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<div class="headlinecontainer" style="font-size:5vw;"><h1>Choice of organism</h1></div>
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Growing conditions between fungal species can vary depending on how they grow in nature. Factors could be temperature, amount of light and the substrate, among other things. With the goal of growing fungal bricks in mind, choosing a suitable organism for our project was one of our very first objectives.
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<h2 class="media-heading" style="text-align: left;margin-bottom: 35px; color:#50C8E8;">Choice of organism</h2>

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We initially researched the fungal species <i>Pleurotus ostreatus</i>, <i>Aspergillus oryzae</i> and <i>Schizophyllum commune</i>. <i>S. commune</i> has been investigated and used for the generation of bricks by the company Ecovative Design (1), whom we collaborated with. In addition, <i>S. commune</i> also allowed us to study genes affecting the structure of mycelium, by using mutants as ΔSC3. This strain is characterized by a deletion of the SC3 gene, which is responsible for the production of hydrophobins. Hydrophobins are small proteins present in filamentous fungi that have the ability to repel water and create a coating on hydrophobic surfaces (2). 
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Secondly, <i>P. ostreatus</i> is a common edible mushroom also known as oyster mushroom that has been widely cultivated. In addition to having a strong mycelium, it is used in industrial processes and mycoremediation. (3). 

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In addition, <i>A. oryzae</i> has been used for fermentation processes for at least 2000 years (4). Moreover, its genome has been sequenced (5), making it an optimal choice for the generation of a genetic toolbox. 
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As our project requires the eventual growth of fungi on Mars, the species used should ideally be able to grow under the conditions there. Besides temperature, radiation is a key element to consider. $\alpha$- and $\beta$-radiation are fairly easily blocked, so even though there is an increased background radiation on Mars (6), the primary concern will be the increased levels of $\gamma$-radiation, since this type of radiation can penetrate very thick walls and inhibit growth of fungi or bacteria (7). To simulate the background gamma-radiation dose that fungi would receive on Mars, which is in the range of 12 rads/year (8), <i>A. oryzae</i> together with another fungal species, <i>Ganoderma resinaceum</i> were exposed to this level of radiation during the first three days of growth. The results suggested that the radiation did not inhibit the growth of any of the species. 

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<h2 class="media-heading"  style="text-align: right;margin-bottom: 35px; color:#F8A05B;">Results</h2>




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In the first experiment, the species were seeded at 3 different concentrations of MEA (47,5 g, 50 g and 52,5), PDA (19,5 g, 39 g and 58,5 g) and YPD. Thereafter, growth pictures were taken every 24 hours and the size of the fungal colonies was compared. In addition, different growth conditions (temperature and light) were tested for each of the species. <i>S. commune</i> was incubated 30ºC at light and 27ºC at light, <i>P. ostreatus</i>  at 28ºC at dark and 25ºC at light, and <i>A. oryzae</i> at 28ºC at dark and 30ºC at dark. <br><br>

In addition, we also investigated the growth of a mutated <i>S. commune</i> strain, ΔSC3. 

<p style="text-align:center;"> <img src="https://static.igem.org/mediawiki/2018/e/e7/T--DTU-Denmark--ResultsSpecies1.png" style="max-width: 100%;" > <figcaption><p style="text-align:center; font-size:14px;"><b>Fig. 1: </b> Growth of <i>A. oryzae</i> on PDA 58.5g (A), <i>S. commune</i> on PDA 39g (B) and  <i>P. ostreatus</i> on PDA 19.5g (C) plates after 24, 48 and 72 hours respectively after inoculation.</p></figcaption>
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<i>S. commune</i>, <i>P. ostreatus</i> and <i>A. oryzae</i> grew at a similar rate on all media. However, the mutant strain of <i>S. commune</i>, <i>Δsc3</i>, was able to grow only at PDA 39 g (Figure 2).

<p style="text-align:center;"> <img src="https://static.igem.org/mediawiki/2018/3/3a/T--DTU-Denmark--results-species2.png" style="max-width: 100%;" > <figcaption><p style="text-align:center; font-size:14px;"><b>Fig. 2: </b>Growth of <i>S. commune</i> <i>Δsc3</i> on PDA 39g plates after 24, 48 and 72 hours respectively after inoculation.</p></figcaption>

<p style="text-align:center;"> <img src="https://static.igem.org/mediawiki/2018/8/83/T--DTU-Denmark--choiceoforg-fig3.png" style="max-width: 100%;" > <figcaption><p style="text-align:center; font-size:14px;"><b>Fig. 3: </b> Growth of <i>A. oryzae</i> on rice to create bricks after 7 days of incubation at 28ºC.</p></figcaption>



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This preliminary experiment indicated several species were interesting for our research, as they were able to grow in common media, producing mycelium that could form fungal bricks possibly of use for making fungal materials. After incubation at the following conditions: <i>S. commune</i> at 30ºC at light, <i>P. ostreatus</i> at 28ºC at dark and <i>A. oryzae</i> at 28 ºC at dark, only <i>A. oryzae</i> showed sporulation despite a similar growing speed. As the protoplastation and transformation protocol available to us relied on spores to produce the protoplasts <i>A. oryzae</i> seemed like the best choice of species to continue work on for the genetic engineering part. Furthermore, comparing the bricks it quickly became clear that <i>A. oryzae</i> produced the most coherent bricks (Figure 3), with the other species producing extremely brittle materials. This might be a feature of the substrates chosen for growth, as they were chosen based on species’  preference in terms of growth rate, so for <i>A. oryzae</i> it was rice and for <i>P. ostreatus</i> and <i>S. commune</i> it was saw dust. Addition of coffee grounds did nothing for the texture of the bricks but a mixture of rice and sawdust made the <i>A. oryzae</i> bricks harder. As shown in Table 1, once again it seemed that <i>A. oryzae</i> was the best choice for further work. 

<p style="text-align:center;"> <img src="https://static.igem.org/mediawiki/2018/thumb/9/98/T--DTU-Denmark--table1.png/1200px-T--DTU-Denmark--table1.png" style="max-width: 100%;" > <figcaption><p style="text-align:center; font-size:14px;"><b>Table 1: </b> Summary of the properties investigated according to every species. </p></figcaption>
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In addition to being the only one to sporulate and to form coherent bricks (Figure 4), <i>A. oryzae</i> is a well-studied species that is used in fermentation processes and has been engineered previously. Therefore, we decided to focus the project on forming bricks and doing genetic engineering of genes of interest on <i>A. oryzae</i>. 

<p style="text-align:center;"> <img src="https://static.igem.org/mediawiki/2018/f/f3/T--DTU-Denmark--incubator.png" style="max-width: 100%;" > <figcaption><p style="text-align:center; font-size:14px;"><b>Fig. 4:</b> Incubator containing the boxes used to grow the bricks.  </p></figcaption>
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Eventually, radiation tests were performed on <i>A. oryzae</i> and  <i>Ganoderma resinaceum</i> (Figure 5). The irradiated samples were kept at a temperature of 20 degrees celsius, controls were prepared and set to grow at the same temperature. The irradiated samples got a combined dose of 63.85 mSv from 28.09.2018, 13.10 to 01.10.2018, 10.45. The irradiated samples had growth radii which were on average twice as large as the controls (Figure 6,7). These results are thought to be spurious, and the most likely candidate is that the sample room was kept at a higher temperature than 20 degrees. The primary result is thus that the fungi were able to grow, but it is unknown if the increased amount of radiation caused an increase in mutations. 
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<p style="text-align:center;"> <img src="https://static.igem.org/mediawiki/2018/e/eb/T--DTU-Denmark--Radiationfig1.png" style="max-width: 100%;" > <figcaption><p style="text-align:center; font-size:14px;"><b>Fig. 5:</b> Agar plates inoculated with species of interest, placed at 4 m from the gamma radiation source at DTU Risø. </p></figcaption>
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<p style="text-align:center;"> <img src="https://static.igem.org/mediawiki/2018/1/10/T--DTU-Denmark--Radiationfig2.png" style="max-width: 100%;" > <figcaption><p style="text-align:center; font-size:14px;"><b>Fig. 6:</b> <i>A. oryzae</i> growth after 3 days incubated under a gamma radiation dose of 63.85 mSv </p></figcaption>
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<p style="text-align:center;"> <img src="https://static.igem.org/mediawiki/2018/c/c1/T--DTU-Denmark--Radiationfig3.png" style="max-width: 100%;" > <figcaption><p style="text-align:center; font-size:14px;"><b>Fig. 7:</b> <i>G. resinaceum</i> growth after 3 days incubated under a gamma radiation dose of 63.85 mSv</p></figcaption>
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<p style="color:#000; font-size:14px;">(1) Flores RM. 2014. Plastic Alternatives: Exploring Mycelium as a Medium. Parsons the New School for Design.<br><br>
(2) van Wetter MA., Wösten HA., Sietsma JH., Wessels JG. 2000. Hydrophobin gene expression affect hyphal wall composition in Schizophyllum commune. Fungal Genet Biol 31:99-104.<br><br>
(3) Sanchez C. 2010. Cultivation of Pleuotus ostreatus and other edible mushrooms. Appl Microbiol Biotechnol 85:1321-37.
<br><br>(4) Rokas A. 2009. The effect of domestication on the fungal proteome. Trends in Genetics 25:60-63.
<br><br>(5) Machida M, Asai K, Sano M, Tanaka T, Kumagai T, Terai G, Kusumoto K-I, Arima T, Akita O, Kashiwagi Y, Abe K, Gomi K, Horiuchi H, Kitamoto K, Kobayashi T, Takeuchi M, Denning DW, Galagan JE, Nierman WC, Yu J, Archer DB, Bennett JW, Bhatnagar D, Cleveland TE, Fedorova ND, Gotoh O, Horikawa H, Hosoyama A, Ichinomiya M, Igarashi R, Iwashita K, Juvvadi PR, Kato M, Kato Y, Kin T, Kokubun A, Maeda H, Maeyama N, Maruyama J, Nagasaki H, Nakajima T, Oda K, Okada K, Paulsen I, Sakamoto K, Sawano T, Takahashi M, Takase K, Terabayashi Y, Wortman JR, Yamada O, Yamagata Y, Anazawa H, Hata Y, Koide Y, Komori T, Koyama Y, Minetoki T, Suharnan S, Tanaka A, Isono K, Kuhara S, Ogasawara N, Kikuchi H. 2005. Genome sequencing and analysis of Aspergillus oryzae. Nature 438:1157–1161.
<br><br>(6) Matt W. (2016) How Bad is the Radiation on Mars? Universe Today. https://www.universetoday.com/14979/mars-radiation1/. Accessed October 15, 2018.
<br><br>(7): Dartnell, L. R., Desorgher, L., Ward, J. M., & Coates, A. J. (2007). Modelling the surface and subsurface martian radiation environment: implications for astrobiology. Geophysical research letters, 34(2).
<br><br>(8): Simonsen. Lisa C. Zeitlin, Cary. (2017) Briefing to NAC HEO/SMD Joint Committee Meeting “Mars Radiation Environment – what have we learned?”, https://www.nasa.gov/sites/default/files/atoms/files/mars_radiation_environment_nac_july_2017_final.pdf

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