Resultados de la variable Sistema de Información Geográfico referente a la

Transformation method was based on methods described by Balance et al (1983), Tilburn et al (1983), John and Peberdy (1984), Yelton et al (1984) and Peberdy (1989). This final method was reviewed in Riach and Kinghorn (1996) and references therein. Mutant constructs were transformed into the appropriate recipient strain and plated out on selective media for growth of transformant colonies. This method was standard for all transformations carried out. For mutant constructs in nrtA, the argB transformation was used as shown in Figure 2.2; while nitA transformations were as described in Chapter Seven.

Mycelia preparation

A single colony inoculum of transformation strain was grown for 4-7 days prior to transformation on agar at 37 ˚C. Plates were left on the bench at room temperature to mature for approximately 7 days. Conidiospores were grown at room temperature on an orbital shaker (250 rpm), for approximately 12 h in liquid media with vitamin supplements, and nitrogen sources. If mycelia had not started to germinate at this time they were re-incubated with orbital shaking at 37 ˚C to produce immature mycelia for protoplast preparation. Mycelia were harvested through an ethanol sterilised Miracloth (Calbiochem) and washed with cold (4

˚C), sterile, 0.6 M MgSO4 for protoplast preparation. The resulting material was placed in a pre-cooled, sterile 250 ml Erlenmeyer flask and re-suspended in 5 ml cold osmotic media (OSMO) (1.2 mM MgSO4, 10 mM Na2HPO4 (pH 7.0), pH 5.8 with 0.2 M Na2HPO4). 2 ml Novozyme 234 (Novoenzyme Prods Ltd) solution (50 mg ml-1 _{OSMO) was added to the cell} suspension and incubated for 5 min on ice. Next 0.25 ml BSA solution (12 mg ml-1 _OSMO) was added to the suspension. The mixture was incubated at 30 ˚C for 3.5 h with gentle orbital

shaking at 80 rpm. The mixture was placed on ice to stop the reaction. Flasks were gently mixed to free protoplasts from the mycelial debris. Using a 10 ml glass pipette, equal volumes of protoplast suspension were placed in two pre-cooled 30 ml corex tubes. The flask was rinsed with 5 ml OSMO to ensure that all the protoplasts had been collected and added to the corex tubes. An equal volume of trapping buffer (0.6 M sorbitol, 100 mM Tris pH 7.0) was overlaid on the suspension ensuring that the two phases did not mix. Protoplasts were separated from the cellular debris by centrifugation at 4100 x g for 20 min in a refrigerated (4

˚C) Sorval RC-5 centrifuge using an HB-4 swing-out rotor. The protoplasts at the interface of the two liquids were removed using a plastic pastette and pooled in a third corex tube. 20 ml cold STC (1.2 M sorbitol, 10 mM Tris pH 7.5, 10 mM CaCl2) was added to wash the protoplasts which were then centrifuged at 10,500 x g for 5 min at 4 ˚C (Sorval RC-5, Rotor HB-4). The protoplast pellet was re-suspended in n x 90 µl STC where n is the number of transformations. DNA concentration is reaction dependant (i.e. depending upon the quality of the protoplasts and the number of transformants required), though for a typical argB transformation 5-7 µg DNA per reaction was used with 90 µl protoplasts.

Transformation

DNA and protoplasts were placed in 15 ml centrifuge tubes in preparation for transformation. 25 µl 60 % PEG 6000 was added to each sample and gently mixed by pipetting, this was left on ice for 20 min. A further 1 ml 60 % PEG 6000 was added to each tube which was then inverted gently to mix and this was left at room temperature for 20 min. 5 ml cold STC was added to each tube and the resulting suspension was centrifuged at 2500 x g for 5 min using a MSE Mistral 1000 centrifuge at room temperature. The supernatant was removed and the pellet re-suspended in 100 µl STC. The suspension was split between two selection plates that were grown at 37 ˚C for up to 7 days. Selection was carried out on transformation medium containing a nitrogen source suitable for the selection of transformants and vitamin supplements. Copy number and plasmid integration was further assessed using Southern analysis of digested genomic DNA.

Strains

Strains used for fungal transformation were JK1060 (nrtA747; nrtB110; argB2; ya2; pyroA4;

pabaA1), VFS50 (nrtA747; nrtB110; nitA26; argB2; yA2) and VFS106 (nrtA747; nrtB110;

yA2; nkuA∆; pyroA4). JK1060 and VFS50 were used for the transformation of nrtA and nitA

mutant constructs respectively for integration at the argB locus. VFS106 was developed for the integration of nitA mutant constructs at the wild-type nitA locus.

Figure 2.2 Integration of mutant constructs at argB. Plasmid is shown in red, chromosomal DNA in pink and

BamHI sites in blue. Both plasmid and chromosomal copies of argB harbour mutations in the argB gene; these are

represented by as dark green and dark blue lines respectively. Southern blotting was used to determine single copy

integrations using BamHI digestion of genomic DNA. (a) Step (I) showing the single crossover event for plasmid

integration, (II) Crossover produces the wild-type argB due to exchange of wild-type genetic material (III)

Southern blotting is performed on BamHI digested transformant DNA. For single copy integration two fragments

are produced (6 and 11 kb). (b) For multiple integrations at argB, the plasmids join prior to transformation. (i)

Single crossover event for plasmid integration, (ii) As the plasmid was joined to another (possibly several i.e. to the

power of n) plasmid, the integration only happens once but the DNA integrated is n times the size. (iii) BamHI

digested Southern blot reveals three bands for multiple copy integration (6 kb, 9 kb, and 11 kb); the intensity of the 9 kb band relative to the others reflects the number of integrations. This integration method was as described by

Unkles et al (2005).