«(Über die Bedeutung der bakteriellen Genomplastizität für die Adaptation und Evolution asymptomatischer Bakteriurie (ABU) Escherichia coli ...»
containing guanidine hydrochloride was used for neutralization, and plasmid DNA was purified from the supernatant by using small spin columns, which were centrifuged at 13,000 rpm for 1 min DNA was eluted in a small volume of dH2O and directly used for further experiments.
4.1.8. Enzymatic digest of DNA with restriction nucleases
The DNA was dissolved in dH2O and mixed with 0.2 vol 10 x reaction buffer and 1 U of restriction enzyme per 1 µg of DNA. The final volume of the sample was 15 µl for plasmid DNA and 50 µl for genomic DNA. The reaction mixture was incubated 37 °C depending of the specific requirements of the enzyme (stated by supplier). Plasmid DNA was digested for one to two hours and genomic DNA at least for 3 hours, eventually overnight. When appropriate, the reaction mix was stopped by heat inactivation (20 min at 65 °C).
4.1.9. Horizontal gel electrophoresis
For routine analytical and preparative separation of DNA fragments, horizontal gel electrophoresis was performed using agarose gels under non-denaturing conditions.
Depending on the size of the DNA fragments to be separated, the agarose concentration varied between 1 and 2 % (w/v) in running buffer (1 × TAE). In order to have a visible running front and to prevent diffusion of the DNA, 0.2 vol loading dye was added to the samples before loading. The electrophoresis was carried out at a voltage in the range between 16-120 V. The gels were stained in an ethidium bromide solution (10 mg ml-1), washed with water and photographed under a UV-transilluminator.
4.1.10. Plus Field Gel Electrophoresis (PFGE) Isolation of high molecular weight genomic DNA Bacteria were grown overnight in 10 ml LB medium at 37 °C, then 4 ml of the culture were harvested by centrifugation and the pellet was washed two times with SE buffer.
After resuspending the cells in 1 ml SE, OD600 was measured (100 μl cells + 900 μl SE buffer). When necessary, the optical density had to be adjusted in the range between 0.6 and 0.7. LGT agarose (2 %) was prepared and cooled to 45-50 °C. 900 μl of the bacterial suspension were mixed with 900 μl agarose and poured into appropriate moulds by pipetting. After solidifying, agarose blocks were cut in pieces with approximately identical size (0.5 × 0.3 × 0.1 cm), and then incubated in 5 ml NDS solution (freshly supplemented with 2 mg/ml proteinase K) overnight at 50 °C with agitation. For the complete removal of the proteinase K, the blocks were washed at least four times for 2 h with TE buffer. So prepared agarose blocks with immobilized DNA were stored at 4 °C in TE buffer for at least one week before they could be used for restriction.
4.1.11. Restriction of high molecular weight DNA LGT agarose blocks containing the high molecular weight DNA were transferred into a new Eppendorf tube. Blocks were pre-incubated 1 h at 50 °C in 1 ml 1 × NEB buffer.
Restriction was carried out for 3 h at 37 °C in an 150 μl overall reaction mixture containing 1 x restriction buffer and 30 U restriction enzyme, when recommended with addition of BSA.
4.1.12. Separation of restriction fragments by gel electrophoresis High molecular weight DNA was separated on an 0.8 % (I-CeuI) and 1 % (XbaI, AvrI) (w/v) agarose gel (1 × TBE buffer with 1 mM urea) by horizontal electrophoresis. The gels were run for 21-24 h with pulse periods of 0.5-50 s. After staining in an ethidium bromide solution (10 g/ml), the gels were photographed on UV-transilluminator.
4.1.13. Isolation of DNA fragments from agarose gels
DNA was purified from agarose gels using the QIAquick Gel Extraction Kit (QIAGEN).
Agarose pieces containing the DNA fragment of interest were cut out of the gel and subsequently melt for 10 min at 50 °C in QG buffer (supplied by the manufacturer). The DNA was separated from the rest of the solution by applying the mixture to QIAquick spin columns followed by centrifugation for 1 min. Columns were washed with 750 µl PE buffer (supplemented with ethanol). Residual PE buffer was removed by centrifugation (2 x 1 min). Finally, DNA was by eluted from the column with 20-50 µl sterile dH2O.
4.1.14. Ligation of DNA fragments
Linearized vector and insert DNA after restriction digest can be ligated either due to the presence of sticky ends or by blunt-end ligation. The modifying enzyme for ligation process was a T4-DNA ligase (New England Biolabs). Best efficiencies were obtained using an insert/vector ratio of 3/1. Reactions were performed over night at 16 °C in a final volume of 15 μl containing 1.5 μl 10 x ligation buffer and 50 U T4 ligase.
4.1.15. Preparation of electrocompetent cells and electroporation 50 ml LB medium were inoculated with 500 μl bacterial overnight culture and grown until OD600 of 0.6-0.8. The cells were collected by centrifugation for 10 min at 2,000 × g at 4 °C. The pellet was left on ice for 30 min and then washed with 50 ml ice-cold dH2O.
After the second centrifugation step in the same conditions, pellet was resuspended in 25 ml 10 % (v/v) glycerol, centrifuged again and finally resuspended in 600 μl of 10 % (v/v) glycerol. Cells were stored as 40 μl aliquots at -80 °C. For electroporation, one aliquot was thawed on ice and mixed with ~ 0.5 μg DNA. The mixture was applied into a “Gene pulser” cuvette (BioRad) with a distance between the electrodes of 0.1 cm and incubated for 10 min on ice. Cells were electroporated using a Gene pulser transfection apparatus (BioRad) at the following conditions: 2.5 kV, 25 μF, and 600 Ω for linear fragments or 200 Ω for plasmids. Immediately after electroporation, 1 ml LB medium was added to the cuvettes. The mixture was transferred into a new tube and incubated at 37 °C (or 30 °C for temperature-sensitive plasmids) for 1 h before the cells were plated on selective agar.
4.1.16 Gene inactivation by λ Red recombinase-mediated mutagenesis using linear DNA fragments The construction of the mutants was performed using linear DNA for recombination, as described by Datsenko and Wanner (Datsenko and Wanner, 2000). This method relies on the replacement of a chromosomal sequence with an antibiotic marker that is generated by PCR using primers with homology extensions to the flanking regions of the target sequence. Recombination is mediated by the Red recombinase derived from the λ phage.
This recombination system consists of three genes (γ, β, exo), which encode the phage recombinases and an inhibitor of the host RecBCD exonuclease V, which normally mediates degradation of linear DNA in the cell. A schematic overview of the procedure is depicted in Fig. 7.
Meanwhile, bacterial cells were first transformed with the pKD46 helper plasmid by electroporation. Transformants were selected at 30 °C on agar plates containing 100 μg ml-1 ampicillin. Of these transformants, electrocompetent cells were prepared from 50 ml LB cultures supplemented with ampicillin and 3 ml of a 0.1 M arabinose solution to induce the Red recombinase on the helper plasmid. 40 μl competent cells were transformed with 5 μl of the linear PCR fragment by electroporation. After the addition of 1 ml LB medium to the cuvette, cells were allowed to recover by incubation for 2 h at 30 °C with aeration. In contrast to normal electroporation, the cultures were then taken out of the incubator and left standing on the bench top over night at room temperature. On the next morning, cells were spun down, resuspended in 300 μl LB medium and distributed onto three agar plates supplemented with the appropriate antibiotic (Cm or Km, respectively). Transformants with confirmed allelic exchange were also re-streaked onto ampicillin-containing agar plates at 37 °C to confirm loss of the temperature-sensitive helper plasmid pKD46.
The antibiotic marker could be removed with the help of the FLP recombinase (encoded on plasmid pCP20), which mediates recombination between the two FRT sites flanking the antibiotic cassette, thus leaving behind a complete deletion of the open reading frame.
Electroporation was performed as described in section 4.1.15. Transformants were first selected on ampicillin-containing agar plates at 30 °C, and then re-streaked onto LB agar
55 Methods – Working with DNA
plates with no antibiotic. These plates were incubated at 37 °C in order to induce the loss of the second helper plasmid pCP20. The deletion mutants now could be used to introduce a second or third mutation by starting the whole procedure from the beginning.
All mutations were confirmed by both PCR and Southern hybridization.
4.1.17. Southern Blot analysis Vacuum blotting For Southern blot analysis, 10 μg chromosomal DNA were restricted with an appropriate endonuclease, harboring 1 to 5-kb DNA fragments containing the target gene. The DNA fragments were separated by horizontal gel electrophoresis. Meanwhile, a nylon membrane (Nytran Super Charge; pore size 0.45 μm; Schleicher&Schuell, Dassel, Germany) of appropriate size was shortly preincubated in dH2O and then soaked for 10 min in 20 × SSC. Afterwards, DNA was transferred from the agarose gel to the membrane using a vacuum blotter (Amersham-Pharmacia) by applying a 50 mbar vacuum. The following solutions were applied on the surface of the agarose gel during
the blotting procedure:
After DNA transfer, the nylon membrane was incubated for 1 min in 0.4 N NaOH and 1 min in 0.25 M Tris-HCl, pH 7.5 for neutralization. The membrane was then shortly dried and the DNA was crosslinked to the membrane by exposure to UV light.
Probe labelling (ECLTM Kit, Amersham Biosciences) For labelling of DNA probes, the ECLTM-Kit (enhanced chemoluminescence) was used.
The binding of a DNA probe to the complementary sequence on the nylon membrane was detected by chemoluminescence. Positively charged horseradish peroxidase molecules were mixed with the negatively charged DNA probe. Addition of glutaraldehyd covalently linked the horseradish peroxidase molecules with the DNA probes. Reduction of H2O2 by the peroxidase requires the oxidation of luminol which results in light emission, which can be detected by suitable light-sensitive films, e.g. the Hyperfilm ECL.
For labeling of the probe, 100 ng DNA per ml hybridization buffer in a final volume of 10 μl in dH2O were denatured for 10 min at 90 °C and cooled for 5 min on ice.
Subsequently, 10 μl labelling reagent and 10 μl glutaraldehyde were added. The mixture was incubated for 10 min at 37 °C and then added to the hybridization reaction.
Hybridization and detection of the membrane
Hybridization of the membrane was carried out over night at 42 °C in hybridization solution (10-15 ml), after the nylon membrane had been pre-incubated at 42 °C in the hybridization solution for 1 h. The next day, the membrane was washed twice for 20 min at 55 °C with wash solution I and two times for 10 min at RT with wash solution II. The membrane was placed on Whatman paper to remove the rest of the wash solution, and then incubated for 5 min in 5-10 ml detection solution I and detection solution II provided with the kit and mixed immediately (1:1) before application to the membrane. The membrane was superficially dried on Whatman paper and packed in saran wrap avoiding air bubbles on the top surface of the membrane. Chemoluminescence was detected by exposure of the membrane to Hyperfilm ECL. The exposure time depended on the signal intensity.
18. Comparative Genome Hybridization Membrane Layout For genomes comparison, an E. coli K12-specific array (Panorama E. coli gene arrays from Sigma-Genosys, Cambridge, United Kingdom) and the E. coli “pathoarray” (Dobrindt et al., 2003) were used in combination. Therefore, by using both types of arrays, it was possible to estimate and compare the genomic content of different ABU strains.
2 µg of total genomic DNA (see section 4.1.1.) was used as a template for direct incorporation of [33P]-dATP (Amersham Pharmacia, Freiburg, Germany) by a randomly primed polymerization reaction using 0.75 µg random hexamer primers (New England Biolabs, Frankfurt (Main), Germany) and 10 U Klenow fragment of the DNA polymerase I (New England Biolabs) according to manufacturer’s recommendations. Unincorporated nucleotides were removed with Microspin S 200 HR spin columns (Amersham Pharmacia). Incorporation rates of labelled nucleotides were determined by measuring radioactivity of the cDNA in the eluate (equals incorporated radionucleotides) and of the radioactivity retained in the column (equals unincorporated radionucleotides).
The obtained value can be used as a marker for the quality of reverse transcription and should be 50 %.
Hybridization and detection Hybridization of the labelled DNA to the membranes was performed as recommended in the technical protocol for the Panorama E. coli K-12 array (Sigma Genosys). After 10 min denaturation at 93 °C in hybridization buffer, labelled DNA was added to the prehybridized membranes and incubated for 16 h at 65 °C in a hybridization oven.
58 Methods – Working with DNA
Membranes were washed three times for 3 min in washing solution at room temperature, followed by three additional washing steps for 20 min at 65 °C. After washing, the membranes were sealed in plastic film and exposed to a PhosphoImager screen (Molecular Dynamics) for 48 h. Membranes could be re-used up to 10 times after stripping (see Panorama-Array manual).
Quantification of hybridization signals Exposed PhosphoImager screens were scanned on a Typhoon 8600 variable mode imager (Molecular Dynamics) at 50 μm resolution. Spot intensities were measured with the ArrayVision software (Imaging Research, St. Catharines, Canada) using the overall spot normalization function of the program (Table 6).