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Material and Methods 4.1.3 Other bacteria Cells of D. chrysanthemi, X. campestris and S. meliloti were grown for 1-3 days at 28°C on LB or KB agar plates. Liquid cultures (20-250 ml) of D. chrysanthemi were grown in Erlenmeyer flask at 28°C by shaking at 280rpm. 4.1.4 Determination of cell density Cell density of bacterial suspensions or liquid culture was determined by photometric measurement. The optical density (OD) of the sample was determined at λ = 600nm. Samples with an OD above 0.6 were diluted in the appropriate medium. It is assumed that an OD 600nm of one correlates to a cell density of 10 9 (Miller, 1992). 4.1.5 Storage of bacterial strains Fresh grow bacterial colonies or lawns from agar plates were resuspended in 15% sterile glycerol solution and stored at -80°C. 4.2 Molecular biology methods 4.2.1 Plasmid DNA isolation 4.2.1.1 Isolation of plasmid DNA by alkaline lysis Small amounts of plasmid DNA were isolated by the 1-2-3 protocol based on alkaline lysis (Sambrook et al., 1989).1-1.5 ml of E. coli overnight culture was harvested by centrifugation at 13,000 rpm for 2-5 min. The pellet was resuspended in 150 µl of pre-cooled buffer P1 (100 µg/ml RNase A, 50 mM Tris/HCl, 10 mM EDTA-Na at a pH of 8.0). Alternative, cells from an overnight LB-plate were resuspended in 150 µl of pre-cooled buffer P1. Subsequently, the cells were lysed upon addition of 150 µl of buffer P2 (200 mM NaOH, 1% SDS) for 5 min, and the lysate was neutralized by addition of 150 µl of precooled 3 M potassium acetate solution (pH 5.5). This fast neutralisation led to the renaturation of small plasmid DNA. Larger molecules as proteins and chromosomal DNA, and cellular debris stay denaturated. Following incubation 29
Material and Methods on ice for 10 min and subsequent centrifugation at 13,000 rpm for 15 min, the supernatant containing plasmid DNA was transferred into another plastic reaction tube. DNA was precipitated by addition of 0.7 volume (300 µl) of 2- propanol and subsequent centrifugation for 30 min. Then the DNA pellet was washed by 400-500 µl of 70% ethanol solution. After centrifugation for 15 min, the DNA pellet was dried in a speedvacuum centrifuge for 5 min, and dissolved in 20 µl of deionised water. 4.2.1.2 Plasmid DNA isolation by NucleoSpin plasmid kit For preparation of larger quantities of plasmid DNA, the NucleoSpin kit provided by Macherey-Nagel (Düren) was applied according to the protocol provided by the manufacturer. The NucleoSpin preparation is based on alkaline lysis, too, but instead of precipitation the plasmid DNA is recovered by binding to a silica-membrane. The application of chaotrophic salts in the buffer system of the kit leads to a high purity of the obtained DNA, thus this method was applied for DNA preparations used for subsequent cloning or sequencing. 4.2.2 Preparation of genomic DNA Genomic DNA of P. syringae was isolated by chloroform phenol extraction. As the pseudomonads produce large quantities of exopolysaccharides that interfere with DNA extraction, a cethylhexatrimethylammoniumbromide (CTAB) extraction of exopolysaccharides was applied (Ausubel et al., 1987). 1.5 ml of a over night culture of P. syringae were harvested by centrifugation. Supernatant was discarded and the cell pellet was resuspended in 567 µl of buffer P1 (4.2.1.1). 30 µl of a 10% SDS solution a 3µl of 20 mg/ml proteinase K were added. The sample was mixed and incubated at 37°C for one hour. 100 µl of 5M NaCl was added to the viscous lysate and mixed thoroughly. Then, 80µl of a 65°C preheated CTAB/NaCl solution (10% CTAB in 0.7 M NaCl) was added. The sample was mixed and incubated at 65°C for 10 minutes. Afterwards, the CTAB/polysaccharide complexes were removed by chloroform- 30
Page 1 and 2: Siderophore production of Pseudomon
Page 3 and 4: Acknowledgements This project was s
Page 5 and 6: Abbreviations Ach achromobactin-lik
Page 7 and 8: Contents 4.1.2 Pseudomonas syringae
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: Material and Methods PCR-Screening
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
Page 65 and 66: Results Nevertheless the site of tr
Page 67 and 68: Results Achr2_KpnI_rev binding site
Page 69 and 70: Results Interestingly the mutant Ps
Page 71 and 72: 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
Page 77 and 78: Results reduced in comparison to th
Page 79 and 80: Results A-E represents single inocu
Page 81 and 82: Results Tab. 10. Development of pop
Page 83 and 84: Results Tab. 11. Distribution of th
Page 85 and 86: 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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