Results

What we accomplished

Assembled and transformed a cassette for expression of amilCP in A. oryzae
Assembled constructs for expression of melA in A. oryzae
Demonstrated nat1 as a selectable marker in A. oryzae
Thoroughly characterised and modeled the properties of our manufactured material
Simulated the stability of a structure created from fungal mycelium
Create an optimal design of experiments for fungal bricks testing
Modelling the design of experiment results, coupling compressive strength and process factors

Choice of organism

Many fungal species were considered to achieve our goals of creating biobricks with tunable properties. To familiarize ourselves with cultivation of fungal cultures and to investigate what species were optimal for our research, Pleurotus ostreatus, Aspergillus oryzae and Schizophyllum commune were tested. The species were grown on Malt Extract Agar (MEA), Potato Dextrose Agar (PDA) or Yeast-extract-Peptone-Dextrose agar (YPD) at different concentrations. In addition, sporulation was assessed for each of the species, although it was only successful for A. oryzae.

Key achievements:

Experimental determination of the best fungal species to generate a toolbox to form brick structures.
Successfully set the growth conditions for A. oryzae needed for the formation of spores.
Familiarised ourselves with cultivation of fungi.

amilCP expression

One aim of our projects was to produce colored bricks. To achieve this we assembled constructs for expression of AmilCP, a blue chromoprotein in A. oryzae.

Key achievements:

Assembled and submitted multiple constructs for expression of amilCP in fungi
Demonstrated integration of the construct into the genome of A. oryzae
Produced evidence indicating change in spore coloration by expression of amilCP

melA expression

In order to provide our fungal materials with increased resistance to UV radiation we aimed to engineer A. oryzae to overexpress melanin. To achieve this we constructed an expression cassette containing melA, which encodes a tyrosinase utilized in the first step of the biosynthetic pathway of melanin.

Key achievements:

Assembled and submitted bricks containing constructs for expression of melA.

nat1 selectable marker

In order to generate a genetic toolbox for this host organism there was a need for a transformant selection. However, A. oryzae has proved to be resistant to the most commonly used antimycotics, geneticin (G418) and hygromycin-B. In order to establish a selection for our plasmids, we tested a less common antibiotic called Nourseothricin (NTC).

Key achievements:

Transformed pDIV079 into A. oryzae and demonstrated selection via NTC.

Ganoderma protoplastation

Protoplast-mediated transformation is a widely used transformation method for fungi. Here, we attempted to generate protoplasts for the species Ganoderma resinaceum. Benefiting from Ecovative Design, who shared their protoplastation protocol with us, we aimed to improve their protocol by making it more affordable and efficient. For that, we avoided using Driselase, a costly lysis enzyme and tried several different concentrations of Glucanex. Although with the variations in the protocol that we tried we did not have any successful protoplasts, we pathed the way to future improvement in this area.

Key achievements:

Testing of a wide range of Glucanex concentrations.
Testing of different incubation times that would make the protoplast protocol more efficient.

Design of Experiments

When testing the many factors involved in the processing of the fungal bricks several combinations are involved. The pilot design involved testing 8 substates, 2 burning temperatures, 2 burning times and 2 incubation times. From a full factorial design this would lead to 216 combinations with no replicates. Applying the optimal design of the fedorov exchange algorithm which utilizes the A-criteria, D-criteria and G-criteria, the design was reduced into 64 trials.
From the optimal substrates which produced the highest compressive strength bricks, the replicated design was created. This involved the testing of substates, 2 burning temperatures, 2 burning times, 2 incubation times and 3 mixing ratios. This would lead to 164 combinations replicated 4 times. The same algorithm was applied and the design was reduced into 17 trials replicated 4 times.

Key achievements:

Successful reduction of pilot design from 216 trials to 64 trials.
Successful reduction of replicated design from 164 trials with 4 replicated to 17 trials with 4 replicates.

Modelling the design

With the data obtained in the pilot study a fixed main effect model was created for the purpose of screening the optimal substrates. From this model post-hoc analysis was conducted and it was identified that the optimal substrates candidates for further testing was blended jasmine rice and a mixture of jasmine rice and sawdust.
From the replicated design the relation between process factors and compressive strength was modeled. It was identified a gamma mixed effect model was the best choice involving the significant factors of incubation time, substrate and burning temperature. It was revealed that the optimal level of the process factors was low incubation time (1 week), substrate 1 (blended jasmine rice) and high burning temperature of 100° celsius.

Key achievements:

Successful identifying the optimal substrate candidates via screening modelling
Successful coupling process factors to gamma mixed effect model
Successfully identifying the optimal levels of substrate, incubation time and burning temperature

Simulation of structure

We used the data from our material tests and COMSOL to simulate the stresses on our structure on Mars. This simulation took into account the stress from gravity and the stress from pressure difference between the inside and the outside of the structures. We wanted to study the structural integrity of the material and not the junctures between the individual bricks. Therefore, we modelled it as one cohesive element. Since we saw large variability in the properties during our measurements, it makes sense to make a simple model. We chose the half sphere, which we have shown can let us use approximately ⅓ the material. For the sphere we have shown that a wall thickness of approximately 0.34 m is enough to ensure structural integrity. Thereby, we have also shown that choosing Aspergillus oryzae is strong enough for our use case. This gives us grounds for choosing this for being a model organism over for instance Ganoderma resinaceum which is used by Ecovative.

Key achievements:

A 0.34 m thick A. oryzae dome is structurally sound
The lowest livable pressure for humans results in a needed thickness of ½
The reduction of stress with increased thickness decreases with added thickness

Team:DTU-Denmark/Results