iGEM 2021 — Team NCHU_Taichung

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NCHU Taichung/Notebook

[![team-logo](https://static.igem.org/mediawiki/2021/c/ce/T--NCHU_Taichung--LOGO_NEW.png)
NCHU\_Taichung](https://2021.igem.org/Team%3ANCHU_Taichung)

* Project
  + [Description](https://2021.igem.org/Team%3ANCHU_Taichung/Description)
  + [Implementation](https://2021.igem.org/Team%3ANCHU_Taichung/Implementation)
  + [Design](https://2021.igem.org/Team%3ANCHU_Taichung/Design)
  + [Endophyte](https://2021.igem.org/Team%3ANCHU_Taichung/Endophyte)
  + [Model](https://2021.igem.org/Team%3ANCHU_Taichung/Model)
  + [Results](https://2021.igem.org/Team%3ANCHU_Taichung/Result)
  + [Engineering success](https://2021.igem.org/Team%3ANCHU_Taichung/Engineering)
  + [Proof of
    Concept](https://2021.igem.org/Team%3ANCHU_Taichung/Proof_Of_Concept)
* Parts
  + [Basic
    Part](https://2021.igem.org/Team%3ANCHU_Taichung/Basic_Part)
  + [Composite
    Part](https://2021.igem.org/Team%3ANCHU_Taichung/Composite_Part)
  + [Contribution&Improvement](https://2021.igem.org/Team%3ANCHU_Taichung/Contribution)
* Notebook
  + [Note](https://2021.igem.org/Team%3ANCHU_Taichung/Note)
  + [Protocol](https://2021.igem.org/Team%3ANCHU_Taichung/Protocol)
  + [Safety](https://2021.igem.org/Team%3ANCHU_Taichung/Safety)
* Human Practices
  + [Integrated
    Human Practices](https://2021.igem.org/Team%3ANCHU_Taichung/Human_Practices)
  + [Education
    and Communication](https://2021.igem.org/Team%3ANCHU_Taichung/Communication)
  + [Collaboration](https://2021.igem.org/Team%3ANCHU_Taichung/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3ANCHU_Taichung/Partnership)
  + [Sustainable](https://2021.igem.org/Team%3ANCHU_Taichung/Sustainable)
* Team
  + [Members](https://2021.igem.org/Team%3ANCHU_Taichung/Members)
  + [Attributions](https://2021.igem.org/Team%3ANCHU_Taichung/Attributions)
* [Criteria](https://2021.igem.org/Team%3ANCHU_Taichung/Criteria)

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NCHU Taichung/Engineering

[![team-logo](https://static.igem.org/mediawiki/2021/c/ce/T--NCHU_Taichung--LOGO_NEW.png)
NCHU\_Taichung](https://2021.igem.org/Team%3ANCHU_Taichung)

* Project
  + [Description](https://2021.igem.org/Team%3ANCHU_Taichung/Description)
  + [Implementation](https://2021.igem.org/Team%3ANCHU_Taichung/Implementation)
  + [Design](https://2021.igem.org/Team%3ANCHU_Taichung/Design)
  + [Endophyte](https://2021.igem.org/Team%3ANCHU_Taichung/Endophyte)
  + [Model](https://2021.igem.org/Team%3ANCHU_Taichung/Model)
  + [Results](https://2021.igem.org/Team%3ANCHU_Taichung/Result)
  + [Engineering success](https://2021.igem.org/Team%3ANCHU_Taichung/Engineering)
  + [Proof of
    Concept](https://2021.igem.org/Team%3ANCHU_Taichung/Proof_Of_Concept)
* Parts
  + [Basic
    Part](https://2021.igem.org/Team%3ANCHU_Taichung/Basic_Part)
  + [Composite
    Part](https://2021.igem.org/Team%3ANCHU_Taichung/Composite_Part)
  + [Contribution&Improvement](https://2021.igem.org/Team%3ANCHU_Taichung/Contribution)
* Notebook
  + [Note](https://2021.igem.org/Team%3ANCHU_Taichung/Note)
  + [Protocol](https://2021.igem.org/Team%3ANCHU_Taichung/Protocol)
  + [Safety](https://2021.igem.org/Team%3ANCHU_Taichung/Safety)
* Human Practices
  + [Integrated
    Human Practices](https://2021.igem.org/Team%3ANCHU_Taichung/Human_Practices)
  + [Education
    and Communication](https://2021.igem.org/Team%3ANCHU_Taichung/Communication)
  + [Collaboration](https://2021.igem.org/Team%3ANCHU_Taichung/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3ANCHU_Taichung/Partnership)
  + [Sustainable](https://2021.igem.org/Team%3ANCHU_Taichung/Sustainable)
* Team
  + [Members](https://2021.igem.org/Team%3ANCHU_Taichung/Members)
  + [Attributions](https://2021.igem.org/Team%3ANCHU_Taichung/Attributions)
* [Criteria](https://2021.igem.org/Team%3ANCHU_Taichung/Criteria)

![](https://static.igem.org/mediawiki/2021/3/31/T--NCHU_Taichung--Engineering_Success.png)

1. Project
2. Engineering success

[Engineering success](#header)

[Plasmid Extraction and PCR](#item-2-1)
[Growth Curve](#item-2-2)
[Gene Expression On *Bacillus subtilis*
RM125/PQQ](#item-2-3)

# Engineering success

Design

Our PQQ plasmid consist of five genes: *pqqA*, *pqqB*, *pqqC*, *pqqD*,  *pqqE*.These are necessary for PQQ synthesis.

Built

We modify pet28a plasmid by adding an f1 ori and replacing the T7 promoter with the PR promoter, so that the PQQ gene can be expressed in Bacillus subtilis.Then we test the gene express by the following experiments.

![](https://static.igem.org/mediawiki/2021/8/82/T--NCHU_Taichung--engineering_fig01.gif)

## Plasmid Extraction and PCR

We conducted plasmid extraction and PCR to verify that the PQQ plasmid was successfully transferred
into *Bacillus subtilis* RM125 (Figure 5).

![](https://static.igem.org/mediawiki/2021/4/41/T--NCHU_Taichung--5new.png)
Figure 5 PQQ plasmid extraction and PCR.

## Growth Curve

The OD600 value of *Bacillus subtilis* with PQQ plasmid is higher than wild type
*Bacillus subtilis* in log
phase and
stationary phase. PQQ can improve the efficiency of nutrient utilization of *Bacillus
subtilis*(Figure 6).

![](https://static.igem.org/mediawiki/2021/5/52/T--NCHU_Taichung--result_fig07.png)
Figure 6 The OD600 value of*Bacillus subtilis* with PQQ
plasmid is
higher.

## Gene Expression On *Bacillus subtilis* RM125/PQQ

### Symbiosis assay of *Arabidopsis*

Comparing with *Arabidopsis* which didn’t be inoculated (CK), the leaves area of
*Arabidopsis* thaliana
inoculated *Bacillus subtilis* RM125 with PQQ Plasmid (RM125/PQQ) increased by 1.5 times and
capsule
number raised by 2 times. Leaves NO. and stalk height also grew significantly. *Bacillus
subtilis*
RM125 with PQQ Plasmid can improve both vegetative and reproductive growth of *Arabidopsis*
(Figure 7B and 7C).

In addition, the value of leaf number, leaves area, stalk height, and capsule of *Arabidopsis*
inoculated with our engineered bacteria *Bacillus subtilis* RM125/PQQ (RM125/PQQ) is higher
than the
*Arabidopsis* inoculated with *Bacillus subtilis* RM125 (RM125), and also more homogenous
(Figure
7E and 7F).

![](https://static.igem.org/mediawiki/2021/a/a0/T--NCHU_Taichung--result_fig08-1.png)
Figure 7-1 Artificial endophyte *Bacillus subtilis* RM125/PQQ promoted
*Arabidopsis*
growth.
![](https://static.igem.org/mediawiki/2021/6/6d/T--NCHU_Taichung--Figure_8.2.png)
Figure 7-2 Artificial endophyte *Bacillus subtilis* RM125/PQQ promoted
*Arabidopsis*
growth.

### Symbiosis assay of *Oryza sativa*

*Oryza sativa* seeds were dehulled and sterilized, and constantly shaked in water under
darkness for one day
then sewed in Murashige & Skoog medium. Thirteen days after inoculation, we found that the value of
leaves width, leaves length, shoot height of seedling inoculated *Bacillus subtilis* RM125 with
PQQ
plasmid (RM125/PQQ) was higher than the seedling inoculated with *Bacillus subtilis* RM125
(RM125), and the
fresh weight was doubled (Figure 8).

![](https://static.igem.org/mediawiki/2021/1/1d/T--NCHU_Taichung--result_fig09.png)
Figure 8 Artificial endophyte *Bacillus subtilis* RM125/PQQ promoted
*Oryza sativa*
growth.

In test on *Arabidopsis* and *Oryza sativa*, we got the same result: *Bacillus
subtilis* RM125 inoculated with PQQ plasmid
can effectively improve plant growth, and the PQQ gene may be the key factor.

### Endophyte Re-isolation

**Re-isolate *Bacillus subtilis* RM125/PQQ**

The persistence and effectiveness of the end product might be affected by the success of in planta
endosymbiotic *Bacillus subtilis* RM125/PQQ. Thus, we re-isolated the strain after inoculation
to check the endosymbiosis of the artificial endophyte, which may be the most intuitive way for the
evaluation. For the *Bacillus* species, selective medium and 16S rDNA amplicon sequencing
checks were used; for PQQ biosynthesis genes, plasmid size, PQQ biosynthesis genes size, and the
follow-up Sanger-sequencing of these genes checks were performed (Figure 9 & 10). Taken all
together, our results directly indicate the success of *Bacillus subtilis* RM125/PQQ in planta
endosymbiosis.

![](https://static.igem.org/mediawiki/2021/2/23/T--NCHU_Taichung--result_pic01.png)
Picture 1. (A)*Bacillus subtilis* RM125 re-isolated from
*Arabidopsis*.
(B)Plant surface sterilization was successful.(Pic B)
![](https://static.igem.org/mediawiki/2021/b/b0/T--NCHU_Taichung--result_pic02.png)
Picture 2. Artificial endophyte.(A)*Bacillus subtilis* RM125/PQQ
re-isolated from
*Arabidopsis*.(B)*Bacillus subtilis* RM125/PQQ re-isolated from *Oryza sativa*.

![](https://static.igem.org/mediawiki/2021/a/ae/T--NCHU_Taichung--result_fig10.png)
Figure 9 Plasmid extraction of *Bacillus subtilis* RM125/PQQ
re-isolate from
plant.(Plasmid didn’t cut by restriction enzyme,so the circular DNA will locate above
10000bp)
![](https://static.igem.org/mediawiki/2021/7/77/T--NCHU_Taichung--result_fig11.png)
Figure 10 PCR result of *Bacillus subtilis* RM125/PQQ re-isolate from
plant

### Inoculate Supernatant of *Bacillus subtilis* RM125/PQQ

Verification of our assumption that PQQ was secreted off to the supernatant needed to be reassured.
We decided to adjust the easiest way, observing the difference after the inoculation by
supernatants. Supernatants from *Bacillus subtilis* RM125 with PQQ plasmid(RM125/PQQ) and
*Bacillus subtilis*(RM125).

In this experiment, we pelleted down the cell and filtered the supernatant. The dilution fold of the
supernatant is equal to the value of OD600 value because assumption was made that the amount of
metabolites is proportional to the OD600 value.

Results showed that the supernatant of *Bacillus subtilis* improved the vegetative and
reproductive growth of
*Arabidopsis*, and the vegetative growth of *Arabidopsis* inoculated with our engineered
*Bacillus subtilis*(RM125/PQQ) was slightly higher than *Arabidopsis* inoculated with
*Bacillus subtilis* RM125
(Figure 11).

![](https://static.igem.org/mediawiki/2021/f/ff/T--NCHU_Taichung--result_fig12-1.png)
Figure 11-1 Supernatant of *Bacillus subtilis* subtilis RM125/PQQ
slightly improved
the growth of *Arabidopsis*.
![](https://static.igem.org/mediawiki/2021/7/7d/T--NCHU_Taichung--result_fig12-2.png)
Figure 11-2 Supernatant of *Bacillus subtilis* RM125/PQQ slightly
improved
the growth of *Arabidopsis*.

* nchutaichung2021@gmail.com
* NCHU iGEM Team
* igem\_nchu\_taichung
* NCHU\_Taichung
* NCHU, Taichung, Taiwan

![](https://static.igem.org/mediawiki/2021/0/0e/T--NCHU_Taichung--Attribution_fig02.png)

![](https://static.igem.org/mediawiki/2021/4/4b/T--NCHU_Taichung--Attribution_fig04.png)

![](https://static.igem.org/mediawiki/2021/c/ce/T--NCHU_Taichung--LOGO_NEW.png)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NCHU Taichung/Experiments

[![team-logo](https://static.igem.org/mediawiki/2021/c/ce/T--NCHU_Taichung--LOGO_NEW.png)
NCHU\_Taichung](https://2021.igem.org/Team%3ANCHU_Taichung)

* Project
  + [Description](https://2021.igem.org/Team%3ANCHU_Taichung/Description)
  + [Implementation](https://2021.igem.org/Team%3ANCHU_Taichung/Implementation)
  + [Design](https://2021.igem.org/Team%3ANCHU_Taichung/Design)
  + [Endophyte](https://2021.igem.org/Team%3ANCHU_Taichung/Endophyte)
  + [Model](https://2021.igem.org/Team%3ANCHU_Taichung/Model)
  + [Results](https://2021.igem.org/Team%3ANCHU_Taichung/Result)
  + [Engineering success](https://2021.igem.org/Team%3ANCHU_Taichung/Engineering)
  + [Proof of
    Concept](https://2021.igem.org/Team%3ANCHU_Taichung/Proof_Of_Concept)
* Parts
  + [Basic
    Part](https://2021.igem.org/Team%3ANCHU_Taichung/Basic_Part)
  + [Composite
    Part](https://2021.igem.org/Team%3ANCHU_Taichung/Composite_Part)
  + [Contribution&Improvement](https://2021.igem.org/Team%3ANCHU_Taichung/Contribution)
* Notebook
  + [Note](https://2021.igem.org/Team%3ANCHU_Taichung/Note)
  + [Protocol](https://2021.igem.org/Team%3ANCHU_Taichung/Protocol)
  + [Safety](https://2021.igem.org/Team%3ANCHU_Taichung/Safety)
* Human Practices
  + [Integrated
    Human Practices](https://2021.igem.org/Team%3ANCHU_Taichung/Human_Practices)
  + [Education
    and Communication](https://2021.igem.org/Team%3ANCHU_Taichung/Communication)
  + [Collaboration](https://2021.igem.org/Team%3ANCHU_Taichung/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3ANCHU_Taichung/Partnership)
  + [Sustainable](https://2021.igem.org/Team%3ANCHU_Taichung/Sustainable)
* Team
  + [Members](https://2021.igem.org/Team%3ANCHU_Taichung/Members)
  + [Attributions](https://2021.igem.org/Team%3ANCHU_Taichung/Attributions)
* [Criteria](https://2021.igem.org/Team%3ANCHU_Taichung/Criteria)

# 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)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NCHU Taichung/Results

[![team-logo](https://static.igem.org/mediawiki/2021/c/ce/T--NCHU_Taichung--LOGO_NEW.png)
NCHU\_Taichung](https://2021.igem.org/Team%3ANCHU_Taichung)

* Project
  + [Description](https://2021.igem.org/Team%3ANCHU_Taichung/Description)
  + [Implementation](https://2021.igem.org/Team%3ANCHU_Taichung/Implementation)
  + [Design](https://2021.igem.org/Team%3ANCHU_Taichung/Design)
  + [Endophyte](https://2021.igem.org/Team%3ANCHU_Taichung/Endophyte)
  + [Model](https://2021.igem.org/Team%3ANCHU_Taichung/Model)
  + [Results](https://2021.igem.org/Team%3ANCHU_Taichung/Result)
  + [Engineering success](https://2021.igem.org/Team%3ANCHU_Taichung/Engineering)
  + [Proof of
    Concept](https://2021.igem.org/Team%3ANCHU_Taichung/Proof_Of_Concept)
* Parts
  + [Basic
    Part](https://2021.igem.org/Team%3ANCHU_Taichung/Basic_Part)
  + [Composite
    Part](https://2021.igem.org/Team%3ANCHU_Taichung/Composite_Part)
  + [Contribution&Improvement](https://2021.igem.org/Team%3ANCHU_Taichung/Contribution)
* Notebook
  + [Note](https://2021.igem.org/Team%3ANCHU_Taichung/Note)
  + [Protocol](https://2021.igem.org/Team%3ANCHU_Taichung/Protocol)
  + [Safety](https://2021.igem.org/Team%3ANCHU_Taichung/Safety)
* Human Practices
  + [Integrated
    Human Practices](https://2021.igem.org/Team%3ANCHU_Taichung/Human_Practices)
  + [Education
    and Communication](https://2021.igem.org/Team%3ANCHU_Taichung/Communication)
  + [Collaboration](https://2021.igem.org/Team%3ANCHU_Taichung/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3ANCHU_Taichung/Partnership)
  + [Sustainable](https://2021.igem.org/Team%3ANCHU_Taichung/Sustainable)
* Team
  + [Members](https://2021.igem.org/Team%3ANCHU_Taichung/Members)
  + [Attributions](https://2021.igem.org/Team%3ANCHU_Taichung/Attributions)
* [Criteria](https://2021.igem.org/Team%3ANCHU_Taichung/Criteria)

# 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)