DNA:DNA Hybridization

2.6.6.2 Ligation

Vector and insert DNA were mixed in proportion 1:3 to assure maximal ligation efficacy.

Two μL T4 buffer, 2 μL ATP (10 mM), 1 μL T4 ligase and ddH2O were added to give a final volume 20 μL. The ligation mixture was incubated for 2 hours at 25 °C and subsequently used for electroporation.

2.6.7 DNA:DNA Hybridization

This method is used for the identification and further analysis of DNA fragments. Hereby, nucleic acids that are separated by gel electrophoresis are transferred to a membrane and immobilized. The easiest transfer method is capillary transfer which was used in this study.

After the adjacent hybridization step the DNA fragments can be detected and analyzed.

Generally, Southern-Blotting is known as the transfer and fixation of nucleic acids to a membrane (Southern, 1975).

2.6.7.1 Generation of digoxigenin-labelled DNA probes

Probe sequences were amplified by PCR (see 2.5.3.1), and isolated and purified from agarose gels (see 2.5.4 and 2.5.5). In the following, labelled DNA fragments were generated by incorporating digoxigenin-linked nucleotides. Therefore, 10 µL of DNA solution and 5 µL ddH2O were heated at 94°C for 4 minutes and immediately cooled on ice. After addition of 2 µL hexanucleotide-mix, 2 µL 10x DIG-dUTP-Labelling mix and 1 µL Klenow polymerase, the reaction mix was incubated at 37°C overnight and afterwards mixed with 280 µL TE buffer and a dye mix (0.8% dextran blue (2x10⁶ g/mol) and 0.5% phenol red (376 g/mol) dissolved in TE buffer).

Before usage the DNA probe was purified by gel filtration through Sephadex G-50 columns (Ø 5 mm, h 4 cm, Pharmacia Fine Chemicals). The column was washed with 2 mL TE buffer and dried by centrifugation (1000 g, 45 sec). The DNA probe was then pipetted onto the gel column and collected by another centrifugation step (1000 g, 30 sec). All unused hexanucleotides, dNTPs and phenol red were retained on the Sephadex column. The DNA probe was stored at -20°C prior to use.

42 2.6.7.2 Restriction digestion of genomic DNA

For DNA:DNA hybridization genomic DNA was digested with a restriction enzyme that cuts the genomic DNA of P. putida relatively often, but not in the DNA fragment of interest. In this study, the enzymes BamHI and PstI (NEB) were used. The reaction mix was incubated at 37°C overnight.

The restriction digestion was examined by agarose gel electrophoresis. The total reaction volume was loaded onto a 1% agarose gel (20 x 20 cm) and separated (1.5 V/cm) at 4°C for 24 hrs. The gel was stained with ethidium bromide, destained and photographed for documentation. Afterwards the DNA was transferred to a nylon membrane (see next part).

2.6.7.3 Transfer and fixation of DNA to a membrane (Southern Blot)

The Southern Blot was performed using a capillary transfer under alkaline conditions (0.4 M NaOH) to transfer and locate the DNA from an agarose gel to a nylon membrane (Hybond N⁺ membrane, Amersham Pharmacia). The gel was placed onto Whatman paper that had contact with the transfer buffer (0.4 M NaOH) reservoir with both ends to ensure the capillary effect.

The nylon membrane was set on the gel and covered by two layers of Whatman paper.

Meanwhile, every single layer was soaked with transfer buffer to avoid air bubbles. For an efficient capillary effect, a thick layer of absorbent paper was placed on top of the construction. The transfer lasted 12 to 16 hours. Afterwards, the membrane was neutralized by washing twice for 5 minutes with neutralization buffer and air-dried. DNA was immobilized onto the membrane by UV irradiation (UV-Crosslinker, Stratagene). If required the membrane can be stored at -20°C prior to use.

2.6.7.4 Hybridization and detection of digoxygenin-labelled DNA

The hybridization reaction allows the detection of specific membrane-bound DNA fragments with digoxigenin labelled DNA probes.

The membrane was placed in a hybridization tube that was filled with prehybridization buffer and incubated in a rotary oven (Bachofer) at 68°C for at least 4 hrs. Afterwards, the labelled DNA probe was dissolved in 10 mL prehybridization buffer and poured onto the membrane

43 which was incubated at 68°C overnight. The probe could be recovered and stored at -20°C.

The membrane was washed at 68°C at least 2 x 20 min in washing buffer to remove unbound probe.

The detection of the DIG-DNA labelled fragments was conducted according to the protocol by Guiliano et al. (1999). The membrane was placed in a plastic basin and treated as follows:

1. Equlibration Buffer I 150 mL 5 min

2. Blocking Buffer II 200 mL 30 min

3. Incubation Antibody solution 50 mL 30 min

4. Washing Buffer I 200 mL 3 x 20 min

In the third step, an anti-DIG antibody that is linked to the alkaline phosphatase, binds to the DIG-labelled probe on the membrane.

The membrane was then transferred to a glass basin that was beforehand washed with buffer III.

5. Equlibration Buffer III 20 mL 5 min

6. Incubation CDP Star solution 10 mL 3 min

In the last step CDP Star is cleaved by the alkaline phosphatase and thus chemoluminescence is released that can be detected by X-ray exposure (X-ray film, X-OMAT^TM AR or Bio-MAX MR, Kodak). Before X-ray exposure, the membrane was laminated.

2.6.7.5 Regeneration of hybridized DNA membranes

DIG-DNA labelled membranes can be recovered for further experiments. For this, they are treated by two washing steps (each for 30 min in Stripping buffer).

The linkage between digoxigenin and dUTP is unstable in alkaline solutions. Thus, the bondage breaks and digoxigenin and the bound antibody are released.

Afterwards, the membrane was washed intensively in H2O to remove SDS, neutralized in blot washing buffer for 5 minutes, laminated and stored at -20°C.

This procedure enables the removal of the DIG-DNA labelling, but not of the DNA fragments from the probe. For this reason, the membrane cannot be regenerated endlessly, as the number of available binding sites for new fragments decreases with every usage.

44 2.6.8 The Y-linker method

The Y-linker method can be used to identify flanking genes of a plasposon insertion as it is independent of the insertion sequence composition. It is PCR-based and depends on the ligation of a designed 40 bp oligonucleotide (Y-linker) to randomly digested mutant chromosomal DNA (200 – 1200 bp). By performing a subsequent PCR with a primer (Y-primer) specific to the non-complementary part of the Y-linker combined with a plasposon specific primer, a PCR product is generated and can be sent for sequencing to identify DNA regions the flanking the insertion (Kwon & Ricke, 2000).

2.6.8.1 Generation of the Y-linker

The Y-linker was generated by ligation of two oligonucleotides (linker strand 1 and 2, see 2.3.3), whereof one part was complementary and built a double-strand and the second part non-complementary and built the y-shape. Linker strand 2 was dephosphorylated by incubating the reaction with a T4-PN-kinase mix at 37°C for 15 minutes and subsequent heating to 95°C for 20 minutes (Thermomixer comfort, Eppendorf). Then, strand 1 was added and the volume was adjusted to 80 µL. The heater was switched off and the ligation mixture was incubated until room temperature was reached slowly. The two y-linker strands annealed and formed the Y-linker.

Finally, another 40 µL ddH20 were added.

Fig. 2.2 Schematic overview about the Y-linker method to generate a PCR product and to identify the flanking region of a plasposon insertion by sequencing (Weinel, 2003; modified).

45

Reaction mix  volume   

strand 2 (3.5 µg/µL)  4 µL 

ATP (10 mM)  4 µL 

10 x T4 PNK buffer (NEB)  4 µL 

4 PN kinase  1 µL 

dd H₂O  27 µL 

Phosphorylation 

37°C for 15 min and 95°C for 20 min   

Strand 2 (3.5 µg/µL)  4 µL 

ddH20  36 µL  Annealing  

Cooling to RT     

ddH2O  40 µL   

2.6.8.2 Y-linker Ligation

Genomic DNA from mutant strains (1 µg) prepared as described in chapter 2.5.5.1 was digested with either NlaIII (5 U) or Sau3AI (5 U) in a final volume of 20 µL. The reaction was incubated at 37°C for 3 hours and immediately used for ligation. For the ligation about 500 µg digested genomic DNA and 5 µL of the annealed Y-linker were incubated at RT for 2 hours together with 2 µL ATP (10 mM), 2 µL T4 buffer and 1 µL T4 ligase (Fermentas) in a final volume of 20 µl adjusted with ddH2O. Approximately 50 ng of the ligation mixture were used as template for PCR using the Y-primer and the specific plasposon primer afterwards.

The PCR product was purified and sent for sequencing.