iGEM 2021 — Team Gunma

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# Team:Gunma/Notebook

Team:Gunma LABNOTES in Proof Of Concept

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LABNOTES

@Lab.

# LABNOTES in Proof Of Concept

## 1. Crystal violet assay

**Day 1**

1.　The *E. coli* RP437 strain was statically grown at 37 °C overnight in Luria-Bertani (LB) medium\*.

\*Luria-Bertani (LB) medium (per 1 L):

* Yeast extract : 5 g
* Tryptone : 10 g
* NaCl : 10 g

**Day 2**

2. The overnight culture (2 μL) was diluted into each Onsen medium\* (200 μL) in 96 well plates, then statically grown at 37 °C for 24 hours.

\*Onsen medium (per 1 L):

* Original onsen water 950 mL
* 20% Glucose 25 mL
* 20% Casamino acid 25 mL

(For Watanoyu and Bandai media, pH was adjusted to 7.0 because they are highly acidic (pH < 2.5).
Then, filtered.

**Day 3**

The optical density at 595 nm (OD595) was measured to monitor the cell growth.

Biofilm cells were washed five times with water to remove floating cells.

Biofilm cells were stained with 0.1% crystal violet for 10 minutes, and washed five times with water.

The crystal violet was eluted from biofilm cells by adding 95% ethanol.

The absorbance at 595 nm (A595) for each well was measured.

The capability for biofilm formation was determined as the A595 normalized to an OD595 of 1.

## 2. Biofilm 3D imaging

**Day 1**

1. The *E.coli* RP437 strain was statically grown at 37 °C overnight in LB medium.

**Day 2**

2. The RP437 overnight culture (30 μL) was diluted into a medium (3 mL), and inoculated on glass coverslips in a 6 well plate.

3. We grew the bacteria at 37 °C for 24 hours, statically.

**Day 3**

4. The biofilm cells on the coverslips were washed with phosphate buffered saline (PBS)\* to remove the floating cells, and stained with 5 μM SYTO-9 (Life Technologies, Carlsbad, CA, USA) for 10 minutes.

5. The coverslips were washed twice, and fixed with 4% paraformaldehyde in PBS.

6. Fluoromount / Plus (Diagnostic BioSystems, Pleasanton, CA, USA) was mounted on a glass slide.

7. Fluorescent images were acquired in the Alexa Fluor 488 laser unit on an Olympus FV1200 IX81 microscope using a 60 X objective and captured with a charge-coupled-device (CCD) camera.

\*Phosphate buffered saline (per 1 L):

* NaCl : 8 g
* Na2HPO4・12H2O : 2.9 g
* KCl : 0.2 g
* KH2PO4 : 0.2 g
* Adjusted to pH 7.4

## 3. Preparation of bacterial cell lysate

1. Bacteria (DH5 α/pSB1C3 and DH5/α pSB1C3/α-gal-RFP) were aerobically pre-cultured overnight at 37 °C in 3 mL of LB medium.
2. The bacterial culture was inoculated into a fresh LB containing 0.2 mM IPTG, then aerobically grown at 25 °C for 24 hours in 5 mL of LB.
3. Bacterial cells were harvested and re-suspended in 800 μL of 50 mM Tris-HCl (pH 7.5).
4. The suspension was sonicated by ultrasound, divided into three 20 second intervals, and centrifuged at 20,000 g for 5 minutes at 4 °C.
5. The resulting supernatant was collected into a clean tube.
6. Proteins in the supernatant were quantified in a Bio-Rad protein assay according to the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA).

## 4. α-Galactosidase assay

1. The reaction was set up in a tube as follows:

* The recombinant α-galactosidase (6.78 mg/mL)　 2 μL
* 100 mM Dithiothreitol (DTT) 50 μL
* 100 mM MnCl2 50 μL
* 4 mg/mL \*Oxidized NAD+ 25 μL
* 50 mM Tris-HCl (pH 7.5) 873 μL
* 4 mg/mL \*PNPG substrate 200 μL

(\* Oxidized nicotinamide adenine dinucleotide [NAD+])
(\* p-Nitrophenyl α-D-Galactopyranoside [PNPG])

The reaction was incubated at 37 °C, 42 °C, 50 °C and 65 °C.
The enzyme reactions were monitored at 2 minutes intervals by measuring the absorbance at 420 nm.

## 5. Inhibition test of biofilm formation using the recombinant enzyme

**Day 1**

1. The *E.coli* RP437 strain was statically grown at 37 °C overnight in LB medium.

### For Crystal violet assay:

**Day2**

2. The overnight culture (2 μL) was diluted into a fresh Ho-shi onsen medium (200 μL) , and statically grown at 37 °C for 24 hours.

**Day3**

3. The biofilm cells in a well were washed with phosphate buffered saline (PBS) to remove the floating cells. Hoshi onsen water containing 5 mM DTT, 5 mM MnCl, and 100 μg/mL oxidized NAD (NAD+) with and without α-Galactosidase (67.8 μg to 678 μg) was added into the biofilm cells, then we incubated it at 37 °C for 24 hours.

Phosphate buffered saline (per 1 L):

* NaCl : 8 g
* Na2HPO4・12H2O : 2.9 g
* KCl : 0.2 g
* KH2PO4 : 0.2 g
* Adjusted to pH 7.4

**Day 4**

4. Biofilm mass in bacterial cultures was measured by the crystal violet assay (as described in section 1)

### For Biofilm 3D imaging:

**Day2**

2. The overnight culture (2 μL) was diluted into a fresh Ho-shi onsen medium (200 μL) , and statically grown at 37 °C for 24 hours.

**Day3**

3. The biofilm cells in a well were washed with PBS to remove the floating cells.

4. Hoshi onsen water containing 5 mM DTT, 5mM MnCl, and 100 μg/mL oxidized NAD (NAD+) with and without α-Galactosidase (67.8 μg to 678 μg) was added into the biofilm cells, then we incubated it at 37 °C for 24 hours.

**Day 4**

5. 3D images of the biofilm were acquired as described in section 2.

## 7. Degradation of biofilm using the bioengineered “suicide biofilm breaker” that overproduces α-galactosidase and along with *SacB*.

### **(a) Preparation for the biofilm breaker strain**

**Day 1**

1. SacB / RFP-fused α-galactosidase-expressing SM10λ (carrying pABB-CRS2/sacB and pSB1C3/α-gal-RFP) and its control strain (SacB / RFP-expressing SM10λ: carrying pABB-CRS2/sacB and pSB1C3/RFP) were aerobically grown at 37 °C overnight in LB medium.

**Day 2**

2. The cultures were inoculated into 5mL LB media, and aerobically cultured in the presence of 0.2 mM IPTG (to induce α-galactosidase production) at 25 °C for 24 hours.

**Day 3**

3. Bacterial cells were harvested and re-suspended in 800 μL of 50 mM Tris-HCl (pH 7.5).

4. The resuspensions were incubated in Hoshi onsen water containing 5 mM DTT, 5 mM MnCl2, and 100 mg/mL oxidized NAD (NAD+) and 30% sucrose (to induce cell lysis) at 25 °C for 2 hours.

### **(b) Biofilm assay**

**Day 1**

5. The *E. coli* RP437 strain was statically cultured at 37 °C overnight in LB medium.

**Day 2**

6. The overnight culture was diluted into a fresh Ho-shi onsen medium, and statically grown at 37 °C for 24 hours.

**Day 3**

7. The biofilm cells were washed with PBS to remove the floating cells.

8. The biofilm breaker and its control strain were added, and incubated for another 24 hours in Hoshi onsen water containing 5 mM DTT, 5 mM MnCl2, and 100 mg/mL oxidized NAD (NAD+) and 30% sucrose.

**Day 4**

9. Degradation of biofilm was estimated by crystal violet and 3D imaging assays (as described in section 1 and 2).

# LABNOTES in Engineering Success

## Expression of α-galactosidase-RFP (from *E.coli*)

We cloned the gene of α-galactosidase-RFP from *E.coli* into the pSB1C3 plasmid. This expression plasmid was transformed using DH5 α, and the transformant was cultured in liquid medium containing chloramphenicol. After culturing the transformant at 37 °C (130 rpm) for 16 hours, the cells were inoculated into a new medium and liquid-cultured until the logarithmic growth phase. IPTG was added to a final concentration of 0.2 mM, and the *E.coli* was cultured at 25 °C (130 rpm) for 20 hours. Cultured cells were sonicated with sonication buffer (phosphate buffer (pH 7.0) + 150 mM NaCl + 10% glycerol) and SDS-PAGE was performed.

The expression of α-Gal-RFP was verified by SDS-PAGE (Figure 1). A competent cell culture medium was used as a control or the wild type (WT).
Our target protein is about 76 kDa in molecular mass. In Figure 2, we observed that there was a thicker band in the vicinity of a band at 72 kDa than that of WT. Thus, we consider that this band represents α-Gal-RFP.

![](https://static.igem.org/mediawiki/2021/1/1f/T--Gunma--SDSPAGE_a-galRFP.png)

**Figure 1.** SDS-PAGE gel for quantification of α-galactosidase-RFP. M, molecular mass markers; WT, wild type; α-Gal-RFP, α-galactosidase-RFP from *E.coli* and coral; S, supernatant; P, pellet.

## Expression of α-galactosidase (from **Thermus thermophilus**)

We cloned the gene of α-galactosidase from  **Thermus thermophilus** into the pET28a plasmid. This expression plasmid was transformed using BL21(DE3), and the transformant was cultured in liquid medium containing kanamycin. After culturing the transformant at 37 °C (130 rpm) for 16 hours, the cells were inoculated into a new medium and liquid-cultured until the logarithmic growth phase. IPTG was added to a final concentration of 0.2 mM, and the *E.coli* was cultured at 25 °C (130 rpm) for 20 hours. Cultured cells were sonicated with sonication buffer (phosphate buffer (pH 7.0) + 150 mM NaCl + 10% glycerol) and SDS-PAGE was performed.

The expression of α-Gal was verified by SDS-PAGE (Figure 2). A competent cell culture medium was used as a control or the wild type (WT).
Our target protein is about 49 kDa in molecular mass. In Figure 2, we observed that there was a thicker band in the vicinity of a band at 52 kDa than that of WT. Thus, we consider that this band represents α-Gal.

![](https://static.igem.org/mediawiki/2021/e/e2/T--Gunma--SDSPAGE_a-gal_T.t.png)

**Figure 2.** SDS-PAGE gel for quantification of α-galactosidase. M, molecular mass markers; WT, wild type; α-Gal (T.t), α-galactosidase from  **Thermus thermophilus**; P, pellet; S, supernatant.

## Protein Dialysis\_ α-Gal-RFP

To dialyze α-Gal-RFP, the protein was put in a dialysis tube (MWCO.50,000, SPECTRUM). The dialysis tube was immersed in dialysis buffer and stirred at 4 °C. The buffer was changed, as below. The composition of the dialysis buffer is as follows.

### Dialysis buffer:

(1)

* 25 mM HEPES-KOH (pH 7.6)
* 500 mM KCl
* 2 mM EDTA
* 1 mM DTT
* 10% (w/v) glycerine

(2)

* 25 mM HEPES-KOH (pH 7.6)
* 500 mM KCl
* 2 mM EDTA
* 150 mM NaCl
* 5 mM MgCl2・6H2O

### Exchanging dialysis buffer:

![](https://static.igem.org/mediawiki/2021/1/1d/T--Gunma--labnote_PS_a-galRFP.png)

### Protein concentration:

67.8 mg/mL

![](https://static.igem.org/mediawiki/2021/a/ae/T--Gunma--Protein_Dialysis_a-galRFP.png)

**Figure 3.** SDS-PAGE gel for quantification of dialyzed α-galactosidase-RFP. M, molecular mass markers; α-Gal-RFP, α-galactosidase-RFP from *E.coli* and coral after dialyzing; S, supernatant.

## Protein Dialysis\_ α-Gal from T.t

To dialyze α-Gal from *Thermus thermophilus*, the protein was put in a dialysis tube (MWCO.10,000, SPECTRUM). The dialysis tube was immersed in dialysis buffer and stirred at 4 °C. The buffer was changed, as below. The composition of the dialysis buffer is as follows.

### Dialysis buffer:

(1)

* 25 mM HEPES-KOH (pH 7.6)
* 500 mM KCl
* 2 mM EDTA
* 1 mM DTT
* 10% (w/v) glycerine

(2)

* 25 mM HEPES-KOH (pH 7.6)
* 500 mM KCl
* 2 mM EDTA
* 150 mM NaCl
* 5 mM MgCl2・6H2O

### Exchanging dialysis buffer:

![](https://static.igem.org/mediawiki/2021/1/16/T--Gunma--labnote_PS_a-galTT.png)

### Protein concentration:

65.36 mg/mL

![](https://static.igem.org/mediawiki/2021/3/35/T--Gunma--Protein_Dialysis_a-gal%28T.t%29.png)

**Figure 4.**SDS-PAGE gel for quantification of dialyzed α-galactosidase. M, molecular mass markers; α-Gal (T.t), α-galactosidase from *Thermus thermophilus* after dialyzing; S, supernatant.

## Colonies of cells harbouring the plasmid expressing α-galactosidase-RFP (from *E.coli*) on agar plates

*E.coli* DH5 α strain was transformed with pSB1C3/ α-galactosidase-RFP onto LB agar plates containing chloramphenicol, which was incubated for 20h at 37 °C until becoming colonies. Moreover, we continued incubating the plates for 24 h at 37 °C until colonies were red.

In Figure 5, we observed both red and white colonies. This fused protein contains RFP, We found that red colonies are usually smaller than white ones.

![](https://static.igem.org/mediawiki/2021/thumb/9/98/T--Gunma--red_and_white_a-gal.png/631px-T--Gunma--red_and_white_a-gal.png.jpeg)

**Figure 5.** Expression of α-galactosidase-RFP in *E.coli* DH5 α strain.

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# Team:Gunma/Engineering

<!DOCTYPE html>

Team:Gunma Engineering Success

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![](https://static.igem.org/mediawiki/2021/a/a3/T--Gunma--Kusatuon2.jpg)

Engineering Success

@Kusatsu

# Overview

What is important and necessary for degradation of biofilms observed in bathtub surfaces and pipes in hot spring facilities?

In this project, we focused on the three points:

* Enzyme for efficient degradation of biofilms
* Bio-carrier of the enzyme to the target biofilm
* Thermal stability of the enzymes

Accordingly, this section is composed of three parts.

# PART-1: Enzyme for efficient degradation of biofilms

## Design

Hot spring facilities all over the word have various problems caused by biofilms. One of the major problems in the facilities is that biofilms protect infectious microorganisms from disinfectants and antimicrobials. Biofilms are communities of microorganisms that irreversibly stick to non-biological surfaces and grow on the surfaces, produce extracellular polymers (mostly of exopolysaccharides). What is more important is that exopolysaccharides facilitate attachment and matrix formation of biofilms We think that degradation of exopolysaccharides on biofilms by a glycolytic enzyme would damages robust biform formation, and thus disinfectants kill microorganisms efficiently.

### Preliminary experiments

To determine which enzyme is more effective for degradation, we performed experiments of biofilm degradation (as described in detail in "Proof of Concept" of the "Gold" section) using five enzymes that were purchased from Sigma-Aldrich and Nacalai Tesque.

The findings showed that the degradation efficiency in descending order is as follows:

Proteinase K > α-galactosidase from green coffee beans > DNase >> β-glucosidase from sweet almonds

![](https://static.igem.org/mediawiki/2021/thumb/e/e4/T--Gunma--engineering_bfgraph.png/1200px-T--Gunma--engineering_bfgraph.png)

Fig. 1

First, we selected a glycolytic enzyme, α-galactosidase, as a strong candidate for degradation enzymes. We designed an expression vector for α-galactosidase, as below. Note that proteinase K cannot be selected because overexpression of the enzyme is toxic to *E.coli*.

* A protein expression vector: Plasmid pSB1C3
* A glycolytic enzyme: α-galactosidase (α-Gal) (E.C. 3.2.1.22) from *E.coli*
* A fluorescent protein fused to the enzyme: Red Fluorescent Protein (RFP) for not only visualization of protein expression but also conformation of binding of the RFP-fused enzyme to biofilms.

Hereafter, we termed the α-galactosidase recombinant protein "α-gal-RFP".

## Build

The DNA encoding the gene of α-galactosidase was amplified by PCR from *E.coli* genomic DNA. This DNA fragment was cloned into the expression vector pSB1C3 using an In-Fusion cloning kit (Takara). This plasmid was transformed into *E.coli* DH5α or BL21(DE3).

![](https://static.igem.org/mediawiki/2021/e/e0/T--Gunma--a-galRFP.png)

Fig. 2

RFP overexpressed in *E.coli* made the cultures light up in red. The *E.coli* cells were visualized using a fluorescence microscope (×100 magnification), showing that the cells were red.

![](https://static.igem.org/mediawiki/2021/3/3e/T--Gunma--experimental_figures.jpg)

Fig. 3

Protein Expression from the vector was confirmed by both color of the transformant and SDS-PAGE, which showed that α-gal-RFP was expressed in *E.coli*. Additionally, most of the expressed protein was soluble in vitro.

![](https://static.igem.org/mediawiki/2021/thumb/5/53/T--Gunma--engineering_SDS76.png/788px-T--Gunma--engineering_SDS76.png)

Fig. 4

## Test

To examine whether α-gal-RFP has an enzymatic activity in vitro, we measured degradation of PNPG that was used as a substrate of α-galactosidase. PNPG (4-Nitrophenyl-β-D-glucopyranoside) is a chromogenic β-D-glucosidase substrate, producing a yellow solution on cleavage.

These results confirmed that a partially purified fraction of α-gal-RFP exhibited an α-gal activity level equal to original α-gal. It is noteworthy that the activity was observed even at 65 °C, although the activity level was about 40% of that at 37°C.

![](https://static.igem.org/mediawiki/2021/6/6e/T--Gunma--a-galRFP_activity.png)

Fig. 5

## Learn

Although we succeeded in expression of α-gal-RFP in the *E.coli*, it was not clear how much time it took to obtain as large an amount of the expressed protein as possible per a given volume of the culture media. Thus, we did experiments to determine when we should finish incubating and collect the *E.coli* strain most appropriately.

The experiments and the results are described in "Contribution" in the BRONZE section.

# PART-2: Bio-carrier of the enzyme to the target biofilm

## Design

If the fractions of the enzyme are poured into bathtubs in hot spring facilities, large amounts of the enzyme will be required for efficient degradation of biofilms. Addtionally, it would cost money to obtain large amounts of the enzyme even if they are partially purified. For these reasons, we got interested in engineering a “bio-carrier” of the enzyme which targets biofilm. What is more important is that bacteriolysis is caused in *E.coli* when we desire. We applied conditional suicide system of *E.coli*. This system uses a suicide vector that produces a protein causing cell lysis, which is induced by addition of sucrose. Note, however, that a ready-made plasmid cannot be used because the ori type of pACYC177 is the same as that of pSB1C3 (See Notes for details)

* A protein expression vector: Plasmid pACYC177
* SacB from Bacillus subtilis

*Note:*

1. Because two kinds of plasmids are transformed into an *E.coli* strain, the origin of replication (ori) differs in kind between the plamids. Thus, we selected pACYC177 (ori, p15A).
2. The ori type of pABB-CRS2 and its control system is specific to *E.coli* SM10 λ, so that the plasmid cannot increase in the other strains.

## Build

The methods are essentially the same as those in Part-1. The SacB gene from plasmid pABB-CRS2 (lab-stock) was cloned into pACYC177, as below.

![](https://static.igem.org/mediawiki/2021/c/c3/T--Gunma--engineering_SacB.png)

Fig. 6

## Test

pACYC177/sacB was transformed into the DH5α strain already harboring pSB1C3/α-gal-RFP. The resultant transformant harboring two kinds of plasmids was grown in LB media until OD600 between 0.8-1.0 in a shaking incubator (37 °C). After sucrose at final concentrations of 30% was added into the culture, the transformant was grown for 15 hours.

We visually conformed apparent low turbidity and precipitates in the 50 mL tube, indicating lysis of the transformant. Moreover, the liquid and precipitates in the tube were reddish, showing expression of α-gal-RFP.

![](https://static.igem.org/mediawiki/2021/5/51/T--Gunma--sac30%25-sashikae.png)

Fig. 7

## Learn

Needless to say, in terms of cost and working hours, the smaller amount, the better; the shorter time, the better.

We examine amounts of sucrose and shaking time of the culture that are required for efficient lysis.

These results showed that concentrations of 15% and culture time of 3 hours, at least, are required for efficient lysis.

![](https://static.igem.org/mediawiki/2021/thumb/7/70/T--Gunma--engneering--final-sucrose.png/800px-T--Gunma--engneering--final-sucrose.png)

Fig. 8

# Part-3: Thermal stability of the enzymes

## Design

The temperature of the water in bathtubs is about 42 °C. The temperature of waste water is sometimes even more than 42 °C. In some hot spring facilities, the spring source temperature is around 90 °C. It is desirable for the enzyme used to possess thermal stability and catalytic activity at high temperatures. Therefore, we tried to use enzymes from an extremely thermophilus bacterium, *Thermus thermophiles* HB8. The bacterium was discovered from a hot spring on the Izu-Peninsula in Japan, and is viable even in the high temperature environment of 85 °C.

## Cycles of Build-Test-Learn

Two plasmids (pET11a) harboring α-galactosidase or β-glucosidase from *T. thermophiles* was provided by the RIKEN BRC through the National BioResource Project of the MEXT/AMED, Japan. Each plasmid with a T7 promoter was transformed into BL21(DE3). Unexpectedly, each enzyme was expressed in the *E.coli* strain; however, both were largely insoluble. Although we changed temperatures as a culture condition, or concentrations of IPTG as an expression inducer, they were not solubilized.

![](https://static.igem.org/mediawiki/2021/b/bf/T--Gunma--engineering_ttplasmid.png)

Fig. 9

![](https://static.igem.org/mediawiki/2021/thumb/1/1b/T--Gunma--engineering_SDS49.png/926px-T--Gunma--engineering_SDS49.png)

Fig. 10

We looked out papers about the two enzymes. As a result, we found that there was a possibility that the α-galactosidase recombinant protein in which a His-tag was added to the N terminus was soluble. The gene from pET11a was cloned into plasmid pET28a and the resultant plasmid was transformed into BL21(DE3). The results showed that the recombinant protein was partially soluble.

Finally, we examined whether the recombinant protein had an activity using the same method as described in the "Part-1 Test" section. The solution containing PNPG became yellow after addition of the recombinant protein. Hence, we concluded that the His-tagged enzyme has the α-galactosidase activity level.

Hereafter, the recombinant protein from *T. thermophilus* is termed α-Gal (T.t.).

![](https://static.igem.org/mediawiki/2021/5/5a/T--Gunma--T.t_activity.png)

Fig. 11

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# Team:Gunma/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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# Team:Gunma/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)