DISCUSIÓN DE RESULTADOS

Southern blotting was performed on transformants harbouring mutant constructs as to confirm site integration and copy number, after phenotypic analysis of these transformants. Single copy strains were sought for biochemical studies. Transforming constructs harbour one

BamHI site allowing enzymatic digests using the corresponding BamHI enzyme. The argB

gene (XbaI fragment, 1 kb) can be used to probe these blots to reveal single copy integrations of the 9.3 kb construct into the argB locus disrupting the local argB fragment (9 kb) to produce two fragments of 11.7 kb and 6.6 kb. For multiple integrations an additional band of 9.3 kb is observed, and its intensity is relative to the number of tandem integrations at argB (Unkles et al., 2005). VFS106 transformants were targeted to the wild-type nitA locus. As a result of high efficiency in this gene targeting system for single copy integrations (Szewqczk

et al., 2006) it was not deemed necessary to screen for single copy integration with the same

vigour of that of argB integrations. As a check on the integrations, BglII genomic digests were performed on sequenced NitA transformants and these were probed with nitA (EcoRI, 1.7 kb) with the expectation of a 3.4 kb fragment for single copy integration. DNA transfer was carried out using the capillary method of Southern (1975) and by Sambrook et al (1989). Blot preparation

DNA samples were incubated at 65 ˚C for 20 min to destroy potential DNAse activity, around 10 µg DNA was then digested and equalised by eye for DNA concentration. Digested fragments were separated by agarose gel electrophoresis on 1 % gels. The gel was viewed on a transilluminator and trimmed to remove the meniscus, wells and top right hand corner for orientation. A photograph of the stained gel was also taken for record (Herolab E.A.S.Y. Photographic Suite, Scotlab). The DNA was then depurinated by washing with 0.25 M HCl for 20 min. DNA was then denatured using denaturing solution (1.5 M NaCl, 0.5 M NaOH) for 30-40 min and finally neutralised in neutralising buffer (1.5 M NaCl, 1 M Tris-EDTA, pH

7.4) for 30-40 min. A long piece of Whattman (Kent, U.K) 3MM paper was wetted with 20 x SSC (3 M NaCl, 300 mM sodium citrate, pH 7) solution and draped over a Perspex stand. Atop this the gel was placed DNA side up and an equal size of nylon Hybond-N membrane (Amersham) was placed on the gel (RH corner cut). On top again 3 pieces of 3MM paper were wetted with SSC and 3 dry pieces of 3MM placed on top. A bundle of cut to size paper towels placed on top of the last pieces of 3MM and finally, a weight. This was left overnight to allow for DNA transfer. The set-up was dismantled the following day and the gel was re- stained with an ethidium bromide solution (0.5 µg ml-1_{). The Hybond-N membrane was} rinsed in 2 x SSC and placed on a piece of 3MM paper to dry. The membrane was placed in a crosslinker (Spectrolinker XL-1500 UV Crosslinker) to crosslink the DNA to the nylon substrate.

32_{P Labelling was carried out by the method of Feinberg and Vogelstein (1983) with high} stringency hybridisations. Double stranded probes (25-30 ng) were denatured in water for 2 min and then chilled on ice before the addition of the remaining reaction ingredients (0.2 mM dCTP, 1x labelling buffer (Promega), Klenow 2.5 u (Fermentas, Yorkshire, U.K.). 2.5 µl of

α32_{P (1 MBq) was added to the reaction, gently mixed and placed on heating block at 37 ˚C} for 30 min. Assessment of the label was carried out using a NICK gel filtration column (GE Healthcare, Buckinghamshire, U.K.) which was washed using TE buffer before assessment. Blue dextran was added to the probe (4 µl of a saturated solution) as a tracker dye. The dyed probe was added directly to the column membrane, followed by a few drops of TE, once the dye had passed through the membrane 4-5 ml TE was applied to the column. The blue label was caught in an Eppendorf. The radioactivity of this sample was compared to the same volume of wash which followed through the column to assess how the label had incorporated to the DNA. Successfully labelled probe was boiled for 5-10 min and then chilled on ice. DNA hybridisation, membrane washing and autoradiography

The nylon membrane was incubated overnight in a 65 ˚C water-bath shaking at 80 rpm in hybridisation buffer (9 % v/v PEG 3000, 0.5 % w/v powdered milk (Marvel) solution, 0.1 % w/v SDS, 0.2 mg ml-1 _{Herring sperm DNA, 5 x SSPE (750 mM NaCl, 50 mM NaH}

2PO4, 5 mM EDTA, pH 7.4 )) with radio-active probe. This was performed at 65 ˚C at 80 rpm.

Membranes were washed in washes of increasing stringency to rinse un-hybridised probe. Specifically, membranes were washed for 20 min in 5 x SSC, then 2 x SSC, then 0.5 x SSC. All washes performed at 65 ˚C at 80 rpm until the background radiation was negligible. Washed filters were placed in an autoradiography cassette against photographic film (Kodak

Biomas MX film) and stored at -80 ˚C. Films were developed in Fugi RG II X-ray film processor with signal dependant exposure time. Phosphoimaging software was later employed to replace the use of photographic film. In this case the Phospho-screens were ‘Kodak Imaging Screen K’, the imager was a Bio-Rad Molecular Imager FX and the software used was Quantity One.

Once single copy transformants were determined they were purified, phenotypically assessed and the coding regions were amplified and fully sequenced. This sequence was then quality control checked by another member of the group.