iGEM 2023 — Team kcis-xiugang-taipei


#

##### Engineering

* [What can we replace animal leather with?](#first)
* [How can we make SCOBY a better leather alternative?](#second)
* [Problems with MaSp1 and MaSp2](#third)
* [How can we bind MaSp1 and MaSp2 into SCOBY’s cellulose membrane?](#fourth)
* [Where can we source CBD?](#fifth)
* [Which promoter system should we use?](#sixth)
* [How can we grow SCOBY effectively?](#seven)
* [How can we test the flexibility?](#eight)
* [Application](#nine)

![](https://static.igem.wiki/teams/4650/wiki/image-1-1.png)

## **What can we replace animal leather with?**

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.

![](https://static.igem.wiki/teams/4650/wiki/hopeengineer/figure-1-eng.png)

## **How can we make SCOBY a better leather alternative?**

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.

![](https://static.igem.wiki/teams/4650/wiki/hopeengineer/figure-2-eng.png)

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](https://2023.igem.wiki/kcis-xiugang-taipei/results).

## **Problems with MaSp1 and MaSp2**

#### *Designing Primer Sets*

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).

#### *No PCR Product Observed After Electrophoresis*

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.

#### *Incomplete Digestion*

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.

## **How can we bind MaSp1 and MaSp2 into SCOBY’s cellulose membrane?**

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.

![](https://static.igem.wiki/teams/4650/wiki/hopeengineer/figure-3-eng.png)

## **Where can we source CBD?**

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.

## **Which promoter system should we use?**

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.

## **Confirmation of Plasmid Design Sucess**

Our engineered parts are documented on the registry: pGal1,10 [BBa\_K4650000](http://parts.igem.org/Part%3ABBa_K4650000); CBD [BBa\_K4650001](http://parts.igem.org/Part%3ABBa_K4650001); MaSp1 [BBa\_K4650002](http://parts.igem.org/Part%3ABBa_K4650002); MaSp2[BBa\_K4650003](http://parts.igem.org/Part%3ABBa_K4650003); pGal1,10-CBD[BBa\_K4650004](http://parts.igem.org/Part%3ABBa_K4650004); pGal1,10-MaSp1-CBD [BBa\_K4650005](http://parts.igem.org/Part%3ABBa_K4650005); pGal1,10-MaSp2-CBD[BBa\_K4650006](http://parts.igem.org/Part%3ABBa_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](https://2023.igem.wiki/kcis-xiugang-taipei/results).

## **How can we grow SCOBY effectively?**

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.

![](https://static.igem.wiki/teams/4650/wiki/hopeengineer/figure-4-eng.png)

![](https://static.igem.wiki/teams/4650/wiki/hopeengineer/figure-5-eng.png)

## **How can we test the flexibility?**

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.

#### *Tensile Strength*

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.

 ![](https://static.igem.wiki/teams/4650/wiki/img-3530.jpg)

![](https://static.igem.wiki/teams/4650/wiki/img-3389.jpg)

 ![](https://static.igem.wiki/teams/4650/wiki/img-3391.jpg)

#### *Shear Flexibility*

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:

![](https://static.igem.wiki/teams/4650/wiki/hopeengineer/figure-7-eng.png)

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:

![](https://static.igem.wiki/teams/4650/wiki/flexibility-machine-twist.png)

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.

![](https://static.igem.wiki/teams/4650/wiki/hopeengineer/figure-9-eng.png)

## **Application**

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.

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#

##### Experiments

* [Experiment Aims](#first)
* [Making SCOBY more flexible](#second)
\* [Creation of our Genetically Engineered Composition Parts](#fourth)
* [Final Proof of Concept Testing: Functioning of Composite Parts](#fifth)
* [Co-culture and creation of engineered SCOBY](#sixth)
* [Protocols (in alphabetical order)](#seven)
* [Works Cited](#eight)

## **Experiment Aims**

Our team’s aim is to create genetically engineered SCOBY that has increased properties
of flexibility and strength so that it is a more viable alternative to traditional
animal skin leather. This will allow a decrease in the production of animal leather.

## **Making SCOBY more flexible**

#### *Choosing the target genes*

Since the SCOBY needs to be able to be used as a leather substitute, it needs to more
closely mimic the properties of animal leather that consumers value most. From our Human
Practice team’s surveys, the team learned that 70.3% of the general public values animal
leather’s “flexibility” as its most important property. Therefore, the team set out to
find genes with the right features to implement into the yeast that cocultures with
SCOBY so that SCOBY which has the desired flexibility could be produced.

#### *MASP1 and MASP2*

We discovered articles that showed the silk produced by spiders has the features we
need. Spider silk mostly consists of large proteins and has highly repetitive spidroins,
which makes it strong and flexible at the same time.[1],[2]

A study by Tian in 2003 showed the repetitive amino acid sequences shown in Figure 1
below.[2] Our team chose MaSp1 and MaSp2, because they are the two shortest amino acids
among all, making it easier for cloning and genetic modification when translating into
DNA sequences, and also easier to design primers for the PCR technique to amplify these
two DNA sequences.

![](https://static.igem.wiki/teams/4650/wiki/figure-12-description.jpg)

Figure 1. Consensus amino acid repeats for known spider silks [2]

#### *CBD*

However, to attach any structure to SCOBY’s cellulose membrane, there has to be a “glue” between the structure and the membrane so that the synthesized MaSp1 and MaSp2 do not just float around in the kombucha and fail to enhance our SCOBY’s properties.

To solve this issue, we were inspired by the 2021 LINKS\_CHINA team’s project of producing durable leather substitutes from bacterial cellulose. To attach proteins and natural pigment dyes to the cellulose membrane, LINKS\_CHINA used a structure called the “Cellulose Binding Matrixes.”[4] This inspired us to also look for similar structures that we can use to bind silk-like proteins to the cellulose, which led us to find “Cellulose Binding Domains” (CBD).

As explained in its name, CBD is a structure that binds to cellulose. CBDs are contained in many organisms, such as fungi, bacteria, and plants, and the major function of CBDs in those organisms is to help enzymes or complexes bind to cellulose for further catalytic activities beneficial for in vivo cellular processes.[5] Many enzymes contain CBDs on their own for cellulose digestion. Those CBDs are conserved, which indicates those CBDs are pivotal for delivering proteins or complexes to bind to cellulose membranes.[5] With in vitro protein purification, scientists could add the desired genes at the 5’- or 3’- end of CBD to form fusion proteins, which could still bind to cellulose.[6] Overall, our team was convinced that using one CBD was enough to do the work that our team needed.

CBD’s property allows our team to use this complex as a connecting line between the cellulose that forms SCOBY and the silk in the modified SCOBY.[7] Without it, the silk cannot connect to the SCOBY and is therefore non-functional. As a result, it is decided the CBD gene needs to be cloned into the plasmid.

One last question remains: many organisms produce CBD, which CBD gene do we choose? Our
answer is the CMB1 from fission yeast, Schizosaccharomyces pombe (S. pombe). We decided
on this particular CBD gene for several reasons. First, we could not source several
specific bacterial strains containing the CBD family locally, however, S. pombe yeast
containing CBD was available through contact at Academia Sinica. Second, several
research papers show the CBDs in fungi are normally smaller than CBDs in bacteria, which
might be a better fit for SCOBY. Finally, our team has already conducted several
experiments using Saccharomyces yeast, which trained our team to be able to work with
yeast better. Therefore, our team has decided to use CBM1 in S. pombe.[5]

#### *Galactose Promoter (pGal1, 10)*

The last piece of DNA sequence that we included in the plasmid uses the pGal1, 10
promoter. The pGal1, 10 promoter is located upstream of any of the DNA sequences we
insert, the promoter can control the expression of the genes downstream through the
presence of galactose: when galactose is not present in the environment, the MaSp1
or MaSp2 downstream of the pGal1, 10 promoter will not be expressed, and no spider
silk protein or CBD will be produced.[8] The galactose can also initiate the
induction faster than other promoters in yeast.

#### *Backbone Plasmid and antibiotic-resistant genes*

The plasmid we acquired, pGal1, 10-SPT5-Streptavidin Binding Protein(SBP)
plasmid, containing 2u origin of replication (ORI) for both yeast and bacteria
DNA replication, was sponsored by Dr. Tien-Hsien Chang at the Genomics Research
Center of Academia Sinica, in Taipei, Taiwan. This plasmid can be transformed
into bacteria and yeast, which was beneficial for our team to finish making
biobricks in bacteria and perform the functional assay in yeast.

The plasmid we acquired also contains ampicillin and kanamycin-resistant genes.
We chose to also acquire these two genes on our plasmids so we could conduct
future bacterial and yeast transformation on LB-amp plates and YPD-Kan plates,
respectively. The kanamycin-resistant gene and YPD-Kan selection plates are
necessary because ampicillin has no effect on yeast, which means we need another
method for incubating only the yeast cells with our plasmid. Having it present
will allow us to use kanamycin in order to incubate our yeast. We will be able
to use Saccharomyces yeast that the team is familiar with from training for the
design.

## **Creation of our Genetically Engineered Composition Parts**

![](https://static.igem.wiki/teams/4650/wiki/experiments/fig-2-process-of-cbd-pcr-product.png)

Fig 2. Process of CBD PCR Product

#### *CBD Extraction: PCR & first enzyme digestion*

We obtained S. pombe fission yeast from Academia Sinica, extracted the
[yeast’s
genomic DNA](https://static.igem.wiki/teams/4650/wiki/experiments/yeast-genomic-dna-extraction-protocol.pdf) , and used
[2X
PCR](https://static.igem.wiki/teams/4650/wiki/experiments/2x-pcr-mix-protocol.pdf),
to amplify CMB1. We engineered two enzyme cut sites (XmaI and KpnI) on primers
for this PCR and obtained the primers from the synthetic bio company Mission
Biotech.

#### *Proof of concept:*

We conducted
[gel
electrophoresis](https://static.igem.wiki/teams/4650/wiki/experiments/gel-electrophoresis-protocol.pdf)
to ensure the PCR was successful.

![](https://static.igem.wiki/teams/4650/wiki/screenshot-2023-10-09-at-5-34-45-pm.png)

Fig 3. The Process of Double Digestion, T4 Ligation, and Bacterial
Transformation

![](https://static.igem.wiki/teams/4650/wiki/experiments/inserting-cbd-into-pgal-plasmid-and-complete-proof-of-concept.png)

Fig 4. The Process of Double Digestion, T4 Ligation, and Bacterial
Transformation

#### *Double-Digestion of CBD*

We then used XmaI and KpnI
[double
enzyme digestion](https://static.igem.wiki/teams/4650/wiki/experiments/neb-enzyme-digestion-protocol.pdf)
to create a 3’ overhang of the CBM1(referred to as CBD) PCR product. The cut
sites, XmaI and KpnI, are designed on the primers so that double enzyme
digestion could be done. The two enzymes create overhangs on the CBD gene for
easier ligation.

#### *T4 ligation, glue CBD to plasmid*

We acquired the backbone plasmids with the pGal1,10 promoter from Academia
Sinica. We first used double
[enzyme
digestion](https://static.igem.wiki/teams/4650/wiki/experiments/neb-enzyme-digestion-protocol.pdf)
with XmaI and KpnI to knock out the original gene downstream of the pGal1,10
promoter, straighten the plasmid called vector, and then used
[DNA ligation](https://static.igem.wiki/teams/4650/wiki/experiments/t4-dna-ligation-protocol.pdf)
with the T4 ligase enzyme to clone the CBD gene downstream of the pGal1,10
promoter.

We chose the enzymes XmaI and KpnI because, first, these two enzymes produce cut
sites with incompatible 3’-overhangs, so the linearized vector would not
re-ligate back together, which makes it easier for ligation; and second, these
enzymes only cut on the vectors once, which ensures our vectors do not break
into pieces.

![](https://static.igem.wiki/teams/4650/wiki/experiments/screenshot-2023-10-09-at-5-37-50-pm.png)

Fig 5. A visualization of kicking out the original gene downstream of the pGal
promoter and replacing it with CBD’s gene sequence.

#### *Proof of concept:*

After running these procedures, we needed to test whether the cloning worked on
a molecular biology level, and that the CBD genes have been cloned to the
downstream of the pGal1, 10 promoter correctly. To confirm this, our team
conducted two tests.

1. After conducting bacterial transformation, we cultured E. Coli transformed
with our plasmids in ampicillin selection plates to generate more of our
pGal-CBD plasmid on LB-amp selection plates. The surviving colonies are
confirmed to carry the pGal-CBD plasmid. We also inoculated more of our pGal-CBD
plasmids through this
[step.](https://static.igem.wiki/teams/4650/wiki/experiments/bacterial-transformation-heat-shock-protocol.pdf)

2. We conducted
[2X
PCR](https://static.igem.wiki/teams/4650/wiki/experiments/2x-pcr-mix-protocol.pdf)
, with CBD forward and reverse primers, and
[gel
electrophoresis](https://static.igem.wiki/teams/4650/wiki/experiments/gel-electrophoresis-protocol.pdf)
to see if the bacterial colonies have the pGal-CBD plasmid.

![](https://static.igem.wiki/teams/4650/wiki/experiments/screenshot-2023-10-09-at-5-43-26-pm.png)

Fig 6. Cloning CBD to form pGal1,10-CBD. Bacterial colonies # 1, 2, and 5 showed
a CBD distinct single PCR product band, BBa\_K4650004 (pGal1, 10-CBD).

#### *Amplifying MaSp1 & MaSp2*

![](https://static.igem.wiki/teams/4650/wiki/experiments/amplify-plasmid-with-masp12-and-perform-electrophoresis.png)

Fig 7. Amplify plasmid with MaSp1/2 and perform electrophoresis.

Our team used Snapgene to convert the two spider silk amino acid sequences into
DNA sequences and then Mission Biotech, synthesized the DNA sequences. They
synthesized MaSp1 and MaSp2 on the T7 promoter and pMA-RQ plasmids,
respectively.

We then conducted
[2X
PCR](https://static.igem.wiki/teams/4650/wiki/experiments/2x-pcr-mix-protocol.pdf)  to produce more copies of our desired genes, MaSp1 and MaSp2.
During this process, we engineered the primers containing one recognition
sequence cut by either SmaI or XmaI, and added the primers to identify the genes
we needed to amplify (containing Masp1&2), and then got more copies through PCR.

From the genes we got through 2X PCR, our team conducted a single enzyme cut
experiment on MaSp1 and MaSp2 PCR products, respectively, for cloning upstream
of the CBD, and downstream of the pGal1, 10 promoter of the pGal-CBD plasmid.

Our team cut MaSp1 using
[single
enzyme digestion](https://static.igem.wiki/teams/4650/wiki/experiments/neb-enzyme-digestion-protocol.pdf) , with the enzyme SmaI. We designed SmaI to have a
similar recognition site (CCCGGG) to a previous enzyme we used, XmaI. The
difference between them is that SmaI creates a blunt end cut site, which we
believed would be easier to work with. However, we found SmaI inefficient as it
could religate and a lot of the product was lost during dephosphorylation. After
advice from Dr. Han, for MaSp2, the team switched to using XmaI to cut out the
gene, which is easier to manipulate in the experiment. So in the future, it
would be best to use XmaI for both MaSp1 and MaSp2.

#### *Proof of concept:*

We conducted
[gel
electrophoresis](https://static.igem.wiki/teams/4650/wiki/experiments/gel-electrophoresis-protocol.pdf) to ensure the PCR was successful.

![](https://static.igem.wiki/teams/4650/wiki/bba-k4650002-masp1-pcr.jpg)

Fig 8. MaSp1 PCR products.

![](https://static.igem.wiki/teams/4650/wiki/bba-k4650003-masp2-pcr.jpg)

Fig 9. MaSp2 PCR products.

#### *pGal-CBD plasmid second enzyme digestion*

![](https://static.igem.wiki/teams/4650/wiki/experiments/inserting-cbd-into-pgal-plasmid-and-complete-proof-of-concept.png)

Fig 10. Opened pGal CBD plasmid with single enzyme digestion, Inserted MaSp1/2
into pGal CBD, then performed gel electrophoresis for proof of concept.

We then extracted the pGal1,10-CBD plasmids from the E.Coli we transformed and
inoculated them to the LB-amp broth.

Once the plasmids were extracted, the pGal1,10-CBD plasmids were cut using
single enzyme digestion with two different enzymes: SmaI to prepare for MaSp1
ligation and XmaI for MaSp2 ligation respectively since these were the enzymes
used for single enzyme digestion of MaSp1 and MaSp2.

#### *T4 ligation of the pGal1,10-CBD plasmid and Masp1/2 gene*

The team conducted
[T4
DNA ligation](https://static.igem.wiki/teams/4650/wiki/experiments/t4-dna-ligation-protocol.pdf) on the pGal1,10-CBD plasmids and MaSp1 or 2 genes to clone
MaSp1 and MaSp2 into pGal1,10-CBD, forming our desired plasmids,
pGal1,10-MaSp1-CBD and pGal1,10-MaSp2-CBD.The team then applied bacterial
transformation on selection plates to replicate our plasmids for further
experiments.

Our team used the forward primer at the 5’ end of the MaSp1 or MaSp2 and reverse
primer at the 3’ end of the CBD to ensure the correct orientation of the
pGal-MaSp1-CBD and pGal-MaSp2-CBD plasmids.

![pcr1](https://static.igem.wiki/teams/4650/wiki/experiments/screenshot-2023-10-09-at-5-52-28-pm.png)

Fig 11. This figure is a visualization of the two possible orientations of Masp1.
Only one of them is correct and will produce amino acid sequecnes downstream of the
pGal promoter.

#### *Bacterial transformation*

Three plasmids are now complete: pGal1,10-CBD, pGal1,10-Masp1-CBD and
pGa1,10l-Masp2-CBD. The team transformed E. coli with the three plasmids separately
and inoculated the modified bacteria to get more copies of the plasmid. The team
used
[heat
shock on the bacteria](https://static.igem.wiki/teams/4650/wiki/experiments/bacterial-transformation-heat-shock-protocol.pdf) to allow the plasmids to enter the membrane, then used
selection plates with ampicillin to select the surviving bacteria.

#### *Proof of concept testing:*

After bacterial colonies were inoculated to perform 2XPCR, the team ran
[gel
electrophoresis](https://static.igem.wiki/teams/4650/wiki/experiments/gel-electrophoresis-protocol.pdf) to check that the PCR product was correct (that MaSp1 in the
plasmids had the right orientation). Successful results proved that our team had
cloned successfully.
We also sent the plasmids to Mission Biotech for further analysis and confirmation.

![pcr1](https://static.igem.wiki/teams/4650/wiki/experiments/screenshot-2023-10-09-at-5-58-43-pm.png)

Fig 12. pGal1,10-MaSp2-CBD PCR products. During ligation of MaSp2, the team used one XmaI single enzyme cut site for the cloning that would give our team two
orientations of MaSp2 cloned between the pGal1,10 promoter and CBD. After bacterial transformation, our
team wanted to detect which bacterial colonies would give us the correct orientation of MaSp2. We used forward primer on the 5’- end of MaSp2, and reverse primer on the 3’- end of CBD to do PCR. Then our team ran our PCR products to detect that both #8 and #13 had a MaSp2-CBD full-length product of 870bp.

![](https://static.igem.wiki/teams/4650/wiki/experiments/screenshot-2023-10-09-at-6-00-23-pm.png)

Fig 13. pGal1,10-MaSp2-CBD PCR products.

#### *Yeast transformation*

Once PCR confirmed our team had the correct orientations of pGal1,10-MaSp1-CBD, and
pGal1,10-MaSp2-CBD bacterial colonies, the bacterial plasmid extraction was
performed to extract the plasmids containing the three composite parts. After the
three new plasmids, pGal1,10-CBD, pGal1,10-MaSp1-CBD, and pGal1,10-MaSp2-CBD, were
created, our team did
[plasmid
transformation](https://static.igem.wiki/teams/4650/wiki/experiments/yeast-plasmid-transformation-protocol.pdf)
to shuffle each of them into the wild-type yeast strain, Saccharomyces cerevisiae,
strain BY4741, respectively. Kanamycin selection plates were used for the
transformation. Kanamycin (also called G418) inhibits the process of the protein
translation so only BY4741 containing our team’s three biobrick plasmids, pGal-CBD,
pGal-MaSp1-CBD, or pGal-MaSp2-CBD, could survive on YPD-Kan plates, since those
plasmids contained Kanamycin resistant genes. After the yeast plasmid
transformation, our team now had pGal-CBD in BY4741, as a control yeast strain for
our team’s project since it doesn’t contain spider silk proteins; and pGal-MaSp1-CBD
in BY4741, and pGal-MaSp2-CBD in BY4741 which are our experimental strains since
they contain our spider-silk proteins, MaSp1-CBD, and MaSp2-CBD, respectively.
All of these parts can be found on the registry: pGal1,10 [BBa\_K4650000](http://parts.igem.org/Part%3ABBa_K4650000); CBD [BBa\_K4650001](http://parts.igem.org/Part%3ABBa_K4650001); MaSp1 [BBa\_K4650002](http://parts.igem.org/Part%3ABBa_K4650002); MaSp2[BBa\_K4650003](http://parts.igem.org/Part%3ABBa_K4650003); pGal1,10-CBD[BBa\_K4650004](http://parts.igem.org/Part%3ABBa_K4650004); pGal1,10-MaSp1-CBD [BBa\_K4650005](http://parts.igem.org/Part%3ABBa_K4650005); pGal1,10-MaSp2-CBD[BBa\_K4650006](http://parts.igem.org/Part%3ABBa_K4650006)

## **Final Proof of Concept Testing: Functioning of Composite Parts**

#### *Time Course samples collection*

After creating the three composite parts, the team did galactose induction with a
time course to prove the desired composite parts could be induced in the presence of
2% YP-galactose to check the induction of the coding regions on the composite parts
via RNA extraction and RT-qPCR technique. The parts discussed in the “Yeast
Transformation” section above were used to perform the time course sample collection
shown in the figure: pGal-CBD in BY4741 as a control, and pGal-MaSp1-CBD in BY4741,
and pGal-MaSp2-CBD in BY4741 as our experimental strains. This process is shown in
the diagram below.

![](https://static.igem.wiki/teams/4650/wiki/screenshot-2023-10-09-at-12-41-42-pm.png)

Fig 14. The team grew yeast in the 2% YP-glucose + G418 medium until it reached
OD~0.2 and collected 35ml as 0’ minute, and then resuspended and rinsed with dH2O to
transfer them into the 2% YP-galactosemedium to collect the time course samples(set
up 30 minutes, 60minutes, 90minutes, 120 minutes, 22 hours) for RT-qPCR to detect
mRNA level of the downstream genes of the pGal1, 10 promoter.

#### *RT-qPCR experiments*

To further verify whether our team’s composite parts had biological functions, our
team used
[RT-qPCR
protocol](https://static.igem.wiki/teams/4650/wiki/experiments/rt-qpcr-protocol.pdf)
to ensure our composite parts functioned properly by detecting mRNA induction levels
of CBD, MaSp1-CBD, and MaSp2-CBD. Our team operated RT-qPCR technique to detect the
mRNA induction of CBD in BY4741 containing BBa\_K4650004, mRNA induction of MaSp1-CBD
in BY4741 containing BBa\_K4650005, and the mRNA induction of MaSp2-CBD in BY4741
containing BBa\_K4650006 from the time course sample collection of 0min, 30min, 60
min,90 min, 120 min, and 22 hours in the presence of the 2%YP-galactose medium. The
BY4741, with each of the respective composite parts, was first grown in
YP-2%glucose, until OD600 0.2-0.4 to allow sufficient growth of the yeast before
transferring it to the 2%YP-galactose medium.

![pcr1]( 	https://static.igem.wiki/teams/4650/wiki/parts-image/bba-k4650004-bargraph.jpg)

Fig15:Induction of CBD showed strong manipulation, above 5 fold induction, in the
presence of galactose at 30 mins, 14 fold induction at 60mins, and the maxium of 19
fold CBD mRNA induction at 90 mins, and then backdown to 10 fold induction at
120mins, finally no detectable CBD mRNA induction at 22hours in the presence of
galactose.

![pcr1](https://static.igem.wiki/teams/4650/wiki/experiments/screenshot-2023-10-09-at-11-03-42-pm.png)

Fig 16. The mRNA induction of MaSp1-CBD showed insignificant change in the presence
of galactose at 30 mins, the maximum of 2 fold induction at 60mins, and and then
backdown to 1.7-1.5 fold induction at 90 and 120 mins, finally no detectable MaSp1-
CBD mRNA induction at 22 hours in the presence of galactose

![pcr1](https://static.igem.wiki/teams/4650/wiki/experiments/screenshot-2023-10-09-at-11-04-41-pm.png)

Fig 17. The mRNA induction of MaSp2-CBD showed insignificant reduction in the
presence of galactose at 30 mins compared to the 0 min sample, the maximum above 2
fold induction at 90 mins, and then backdown to 1.7 fold and 1.3 fold induction at
120 mins and 22 hours in the presence of galactose.

## **Co-culture and creation of engineered SCOBY**

![pcr1]( 	https://static.igem.wiki/teams/4650/wiki/experiments/overall-diagram-3.png)

Fig 18. The overall diagram of the growing the modified SCOBY and performing the flexibility test.

#### *Growing a new batch of SCOBY*

The team now needed to start working with real SCOBY samples. But before any
experiments could be conducted, the team first grew two
[new
batches of SCOBY](https://static.igem.wiki/teams/4650/wiki/experiments/scoby-growth-protocol.pdf) as trials to find the thickness of SCOBY grown in different
time periods. The team used black tea, the popular traditional recipe, as our base
for making SCOBY, due to its high content of caffeine, which would provide the
bacteria groups in the kombucha a nitrogen source. Nitrogen, a nutrient yeasts
assimilate when performing alcholic fermentation, promotes the metabolism of yeasts,
which would assist the growth of SCOBY.[9]

After dissolving sugar in the black tea, we added starting fluid and mother SCOBY
into the sweet tea and then fermented it at 23 degrees Celsius for 7 days.
Originally, the SCOBY grown for 14 days became too thick and strong for the machines
we designed for flexibility tests (mentioned later in “Flexibility Test”) to break
them, therefore unable to yield any usable data. Therefore, the team shortened the
fermentation period to 7 days to make the SCOBY thinner and able to be torn apart by
our machines used in the flexibility tests, which produces data that can be compared
with our modified SCOBY.

#### *Co-culturing SCOBY with BY4741 yeast*

After being grown, the SCOBY was rinsed with DI water and the team co-cultured our
SCOBY with a mix of our genetically modified yeast (BY4741 containing each of the
respective plasmids, and a control yeast), 2% YP-galactose, and 100ug/ml Kanamycin
(G418).
The BY4741 yeast contains the plasmids we genetically modified in previous steps,
either our pGal-Masp1-CBD, pGal-Masp2-CBD, or pGal-CBD.
The 2% YP-galactose provides nutrients for our modified yeast and the most important
function is to turn on the pGal1,10 promoter’s switch to activate the transcription
of downstream of genes. Kanamycin kills the original yeasts in the SCOBY since it is
no longer needed in the experiment. Our plasmids contain kanamycin-resistant genes,
which protect them from this chemical. By doing this, we can make sure that only our
modified yeast is generating proteins.

#### *Cleaning and drying SCOBY*

After three days of co-culturing with our genetically engineered yeast, the team
first washed the SCOBY with DI water to get rid of any impurities on the surface.
Then, the team researched how to properly dry the SCOBY. Since working with SCOBY as
an alternative material is a rather new and unexplored method, there is no standard
protocol on how to dry it. Instead, the team only discovered protocols from blog
posts about making SCOBY at home, with no certain range of temperature sets and
times.[10] This pushed us to experiment with suitable times and temperatures by
ourselves and then give the result. Based on our results, we wrote our own SCOBY
drying protocol, which is to dry SCOBY
[dry
scoby.](https://static.igem.wiki/teams/4650/wiki/experiments/dehydrating-scoby-protocol.pdf)
at 40 degrees Celsius for 20 hours.

#### *Flexibility and Strength test*

Subsequently, the team conducted an experiment to [test flexibility](https://static.igem.wiki/teams/4650/wiki/experiments/flexibility-test-protocol.pdf) to confirm if the
genetic modification improved the dried SCOBY membrane’s performance.

For the tensile strength, we built a machine that holds the SCOBY on its sides and
pushes it on the center to test how much force it can hold before breaking, as shown
in the diagram below.

![pcr1]( https://static.igem.wiki/teams/4650/wiki/img-3530.jpg)

Fig 19. The design of tensile strength flexibility machine.

The wooden frame holds the basic structure of the machine. There are two mini vice
that would hold the SCOBY tightly without causing much damage while the force gauge
would be push down on the center of the SCOBY and record the highest peak value of
force when the SCOBY breaks. The more force it can support, the stronger and more
flexible the membrane is.

For shear strength, we have a different set of equipment. We designed a 3D-printed
wheel that is connected with wood block, which will be paired with another wood
block and a second pair of wood blocks, as shown in the diagram below.

![pcr1]( https://static.igem.wiki/teams/4650/wiki/experiments/the-design-of-shear-strength-flexibility-machine.png)

Fig 20. The design of shear strength flexibility machine.

After setting up the machine, the bottom pair of wood blocks would allow the SCOBY
membrane to twist as the blocks turn on the track of the wheel while the top pair is
fixed. By looking at how much force it takes for the SCOBY to turn a certain amount,
we can learn about its flexibility. The less force needed, the higher its
flexibility. We twist all SCOBY for 40 degrees and did three trials for each of the
different membranes.

## **Protocols (in alphabetical order)**

 [2X PCR Mix Protocol](https://static.igem.wiki/teams/4650/wiki/experiments/2x-pcr-mix-protocol.pdf)

 [Bacterial Plasmid Extraction Protocol](https://static.igem.wiki/teams/4650/wiki/experiments/bacterial-plasmid-extraction-protocol.pdf)

 [Bacterial Transformation Heat Shock Protocol](https://static.igem.wiki/teams/4650/wiki/experiments/bacterial-transformation-heat-shock-protocol.pdf)

[Dehydrating Scoby Protocol](https://static.igem.wiki/teams/4650/wiki/experiments/dehydrating-scoby-protocol.pdf)

[Flexibility Test Protocol](https://static.igem.wiki/teams/4650/wiki/experiments/flexibility-test-protocol.pdf)

[Gel Electrophoresis Protocol](https://static.igem.wiki/teams/4650/wiki/experiments/gel-electrophoresis-protocol.pdf)

[NEB Enzyme Digestion Protocol](https://static.igem.wiki/teams/4650/wiki/experiments/neb-enzyme-digestion-protocol.pdf)

[RT-qPCR protocol](https://static.igem.wiki/teams/4650/wiki/experiments/rt-qpcr-protocol.pdf)

[SCOBY and Yeast Co-Culture Protocol](https://static.igem.wiki/teams/4650/wiki/experiments/scoby-and-yeast-co-culture-protocol.pdf)

[SCOBY Growth Protocol](https://static.igem.wiki/teams/4650/wiki/experiments/scoby-growth-protocol.pdf)

[T4 DNA Ligation Protocol](https://static.igem.wiki/teams/4650/wiki/experiments/t4-dna-ligation-protocol.pdf)

[Yeast Genomic DNA Extraction Protocol](https://static.igem.wiki/teams/4650/wiki/experiments/yeast-genomic-dna-extraction-protocol.pdf)

[Yeast Plasmid Transformation Protocol](https://static.igem.wiki/teams/4650/wiki/experiments/yeast-plasmid-transformation-protocol.pdf)

## **Works Cited**

[1] Römer L, Scheibel T. The elaborate structure of spider silk: structure and function of a natural high performance fiber. Prion. 2008; 2(4): 154-61. Available from: doi:10.4161/pri.2.4.7490.

 [2] Tian M. Expression and Purification of A Synthetic Spider Silk Protein In Arabidopsis thaliana. Master thesis. University of Wyoming; 2003.

 [3] LINKS\_CHINA. Description. Available from: https://2021.igem.org/Team:LINKS\_China/Description [Accessed September 28 2023].

 [4] Malay AD, Arakawa K, Numata K. Analysis of repetitive amino acid motifs reveals the essential features of spider dragline silk proteins. PLoS One. 2017;12(8): e0183397. Available from: doi:10.1371/journal.pone.0183397.

 [5] Doi RH, Park JS, Shin HS. Fusion proteins containing cellulose-binding domains. Methods in Enzymology. 2000;326: 418-429. Available from: doi:10.1016/S0076-6879(00)26067-9.

 [6] Levy I, Shoseyov O. Cellulose-binding domains: Biotechnological applications. Biotechnology Advances. 2002;20: 191–213. Available from: doi:10.1016/S0734-9750(02)00006-X.

 [7] Linder M, Teeri TT. The cellulose-binding domain of the major cellobiohydrolase of Trichoderma reesei exhibits true reversibility and a high exchange rate on crystalline cellulose. PNAS. 1996; 93(22): 12251-12255. Available from: doi:10.1073/pnas.93.22.12251.

 [8] O’Connor CM. 13.1: Regulation of the GAL1 promoter. California: Libretexts. https://bio.libretexts.org/Bookshelves/Cell\_and\_Molecular\_Biology/Book%3A\_Investigations\_in\_Molecular\_Cell\_Biology\_(O%27Connor)/13%3A\_Protein\_overexpression/13.01%3A\_Regulation\_of\_the\_GAL1\_promoter.

 [9] Roca-Mesa H, Sendra S, Mas A, Beltran G, Torija M-J. Nitrogen preferences during alcoholic fermentation of different non-saccharomyces yeasts of oenological interest. U.S. National Library of Medicine. U.S. National Library of Medicine; Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7074775/ [Accessed September 28 2023].

 [10] How to Make SCOBY Fruit Leather. [Online] KOMBUCHA TO THE PEOPLE. KOMBUCHA TO THE PEOPLE; Available from: https://www.kombuchatothepeople.com/blog/how-to-make-scoby-fruit-leather [Accessed September 28 2023].

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#

##### Proof of Concept

* [Confirmation of each cloning step](#first)
* [Confirmation of part collection function](#second)
* [Product Testing](#third)
* [Conclusion](#fourth)
* [Futher Extension](#fifth)

Our team’s aim is to create genetically engineered SCOBY that has increased properties
of flexibility and strength so that it is a more viable alternative to traditional
animal skin leather. This will allow a decrease in the production of animal leather.

## **Confirmation of each cloning step**

#### *CBD*

Our team performed PCR technique to amplify CBD using the S.pombe genome as the DNA
template and then confirmed the success of the step by observing the right size bands of
the CBD PCR products on 1% agarose gel.

![pcr1](https://static.igem.wiki/teams/4650/wiki/data-and-poc/2023-10-09-4-33-03.png)

Fig 1: After extracting genomic DNA from S. Pombe, the team amplified CBD with two
enzyme cut sites, Xma1 and Kpn1, flanked on the two sides through PCR. After PCR, the
team ran a sample through electrophoresis on 1% agarose gel, CBD PCR product showed a
single distinct DNA band with a length of 594 base pairs+20 bp of enzyme cut site on
primers, and showed success after amplification.
. The DNA fragment corresponds with DNA ladders according to the gel electrophoresis.

#### *PGal1,10-CBD*

The team conducted bacterial transformation on the LB-Amp selection plates and
inoculated several bacterial colonies to conduct PCR technique which showed that our
team had successfully created a composite part, BBa\_K4650004 (pGal1,10-CBD).

![pcr1](https://static.igem.wiki/teams/4650/wiki/data-and-poc/b089c8b2-71fe-4355-b510-ed2668d9e745.png)

Fig 2: After cloning CBD to the downstream of the pGal1,10 plasmid, bacterial colonies
were grown on the LB-Amp selection plates, and then were directly inoculated to perform
the PCR technique via CBD primer set. The team conducted PCR with CBD forward and
reverse primers and gel electrophoresis to prove that bacterial colonies # 1, 2, and 5
showed a distinct CBD single PCR product band, which indicated that the bacterial
colonies contained our team’s composite part, BBa\_K4650004 (pGal1, 10-CBD).

#### *MaSp1*

Our team performed a PCR technique to amplify MaSp1 synthesized by the Mission Biotech
company as described in Experiment, and then confirmed the success of the step by
observing the right size bands of the MaSp1 PCR products on 3% agarose gel.

![pcr1](https://static.igem.wiki/teams/4650/wiki/data-and-poc/2023-10-09-4-13-59.png
						)

Fig 3: For cloning of MaSp1, the team first performed PCR and gel electrophoresis to the
MaSp1 PCR products. As the size of MaSp1 is only 102 base pairs, electrophoresis is
performed on 3% agarose gel. MaSp1 PCR products showed a single distinct DNA band with a
length of 102bp of MaSp1+20 bp of enzyme cut site on primers, and showed success in
amplification.

#### *pGal1,10-MaSp1-CBD*

The team conducted bacterial transformation on the LB-Amp selection plates and
inoculated several bacterial colonies to conduct PCR technique via forward primer at the
5’ end of MaSp1 and reverse primer at the 3’ end of CBD, the results determined that our
team had successfully created a composite part, BBa\_K4650005 (pGal1,10- MaSp1-CBD).

![pcr1](https://static.igem.wiki/teams/4650/wiki/data-and-poc/066efaa8-bf42-43ef-be00-619b67317ea5.png)

Fig 4: During ligation of MaSp1, the team used one SmaI single enzyme cut site for the
cloning that would give our team two orientations of MaSp1 cloned between pGal1,10
promoter and CBD. Therefore, after bacterial transformation, our team wanted to detect
which bacteria colonies would give us the correct orientation of MaSp1, our team used
forward primer on the 5’- end of MaSp1, and reverse primer on the 3’- end of CBD to do
PCR. Then our team ran our PCR product on 1%agarose gel, and our team detected bacterial
colony # 8 had one distinct PCR band between 700-800 bp, which aligned with the
theoretical size of MaSp1 plus CBD of 720bp, indicating MaSp1-CBD PCR product and
cloning success.

#### *MaSp2*

Our team performed PCR technique to amplify MaSp2 synthesized by the Mission Biotech
company, as described in Experiment, and then confirmed the success, as the right size
bands of the MaSp2 PCR products on 3% agarose gel were shown.

![pcr1](https://static.igem.wiki/teams/4650/wiki/data-and-poc/03fe236e-3f91-4d7b-a434-8e1ac37915fb.png)

Fig 5: For cloning of MaSp2, the team first performed PCR and gel electrophoresis on the
MaSp2 gene. As the size of MaSp2 was the size of 255bp, gel electrophoresis was
performed with 3% agarose gel. MaSp2 PCR products showed a single distinct DNA band with
a length of 255bp of MaSp2+20 bp of enzyme cut side designed on the primers in addition
to showing success in amplification.

#### *pGal1,10-MaSp2-CBD*

The team conducted bacterial transformation on the LB-Amp selection plates and inoculated several bacterial colonies to conduct PCR technique via forward primer at the 5’- end of MaSp2 and reverse primer at the 3-’ end of CBD, which determined that our team successfully created a composite part, BBa\_K4650006 (pGal1,10- MaSp2-CBD).

![pcr1](https://static.igem.wiki/teams/4650/wiki/data-and-poc/dcc1b243-f1fc-43c7-bf8c-e3b45038d600.png)

Fig 6: During ligation of MaSp2, the team used one XmaI single enzyme cut site for the cloning that would give our team two orientations of MaSp2 cloned between the pGal1,10 promoter and CBD. After bacterial transformation, our team wanted to detect which bacterial colonies would give us the correct orientation of MaSp2. We used forward primer on the 5’- end of MaSp2, and reverse primer on the 3’- end of CBD to do PCR. Then our team ran our PCR products to detect that both #8 and #13 had a MaSp2-CBD full-length product of 870bp.

After finishing these 3 composite parts, our team also sent them out to Mission Biotech to do DNA sequences in order to confirm them as 100% correct DNA sequences.

Our contributed parts can be found on the registry: pGal1,10 [BBa\_K4650000](http://parts.igem.org/Part%3ABBa_K4650000); CBD [BBa\_K4650001](http://parts.igem.org/Part%3ABBa_K4650001); MaSp1 [BBa\_K4650002](http://parts.igem.org/Part%3ABBa_K4650002); MaSp2[BBa\_K4650003](http://parts.igem.org/Part%3ABBa_K4650003); pGal1,10-CBD[BBa\_K4650004](http://parts.igem.org/Part%3ABBa_K4650004); pGal1,10-MaSp1-CBD [BBa\_K4650005](http://parts.igem.org/Part%3ABBa_K4650005); pGal1,10-MaSp2-CBD[BBa\_K4650006](http://parts.igem.org/Part%3ABBa_K4650006)

## **Confirmation of part collection function**

Overall,the purpose of making these composite parts is that the CBM1 in yeast (discussed as CBD) protein cloned to the downstream the pGal1,10 promoter, an inducible promoter to manipulate the transcription of CBD in the presence of galactose, and then MaSp1 and MaSp2 were cloned between the pGal1,10 promoter and CBD, respectively, to make MaSp1-CBD and MaSp2-CBD fusion proteins. The purpose of cloning this CBM1 (referred to as CBD) is to carry our spider silk proteins, MaSp1, and MaSp2 to bind to SCOBY membranes to make our team’s scoby membranes more flexible and softer.

To manipulate our team’s spider silk proteins, MaSp1, MaSp2, and CBD, 3 different composite parts were generated. BBa\_K4650004 contained BBa\_K4650000+BBa\_K4650001 (pGal1,10-CBD); BBa\_K4650005 contained BBa\_K4650000+BBa\_K4650002+BBa\_K4650001 (pGal1,10-MaSp1 -CBD); BBa\_K4650006 contained BBa\_K4650000+BBa\_K4650003+ BBa \_K 4650001(pGal1,10- MaSp2-CBD). BBa\_K4650004, BBa\_K4650005, and BBa\_ K4650006 were then transformed into wild-type Saccharomyces yeast strain, BY4741, respectively. After finishing these 3 composite parts, our team also sent them out to Mission Biotech to do DNA sequences in order to confirm them as 100% correct DNA sequences.

Before explaining the RT-qPCR data results, our team briefly describes each basic part’s function to generate these 3 composite parts below:

1.BBa\_K4650004 contained BBa\_K4650000+BBa\_K4650001 (pGal1,10-CBD) and was transformed into BY4741 to make the BY4741 containing BBa\_K4650004 used as a control in our project. The CBM1 in S. Pombe encodes for the cellulose binding domain (CBD) protein cloned downstream of the galactose promoter, an inducible promoter to manipulate the transcription of CBD in the presence of galactose. The purpose of manipulating CBD is to be a carrier for our team’s spider silk proteins to scoby membranes in the composite parts, BBa\_K4650005, and BBa\_K4650006.

2.BBa\_K4650005 contained BBa\_K4650000+BBa\_K4650002+BBa\_K4650001 (pGal1,10-MaSp1-CBD) and was transformed into BY4741 to make the BY4741 containing BBa\_K4650005 as one of the experimental strains in our project. MaSp1 encodes for spider silk, which is a highly repetitive amino acid sequence and has the properties of being strong, flexible, and elastic. Our team created MaSp1-CBD fusion protein so CBD can deliver MaSp1 to the SCOBY membrane.

3.BBa\_K4650006 contained BBa\_K4650000+BBa\_K4650003+BBa\_K4650001(pGal1,10- MaSp2-CBD) and was transformed into BY4741 to make the BY4741 containing BBa\_K4650006 as one of the experimental strains in our project. MaSp2 encodes for spider silk, which is a highly repetitive amino acid sequence and has the properties of being strong, flexible, and elastic. Our team created MaSp2-CBD fusion protein so CBD can deliver MaSp2 to the SCOBY membrane.

#### *RT-qPCR time course data for Galactose Induction*

To further verify whether our team’s composite parts were biological function, RT-qPCR technique was operated to detect the mRNA induction of CBD in BY4741 containing BBa\_K4650004, the mRNA induction of MaSp1-CBD in BY4741 containing BBa\_K4650005, the mRNA induction of MaSp2-CBD in BY4741 containing BBa\_K4650006 via timecourse sample collection, 0min, 30min, 60min,90min, 120min, and 22hours in the presence of the 2%YP-galactose medium. The internal control gene, ACT1, was used to detect the ACT1 mRNA level at different time courses as standard. The BY4741 contained different composite parts, respectively, were grown in 2%YP-glucose, until OD600 0.2-0.4, and then 35 ml of yeast culture medium was taken out as 0 min as a control. Without galactose, the pGal promoter would not be switched on to perform the downstream of genes’ inductions. Then transferring the 3 composite parts of the yeast culture samples from 2%YP-glucose to 2%YP-galactose medium, our team took out 30ml of yeast culture medium samples at different time courses in the presence of galactose.

#### *RT-qPCR technique showed the maximum mRNA induction of CBD close to 20 fold in the presence of the galactose at 90 mins*

In order to detect CBD mRNA induction, our team has designed a pair of CBD primers within the CBD gene and a common primer set of ACT1 to detect ACT1 mRNA induction as an internal control. In the fig7, at 0 min timecourse, BY4741 yeast contained BBa\_K4650004(pGal1,10-CBD) showed no CBD mRNA induction, which indicated the pGal1,10 promoter didn’t switch on to trigger the downstream gene’s (CBD) transcription in the absence of galactose. At 30 mins in the presence of galactose, the induction of CBD mRNA was close to 5-fold induction, increased to 13-fold CBD mRNA induction at 60 mins, the maximum of CBD mRNA induction close to 19-20 fold induction was observed at 90 mins in the presence of galactose, at 120 mins in the presence of galactose, CBD mRNA induction was declined to 10-fold, and finally, the CBD mRNA induction was not detectable at 22 hours in the presence of galactose. Overall, the data indicated that the pGal1,10 promoter has done its job properly to manipulate the downstream gene, CBD, transcription in the presence of galactose. Now, our team can use BY4741 yeast containing BBa\_K4650004(pGal1,10-CBD) as our control yeast strain.

![pcr1](https://static.igem.wiki/teams/4650/wiki/data-and-poc/2023-10-09-4-33-28.png)

Fig7: The mRNA induction of CBD showed strong manipulation, above 5-fold induction, in the presence of galactose at 30 mins, 14-fold induction at 60mins, and the maximum of 19-fold CBD mRNA induction at 90 mins, and then back down to 10-fold induction at 120 mins, finally no detectable CBD mRNA induction at 22 hours in the presence of galactose.

#### *RT-qPCR technique showed the maximum of the mRNA induction of MaSp1-CBD above 2-fold induction in the presence of the galactose at 60 mins*

In order to detect MaSp1-CBD mRNA induction, our team has designed the forward primer at the 5’end of the MaSp1, and the reverse primer within the CBD gene, and also used a common primer set of ACT1 to detect ACT1 mRNA induction as an internal control.

On the fig8, at 0 min timecourse, BY4741 yeast contained BBa\_K4650005 (pGal1,10-MaSp1-CBD) showed no MaSp1-CBD mRNA induction, which indicated the pGal1,10 promoter didn’t trigger the downstream gene’s (MaSp1-CBD) transcription in the absence of galactose. At 30 mins in the presence of galactose, the induction of MaSp1-CBD mRNA was not impressed, however, the maximum increased to above 2-fold MaSp1-CBD mRNA induction at 60 mins, but then it declined to 1.7 fold and 1.5 fold of MaSp1-CBD mRNA induction at 90 mins and 120 mins in the presence of galactose. Finally, the MaSp1-CBD mRNA induction was not detectable at 22 hours in the presence of galactose.

On the fig8, at 0 min timecourse, BY4741 yeast contained BBa\_K4650005 (pGal1,10-MaSp1-CBD) showed no MaSp1-CBD mRNA induction, which indicated the pGal1,10 promoter didn’t trigger the downstream gene’s (MaSp1-CBD) transcription in the absence of galactose. At 30 mins in the presence of galactose, the induction of MaSp1-CBD mRNA was not impressed, however, the maximum increased to above 2-fold MaSp1-CBD mRNA induction at 60 mins, but then it declined to 1.7 fold and 1.5 fold of MaSp1-CBD mRNA induction at 90 mins and 120 mins in the presence of galactose. Finally, the MaSp1-CBD mRNA induction was not detectable at 22 hours in the presence of galactose.

Overall, the data indicated that the pGal1,10 promoter has done its job properly to manipulate the downstream gene, MaSp1-CBD, transcription in the presence of galactose, however, the MaSp1-CBD mRNA induction level was not as drastic as the CBD mRNA induction (Fig 7) even though they had the same inducible promoter, pGal1, 10. One of the reasons our team discussed this occurring was that MaSp1-CBD has become a fusion gene, which might have changed its mRNA transcription timing even though our team did see 2-fold MaSp1-CBD mRNA induction. Next time. Our team might be able to collect more intense time course samples, for example, every 15 minutes, to closely monitor the MaSp1-CBD mRNA induction pattern. Now, our team can use BY4741 yeast containing BBa\_K4650005 (pGal1,10-MaSp1-CBD) as one of our team’s experimental yeast strains.

![pcr1](https://static.igem.wiki/teams/4650/wiki/data-and-poc/6a9b776d-6999-4ac9-a810-56cbb99bd2d0.png)

Fig8: The mRNA induction of MaSp1-CBD showed insignificant change in the presence of galactose at 30 mins, the maximum of 2-fold induction at 60 mins, and then back down to 1.7-1.5 fold induction at 90 and 120 mins, finally, no detectable MaSp1-CBD mRNA induction at 22 hours in the presence of galactose.

#### *RT-qPCR technique showed the maximum mRNA induction of MaSp2-CBD above 2-fold induction in the presence of the galactose at 90 mins*

In order to detect MaSp2-CBD mRNA induction, our team has designed the forward primer at the 5’ end of the MaSp2, and the reverse primer was within the CBD gene, and a common primer set of ACT1 to detect ACT1 mRNA induction as an internal control. On the fig9, at 0 min timecourse, BY4741 yeast contained BBa\_K4650006 (pGal1,10-MaSp2-CBD) showed no significant MaSp2-CBD mRNA induction, which indicated the pGal1,10 promoter didn’t trigger the downstream gene’s (MaSp2-CBD) transcription in the absence of galactose. At 30 mins in the presence of galactose, the reduction of MaSp2-CBD mRNA was negligible, and the Masp2-CBD induction at 60 mins in the presence of galactose was close to the one at 0 mins, however, the maximum increased to above 2-fold of MaSp2-CBD mRNA induction was at 90 mins, but then it declined to 1.7 fold and 1.5 fold of MaSp1-CBD mRNA induction at 120 mins and 22 hours in the presence of galactose.

Overall, the data indicated that the pGal1,10 promoter has manipulated the downstream gene, MaSp2-CBD, transcription in the presence of galactose, however, the MaSp2-CBD mRNA induction pattern was not the same as the MaSp1-CBD mRNA induction (Fig 8) even they had the same inducible promoter, pGal1,10. One of the reasons our team discussed this occurring was that MaSp2-CBD has become a longer fusion gene compared to the one of MaSp1-CBD, which might have changed its mRNA transcription timing even our team did see 2-fold MaSp1-CBD mRNA and MaSp2-CBD mRNA inductions, but the MaSp2-CBD mRNA induction time lasted longer compare the one in MaSp1-CBD. Now, our team can use BY4741 yeast containing BBa\_K4650006 (pGal1,10-MaSp2-CBD) as one of our team’s experimental yeast strains.

However, our team only did one set of data for the RT-qPCR results, which is not sufficient to show the correct mRNA induction. However, our team did show that in the presence of the galactose, pGal1.10 did trigger the downstream genes, CBD, MaSp1-CBD, and MaSp2-CBD, mRNA inductions, and no gene induction occurred without galactose. This important set of RT-qPCR data encouraged our team to continue this project to the next level, coculturing the respective yeasts with SCOBY and doing flexibility and strength tests on the resulting “leather”.

![pcr1](https://static.igem.wiki/teams/4650/wiki/data-and-poc/b3c5920a-7c18-4a92-8b47-9fc169d869ec.png)

Figure 9: The mRNA induction of MaSp2-CBD showed an insignificant reduction in the presence of galactose at 30 mins compared to the 0 min sample, the maximum above 2 fold induction at 90 mins, and then back down to 1.7 fold and 1.3 fold induction at 120 mins and 22 hours in the presence of galactose.

#### *Conclusion*

From the beginning of making basic parts in order to generate composite parts, our team has recorded all of the PCR data to prove that each step of the process was done correctly and also sent out three composite parts to the Mission Biotechnology company for the confirmation of the DNA sequences.

Overall, our team has shown convincing evidence that three composite parts, BBa\_K4650004 contained BBa\_K4650000+BBa\_K4650001 (pGal1,10-CBD); BBa\_K4650005 contained BBa\_K4650000+BBa\_K4650002+BBa\_K4650001 (pGal1,10-MaSp1-CBD); BBa\_K4650006 contained BBa\_K4650000+BBa\_K4650003+ BBa \_ K 4650001(pGal1,10-MaSp2-CBD) have proper function via RT-qPCR. In the presence of galactose, which induced pGal1, 10 promoter’s switch, the downstream genes, CBD, MaSp1-CBD, MaSp2-CBD were transcribed at least 2-fold mRNA inductions of MaSp1-CBD and MaSp2-CBD as our experimental samples, and highly induced at least 20 fold mRNA of CBD as our control sample. However, our team only did one of the RT-qPCR experiments so repeating the RT-qPCR experiment is necessary. However, if only one BY4741 yeast containing one composite part showed mRNA induction, but not the other two, it would still make the data unreliable since only one composite part was induced.

## **Product Testing**

#### *Strength*

To enable our team to test the strength of the modified SCOBY, the team created a strength testing machine to hold the SCOBY and test how much force it can hold. The wooden frame would be a hollow square with two sides attached with a vice. The vice is operated by spinning the handle clockwise to clamp the scoby tight and firm. The force gauge will be used to contact the scoby with a 1.54 cm2 circular area. The machine would record the applied force’s first peak value in newtons as the SCOBY breaks, as described in Experiment.

As shown in figure 10, genetically modified SCOBY with MaSp2-CBD protein gave the highest weighting capacity (258N), and genetically modified SCOBY with MaSp1-CBD protein was the second highest (118.8N). Therefore we can conclude that genetically modified SCOBY that had been co-cultured with MaSp1-CBD, and MaSp2-CBD, respectively, increases its strength and durability.

![pcr1](https://static.igem.wiki/teams/4650/wiki/data-and-poc/26fcec60-c28f-450d-a12f-2ed3472bc369.png)

Fig10: Genetically modified SCOBY with MaSp2-CBD protein showed a significantly higher weighting capacity among all, with a carrying capacity of 258 newtons. Followed by genetically modified SCOBY with MaSp1-CBD protein with 118.8 newtons and genetically modified SCOBY with CBD protein with 105.7 newtons. The SCOBY that was co-cultured with BY4741 only performed the lowest carrying capacity of 52.2 newton.

#### *Flexibility*

Next, in order to assess the flexibility of our SCOBY, our team created a device that consists of a 3D-printed wheel attached to a piece of wood that will be paired with another wood block and a second pair of wood blocks. Once the device was set up, the lower set of wooden blocks allowed the SCOBY membrane to twist as the wheel turned on its path, while the upper set of wooden blocks remained stationary. By measuring the amount of force required for the SCOBY to twist a certain angle, the team can determine how flexible it is. If less force is applied, it means the SCOBY is more flexible, as elaborated on in Experiment. 3 trials were performed.

Fig 11 summarizes our findings. The negative control has an average force of 1.292 N applied for a 40° rotation. With the same degree rotated, pGal-CBD 0.401 N more force than the negative control, indicating that CBD protein is slightly more rigid. MaSp1-CBD protein needs an average force of 1.004 N, which is 0.288 N less than the negative control. Since it requires 22.29% less force, MaSp1-CBD protein is more flexible than the negative control. MaSp2-CBD protein needs an average force of 0.985 N, which is 0.305N less than for the negative control. In addition, the error bars did not overlap, indicating a significant difference. The 23.76% decrease indicates that the MaSp2-CBD SCOBY is more flexible than the unmodified SCOBY, or negative control.

![pcr1](https://static.igem.wiki/teams/4650/wiki/data-and-poc/20c86325-c36d-4fda-8ac7-1cef30b1ce10.png)

Fig 11: The genetically modified SCOBY with MaSp1-CBD protein, and MaSp2-CBD protein, separately, required less strength for 40 degree turn. The SCOBY, co-cultured with BY4741 only, required the most strength of 1.292 newton to make a 40 degree turn, similar to the one with CBD protein only (1.378N), followed by genetically modified SCOBY with MaSp2-CBD protein of 0.985 newton and genetically modified SCOBY with MaSp1-CBD protein of 1 newton.

While preliminary results indicate the spider silk genes, MaSp1 and MaSp2 have achieved the result of a more flexible SCOBY, during the experiment, we noticed many factors that might affect the results. First, the thickness of the membrane directly affects the amount of force needed to twist the SCOBY: thicker SCOBY needs more force. Although we attempted to control this by growing SCOBY for the same amount of time, the thickness still has obvious variations, which may also be caused by how dry the SCOBY actually is. Second, the speed of twisting directly affects the amount of force measured: higher speed will result in higher force measured. We tried to control this by twisting the SCOBY for the same amount of time, which may help keep the speed constant. But still, it is difficult to control it completely, leading to errors in the data. So we also did many trials to minimize the effect the errors bring. Finally, we realized that twisting the SCOBY many times causes the inelastic membranes to deform, so there will be less restoring force pulling the force sensor, leading to a decreased measured force, which may be misinterpreted as higher flexibility. This factor is difficult to control unless we only do one trial on each SCOBY, which is not an ideal solution as it will require a lot of labor to grow so many of them. Therefore, further investigation and trials still need to be done on this experiment to make sure our data is reliable.

## **Conclusion**

To test the outcome of genetically modified SCOBY with different spider silks fused with CBD, the team designed two machines to test the strength and flexibility via different forces. The data in fig10 and 11 clearly show that the strength and flexibility of our genetically modified SCOBY with MaSp1-CBD,and MaSp2-CBD fusion proteins, respectively, were both stronger and more flexible compared to the control SCOBY only or SCOBY with CBD. Overall, our team has shown convincing preliminary evidence that genetically modified SCOBY with MaSp1-CBD and MaSp2-CBD protein can hold significantly stronger strength and are more flexible to generate useful products in the future.

## **Further Extension**

The team noticed some weaknesses in our data. First of all, our team only had time to finish one trial of the RT-qPCR experiment, and even though the data is convincing, showing the downstream genes of the pGal1,10 promoter did induce mRNA transcription in the presence of galactose, repeated RT-qPCR trials will be necessary to validate our team’s biological functional assay’s data. Second, the SCOBY was dried directly inside the oven, resulting in a wrinkled surface which may cause inaccuracy in measuring data. Therefore the team decided to flatten SCOBY by placing a heavy object above it when drying, aiming to completely flatten SCOBY. Third, since we only include 1 batch of tested samples, to increase the validity of our project, the team aims to conduct more batches of testing regarding the modified SCOBY’s flexibility.

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