Difference between revisions of "Team:Stanford-Brown-RISD/Results"

Our overall project can be broken down into four sub-projects. These are characterized as the following: Mycelium Material & Habitat Development, Mycelium Glue Project, Mycelium Filter Project, and finally the Mission Architecture that ties everything together. Below are the Experiments, Materials, and Methods corresponding to each subproject.

To view the methods and experiments that correspond to the following results, please see our Experiments page linked here.

Mycelium Glue

Figure 1: Mycelium Lap Shear Adhesive Testing Results

As shown in figure 1, all of our purified bioadhesive proteins successfully bound to baked mycelium substrate and produced non-negligible adhesive strengths. The CsgA-CBD and CBD4x fusion proteins were the most successful, with both reaching strengths of 28.2 MPa which is comparable to the tested strength of Elmer’s glue on mycelium at 32.1MPa. Non-fusion CsgA was slightly weaker at 27.1Mpa on, and fp151 gave the poorest adhesive strength on mycelium at 16.8MPa.
On a cardboard substrate (Figure 2) there are several notable differences from the mycelium substrate testing. Elmer’s Glue was again the strongest and more than doubled its bonding strength from the mycelium substrate test, to 78.1MPa. CBD4x was surprisingly stronger than on mycelium, improving to 46.5Mpa. The fp151 protein also more than doubled in strength, reaching 40.6Mpa in the cardboard testing. Csga-CBD was weaker in the cardboard testing, at 22.7Mpa. The sample of csgA bound to cardboard was unfortunately dropped and was not salvageable for lap-shear testing.

Figure 2: Relative Mycelium Adhesive Strength

By taking the ratio of adhesive strength on mycelium to the adhesive strength on cardboard, we can find the relative efficiency of the adhesives on the two substrates. Every adhesive except for csgA-CBD bonded stronger on cardboard substrate than the mycelium substrate. This could be due to cardboard having a more uniform surface and thus allowing better molecular bonding and glue function. This makes it very notable that csgA-CBD was a stronger adhesive on mycelium, and provides evidence that by adding the chitin binding domain to csgA we were able to successfully make the protein a mycelium-specific glue. CBD4x, while still performing better on cardboard, did not decrease in efficiency as much as Elmer’s glue or fp151 which shows, albeit less dramatically, that chitin binding domains can be functionalized as mycelium specific bioadhesives.
In total, from the lap shear testing data it can be concluded that the fusion proteins that were designed for this project, CBD4x and csgA-CBD, successfully act as biological adhesives for mycelium materials. By outperforming fp151, which has been reported to be an extremely strong bioadhesive[9], and almost reaching the adhesive strength of Elmer’s glue both csgA-CBD and CBD4x show promise in future use as structural binders of mycotecture in Mars or other extraplanetary colonization efforts. As Elmer’s glue requires raw materials like natural gas and petroleum that are plentiful on Earth but are often rare to non-existent on other planets, the slight adhesive advantage it shows in comparison is greatly outweighed by the mass producibility of our bacterially synthesized bioadhesives[12]. In further research, the characterization of alternate csgA-CBD fusion proteins implementing different spatial conformations and/or multiples of csgA of CBD polypeptides could yield even better mycelium-specific bioadhesives. The inclusion of a third type of polypeptide in the fusion protein could theoretically create multipurpose mycelium-specific glue proteins, able to bind mycelium together while also performing some other useful task such as molecular sensing.

Filter

ITC Analysis: Data, Modeling, and Binding Parameters

The results from the ITC experiment to determine binding parameters for peptides 1x-, 2x-, and 3x-HHTC-Re alongside the predicted structure (QUARK Ab Initio program) follows [6]. This program uses Monte Carlo simulations and knowledge of atomic force fields to construct the most probable structural conformation of a protein from just the linear amino acid sequence.

Figure 3:ITC data for the three HHTC-Re peptides. The binding affinity (K_a) was assessed to determine the strength of the interaction, and the results follow: 1x-HHTC-Re K_a = (1.55 ± 0.21) x 10⁶ M^-1; 2x-HHTC-Re K_a = (3.73 ± 0.53) x 10⁵ M^-1; 3x-HHTC-Re K_a = (1.50 ± 0.05) x 10⁵ M^-1. Figure design credits to Jesica Urbina.

Mission Architecture

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!