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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.

Cloning

G-blocks

To avoid all the illegal restriction found inside the different domains of lovb gene, G-blocks for the metabolic pathway of lovastatin were ordered from IDT, which comes in with 200 ng of either 500 bp or 750 bp double stranded DNA. The G-blocks were designed in a way that allows it to assemble with a linearized pJet1.2 blunt end vector by adding overlap extended sequences with the pJet1.2 vector at both the 5’ and ‘3 end.

Gibson Assembly

Gibson Assembly master mix (NEB #2611) kit was used for all of our gibson assembly, as this master mix was suited for the assembling of more than 3 fragments. The protocols are as followed:

50 ng of insert fragments (G-blocks) + 25 ng of backbone vector (pJet1.2 blunt end), and fill the rest with Milli-Q H2O untill 10 μl. Afterwards, the mixture was added with 10 μl of Gibson Assembly master mix, ending up with a total volume of 20 μl. Subsequently, the mixture was incubated for 1 hour at 50°C and electroporated with 50 μl of DH5-α electro-competent cells (E.coli). After 1 hour of recovery time in SOC medium, the culture was then centrifuged (3000 rpm, 5 minutes) and 800 μl supernatant was discarded. The remaining 200 μl was resuspended and plated on agar plate with their corresponding antibiotic.

PCR

For the amplification of a gene, polymerase chain reaction were performed using different primer pairs. Generally, the PCR mix consisted of ±50 ng templates DNA, 0.5-1.0 μl forward primer (10mM), 0.5-1.0 μl reverse primer (10mM), 4μl 5X buffer, 1.25μl MgCl2 (25mM), 0.25μl dNTPs (10mM) and filled the rest of the mix up until 19.5 μl with sterilized Milli-Q H2O. Before transferring the PCR mix to the thermocycler (applied biosystems 2720 thermal cycler), add either 0.25 μl of Q5® High-Fidelity DNA polymerase or 0.05 μl DreamTaq DNA polymerase (Thermo Scientific) into the PCR mix.

Q5 PCR

20 µl reaction mix contained 4 µl 5x Q5 reaction buffer, 1 µl dNTPs (10mM), 1 µl Forward primer (10µM), 1 µl Reverse primer (10µM), 200 ng template DNA, 0.25 µl Q5® High-Fidelity polymerase and filled the rest with Milli-Q H2O.

Thermal cycle Q5-PCR

Colony PCR

20 µl reaction mix contained 2 µl 10x Dream Taq green buffer, 0.5 µl forward primers (10µM), 0.5 µl reverse primers (10µM), 0.25 µl dNTPs (10mM), 1.25 µl MgCL2 (25mM), 0.05 µl Dream Taq polymerase, 50 ng plasmid DNA, and filled the rest with Milli-Q H2O untill 20 µl.

Thermal cycle Colony-PCR

Digestion

Plasmid DNA are generally linearized by digesting with the selected restriction enzymes. Per 10 µl of digestion mixture, consisted of 1 µl of 10x appropiate restriction buffer, 0.5-1 μg DNA template, filled the rest with Milli-Q H2O. lastly, add 0.5 µl of the appropiate restriction enzyme and incubate it for 1-2 hours in a waterbed of 37°C. The mixture was loaded and run on gel for further analysis or purification.

Ligation

The 10 µl mixture consisted of 1 µl 10x ligation buffer, 1 µl T4 ligase, molar ratio of 1:3 of linearized vector and DNA insert, respectively, and the rest was filled with Milli-Q H2O untill 10 µl. Molar ratio was calculated with ligation calculator (Gibthon). The ligation mix was incubated for 4 hours at room temperature for 1:30 hours. The ligase was inactivated by 10 minutes incubation at 65°C and then the ligation mix was run through a column for removing salt contamination. Finally, 1-2 µl could be used for the transformation of DH5-α competent cells.

Electroporation

50 µl of aliquot of -80°C DH5-α electro-competent cells (E.coli) was first thawed on ice. Then 1-2 µl of purified plasmid solubilized in either Milli-Q H2O or TE buffer was added to the thawed electro-competent cells (E.coli), in which the mixture was electroporated with a shock of 200 Ω at 2500 volt inside an electroporation cuvette. After electroporation, 1mL of SOC medium without antibiotic was immediately added for recovery and was incubated for 1 hour at 37°C. Afterwards, 200 µl of the mixture was plated on an appropriate antibiotic LB agar plate, while the remaining 800 µl was concentrated by centrifuging (3000 rpm, 5 minutes) and 600 μl of supernatant was discarded. The remaining 200 μl was resuspended and plated on new LB agar plate. Colonies were formed after the plates were incubated overnight at 37°C and can be handpicked by a toothpick, and inoculate it in a 10 mL blue capped tube filled with 3mL LB medium with the appropriate antibiotic. After another overnight incubation at 37°C, isolation of DNA can be performed.

Heatshock

50 µl of aliquot of -80°C DH5-α chemical-competent cells (E.coli) was first thawed on ice. 5 µl of purified plasmid solubilized in either Milli-Q H2O or TE buffer was added to the thawed chemical-competent cells (E.coli). The mixture was incubated in ice for 20 minutes and was subsequently incubated at exactly 42°C for 45 seconds. Afterwards, 500 µl of SOC medium was added and incubated at 37°C for 1:30 hours. After incubation, the culture was then centrifuged (3000 rpm, 5 minutes) and 800 μl supernatant was discarded. The remaining 200 μl was resuspended and plated on agar plate with their corresponding antibiotic.

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 1) Different Aspergillus niger strains expressing GFP

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

Antibiotic concentrations

An appropriate concentration for each antibiotic need to be added to either liquid LB medium or agar LB plate, see table below.

Antibiotic Concentrations

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

Materials:
Sterile Pipettes and Pipetting tips
Sterile saline-Tween solution (0.9 % NaCl + 0.005 % (v/v) Tween-80
Sterile saline solution (0.9 % NaCl)

Method for plates:
1. Inoculate on Thursday six 15 cm CM plates (protocol 30) with a total number of 4x105 spores (this is ~22 spores per mm2 on a 15 cm plate; for 9 cm plates, add total 1.4x105 spores). Make sure to pour the CM-agar while it is relatively cold, as this will enhance the spore scraping procedure. Do not add too many spores: with >50 spores per mm2 the plates will wrinkle and spore concentration will decline since it is more difficult to harvest the spores!
2. Place the plates for 4 days at 30 °C (until Monday).
3. Put the spore plates overnight in the cold room @ 4 °C for maturation (Monday overnight).
4. Pipette 10 ml saline-Tween solution on the spore-mat.
5. Scrape off the spores using a Drigalski spatula.
6. Transfer the spore suspension to a sterile 50 ml Greiner tube with a sterile 10 ml pipet.
7. Mix 30 seconds on Vortex and filter over funnel with glass wool. Centrifuge 10 minutes, 5000 rpm at room temperature.
8. Pour off supernatant and resuspend in 40 ml saline solution, mix 30 seconds and repeat the wash once more.
9. Prepare a 50-fold dilution (20 µl spore suspension + 980 µl saline) and determine the concentration of spores by counting the diluted sample in a heamocytometer (see remarks).
10. Calculate the concentration of spores.

Neubauer improved heamocytometer:

depth: 0.1 mm
Minimal area (A): 1/400 mm2

Pipette spore dilution between coverslip and slide enough liquid until the space is flooded (~20 ul). NB If the coverslip is chipped or in any way damaged, it must be replaced- it is no longer functional.
Count the spores (magnification 400 X) in 16 fields.
Calculate the spore concentration in the spores suspension:
spores/ml = count x d x 2.5 x 105 (d = dilution)

B. Preparation and storage of Aspergillus glycerol stocks

1. Harvest spores from a plate.
2. Transfer the spore solution in ST into a sterile glass tube with aluminium cap
3. Spin down the spores at low speed (3000 RPM in table top centrifuge in rec lab)
4. Resuspend the spores in 1 ml sterile glycerol medium (1 part glycerol 87% : 2 parts 1% pepton).
5. Divide over two Eppendorff tubes and label well
6. Store immediately in the -70⁰C freezer and update the collection database
7. Sterilize all tubes used and fungal material left

Taking a strain from glycerol stock:
1. To take a strain from the stock use a sterile platinum needle.
2. Take an inoculum from the stock and transfer to a plate
3. Distribute the spores with a sterile Drigalski spatula
4. Return the stock to the freezer as quickly as possible

C. Media for Aspergilli

Minimal medium salts(MMS) for 1000 ml:
6.0 g NaNO3
1.5 g KH2PO4
0.5 g KCl
0.5 g MgSO4. 7 H2O

when 10xMMS is adjusted to pH=5.45, a 1x MM solution will have pH ~6.0

Minimal Medium for 1000 ml:

100 ml Minimal medium salts 10x

pH adjusted to 6.0
For solid medium 15 g agar is added
autoclave

Before use:

50 mM carbon source and appropriate supplements, 1 ml Vishniac trace-elements solution (lab fridge)

Complete Medium for 1000 ml:
100 ml Minimal medium salts 10x +
2 g meat peptone (pepton 100)
1 g yeast extract
1 g pepton 140 or Casamino acids (vitamins free)
0.3 g yeast ribonucleic acids (cold room shelf)
2 ml vitamins solution (fridge)

pH adjusted to 6.0
For solid medium 15 g agar is added
autoclave

Before use:

50 mM carbon source and appropriate supplements (added after sterilization) 1 ml Vishniac trace elements solution

Vishniac solution (Vishniac and Santer, 1957): 1 litre
10 g EDTA
4.4 g ZnSO4.7H2O
1.0 g MnCl2.4H2O
0.32 g CoCl2.6H2O
0.32 g CuSO4.5H2O
0.22 g (NH4)6Mo7O24.4H2O
1.47 g CaCl2.2H2O
1.0 g FeSO4.7H2O

pH adjusted to 4.0 and stored at 4°C.

Vitamins solution: 100 ml, store in protected from light
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

D. HPLC, sample preparation

Materials:
Samples and if necessary defrost them on time
HPLC vials and caps
micro centrifuge vials
Demi water
HPLC standards

Media

For Organic acids:
Crotonate Internal Standard (MICFYS): 30 mM crotonate in 0,3 n H2SO4
per liter: 2,58 g crotonate + 8,4 µl (0,96%) H2SO4

External Standards organic acids: (example) 100mM stock concentration
#1; oxalic acid, a-ketoglutaric acid, sorbitol, fumaric acid
#2; cis-aconitic acid, citric acid, xylose, , itaconic acid
#2; oxaloacetic acid, glucose, malic acid, succinic acid
#4; pyruvic acid, formic acid

External Standards sugar: (example) 100mM stock concentration
#1; sorbitol, Xylose (for Carbopac-MA1)
#2; sorbitol, arabinose, galactose, glucose, xylose, mannose (for Carbopac-PA20 max 100 µM)

Protocol
1. Take your samples, if necessary defrost them, centrifuge for 15 min max. speed
2. Label your HPLC vials or 96 well plate (ICS 5000)
3. Make a H2O HPLC vial
4. Prepare the external standards in the following concentrations in a micro centrifuge

Table 2)

5a.
For the HPLC vials take 800 µl standard dilution, add that to the HPLC vial + 200 µl Crotonate I.S.
Do this also with the samples.

Take sample/controls + Crotonate I.S. in a 4:1 ratio,
The minimal total volume in the HPLC vial should be 500 µl

5b.
For 96 well plate take 200 µl standard dilution, add that to the 96 well plate + 50 µl Crotonate I.S.
Do this also with the samples. When done cover the plate with a sheet

Take sample/controls + Crotonate I.S. in a 4:1 ratio,
The minimal total volume in the 96 well plate should be 250 µl

6. For HPLC-vials, Cap the vials
7. Mix the HPLC vials well, not necessary for a 96 well plate

The samples are now ready for measurement on HPLC to measure organic acids.

E. DAPI staining of A. niger

Made using A. niger strain N400

Materials:
• Gloves
• 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen D1036)
• pH meter
• Fluorescence microscope

Protocol
1. Use gloves! DAPI is carcinogenic. Prepare a 10x Hoechst stock solution (1,0 g/ml) in demi water and store protected from light at 4°C.
2. Sample a minimum of 200 µl of mycelium in an eppendorf.
3. Correct the pH to 7 (between pH 4 and 11 fluoresence remains unchanged)
4. Cultured cells are examined immediately after incubation with DAPI at
0.1g/mL in phosphate buffered saline at 30 °C for 15-30 minutes.
5. Use the fluorescence microscope with block A. Consider using Vectashield mounting medium if bleaching of the stain occurs too quickly.

F. Calcofluor staining

Calcofluor white (fluorescent brightener 28, Sigma; 100 μl) (0•3 g l−1) was added to 1 ml diluted sample in a 1•5 ml Eppendorf vial covered with tin foil. Samples were mixed and incubated at room temperature for 5 min. A few drops of the solution were placed on a glass slide and covered with a coverslip for analysis. The dye fluoresces when bound to chitin and glucans, and thus stains cell walls and septa. Images were obtained using fluorescence microscopy on a Leika microscope using a mercury burner and a filter block with 330–380 nm excitation wavelength and dichroic mirror with emission >420 nm.

G. Experimental evolution protocol “Mycelium to single cell”

Daily transfers

Aim

Obtain genotype for single cell morphology from mycelium

Method:
1. Inoculate 10 ml CM to 1x106 spores/ml
2. Grow O/N @ 30⁰C
3. Disturb culture by 30s of vortexing
4. Filter culture and determine mass of filtrate (pellet) and retentate (dry)
5. Resuspend filtrate (in 1 ml) and inoculate fresh medium
6. Repeat step 2-6

NB: Take a more stringent method/smaller pore size when retentate ≤ filtrate

Requirements
• Filters with a range of pore sizes
• 30⁰C climate shaker
• Glass tubes + medium

When no transfers can be done during weekends, spore plates are inoculated on Friday and harvested on Monday according to protocol 31 to inoculate new liquid cultures on Monday for continuation of the experiment.

H. RNA extraction protocol

• DEPC-treated H2O: Use DEPC (Diethylpyrocarbonate) to make the H2O RNase free. Add DEPC to water (final concentration 0.1%) and autoclave at least once. This DEPC-treated H2O can be used to prepare 75% EtOH: add 75 mL EtOH to 25 mL autoclaved DEPC H2O.
• Organic liquids (chloroform, phenol, isopropanol) do not have to be treated with DEPC,
• RNase free filter tips and Eppendorf tubes can be ordered. To make other material RNase free, soak it in 0.1% DEPC (in demi-H2O) in a beaker glass, cover it with foil, and leave O/N. Autoclave the next day and discard the liquid. After drying, the material can be used.
• Wear clean gloves all the time (human and water, also ice, are the main source of RNases)

Materials
-80°C freezer
Thermoblock
Beadbeater (MP FastPrep-24)
Liquid N2
Glass beads
RNase free filter tips
Screw cap vials 2.0 mL (Sigma Aldrich, Lot. 2215479)
1.5 mL microcentrifuge tubes RNase Free (Ambion, Lot. 1004034)
RNA extraction solution (peqGOLD TriFast, PeqLab, Lot 131012)
Chloroform
Isopropanol
DEPC-treated H2O
75% EtOH (RNase free)

Procedure:
1. Fill screw cap vial with glass beads

2. Add 1 mL peqGOLD TriFast. Flush tip to avoid dripping. Filter tip is not necessary, but you can use the tip for all samples.
Work in the fumehood, peqGOLD contains phenol. The peqGOLD should be protected from light as much as possible. If the extraction did not work, this might be because the peqGOLD was not stable anymore, replace it.
3. Add small piece of mycelium (not too much!) directly into the peqGOLD. Use tweezers which are pre-cooled in liquid N2.
Because of phenol, vials have to be tightly close, also take care of the rubber ring.
4. Place all vials in the same position in the Beadbeater and distribute them evenly over the rotor. Tighten well! Use Setting 6, 30 sec. (Press RUN 2x)
5. Keep the samples at RT, 5 min
Usually transport and unscrewing is long enough
6. Add 200 μL chloroform to each sample. Flush tip to avoid dripping. Filter tip is not necessary, but you can use the tip for all samples.
7. Vortex each sample exactly 15 sec (use a timer!)
8. Leave samples 3-10 min at RT
9. Centrifuge 5 min (12000×g, RT)
10. Transfer the aqueous (upper) phase to 1.5 mL tube using filter tips (app. 4-5 time 100 μL). Be careful not to contaminate the sample with DNA or proteins (in organic and interphase). If this accidently happens: Centrifuge again! Put samples on ice.
Organic and interphase should be discarded in phenol-wast. Also see manual, this can also be used for protein extraction.
11. Take samples from ice to add 0.5 mL isopropanol. Flush tip to avoid dripping. Filter tip is not necessary, but you can use the tip for all samples. Place samples on the ice again.
12. Leave samples 5-15 min on ice.
13. Centrifuge 10 min (12000×g, 4oC).
14. Remove the supernatant.
15. Wash the pellet with 0.7-1.0 mL 75% EtOH.
16. Vortex briefly.
17. Centrifuge 10 min (12000×g, 4oC).
18. Remove the supernatant
The easiest is to remove the supernatant in 2 rounds, once roughly with a big tip and afterwards with a small tip more carefully.
19. Air dry the samples.
20. Place samples 2-5 min in thermo-block at 65oC to remove the EtOH (leave caps open).
21. Dissolve the pellet in 50 μL DEPC-treated H2O by shaking the samples for app. 10 min at 65oC and vortexing once in a while.
22. Place samples back on ice.
23. When completely cooled down, vortex the samples, and spin them down.
24. Measure the RNA concentration.
25. Store the RNA at -80oC (Put samples directly in -80oC).