iGEM 2022 — Team nchu-taichung

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1. Notebook
2. Note

[Timeline](#Overview)
[Labwork Timeline](#Lab)

# Timeline

![](https://static.igem.wiki/teams/4358/wiki/note/timeline.png)

# Labwork Timeline

* Our labwork can be divided into (i.)plasmid transformation group and (ii.)cell experiment group

1. Plasmid transformation group

![](https://static.igem.wiki/teams/4358/wiki/note/transmutation.png)

2. Cell experiment group

![](https://static.igem.wiki/teams/4358/wiki/note/cell.png)

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1. Projects
2. Engineering success

[Electrophoresis](#Overview)
[PCR](#PCR)
[Antibiotics resistance bacteria selection](#Antibiotics)
[Growth curve](#Growth)

# Electrophoresis

After transformation, we extract the PQQ plasmid from EP2 (*Bacillus subtilis* natto PQQ) and amplify the DNA, then run the electrophoresis. The result illustrates the successful transformation based on the band all on the same height level.

![](https://static.igem.wiki/teams/4358/wiki/engineering-success/engineering-success/2022igem-nchu-taichung-engineer-success-1.png)

Figure 1. Electrophoresis of PQQ plasmid.

Comparing the plasmid(BBa\_K3830012, left) stored in *E.coli* JM109 to plasmid extracted from modified

*Bacillus subtilis* natto(right).

# PCR

Also, we run PCR to make multiple copies of a segment of the plasmid, to sequence the gene of PQQ.

![](https://static.igem.wiki/teams/4358/wiki/engineering-success/image3.jpg)

1. *B. subtilis* natto PQQ
2. *E. coli* JM109
3. Positive control (Pure plasmid)

# Antibiotics resistance bacteria selection

As the kanamycin resistant gene in our PQQ plasmid but in the wild-type *Bacillus subtilis* natto, we carry out the plate selection with kanamycin to confirm the transformation is successful.

![](https://static.igem.wiki/teams/4358/wiki/engineering-success/2022igem-nchu-taichung-engineering-success-2.png)

# Growth curve

![](https://static.igem.wiki/teams/4358/wiki/engineering-success/engineering-success/2022igem-nchu-taichung-engineer-success-4.png)

By the growth curve, we could see the differences between the wild-type and the *Bacillus subtilis* natto PQQ. And also, the EP2 (*Bacillus subtilis* natto PQQ), with the PQQ plasmid, grows more fast and more quickly after entering the log phase and stationary phase.

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{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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## What should this page contain?

---

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).

## Inspirations

---

* [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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1. Projects
2. Results

[Overview](#Overview)
[The source of B. subtilis natto](#Source)
[Transfer PQQ plasmid into B. subtilis natto](#Transfer)
[Design PQQ plasmid](#Design)
[PQQ Separation and Purification](#Purification)
[Cell Experiment](#Cell)
[Reference](#Reference)

# Overview

The principal goal of the project is to design a wound repair-promoting dressing for the people who suffering from diabetes mellitus. The first step is to mass-produce PQQ, a compound proven to improve wound repair. To reach the goal , we transferred the PQQ plasmid shown in 2021 NCHU\_Taichung into *Bacillus subtilis* natto. (*B. subtilis* natto PQQ) In order to further improve the production of PQQ, we redesign the PQQ plasmid with a high expressible promoter.

# The source of *B. subtilis* natto

### Isolated *B. subtilis* natto from merchant natto

The *B. subtilis* natto was seperated and purified from merchant natto using LB agar and finally received a single strain for further experiments.

![](https://static.igem.wiki/teams/4358/wiki/result/image10.png)

Figure 1.

### Natto fermentation

To preliminarily identify that *B. subtilis* natto isolated from merchant natto work, we ferment soybeans with the bacteria at 25°C for 24 hours. As the picture shows, soybeans become stringy, just like merchant natto. (Figure2) The metabolic-engineered *B. subtilis* natto are implemented in our following experiment.

![](https://static.igem.wiki/teams/4358/wiki/result/2022igem-nchu-taichung-result-1.jpg)

Figure 2. Soybean fermented with isolated *B. subtilis* natto.

# Transfer PQQ plasmid into *B. subtilis* natto

### Plasmid extraction

On the gel electrophoresis diagram, the first and second columns contain PQQ plasmid as a positive control. The third and the fourth lanes contain PQQ plasmid from *B. subtilis* natto PQQ. As the picture shows below, our positive control and the PQQ plasmid from *B. subtilis* natto PQQ are on the same molecular size. (Figure 3)

![](https://static.igem.wiki/teams/4358/wiki/result/2022igem-nchu-taichung-result-2.png)

Figure 3. (1) Ladder; (2, 3) Positive control; (5, 6) Plasmid extracted from *B. subtilis* natto PQQ.

### Growth Curve

We measure 5 growth curves, *B. subtilis* natto wild-type, *B. subtilis* natto PQQ with and without kanamycin (Kan), *B. subtilis* RM125, and *B. subtilis* RM125 PQQ by culturing in lysogeny broth (LB) and measuring OD600 every hour.

*B. subtilis* RM125 PQQ was from the previous iGEM team, compared with non-treat *B. subtilis* RM125, the growth curve shows a very significant difference with a higher OD value in the end point (Figure 4A).The growth of *B. subtilis* natto PQQ (Ep2) is significantly greater than *B. subtilis* natto wild-type in log and stationary phase. (Figure 4B) It showed that PQQ would promote the growth of *B. subtilis* natto PQQ. The difference between their growth also indicated we successfully transformed PQQ plasmid into *B. subtilis* natto.

As PQQ plasmid is an exogenous plasmid, *B. subtilis* natto PQQ might be exocytosis. The antibiotic resistance gene in PQQ plasmid plays an important role. While there is an antibiotic, kanamycin, in the culture medium, *B. subtilis* natto PQQ wouldn’t lose the plasmid. However, it’s not enough. In the aim of increasing the stability of PQQ plasmid maintained in the cell, we commit to optimizing PQQ plasmid. To verify our achievement, we culture *B. subtilis* natto PQQ with and without kanamycin treatment. Our data illustrates that there is almost no difference between the conditions of adding kanamycin or not. (Figure4 B.) The PQQ plasmid can still be stored stably in *B. subtilis* natto PQQ even no kanamycin in the environment. It represents that our optimization to plasmid with PQQ biosynthetic gene domain is successful.

*B. subtilis* RM125 is the bacteria 2021 NCHU\_Taichung team chose.
We compare the growth of *B. subtilis* RM125 PQQ to *B. subtilis* natto PQQ. In the log phase, *B. subtilis* natto PQQ grows faster and more quickly (Figure4 D.)

![](https://static.igem.wiki/teams/4358/wiki/result/results-figure4.png)

Figure 4. The growth curve of different bacteria strain. Ep2: *Bacillus subtilis* natto PQQ. (A) All the experiments were performed triplicate, and the comparison of the end points OD value was using one-way ANOVA with p < 0.05.

We cultured the *B. subtilis* natto wild-type with and without kanamycin and *B. subtilis* natto PQQ with kanamycin in LB for 6 hours. As the picture shows below, we can see that their turbidity is different. (Figure 4)

![](https://static.igem.wiki/teams/4358/wiki/result/2022igem-nchu-taichung-result-6.jpg)

Figure 5. From left to right: *B. subtilis* natto wild type with kanamycin, *B. subtilis* natto wild type without kanamycin, and *B. subtilis* natto PQQ.

# Design PQQ plasmid

From literature review, various *pqq*A genes were proved to play important roles in PQQ biosynthesis [1]. Hence, we designed a new plasmid with doubled *pqq*A genes to improve the biosynthesis of PQQ. From another literature, *pq*A and *pqq*B genes dominate the biosynthesis of PQQ [2]. In order to express considerable PqqA and PqqB, we design another new plasmid separating *pqq*A and *pqq*B from *pqq*C, D, E by subcloning xylose operon to regulate *pqq*C, D, E expression, which do not need that much. Our test keeps going.

# PQQ Separation and Purification

Supramolecular solvent, SUPRAS in abbreviations, is the method to separate and purify PQQ out of the supernatant of Ep2 culturing. Subsequently, we quantify the concentration of PQQ after SUPRAS extraction by LC/MS, Liquid chromatography/Mass spectrometry. The result is ongoing and will be demonstrated in the presentation.

# Cell Experiment

### Diabetic-like cell induced

The HaCaT cell will be induced using glucose (50 mM) and insulin (5 nM) for at least 5 days. After 5 days of inducing, the cell would harvest for further investigation.

### The viability of diabetic HaCaT cell in different compound treatment

To understand if the PQQ will promote cell proliferation, the CCK-8 assay would evaluate the cell viability. After 48 hours of treatment, the H PQQ group show the most significant change (Figure 6)

![](https://static.igem.wiki/teams/4358/wiki/result/image4.png)

Figure 6. CCK-8 method for detecting the viability of diabetic HaCaT cell in different environments Natto: 50 μg/mLNatto extract; L PQQ: 5 nMPQQ; H PQQ: 50nM PQQ; Mix: 50 μg/mL natto extract and 50nM PQQ

### Wound healing assay (Normal HaCaT cell and Diabetes induced HaCaT cell)

To verify whether the PQQ would enhance wounds healing, we prepared HaCaT cells in two conditions, normal HaCaT cell and diabetic-like HaCaT cell, the induced step of diabetic-like cell was shown in 5.1.

Both cells would seed in a silicone mold until the cell attached to the surface, the mold would remove after a compact monolayer was formed. Cells would be washed using PBS twice, then 1 mL of DMEM containing test samples would be added and cultured in the incubator. The observation of the cell was using a microscope after 24 hours.

From Figure 7 and 8, the recovery rate of diabetic-like cells is seemingly slower compared to normal cells. It is found that the treatment of PQQ can significantly increase the recovery rate, which provides solid evidence that PQQ can accelerate cell growth and shows a great potential in promoting wound healing.

![](https://static.igem.wiki/teams/4358/wiki/result/fig-7.png)

Figure 7. Wound healing assay of normal HaCat cell in control, Natto (natto extraction 50 μg/mL) , PQQ (50 nM) and Mix (natto extraction 50 μg/mL with PQQ 50 nM.

![](https://static.igem.wiki/teams/4358/wiki/result/fig-8.png)

Figure 8. Wound healing assay of diabetic-like induced HaCaT cell in control, Natto (natto extraction 50 μg/mL) , PQQ (50 nM) and Mix (natto extraction 50 μg/mL with PQQ 50 nM.

### q-PCR analysis

To understand the effect of PQQ on HaCaT cells, q-PCR was performed to analyze the mRNA expression level change. PQQ targeting protein SIRT1, PGC-1a, and mitochondrial ribosomal 18S rRNA were chosen as targets, and used GAPDH as internal control. However, the results didn’t show significant change in mRNA expression level, which indicated that PQQ may not affect the cell motility in targeted genes, the detailed mechanisms of PQQ in mammalian cells remained for further investigation.

# References

1. Ge, X., Wang, W., Du, B., Wang, J., Xiong, X., & Zhang, W. (2015). Multiple pqqA genes respond differently to environment and one contributes dominantly to pyrroloquinoline quinone synthesis. *Journal of basic microbiology, 55*(3), 312–323.

   <https://doi.org/10.1002/jobm.201300037>
2. Li, L., Jiao, Z., Hale, L., Wu, W., & Guo, Y. (2014). Disruption of gene pqqA or pqqB reduces plant growth promotion activity and biocontrol of crown gall disease by Rahnella aquatilis HX2. *PloS one, 9*(12), e115010.

   <https://doi.org/10.1371/journal.pone.0115010>

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