Team:Stanford-Brown-RISD/Experiments

In designing our mycelium specific bioadhesive candidates, we looked for protein-based adhesive molecules that could be easily produced in E. coli and B. subtilis. We excluded highly glycosylated adhesive proteins as well as proteins with other significant post translational modifications (PTMs) simply because coordinating large PTMs in B. subtilis in space would be exceptionally challenging and beyond our capabilities for the summer. The last component of our criteria for identifying mycelium specific bioadhesive candidates revolved around the composition of mycelium itself. All fungus, mycelium included, contains an outer cell wall composed of chitin, which makes up the surface that would interface with the adhesive [3].

In nature, various organisms have evolved chitin-binding-domains (CBDs) that allow them to bind tightly to the fungal cell wall [4]. One of these organisms is B. circulans from which we borrowed a CBD sequence [4]. Our first glue candidate uses this CBD in a fusion protein of four CBD’s with GSGGSG linkers in between each; our hope was that this could act as a mycelium specific “cross linker” in a sense. Our construct also included a six residue polyhistidine tag and a Lumio fluorescent sequence (CCPGCCGAGG) for detection with Thermo Fisher Scientific’s Lumio Detection Kit [5].

Another protein-based adhesive candidate we investigated was csgA, the major subunit of the bacterial curli system which is secreted in monomers and self-assembles into longer polymer fibers that contribute to the biofilm formation[6]. The csgA protein has been known to be highly adhesive and have amyloid-forming properties, so it was reasoned that it would be a good starting point for bioadhesive proteins [6]. Furthermore, the manner in which csgA fusion proteins express the functionality of the introduced polypeptides while retaining their self-assembly behavior has made them a molecule of extreme interest in engineering functional biomaterials [7].

Our third glue candidate was a combination of the first two: a csgA-CBD fusion protein. We hoped to take advantage of the self-assembling nature of the csgA as well as its known tendency to aggregate under isoelectric conditions [5, 8]. The rationale for this fusion protein was that csgA has a strong affinity for binding its own monomers and CBD of course has a strong affinity for binding chitin. We theorized that a fusion protein with both of these domains would bond tightly to itself and to the mycelium surface.

Lastly, we decided to test the mussel foot fusion protein fp151 developed by researchers at Pohang University of Science and Technology [9]. The fp151 fusion protein is comprised of repeats of the most adhesive segments of marine mussel adhesive proteins (MAPs) 1 and 5 [9]. MAPs are the proteins responsible for adhering marine mussels to the rocks and other hard surfaces they live on in marine ecosystems [9]. It is thought that much of MAPs adhesion properties’ can be attributed to the high number of 3,4-dihydroxyphenyl-alanine (L-DOPA) residues, which require the hydroxylation of tyrosine [9]. To facilitate this post translational modification, we ordered an additional DNA construct encoding the tyrosinase enzyme, which would be able to hydroxylate tyrosine residues in vivo. We also ordered mushroom tyrosinase which has been shown to be able to hydroxylate tyrosine in vitro post-purification [10].

Once all five constructs were ordered, we ligated the linear DNA constructs to the PSB1C3 iGEM backbone using Gibson Assembly Mastermix 2x, transformed NEB T7 competent E.coli with our new plasmids, and plated the colonies on chloramphenicol selective LB plates. The colonies were incubated at 37℃ overnight or until there were distinct, visible colonies and never longer than 72 hours. The existence of our DNA constructs in the colonies were confirmed using verification primers in a colony PCR. We then performed his-tag purification on crude cell extracts from our colonies using Thermo Scientific HisPur Ni-NTA spin columns. Following protein purification with the Thermo Scientific HisPur Ni-NTA spin columns, we confirmed the presence of our protein in the final elution using Thermo Fisher Scientific’s Lumio Tag Protein Detection Kit. We then performed a standard BCA Assay to determine our total protein concentration in each elution.

To quantitatively test the strength of each purified glue candidate we performed a lap-shear test using the Instron 5565 in the Stanford Soft & Hybrid Materials Facility. To do this, we grew Ganoderma Lucidum mycelium on sawdust into the specified dimensions of the ASTM D3163 rectangle, which is used as a standard for lap-shear adhesive tests [11]. We also cut cardboard into these dimensions to use as a rough control that would help us assess whether or not our glue candidates were truly specific to mycelium material. Before applying our various glue candidates to the mycelium and cardboard specimens, we incubated our purified protein solutions at their isoelectric points at 4℃ for 2 days, spun them down, and discarded the supernatant. This was done to allow aggregate forming proteins (csgA, csga-CBD) to form amyloid fibers and non-amyloid-forming proteins (CBD4x, fp151) to reach their optimal pH [8].

Mission Architecture

On another planet, mycelia will grow and expand to provide structural integrity for the house, inside which the astronauts will live. However, the mycelia requires a substrate (food) and oxygen to grow. Where will these supplies come from? Cyanobacteria.

Cyanobacteria are self-replicating, photosynthetic organisms that can convert the abundant carbon dioxide from the Martian atmosphere into oxygen. This oxygen can be used to grow the mycelia, as well as to keep astronauts healthy and alive inside the habitat. Moreover, it has been shown that mycelia can use cyanobacteria as a substrate (food) to grow (at normal Earth gas concentrations). Our goal in this experiment is to demonstrate and quantify the oxygen production capabilities of a specific strain of cyanobacteria, known as Anabaena variablis. Achieving Earth-like percentages of oxygen (~20%) solely from the Anabaena, in combination with existing knowledge, would demonstrate the feasibility of cyano-based mycelial growth and Astronaut sustenance.

All experimentation was conducted inside an airtight, Nasco Whirl-Pak bag (total volume: 1627 mL). Two BD Gaspak Anaerobe Sachets were used to help establish an anoxic environment (down to .8% O2); Dry ice was used to establish a 90% + CO2 atmosphere, mimicking the Martian atmosphere. Wireless CO2 and O2 sensors measured the gas compositions over time, for the respective gases.

Using 425 ml of Anabaena liquid culture (@ end, OD600 is 1.449; OD750 is 1.035), oxygen production went from .8% to ~5% in 60 seconds. Then, oxygen gained 2.7% in 73,560 seconds (with an almost perfectly linear increase).

Given the empirically established rate, it will take ~92 hours (4 days) to achieve 20% oxygen, mimicking Earth's atmosphere. This time-frame is certainly reasonable in the context of our mission architecture. Therefore, we have empirically quantified oxygen production for mycelial growth and astronaut sustenance, demonstrating promising results from cyanobacteria alone!

References

[0] Folger, J. (2012, August 20). Why curiosity cost $2. 5 billion.https://www.investopedia.com/financial-edge/0912/why-curiosity-cost-2.5-billion.aspx.

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[2] Horneck, G., Klaus, D. M., & Mancinelli, R. L. (2010). Space microbiology. Microbiology and Molecular Biology Reviews : MMBR, 74(1), 121–156. https://doi.org/10.1128/MMBR.00016-09.

[3] Fungal cell wall - an overview | sciencedirect topics. (n.d.). https://www.sciencedirect.com/topics/immunology-and-microbiology/fungal-cell-wall

[4] Hashimoto, M., Ikegami, T., Seino, S., Ohuchi, N., Fukada, H., Sugiyama, J., … Watanabe, T. (2000). Expression and characterization of the chitin-binding domain of chitinase a1 from bacillus circulans wl-12. Journal of Bacteriology, 182(11), 3045–3054. https://doi.org/10.1128/JB.182.11.3045-3054.2000.

[5] Lumio green in-cell detection kit - thermo fisher scientific. (n.d.). https://www.thermofisher.com/order/catalog/product/12589057.

[6] Barnhart, M. M., & Chapman, M. R. (2006). Curli biogenesis and function. Annual Review of Microbiology, 60, 131–147. https://doi.org/10.1146/annurev.micro.60.080805.142106.

[7] Nguyen, P. Q., Botyanszki, Z., Tay, P. K. R., & Joshi, N. S. (2014). Programmable biofilm-based materials from engineered curli nanofibres. Nature Communications, 5, 4945. https://doi.org/10.1038/ncomms5945.

[8] Zhong, C., Gurry, T., Cheng, A. A., Downey, J., Deng, Z., Stultz, C. M., & Lu, T. K. (2014). Self-assembling multi-component nanofibers for strong bioinspired underwater adhesives. Nature Nanotechnology, 9(10), 858–866. https://doi.org/10.1038/nnano.2014.199.

[9] Hwang, D. S., Gim, Y., Yoo, H. J., & Cha, H. J. (2007). Practical recombinant hybrid mussel bioadhesive fp-151. Biomaterials, 28(24), 3560–3568. https://doi.org/10.1016/j.biomaterials.2007.04.039.

[10] Marumo K, Waite JH. Optimization of hydroxylation of tyrosine and tyrosine-containing peptides by mushroom tyrosinase. Biochemistry Biophysics Academy. 1986;872:98–103. https://www.ncbi.nlm.nih.gov/pubmed/3089286.

[11] Lap shear strength astm d3163 and lap shear adhesion astm d5868. (n.d.). http://www.ptli.com/testlopedia/tests/lap_shear-d3163.asp.