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Our team was confronted with a pressing dilemma rooted in ethical and environmental concerns surrounding the leather industry. To address this challenge, our team explored various alternative materials. These included: plastics, mycelium, SCOBY, and fruit peels. Plastic was initially considered but rejected due to its widespread environmental pollution and detrimental effects on ecosystems. Mycelium, a fungal material showing promise as a sustainable leather substitute, is not a possible option as our laboratory skills lack prior experience working with fungi. Fruit peel has prolonged drying processes, susceptibility to environmental contamination, reliance on weather conditions, and the necessity for manual labor, so it was ruled out. Lastly, SCOBY emerged as a compelling choice, primarily due to its potential to address the ethical and environmental concerns associated with traditional leather production. Research showed us SCOBY exhibited a significantly lower carbon footprint compared to cattle-derived leather and since we have experience working with yeast, we thought it may be a viable material for genetic modification.
The challenge at hand was to address the brittleness and inflexibility of SCOBY when dehydrated. This was a crucial issue identified through a survey where 70.3% of respondents prioritized “flexibility” in leather goods.
Therefore, we did research to explore various options and found a paper by Tien et al. about spider silk protein. Thus, we considered the consecutive amino acid repeats shown by MaSp1, MaSp2, MiSp1, and MiSp2. We have decided to conduct our experiment by converting MaSp1 and MaSp2 into DNA sequences, as these repeating amino acids are shorter than the other identified amino acids and, therefore, easier to perform experiments on.
Preliminary testing of our final product, using the strength and flexibility testers discussed below, has shown our design incorporating MaSp1 and MaSp2 have resulted in SCOBY with increased strength and flexibility. This is further discussed in Results.
The MaSp1 and MaSp2 amino acids had several repeating sequences, so the primers would bind to several places on them, converting them into DNA sequences. Therefore, it is difficult to design a unique primer set that will bind to the required area of the DNA to amplify the genes.
Our team designed primer sets via the Snapgene app to amplify MaSp1 and MaSp2, respectively, via the PCR technique. However, the primer sets kept annealing to numerous places on the DNA sequences. After consulting with Mission Biotech Company, they have suggested our team use 3 different nucleotide codons for the same amino acid to make the DNA sequence not repeat much. Our team also added the start codon, ATG, at the 5'- end of the MaSp1, and MaSp2 DNA sequences so the RNA transcription would start from ATG on MaSp1, and MaSp2 to make fusion proteins with CBD in the presence of galactose (BBa_K4650005 and BBa_k4650006).
At the beginning of working on the MaSp1 PCR, we didn’t see any PCR product after electrophoresis. After consulting with several professors, Dr. Chen Shao-Kuan and Dr. Pei-Wen Chu at the Institute of Neuroscience, NCCU suggested that our team should try to add less DNA template since MaSp1 DNA was synthesized by the MB (Mission Biotech) company and cloned into T7 promoter plasmid. When our team did PCR, 200 ng of DNA template was used, which was a high concentration for using plasmid as a DNA template. After our team reduced the amount to 50 ng of DNA template, we could see 102bp of MaSp1 product.
Regarding the bacterial transformation, on the negative control plate without MaSp1 inserted DNA, we observed that a decent amount of bacterial colonies grew on the LB-Amp plate, indicating that the digestion of the pGal1,10-CBD plasmid was not complete. We noticed an increased number of colonies, which suggested that SmaI was not as efficient. The negative controls indicated that incomplete SmaI digestion had happened. Our MaSp1-CBD plates had some bacterial colonies for us to continue, but this was not guaranteed in the future. Therefore, we consulted with Dr. Han, who suggested using Xma1 as it has the same recognition site; when used on our MaSp2, it showed that the Xma1 enzyme was more efficient at the digestion step.
If our team only generated MaSp1 and MaSp2, these two spider silk proteins would not be able to leave the yeast cell and bind to the SCOBY. Thus, we looked for inspiration from the project by iGEM team LINKS China, in which they used several different types of plasmids and a cloned cellulose binding domain family to create a cellulose binding matrix for the silk protein to bind to SCOBY. However, this method was impractical for our team, as it would be too time-consuming and require more than one kind of antibiotic to check modified yeast function. Therefore, we did research, and it indicated that in previous experiments using in vivo and in vitro techniques, CBM1 (referred to in our project as CBD) alone would have the desired function.
Cellulose Binding Domain proteins can be found in fungi, bacteria, and plants. We had aimed to obtain the gene through bacteria; however, when we tried to source it, we found it was not found locally in Taiwan. We overcame this problem as we found the fission yeast (S. Pombe), which we received from Academia Sinica. This was a better option than bacteria as our team had done several experiments with yeast before, using Saccharomyces cerevisiae. In addition, we found that fungi’s CBD genes are shorter than bacteria’s after research, which is easier to manipulate.
The plasmid requires a promoter to initiate transcription of our inserted gene. To be able to amplify our inserted gene in the plasmid, regions in the binding of the RNA polymerase and transcription factors need to be controlled, or else genes of expression would not be known. For this reason, we applied the pGal1,10 promoter to regulate the expression of genes in the presence or absence of the galactose sugar to turn on or off the pGal1,10 promoter switch, which triggered the transcription of the gene cloned downstream of the pGal1,10 promoter.
After that, the team performed RT-qPCR to check the mRNA transcription level, and the results showed that our team had successfully cloned MaSp1, and MaSp2 downstream of the pGal1,10 promoter. By using this advantage of switching on and off of the pGal1, 10 promoters via the presence or absence of the galactose sugar, our team could manipulate our spider silk proteins whenever our team needed it.
Our engineered parts are documented on the registry: pGal1,10 BBa_K4650000; CBD BBa_K4650001; MaSp1 BBa_K4650002; MaSp2BBa_K4650003; pGal1,10-CBDBBa_K4650004; pGal1,10-MaSp1-CBD BBa_K4650005; pGal1,10-MaSp2-CBDBBa_K4650006 .Electrophoresis has confirmed each of the cloning steps, we also sent our final plasmids to Mission Biotech who confirmed our design was successful through sequencing. The team also performed RT-qPCR to check the mRNA transcription level, and the results showed that our team had successfully cloned MaSp1, and MaSp2 downstream of the pGal1,10 promoter. By using this advantage of switching on and off of the pGal1, 10 promoters via the presence or absence of the galactose sugar, our team could manipulate our spider silk proteins whenever our team needed it. The RT-qPCR confirmed the team’s plasmids did have biological function! This is further described in Results.
To optimize SCOBY production, we consulted Mr. Andrew Nicholls. He advised us to make key adjustments to our cultivation process. We lowered the growth temperature from 23°C to 28°C to influence the SCOBY's microbial balance, aiming for healthier and more consistent cultures; he also stressed the importance of using consistent parent SCOBYs to maintain stable microbial populations. Moreover, Mr. Nicholls emphasized maintaining a constant tea-to-sugar ratio to provide the necessary nutrients for robust SCOBY growth. These changes are expected to improve efficiency and maintain product quality and consistency in SCOBY production.
In further strength tests of the SCOBY, we discovered the SCOBY became overly rigid and wouldn’t break when tested. For this reason, we shortened the growth phase from two weeks to one week. The primary rationale was also to produce more SCOBY in a shorter time frame and enhance overall efficiency for testing.
After we grew our SCOBY, we realized that there was no available machine for us to test the strength of the membranes. Without this, we would not be able to prove that our cloning actually affects the flexibility of SCOBY. Therefore, we decided to create our own machines. We built two machines to test both the tensile strength and shear flexibility.
We initially got our idea from a water well with an axle at the top, rolling the object down using a rope: the object would be 3D printed, and we designed the rectangular prism on Tinkercad; the wooden frame would be a square with two sides attached with vices to hold the SCOBY. Kurt Chen, a teacher with woodworking experience, helped make the structure. Then, we thought the Arduino motor would work, so we drew a blueprint, identifying how to use the Arduino motor to control the 3D-printed object.
On the first try, we realized it's very hard to control the rectangular prism, it's too heavy and shaky when lowering down; Arduino step motors aren't powerful enough. As the first test failed, we altered our design with the help of two physics students, Bill and Teddy, they suggested having a crossbar on top of the SCOBY and letting the Arduino motors work as rope tows. We also changed the step motors to gear motors with advice from Andy Shao, the VEX robotics teacher at our school.
Performing the second test, we realized the contact area was too large, and it was hard to apply pressure on SCOBY with a crossbar; unfortunately, the gear motor did not work as intended. We therefore abandoned the Arduino design and bought a force gauge that can hit with one point and measure force in Newton very accurately. In test three, the force gauge worked well and would be used for the experiment.
Our second machine’s focus was to test the flexibility of the leather. When we were designing the machine, the first problem that came to mind was how to hold the SCOBY membrane without causing it to rip accidentally due to being held by holes in the middle of the experiment. Therefore, we decided to create our own clip for holding the membrane with two wide pieces of wood and two sets of screws and nuts. We tested the clip by putting the membrane in and pulling on it. It held the membrane well.
Then, we thought about how to twist the membrane on a fixed track and a fixed distance between the two sets of clips. Initially, we came up with this sketch:
However, since this machine will be hung, there will be a significant gravitational pull that will vertically stretch the membrane, which may affect the procedure during the experiment. Therefore, we wanted to conduct the experiment on a surface. After discussing, we came up with this second design:
We planned to 3D-print the wheel, which is made of an outer circle, several spheres, and an inner circle. The spheres will be placed between the circles while the outer circle is fixed to the table, which will allow the inner circle to spin on a fixed path without experiencing significant friction.
However, after designing the materials on Tinkercad and printing them, we noticed that the tracks within the two circles were too wide to keep the spheres in place. To solve this problem, we added clay to the tracks to narrow it. After we did this, the spheres could roll properly. Then, another problem emerged: how to connect the wood with the 3D-printed material. After considering using a small wooden stick between the two, we soon realized that it was too complicated to solve a simple problem. Eventually, we directly stuck one of the wood pieces to the inner circle. When the glue dried, we tested it and there was no problem with the rotation.
Next, we noticed that the top of our design is difficult to function as we need to connect it all the way to the ceiling with a really long rope and it will rotate with the bottom set of wood blocks, affecting the measurement. Instead, we set up a stand with clamps to hold the top set of wood blocks up and secure them together with ropes. This way, we’ll just need a person to press down the track to keep the distance between the two clips constant. We tested this with a piece of paper towel and it works perfectly.
Finally, we have two experiments that need to be done, including this one and the tensile strength experiment, which would require breaking the membrane. It would be inefficient for both experiments to use so much SCOBY. As a result, we decided to measure the result in another way. We would look at how much force is needed to twist the SCOBY membrane by a certain degree, based on the idea that less force would be needed to twist a flexible SCOBY. To help us do the experiment this way, we added a rope beside the bottom set of wooden blocks so that a force sensor could hook on it and measure the force. We tested this with pieces of SCOBY and realized that the design worked.
Therefore, this is our final design.
Our team initially aimed to make synthetic leather from SCOBY material and consulted with three specialists for advice. Dr. Jurgita suggested working with industrial kombucha fermentation companies, but Dr. Chen explained that it wouldn't work due to the industrial process. So, we shifted our focus to collaborating with small-scale kombucha producers. We met with Mr. Nicholls to learn about homemade kombucha production and using cellulose waste for creating products. We also explored eco-friendly leather companies like Voome. Our goal is to establish a SCOBY farm with standardized formulas for synthetic leather production, which can be supplied to other companies for further use.
