2 MATERIALS AND METHODS
2.1.2.1 Pseudomonas putida Genome Oligonucleotide Array
The P. putida Genome Oligonucleotide Array (Progenika Biopharma, S.A, Derio, Spain) was developed in collaboration with the company Progenika Biopharma and several Spanish scientists working in the field of P. putida (Dr. Fernando Rojo, Madrid; Dr. Juan Luis Ramos, Granada; Dr. Eduardo Díaz, Madrid; Dr. Victor de Lorenzo, Madrid and Dr. Eduardo Santero, Seville).
It is a two-dye gene expression array, whereby the comparison of the binding efficiencies of two samples to one array provides an insight into gene expression changes in a single experiment. In this protocol the two fluorescent cyanine dyes cy3 and cy5 are used for indirect labelling by coupling to aa-dUTP, an amine-modified nucleotide incorporated into cDNA during reverse transcription (see 2.7.4.1 and 2.7.5.1).
The array is designed as listed below:
• γ-aminosilane treated glass slides
• single-stranded oligonucleotides in repeating spots
• 50mer oligonucleotides
• One oligonucleotide representative for one ORF
• 5539 P. putida KT2440 ORFs
• Homogeneity control with 2 ORFs (20 replicates each) distributed over entire array
• Negative control with DMSO (50%), 406 spots, for background hybridization correction distributed over entire array
17 2.1.2.2 Affymetrix microarray
The Affymetrix high-density oligonucleotide microarray was based on the annotated genome of P. putida KT2440 (NC_002947.3) and was designed with a pair-wise configuration of 13 perfect match (PM) and mismatch (MM) 25mer oligonucleotides per probe set. In total 8047 probe sets were spotted that represent 5330 annotated ORFs, 207 ORFs not present on the Pseudomonas homepage (www.pseudomonas.com), 22 genes encoded on the TOL plasmid (xyl cluster), 2443 intergenic regions and 45 control sequences from other organisms not in the genus Pseudomonas.
Preparation of RNA, cDNA and cDNA fragmentation and biotin-labelling are described in chapters 2.7.2., 2.7.4. and 2.7.5.
2.1.2.3 Consumables
A selection of further consumables used in this study is given below:
Consumable Manufacturer
CryoTubeTM Vials NUNC
Electroporation cuvettes 1 mm BioRad
Filter Celluloseester HA, 0.45 μM pore size Millipore
Hybond N+ Nylon Membrane Amersham Biosciences
MicroCons YM-10 Millipore
Qiaprep Spin Miniprep Kit Qiagen
QiaQuick PCR Purification Kit Qiagen
RNeasy Mini Kit Qiagen
Whatman paper Schleicher & Schüll
X-ray film X-Omat AR Qiagen
96 well plates Greiner
18 2.1.2 Chemicals
Chemical Manufacturer
Agarose Eurogentec Antifoam Struktol SB2121 Schill & Seilacher
Biotin, Streptavidin
(anti-streptavidin (goat), biotylilated) Vector Laboritoiries
BSA New England Biolabs
CDP-Star Tropix
Coomassie Brilliant Blue R250 Serva
Cyanine-3, -5 GE Healthcare
λ-DNA, BstEII digested NEB
50bp-, 100bp-ladder Fermentas
Ethanol J.T. Baker
Formaldehyde Merck
GeneChip DNA Labelling Affymetrix
Gentamicin Serva
Glycerol AppliChem
β-Mercaptoethanol Merck
Neutravidin ThermoScientific Oligonucleotides MWG
10x One-Phor-All Buffer Amersham
Phenolrot Merck
RNAprotect Bacteria Reagent Qiagen
Rnase Inhibitor Ambion
R-Phycoerythrin Streptavidin (SAPE) DIANOVA
Sephadex G-50 Pharmacia Fine Chemicals
Sucrose loading dye Amresco
SYBR Gold Invitrogen
All chemicals not listed were either purchased from Fluka, Roche, Roth or Sigma-Aldrich.
19 2.1.3 Enzymes
A list of enzymes with their corresponding buffer system is listed below:
Enzyme Manufacturer
Alkaline phosphatase, calf intestine (CIP) NEB Anti-digoxigenine alkaline phosphatase Roche
DNase I Qiagen
FailSafeTM PCR Premix Selection Kit Epicentre Technologies
Goldstar-Taq-Polymerase Eurogentec
Invitek-Taq-Polymerase Invitek
Klenow-Poylmerase Boehringer Restriction endonucleases + buffer systems NEB
Acc65I, BamHI, EcoRI, HindIII, NlaIII, NsiI, PstI, Sau3AI, SphI, XbaI
RNase A (10 mg/ml) Qiagen
SuperScriptII reverse transcriptase Invitrogen
Terminal transferase Promega
T4 DNA ligase NEB
T4 PN kinase NEB
20 2.2 Media and Solutions
Media and consumables were sterilized by autoclaving at 121 °C for at least 30 minutes, unless otherwise specified. The reagents were of high purity (“pro analysis”) and purchased from Fluka, Merck, Roche, Roth or Sigma-Aldrich.
2.2.1 Media
Luria-Bertani Medium (LB Medium)
Tryptone 15.0 g/L
Yeast Extract 5.0 g/L
NaCl 10.0 g/L
pH 7.0
LB-Gm: LB medium/ agar with 30 µg/mL Gentamicin LB-Amp: LB medium/ agar with 100 µg/mL Ampicillin LB-Car: LB medium/ agar with 1000 µg/mL Carbenicillin
LB Agar: LB medium was solidified by adding 20 g/L agar and subsequent autoclaving.
Glycerol Medium
For long-term storage of bacterial strains LB medium was supplemented with glycerol to a final concentration of 15%.
ABC Minimal Medium
Na2HPO4 6.0 g/L
KH2PO4 3.0 g/L
NaCl 3.0 g/L
(NH4)2SO4 2.0 g/L
pH 7.0
Medium was supplemented with 15 mM or 45 mM sodium benzoate for phenotypical verification growth experiments.
21
Minimal Medium for growth experiments
stock concentration end concentration
Stock solutions were adjusted to a pH of 6.8, filter sterilised through a 0.2 µm nitrocellulose filter and stored aseptically prior to use. FeSO4-solution was always prepared fresh. Solutions were mixed immediately before usage.
2.2.2 Solutions
Antibody-Solution
Antibody-solution was made by adding 10 µL Anti-Digoxigenin AP Fab (150 U/200 µL) to 50 mL Buffer II (1:5000 dilution)
Antibody solution for Affymetrix microarray hybridization 2x staining buffer 315 µL
DEPC-treated H2O 279.7 µL BSA (50 mg/mL) 25.2 µL
22 Blot washing buffer
sodium phosphate 50 mM pH 6.5
Buffer I
Tris/HCl 100 mM
NaCl 150 mM
pH 7.5
Buffer II
Buffer II was freshly made from Buffer I by adding 0.5% blocking reagent (Roche). The solution was stirred on a heater to ensure solubility.
Buffer III
Tris/HCl 100 mM
NaCl 100 mM
MgCl2 50 mM
pH 9.5 CDP Star
A 12.5 mM stock solution of CDP Star was stored at 4°C and diluted 1:500 in Buffer III prior to use.
Coomassie solution
Coomassie Brilliant Blue R250 0.05%
Methanol 50%
Acetic acid 10%
Denhardt’s solution (50x)
BSA 1% (w/v)
Ficoll 1% (w/v)
PVP 1% (w/v)
Solution was sterile filtrated ( Ø 0.2 µm) and stored at 4°C.
Elution-buffer
Potassium Phosphate Buffer 5 mM pH 8.5
23 Hybridization buffer
Formamide (deionised) 50% (v/v) Denhardt´s solution 5% (v/v)
SDS 1% (w/v)
SSC 3% (v/v)
dextransulphate 5% (w/v)
Hybridization buffer (2x) for Affymetrix mmicroarrays
12x MES 8.3 mL Bromophenol Blue 0.25% (w/v) Xylene cyanol 0.25% (w/v)
MES free acid monohydrate 17.6 g MES sodium salt 48.33 g
ddH2O add to 250 mL
pH 6.5 – 6.7
The solution was sterile filtrated and stores at 4°C in the dark.
24
25 Solution III
Potassium acetate 3 M
Acetic acid 2 M
20x SSPE solution
Sodium chloride 3 M Sodium phosphate 200 mM
EDTA 20 mM
pH 7.7
2x Staining buffer for Fluidic station (Affymetrix)
12x MES 41.65 mL
NaCl (5 M) 92.5 mL
Tween 20 (10%) 2.5 mL
ddH2O 112.8 mL
Standard saline citrate solution (20%) (SSC) Sodium chloride 175.3 g
Sodium citrate 88.2 g pH 7.0
Stripping buffer
NaOH 0.2 M
SDS 0.1% (w/v)
TBE-Buffer (10X)
Tris/HCl 0.9 M
Boric Acid 0.9 M
EDTA 0.02 M
pH 8.3 - 8.5
TE Buffer
Tris/HCl 10 mM
EDTA 1 mM
pH 8.0
26
Washing Buffer for Southern Blot
NaH2PO4 40 mM
SDS 1% (w/v)
1 mM EDTA pH 7.2
Washing-buffer for cDNA labelling Potassium phosphate buffer 4 mM
Ethanol 80% (v/v)
pH 8.0
Washing buffers for microarray hybridization (Progenika)
WB1 SSC 2%, SDS 0.1%
WB2 SSC 1%
WB3 SSC 0.2%
WB4 SSC 0.1%
Washing buffers for Fluidic Station (Affymetrix) Buffer A
27 2.3 Biological Materials
The P. putida KT2440 mini Tn5 plasposon mutant library was generated by Christian Weinel (2003). Insertion point and flanking genes were determined either by plasmid rescue or Y-linker method. Plasposon mutants used in this study were already described for their stress sensitive phenotype (Reva et al., 2006).
2.3.1 Strains
Tab. 2.1 Overview of strains used in this study for either cold or benzoate stress experiments.
Strain Genotype Reference
Pseudomonas putida
KT2440 (DSM 6125) hsdR1, hsdM+, Ben+ Bagdasarian et al., 1981
#cysM pTnMod‐OGm::KTcysM:92* Reva et al., 2006
F– 80lacZ M15 (lacYZA‐argF)U169 recA1 endA1 hsdR17 (rK– mK+) supE44 thi‐1 gyrA relA1 (Nalr)
Ausubel et al., 1987
* Number indicates insertion site within the gene.
2.3.2 Plasmids
Tab. 2.2 Overview of plasmids used in this study.
Plasmid Genotype Reference
pUCP20 Escherichia – Pseudomonas shuttle vector; Apr Garrity‐Ryan et al., 2000 pUCP20::KTcysM pUCP20 plasmid carrying the XbaI/HindIII the PCR
product bearing the cysM gene This study pUCP20::KTkgdA pUCP20 plasmid carrying the XbaI/HindIII the PCR
product bearing the kgdA gene
pUCP20::KTPP4646 pUCP20 plasmid carrying the XbaI/HindIII the PCR product bearing the PP4646 gene pUCP20::KTomlA pUCP20 plasmid carrying the SphII/HindIII the PCR
product bearing the omlA gene
28 2.3.3 Oligonucleotides
Tab. 2.3 Summary of oligonucleotides used in this study.
Primer in 5’ – 3’ direction Reference
Verification of plasposon mutants
29 2.4 BioFlo 110 Modular Benchtop Fermenter
Growth experiments for comparative analysis of transcriptome, metabolome and proteome in P. putida wt and respective mutants were performed in a bioreactor with 1.5 L operating volume.
The BioFlo 110 fermenter (New Brunswick Scientific Co., Inc., Edison, NJ, USA) included a vessel with an external heat blanket and a headplate for variable port usage. pH and dO2 -probe were provided by Mettler-Toledo GmbH (Gießen). The process was controlled by the Primary Control Unit (PCU), including agitation, aeration, pH, dO2 and antifoam. The BioCommand software was used for documentation and visualization of the fermentation process (both New Brunswick Scientific Co., Inc., Edison, NJ, USA).
Media was maintained to 30°C, agitated with 600 rpm and streamed with pressure air (~ 21%
oxygen) with an air flow of 1.5 L/min. After dO2-probe calibration, oxygen saturation was set to 100%. Agitation, aeration and pH were kept constant during fermentation. pH was adjusted automatically to 6.8 ± 0.1 with 5 % H3PO4 or 1 M NaOH. Antifoam was provided with the medium (0.025%).
The vessel containing pure M9 medium with 0.025% antifoam and all required probes were autoclaved at 121°C for 30 min ahead of fermentation, carbon source and other components of the media were aseptically added afterwards.
To check for contamination, prior to inoculation, 1 mL media were collected and screened for bacterial growth on LB agar plates.
Fig. 2.1 BioFlo 110 Modular Benchtop Fermenter.
30 2.5 Microbiological Methods