). Mech
Page 99 and 100:
Literature Schubert, S., Rakin, A.,
Page 101:
Literature Zou, J., Rodriguez-Zas,
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School of Engineering and Science - Jacobs University

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