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Materials

Standard lab procedures are explained in Appendix A, B, C and D. Novel protocols are mentioned and explained at the specific topic pages, since they cannot be explained without the mention of intermediate results and can be considered results. A detailed version of these protocols is also given here.

Strains

A. niger N593
This strain is cspA and pyrA deficient. Media for N593 are supplemented with 5 mM uridine. Unlike citric acid producers, this strain produces mainly oxalic acid.

A. niger strains expressing GFP
Three strains have been applied that express Green Fluorescent Protein (GFP) in various constructs. GFP is widely used as a localisation tool. These strains have been constructed using strain AB4.1 (Vinck, A., M. Terlou, et al. (2005). "Hyphal differentiation in the exploring mycelium of Aspergillus niger." Molecular Microbiology 58(3): 693-699). The GFP constructs are made using the sGFP gene. This mutated GFP variant has an excitation peak at 488 nm and an emission peak at 510 nm. Information is given in table 1.

**TABLE HERE**

A. nidulans WG505
This is a pyr-deficient strain, so 5 mM uridine was added to the medium. Use of this strain is mentioned by Nyyssola (Nyyssola, A., R. Heshof, et al. (2012). "Methods for identifying lipoxygenase producing microorganisms on agar plates." AMB Express 2(1): 17). No further data available.

Media

The standard protocol for media for the SSB group is provided as Appendix D. Media and supplements used here are:

Uridine solution
Per 100 ml; 12.2 g uridine. Filter-sterilised using a 0.2 µm filter.

Vishniac solution
Per litre; 10 g EDTA; 4.4 g ZnSO4_7H2O; 1.0 g MnCl2_4H2O; 0.32 g CoCl2_6H2O; 0.32 g Cu¬SO4_5H2O; 0.22 g (NH4)6Mo7O24_¬4H2O; 1.47 g CaCl2_2H2O; 1.0 g FeSO4_7H2O. pH adjusted to 4. Filter-sterilised using a 0.2 µm filter.

Vitamins solution
Per 100 ml; 10 mg thiamine; 100 mg ribovlavine-5P; 10 mg p-aminobezoic acid; 100 mg nicotinamide; 50 mg pyridoxine-HCl; 10 mg panthothenic acid; 2 mg biotine. Filter-sterilised using a 0.2 µm filter.

Minimal medium salts (MMS)
For preparation of medium, 10x MMS stock solution is used. This contains per 1000 ml; 60 g NaNO3; 15 g KH2PO4; 5 g KCl; 5 g MgSO4_7 H2O and is adjusted to pH 5.45.

Minimal medium (MM)
Stock 10x MMS solution is diluted 10x, autoclaved for 20 minutes at 121°C and supplemented with 50 mM carbon source, appropriate supplements (e.g. 5 mM uridine) and 1 ml/l Vishniac trace element solution.

Complete medium (CM)
Stock 10x MMS solution is diluted 10x, supplemented per litre with 2 g meat peptone; 1 g yeast extract; 1 g Casamino acids; 0.3 g yeast ribonucleic acids and then autoclaved for 20 minutes at 121°C. After autoclaving it is supplemented with 50 mM carbon source, appropriate supplements (e.g. 5 mM uridine), 1 ml Vishniac solution and 2 ml vitamins solution.

Cultures

Microscopes

Appendices

A. Preparation spores suspension/inoculation cultures and making spore plates
B. Preparation and storage of Aspergillus glycerol stocks
C. Media for Aspergilli
D. HPLC, sample preparation
E. DAPI staining of A. niger
F. Calcofluor staining of A. niger
G. Experimental evolution protocol “Mycelium to single cell”
H. RNA extraction protocol

A. Preparation spores suspension/inoculation cultures and making spore plates