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Material and Methods 4.2.5.11 Preparation of electrocompetent P. syringae cells One inoculation loop of P. syringae cells was scratched from an over night KB agar plate. The cells were resuspended in 1ml of ice-cold water and kept on ice for 15 to 30 minutes. Then the cells were pelleted by centrifugation and washed with 1 ml of ice-cold water two times. Thereafter the cells were washed another two times in 0.5 ml of ice cold 10% glycerol. After the final washing step, the cell pellet was resuspended in 200 µl of 10% glycerol. The cells were not stored but directly used for electroporation. 4.2.5.12 Electroporation of DNA 40 µl of electrocompetent cells (either E.coli or P. syringae) were mixed with 0.1-1 µg of plasmid DNA or 1-2 µl of ligation mixture, and exposed to electric shock in a pre-chilled electroporation cuvette. The GenePulser-Apparatus (BioRad) used for this experiment was set to 2.5 kV and 200 Ω. Directly after the electric pulse, cells were resuspended in 1 ml of the appropriate medium (LB medium for E. coli and KB medium for P. syringae) The cells were then incubated for 60 min. by shaking at 280 rpm at the respective growth temperature, and plated onto agar plates (LB medium or KB medium) containing the appropriate antibiotic(s). The plates were incubated until single colonies became visible. 4.2.5.13 Conjugation by triparental mating The native mechanism of horizontal gene transfer by plasmid conjugation via ‘triparental mating’ was used to transfer broad-host range plasmids into P. syringae. Donor plasmids with the mob function are mobilized and transferred into the recipient P. syringae strain by the use of the helper strain E. coli HB101 (pRK2013) carrying the transfer function. Recipient P. syringae strains were grown for 2 days on MG agar plates prior to the conjugation. Approximately one inoculation loop of recipient bacteria was resuspended in 1 ml of sterile water. Subsequently, 1/3 of an inoculation loop each of the donor and helper E. coli 37
Material and Methods strains were scratched from an over night grown plate and resuspended in the same volume. Then 2 µl of each bacterial suspension was mixed together. The resulting 60µl of bacterial suspension was divided into three aliquots of 20 µl and spotted onto an LB plate. The suspension was allowed to dry and the plate was subsequently incubated over night at 28°C. Then the mating spots were scraped off the plate and resuspended in 1 ml sterile water, each. This cell suspension was diluted from 10 -1 to 10 -5 . 200-300 µl of each dilution step were plated onto MG plates with the appropriate antibiotic(s) to select for transconjugants. Subsequently, plates were incubated at 28° for 3-5 days until single colonies of P. syringae became visible. The selected transconjugants of P. syringae were restreaked twice on MG agar plates in order to eliminate residual E. coli cells. 4.2.6 DNA-analysis by Southern blot technique 4.2.6.1 Generation of digoxigenin labelled probes Digoxigenin labelling of DNA-probes for DNA-DNA hybridisation was performed according to the manual of the “DIG DNA labelling and detection kit”. 15 µl of sample DNA was incubated at 95°C for 10 min. and subsequently shock-cooled in an ethanol/ice-bath. The denaturated sample DNA was the mixed with 2 µl 10x hexanucleotidemix, 2 µl of dNTP mixture (1 mM dATP/dCTP/dGTP; 0.65 mM dTTP and 0.35 mM DIG-dUTP) and 1 µl Klenow fragment. The reaction mixture was incubated at 37°C over night. In order to remove residual nucleotides, the labelled DNA probe was precipitated by addition of 1 µl 0.5 M EDTA, 2.5 µl of 4M LiCl and 75 µl of pre-cooled EtOH (96%). The mixture was incubated for 30min. at –80°C and subsequently centrifuged. The pelleted DNA was washed with 1 ml of cold 70% EtOH and dried completely. The labelled DNA was resuspended in 50 µl of water and stored at –20°C. 38
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 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: Material and Methods 4.2.5.9 Ligati
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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