Introduction

To engineer an Aspergillus niger strain capable of detecting and converting furfural, it is first necessary to develop a functional furfural biosensor and to identify furfural-converting enzymes that retain their activity when heterologously expressed in A. niger. To this end, we divided our experimental strategy in three distinct, yet deeply interconnect, experimental tracks:

The design and testing of synthetic transcription factor (sTF) paired with a minimal promoter that could be used to build furfural-inducible expression systems; the testing of activity in A. niger of three fungal promoters which were identified as potentially induced by furfural; the heterologous expression in A. niger of both prokaryotic and eukaryotic furfural-converting enzymes, and the subsequent evaluation of their activity in vivo.

Alongside the main experimental tracks, we performed a smaller experiment to further our partnership with TU Delft, and an Adaptive Laboratory Evolution (ALE) experiment to evaluate if a furfural-tolerant A. niger strain could be obtained without any rational engineering.

sTF-promoter pairs

The sTFs were designed to be modular proteins, each containing a DNA-binding domain, a putative furfural sensing domain for allosteric regulation and an activator domain. Initially, six sTFs were designed utilising a combinatorial approach to mix and match the different domains (as described in details here). Subsequently, new sTFs were designed to include the mutations identified as beneficial by the in silico modelling. We also designed a minimal promoter containing LexO binding sites for our sTFs utilising the Tet-ON promoter as a blueprint (Wanka et al.).

Four of the initial sTFs were synthesised de novo, and their DNA sequences used as templates for the PCR amplification of their modular domains. These PCR products were used to assemble the remaining two possible sTFs by USER cloning. Similarly, DNA fragments of the modular domains containing the modelled mutations were obtained by PCRs and assembled by USER fusion to build new sTFs.

To test the functionality of these sTFs, we assembled test-device plasmids for transient expression in A. niger. Each test-device plasmid carries a constitutively expressed sTF cassette, the minimal promoter regulating the expression of a mCherry cassette, an AMA sequence for replication in A. niger, and an auxotrophic selection marker (PyrG). All the test-devices were assembled by USER cloning, replicated in DH5α Escherichia coli, and purified using the GenElute™ Plasmid Maxiprep Kit (Sigma-Aldhrich) according to the manufacturer's instructions. Following validation by restriction digestion and Sanger sequencing, the test-devices were transformed into PyrG deficient A. niger. These strains were used to carry out an experiment on solid media to obtain a qualitative measure of the sTFs’ activity and one in liquid media for a quantitative measure. For both experiments, the A. niger strains were grown either in minimal media, minimal media with furfural, or minimal media with benzoic acids. Subsequently, their mCherry fluorescence was monitored with an imaging system (solid media) or with a plate reader (liquid media).

Putative inducible fungal promoters

A previous transcriptomic study (Li et al.), showed that the genes Formate Dehydrogenase 1 and 2 (scFDH1 and scFDH2) are upregulated in response to furfural in Saccharomyces cerevisiae. Therefore, we decided to test if the promoters of these genes are responsive to furfural in A. niger as well. Through NCBI’s BLAST we identified the homologous gene to scFDH1 and scFDH2 in A. niger, and decided to test its promoter too. We amplified the 1001 bp upstream of the start codon of the three genes by PCR using genomic DNA as a template. The fragments were subsequently used to assemble test-device plasmids containing an AMA sequence, a PyrG auxotrophic selection marker, and an mCherry expression cassette under the regulation of the promoters to be investigated. The test-devices were replicated in DH5α E. coli, validated by restriction digestion and Sanger sequencing, and transformed in PyrG deficient A. niger. These A. niger strains were grown in liquid minimal media, induced with furfural and their fluorescence and absorbance were monitored in a plate reader to characterise the promoters’ activity.
The activity of the anFDH promoter was also investigated by qPCR, as we quantified the FDH transcript from a WT strain and the mCherry transcript from our test-device strain following growth in minimal media or furfural supplemented media. The advantage of using qPCR is twofold: when quantifying FDH transcript, it allows characterization of the anFDH promoter in its native regulatory environment; when quantifying mCherry, it reduces noise from background fluorescence and it is more sensitive to small changes in expression.

Furfural converting enzymes

Several organisms exist that are either resistant or capable of converting furfural to combat the toxic effects. After an extended literature search we decided to try out three separate genes: fucO, hmfH and arz_7774 from the organisms Escherichia coli, Cupriavidus basiliensis and Amorphotheca resinae Zn1 respectively. All three genes convert furfural into a less toxic derivative; for FucO, furfuryl alcohol is formed, while HmfH and Arz_7774 convert furfural into 2-furoic acid.

We were unable to successfully clone FucO, and as such chose to continue only with Arz_7774 and HmfF for further testing. Next we tried to integrate the two genes into the genomes of a PyrG deficient A. niger strain. All transformation attempts failed, and as such we switched to a transient expression system through a plasmid containing an AMA sequence and a PyrG auxotrophic selection marker (BBa_K4129025). Several growth experiments were conducted with the two A. niger strains transiently expressing furfural converting enzymes and the wild type A. niger strain, where growth rates and germination capabilities under various levels of furfural were tested. Subsequently, a HPLC analysis was done to investigate if the engineered strains were capable of converting furfural to 2-furoic acid.

Partnership with TU Delft

We aimed at sending purified FunsTF01-FunsTF06 to the TU Delft team, so that they could investigate the binding affinity of our sTFs to our synthetic promoter in vitro. We assembled vectors expressing our sTF with 6xHisTag into a pET28a backbone, and transformed them into E. coli BL21(DE3) cells. The sTF were produced in E. coli, extracted, and purified using ThermoFisher NiNTA spin columns. However, a subsequent SDS page showed that the proteins had not been purified. Due to time restraints we were not able to repeat the production and purification of our sTFs and never sent them to the TU Delft team for analysis.

Adaptive Laboratory Evolution

To evaluate if a furfural-tolerant A. niger strain could be obtained without any rational engineering, we carried out an ALE experiment. WT A. niger was grown in liquid media containing 0.6 g/L of furfural. When pellets started to form, the sample was used to inoculate a new media containing furfural. This process was repeated several times while slowly increasing the furfural concentration in the media. We expected that furfural tolerant mutants would evolve and outcompete the previous A. niger strains. After 4 months, however, a bacterial contamination was detected. The contaminating organisms successfully outcompeted A. niger forcing us to interrupt the experiment.

References

• Wanka F. et al. Tet-on, or Tet-off, that is the question: Advanced conditional gene expression in Aspergillus. Fungal Genetics and Biology 89,72-83 (2016)
• Li B. et al.Improving Acetic Acid and Furfural Resistance of Xylose-Fermenting Saccharomyces cerevisiae Strains by Regulating Novel Transcription Factors Revealed via Comparative Transcriptomic Analysis. Applied and Environmental Microbiology 87 (2021)

Protocols

PCR

Materials:

• Forward primer (10 µM)
• Reverse primer (10 µM)
• Template DNA (10-25 ng for plasmid DNA, 250 ng for genomic DNA)
• PCR grade water
• PCR tubes

Procedure:
 1. Prepare the PCR reaction as indicated below:

Component	Volume
Phusion U Multiplex Mix	25 μL
Forward primer	5 μL
Reverse primer	5 μL
Template DNA	10-25 ng (plasmid) 250ng (gDNA)
PCR grade water	to 50 µL of total volume

 2. Put the sample(s) in a PCR machine and run the following protocol:

Step 1 (1x)	3 min.	at 98°C
Step 2 (30x)	30 sec.	at 98°C
	30 sec	at *°C
	** sec.	at 72°C
Step 3 (1x)	5 min.	at 72°C
Hold		at 10°C

*note: this temperature depends on your primer pair
**note: depends on the length of the amplified piece (around 30 sec./kb)

Gel Electrophoresis

Note: SYBR Safe and gels containing SYBR Safe should always be handled with gloves

Materials:

• 1% or 2% agarose in TAE buffer
• SYBR Safe (10 000x)
• Gel tray and comb
• Gel electrophoresis chamber
• TAE buffer
• PCR-amplicon
• Loading dye (6x)
• DNA ladder (100 bp or 1 kbp)
• Gloves

Procedure:

• Set up a gel tray and comb on a levelled surface
• Pour 1% or 2% agarose in TAE buffer (at 60°)
• Add SYBR Safe 10 000x to a final concentration of 1x
• Wait until the gel is set (15-20 min.)
• Move the gel to a gel electrophoresis chamber filled with TAE buffer
• Add loading dye (6x) to the samples to a final concentration of 1x
• Load samples in the gel
• Run the gel at 100V for 20min
• Remove gel from the chamber and place it in the Bio-Rad Imager to take pictures

DNA fragment purification

Note: Gels containing SYBR Safe should always be handled with gloves

Materials:

• All necessary material for gel electrophoresis
• DNAfragments / PCR products
• Scalpel
• 96% ethanol
• Gloves
• Gel Imager/excision plate and goggles
• Gloves
• Gel DNA extraction kit
• 2 ml Eppendorf tubes
• Zymoclean Gel DNA Recovery Kit

Procedure:

• Run the DNA fragments / PCR products on a 1% agarose gel (see Gel Electrophoresis protocol)
• Place gel on the gel imager, turn on gel imager while wearing the goggles
• Cut the desired band out using a scalpel and place it in a pre-weighted 2 ml Eppendorf tube
• When excising multiple bands, clean the scalpel between bands with 96% ethanol
• Use the Zymoclean Gel DNA Recovery Kit following the manufacturer protocol to purify the DNA

USER cloning

Materials:

• Purified USER fragment
• Linearized USER vector
• 10x Cutsmart buffer (NEB)
• USER enzyme (NEB)
• PCR grade water

Procedure:
 1. Prepare the USER reaction as indicated below:

Component	Volume
10x Cutsmart buffer (NEB)	1 μL
USER enzyme (NEB)	1 μL
Linearized USER vector	1 μL
USER fragments	equal amount of each in ng
PCR grade water	to 10 µL of total volume

 2. Put the sample(s) in a PCR machine and run the following protocol:

Step 1 (1x)	30 min.	at 37°C
Step 2 (8x)	3 min.	at *°C
Step 3 (1x)	5 min.	at 16°C
	10 min.	at 10°C
Hold		at 4 °C

*note: this temperature starts at 28 C° and decreases by 1°C with each cycle
 3. Cool the USER reaction on ice if transforming them into E. coli right away or store them at -4°C

Competent DH5α E. coli transformation

Materials:

• Competent DH5α E. coli cells
• USER cloning product / plasmid
• Ice
• Heat block
• Selective media plates
• Spreader
• Gloves
• 96% ethanol
• Bunsen burner

Procedure:

• Thaw the competent cells on ice
• Set the heat block at 42°C
• Aliquot 50 μl of competent cells to sterile 1.5 ml Eppendorf tubes
• Add 10 μl of USER cloning product or 500 ng of plasmid DNA to the tube and mix gently
• Incubate on ice for 15 min
• Transfer the tubes to the heat block for 45-60 sec
• Incubate on ice for 15 min
• Plate the cells on selective media. When plating, work near the Bunsen burner flame to prevent contaminations
• Sterilise the spreader by dipping it in ethanol and passing it in the flame between plating samples

E. coli colony PCR

Materials:

• Taq DNA Polymerase Master Mix RED (2x)
• Forward primer (10 µM)
• Reverse primer (10 µM)
• Milli-Q water
• PCR grade water
• Agar plate with colonies to be tested

Procedure:
 1. Transfer a colony with a pipette tip to a PCR tube with 30 µl MQ and resuspend the colony
 2. Prepare the PCR reaction as indicated below:

Component	Volume
Taq DNA Polymerase Master Mix (2x)	5 μL
Forward primer	1 μL
Reverse primer	1 μL
Resuspended colony	1 μL
PCR grade water	2 µL

 3. Put the sample(s) in a PCR machine and run the following protocol:

Step 1 (1x)	3 min.	at 98°C
Step 2 (30x)	30 sec.	at 98°C
	30 sec.	at *°C
	** sec.	at 72°C
Step 3 (1x)	5 min.	at 72°C
Hold		at 10 °C

*note: this temperature depends on your primer pair
**note: depends on the length of the amplified piece (around 20 sec./kb)

Plasmid validation by restriction digestion

Materials:

• Plasmid
• Relevant restriction digestion enzyme
• Relevant buffer (10x)
• PCR tubes
• Gel imager

Procedure:
 1. Choose a restriction digestion enzyme that will cut the plasmid in a recognisable pattern
 2. Identify the buffer associated with the restriction enzyme that was chosen
 3. Prepare the restriction digestion reaction as indicated below:

Component	Volume
Restriction enzyme	0.5 μL
Buffer (10x)	1 μL
Plasmid DNA	500 ng
Milli-Q water	to 10 µL of total volume

 4. Incubate the reaction at the temperature indicated by the manufacturer for 1 hour
 5. Run the restriction digestion product on a 1% agarose gel (see Gel Electrophoresis)
 6. Take pictures of the gel using the gel imager
 7. Compare the observed and expected restriction pattern

A. niger protoplastation

Materials:

• Plate with desired A. niger strain culture
• Sterile 500 ml shake flasks
• Sterile Mira cloth
• YPD with required supplements (e.g. Uridine for PyrG deficient strains)
• Aspergillus protoplastation buffer (APB)
  1.1 M MgSO4 and 10 mM Na-phosphate buffer, pH adjusted to 5.8 with2 M NaOH
• Aspergillus transformation buffer (ATB)
  1.2 M Sorbitol; 50 mM CaCl2·2 H2O; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 M HCl to 7.2
• Sterile Milli-Q water
• Sterile 50 ml falcon tubes
• Sterile 1.5 ml Eppendorf tubes

Procedure:

• Collect the conidia from a plate by adding 5 mL of sterile liquid YPD with required supplements and firmly rubbing the colonies with a sterile Drigalski spatula. The conidial suspension is withdrawn from the plate and added to the 500 ml shake flask containing 100 mL of YPD
• The culture is incubated for 2 days at a30 C° and 150 rpm
• Harvest the mycelia and germlings by using Miracloth
• Wash the mycelia with APB to remove the liquid media from the mycelia
• Resuspend the mycelium in 10-20 ml APB solution containing 40 mg Glucanex/ml APB
• Homogenize mycelial and enzyme suspension gently to obtain the best possible digestion of the fungal cell wall
• Shake at 30°C and 150 rpm for 2-3 hours
• Filter through Miracloth and collect the flow through in a Falcon tube
• Add APB up to the total volume 40 ml
• Carefully make an overlay with 5 ml of 2 fold diluted Aspergillus transformation buffer (ATB)
• Dilute with sterile Milli-Q water
• Centrifuge at 3000xg (acceleration 9, deceleration 4) for 12 min
• Upon a successful protoplastation, a halo of white protoplast slurry is caught just below the surface. Collect protoplast slurry and transfer it to a new tube
• Add ATB up to the total volume 40 ml/li>
• Centrifuge at 3000xg (acceleration 9, deceleration 9) for 12 min. Discard supernatant
• Resuspend the protoplasts in approximately 1 ml ATB
• Aliquot 150 μL protoplasts into 1.5 Eppendorf tubes and store at -80°C

A. niger transformation

Materials:

• Aspergillus niger protoplasts
• Plasmids
• Aspergillus protoplastation buffer (APB)
  1.2 M Sorbitol; 50 mM CaCl2·2 H2O; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 M HCl to 7.2
• PCT
  Final conc: 50 % w/vol PEG 8000; 50 mM CaCl2; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 N HCl to 7.5. Store PCT at 4°C
• Plates of osmotically stabilised selective media
• Sterile 1.5 ml Eppendorf tubes

Procedure:

• Gently mix 500 ng of plasmid DNA and 50 μL protoplast in an Eppendorf tube
• Add 150 μL PCT
• Mix by inversion of the tube
• Incubate 10 min at room temperature
• Add 250 μL ATB and mix
• Plate on osmotic stabilized, selective media

Fungal tissue PCR

Materials:

• AQ97 High Fidelity DNA Polymerase Master Mix (2x) OR Taq DNA polymerase Master
• Mix Red (2x)
• Primers
• PCR grade water
• Sterile PCR tubes
• Sterile 10 µl tips and pipette

Procedure:
 1. Prepare the PCR reactions as indicated below:

Component	Volume
Polymerase Master Mix (2x)	12.5 μL
Forward primer	2.5 μL
Reverse primer	2.5 μL
PCR grade water	7.5 μL

 2. Put the sample(s) in a PCR machine and run the following protocol:

Step 1 (1x)	30 min.	at 98 °C
Step 2 (30x)	30 sec.	at 98°C
	30 sec.	at *°C
	** sec.	at 72°C
Step 3 (1x)	5 min.	at 72°C
Hold		at 10°C

*note: this temperature starts at 28 C° and decreases by 1°C with each cycle
**note: depends on the length of the amplified piece (around 30 sec./kb)
 3. Two reactions per sample should be prepared, and will be run with different quantities of tissue
 4. Using a 10µl pipette tip and compatible pipette set to highest volume, penetrate the mycelium and agar (partially) and transfer pipette tip to the first PCR reaction and wash the pipette tip in the PCR solution
 5. Then move the tip (empty) to the second PCR reaction and wash the pipette tip in the PCR solution:

• Work on ice, after taking the tissue sample. Don't forget to mark the sampled colony and PCR tube, so you can find the colony later
• Avoid sampling spores and too much mycelium. This will inhibit the PCR reaction

Measuring mCherry fluorescence in A. niger (liquid)

Materials:

• Spore suspension (\(10^9\) spore/ml)
• CELLSTAR 48 well suspension culture plate
• Minimal media (liquid)
• Inducer (furfural, benzoic acid)
• Plate reader (ClarioStar)

Procedure:

• Fill each well with 790 µl of minimal media
• Add 10 µl of spore suspension to each well
• Incubate at 30°, 150 rpm for 20h
• Prepare minimal media with 5x inducer:
• 1x for furfural is 0.6 g/L
• 1x for benzoic acid is 2mM
• Add 200 µl of minimal media with the 5x inducer
• Incubate at 30°C, 150 rpm for 45 minutes
• Measure mCherry fluorescence and absorbance of the wells
• Repeat step 7 and 8 three more times (4 in total)
• Incubate at 30 C°, 150rpm, for the desired duration of the experiment (e.g. 120)
• Measure fluorescence and absorbance at regular intervals (e.g. 20h)

Measuring mCherry fluorescence in A. niger (solid)

Materials:

• Spore suspension (\(10^9\) spore/ml)
• Minimal media (liquid)
• Inducer (furfural, benzoic acid)
• Plastic petri dishes
• Imager system (Vilber Fusion FX)

Procedure:

• Prepare minimal media agar plates with the desired inducer with inducer:
• for furfural is 0.6 g/L
• for benzoic acid is 2mM
• Place 4 µl droplets of spore suspensions in three point of each plate
• Incubate at 30° for 3 to 5 days
• Take pictures of the plates with an mCherry fluorescence filter (maintain the same exposure time to ensure the pictures can be directly compared to each others)

Testing furfural resistance in germinated wild type cells (BioLector)

Note: Wear gloves and work in the LAF bench when handling furfural

Materials:

• BioLector
• FlowerPlate 48 well MTP
• YPD media
• Furfural
• A. niger spore suspension
• 1 Shake flask with baffles

Procedure:

• Inoculate 100 mL of YPD in a shake flask with wild type A. niger
• Grow cell culture shaking at 30°C
• Allow cells to reach a concentration of \(~5\times10^6\) cells per mL
• Once the desired concentration is reached, prepare the Biolector plate:
• Add 750µL of cell culture to 750µL YPD in each well, and add the desired furfural concentration to each well
• Run the BioLector at settings:
• 85% humidity
• 30°C
• Shaking at 800 RPM
• Biomass measure at gain = 3
• Stop run after 48 hours and analyse the results

Testing furfural resistance in mutants (BioLector)

Materials:

• BioLector
• FlowerPlate 48 well MTP
• Minimal media
• Furfural
• Wild type A. niger spore suspension
• Mutant A. niger strains spore suspensions

Procedure:

• Prepare the FlowerPlate 48 well MTP plate with 1.3 mL minimal media, with \(~5\times10^6\) spores per mL in the various wells (except for the blanks)
• Run the BioLector at settings:
• 85% humidity
• 30°C
• Shaking at 800 RPM
• Biomass measure at gain = 3
• Once the exponential phase has started and been going for at least 3 hours, pause the BioLector and add the desired furfural concentrations you wish to test
• Stop run once stationary phase has been reached

qPCR

Materials for cultivations and sample preparations:

• Shake flask with baffles
• Minimal media
• Furfural
• Wild type A. niger spore suspension
• Mutant A. niger spore suspension
• Shaking incubator
• Sterile miracloth filters
• Paper towel
• Liquid nitrogen
• Motor
• Aluminium foil
• RNAse-free 2 mL tubes.

Materials for RNA extraction:

• RNeasy® Mini Kit (50), Cat. #74104, QIAGEN
• Gloves
• RNaseZap™
• NanoDrop™ machine for RNA quantification
• Filter tips and pipettes for RNA

Materials for cDNA synthesis:

• BIO-RAD iScript Select cDNA Synthesis Kit, Cat. #1708896
• Filter tips
• Sterile PCR tubes
• PCR machine

Materials for cDNA synthesis:

• BIO-RAD iScript Select cDNA Synthesis Kit, Cat. #1708896
• Filter tips
• Sterile PCR tubes
• PCR machine

Materials for qPCR:
• Primers pair (designed on NCBI primer-BLAST tool) for each gene of interest as well as a house hold gene, e.g. actin.
• Agilent Brilliant III Ultra-Fast SYBR®, Green QPCR Master Mix, Cat. #600882
• Filter tips
• qPCR plates and seals
• qPCR machine

Procedure for cultivations and sample preparations:

Prepare cultivation of samples in each condition in triplicates. Inoculate approximately 4x10^6 spores into each shake flask containing 150 mL minimal media. Grow for approximately three days, or until an appropriate amount of biomass has been achieved for all samples. Add 1 g/L furfural to the samples corresponding to that condition. After furfural has worked for 1.5 hours, filter off the biomass using the miracloth filters. Squeeze the fluid out of the biomass by pressing it with paper towels. Wrap the biomass in aluminium foil and flash freeze it using liquid nitrogen. When the biomass is completely frozen, cool a motor with liquid nitrogen while crushing the biomass into a dusty powder. Take a precooled RNase-free 2 mL tube and collect approximately 0.5 mL biomass powder. Proceed immediately to RNA extraction or store samples at -80C until needed.

Procedure for RNA extraction, cDNA synthesis and qPCR: Clean your workspace using 70% ethanol and RNaseZap™. Extract the RNA as described in the RNeasy® Mini Kit, use gloves and filter tips at all times . Quantify RNA using NanoDrop and note the concentrations and the A60/A80 values to ensure sufficient quality. Synthesise cDNA using BIO-RAD iScript Select cDNA Synthesis Kit. Before running the real qPCR, test at what primers work best and at which concentrations. Prepare a small amount of cDNA from each sample and make a test qPCR (using Agilent Brilliant III Ultra-Fast SYBR® kit) with primer concentrations of 100 mM, 300 mM and 600 mM. Pick the best primers and concentrations and run all your samples as described in the Agilent Brilliant III Ultra-Fast SYBR® Kit. The relative expression levels were calculated according to the formula 2^(−ΔΔCT).

Preparing BioLector samples for HPLC analysis

Materials:
• HPLC vials
• BioLector plate with cells culture you wish to sample
• 2 Eppendorf tubes for each well to sample
• Ice
• Acetonitrile (HPLC grade)

Procedure:
• Once a BioLector run has concluded, transfer the BioLector plate to ice
• Transfer all liquid from each well to a Eppendorf tube kept on ice (If you wish to test biomass as well, carefully scrape the sides of each well and transfer the biomass to the eppendorf tube as well)
• Spin down all samples in a centrifuge at >10.000 RPM at 4°C
• Transfer the supernatant to a new eppendorf tube
• Mix 125µL sample with 125µL of HPLC grade acetonitrile
• Samples are now ready for HPLC analysis
• An Agilent 1290 Infinity II LC system coupled to an Agilent 6545 QTOF MS (Agilent Technologies, Santa Clara, CA, USA) was used to perform UHPLC-HRMS. The column used was an Agilent Poroshell phenyl-hexyl column (2.1 x 150 mm, 1.9µm). MS detection was performed in positive mode on the QTOF MS equipped with Agilent Dual Jet Stream electrospray ion source. The Agilent MassHunter Qualitative Analysis B.07.00 software tool was used for processing and data analysis of MS data



Testing germination times in mutants during furfural exposure (BioLector)

Materials:
• BioLector
• FlowerPlate 48 well MTP
• Minimal media
• Furfural
• Wild type A. niger spore suspension
• Mutant A. niger strains spore suspension

Procedure:
• Prepare the FlowerPlate 48 well MTP plate with 1.3 mL minimal media, with \(~5\times10^6\) spores per mL in the various wells (except for the blanks)
• Add the varying levels of furfural you wish to test germination potential at to the wells
• Run the BioLector at settings:
• 85% humidity
• 30°C
• Shaking at 800 RPM
• Biomass measure at gain = 3
• Stop run after 48 hours and analyse the results



Adaptive laboratory evolution

Materials:
• Shake flask with baffles
• YPD media
• Furfural
• Wild type A. niger spore suspension
• Shaking incubator

Procedure:
• Inoculate 100 mL of YPD with \(~1\times10^6\) A. niger spores
• Grow the cells in the flask until the first pellets start to form
• Transfer 20 mL cell broth to 80 mL fresh YPD in a new sterilised shake flask with baffles
• Add furfural to a final concentration of 0.6 g/L and let the cells grow for 5 days
• Repeat the transfer and growth step, while slowly increasing the furfural concentration in the shake flasks by 0.05 g/L every month



SDS-page

Note: SDS gels NuPage Sample reducing agent (10x) contain denaturing agents and should always be handled with gloves

Materials:
• Tris-glycine SDS sample buffer (2x)
• NuPage Sample reducing agent (10x)
• Protein sample
• Tris-Glycine SDS-running buffer (10x)
• Precast SDS gel
• Appropriate gel chamber (need to match the pre cast gel specifications)
• Mark12 protein ladder
• simplyBlue staining agent
• Gloves

Procedure:
• Mix the following in a PCR tube:
• 20 µL Tris-glycine SDS sample buffer (2x)
• 4 µL NuPage Sample reducing agent (10x)
• 16 µL of the sample
• Incubate for 5 min at 95°C
• Dilute 10x Tris-Glycine SDS-running buffer to 1x
• Remove the plastic comb and bottom tape from the precast gel
• Assemble the gel in the electrophoresis chamber
• Fill the chamber with buffer until the line on the chamber
• Load 10 µL Mark12 protein ladder
• Load the gel with 15 µL of sample for well
• Run the SDS-PAGE for 35-45 min at 225 V constant
• Rinse the gel with milli-Q water
• Crack the plastic case and extract the gel
• Place the gel in a shallow tray and add simplyBlue until the gel is covered
• Incubate for 1-3 hours at room temperature with gently shaking
• Discard the staining agent and wash the gel in milli-Q water for 1 hour with gently shaking
• Discard water and take a picture of the gel