iGEM 2020 — Team SHSBNU_China

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# Team:SHSBNU China/Notebook

Document

* ![](https://static.igem.org/mediawiki/2020/a/a6/T--SHSBNU_China--SHSBNU_China_logo.jpeg)
* [Home](https://2020.igem.org/Team%3ASHSBNU_China)
* [Project](https://2020.igem.org/Team%3ASHSBNU_China/Description)

  、+ [Description](https://2020.igem.org/Team%3ASHSBNU_China/Description)
  + [Design](https://2020.igem.org/Team%3ASHSBNU_China/Design)
  + [Proof of Concept](https://2020.igem.org/Team%3ASHSBNU_China/Proof_Of_Concept)
  + [Proposed Implementation](https://2020.igem.org/Team%3ASHSBNU_China/Implementation)
  + [Hardware](https://2020.igem.org/Team%3ASHSBNU_China/Hardware)
* [Lab](https://2020.igem.org/Team%3ASHSBNU_China/Experiment)

  + [Experiment](https://2020.igem.org/Team%3ASHSBNU_China/Experiment)
  + [Notebook](https://2020.igem.org/Team%3ASHSBNU_China/Notebook)
* [Team](https://2020.igem.org/Team%3ASHSBNU_China/Members)

  + [Members](https://2020.igem.org/Team%3ASHSBNU_China/Members)
  + [Attributions](https://2020.igem.org/Team%3ASHSBNU_China/Attributions)
  + [Collaboration](https://2020.igem.org/Team%3ASHSBNU_China/Collaborations)
  + [Partnership](https://2020.igem.org/Team%3ASHSBNU_China/Partnership)
* [Parts](https://2020.igem.org/Team%3ASHSBNU_China/Part%20Overview)

  + [Part Overview](https://2020.igem.org/Team%3ASHSBNU_China/Part%20Overview)
  + [Contribution](https://2020.igem.org/Team%3ASHSBNU_China/Contribution)
  + [Engineering Success](https://2020.igem.org/Team%3ASHSBNU_China/Engineering)

  * [HP](https://2020.igem.org/Team%3ASHSBNU_China/Human%20Practice)

    + [Human Practices](https://2020.igem.org/Team%3ASHSBNU_China/Human_Practices)
    + [Education](https://2020.igem.org/Team%3ASHSBNU_China/Education)
  * [Model](https://2020.igem.org/Team%3ASHSBNU_China/Model)
  * [Judging](https://2020.igem.org/Team%3ASHSBNU_China/Judging)
  * [Safety](https://2020.igem.org/Team%3ASHSBNU_China/Safety)

# SHSBNU\_China' Notebook

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:SHSBNU China/Engineering

Document

* ![](https://static.igem.org/mediawiki/2020/a/a6/T--SHSBNU_China--SHSBNU_China_logo.jpeg)
* [Home](https://2020.igem.org/Team%3ASHSBNU_China)
* [Project](https://2020.igem.org/Team%3ASHSBNU_China/Description)

  + [Description](https://2020.igem.org/Team%3ASHSBNU_China/Description)
  + [Design](https://2020.igem.org/Team%3ASHSBNU_China/Design)
  + [Proof of Concept](https://2020.igem.org/Team%3ASHSBNU_China/Proof_Of_Concept)
  + [Proposed Implementation](https://2020.igem.org/Team%3ASHSBNU_China/Implementation)
  + [Hardware](https://2020.igem.org/Team%3ASHSBNU_China/Hardware)
* [Lab](https://2020.igem.org/Team%3ASHSBNU_China/Experiment)

  + [Experiment](https://2020.igem.org/Team%3ASHSBNU_China/Experiment)
  + [Notebook](https://2020.igem.org/Team%3ASHSBNU_China/Notebook)
* [Team](https://2020.igem.org/Team%3ASHSBNU_China/Members)

  + [Members](https://2020.igem.org/Team%3ASHSBNU_China/Members)
  + [Attributions](https://2020.igem.org/Team%3ASHSBNU_China/Attributions)
  + [Collaboration](https://2020.igem.org/Team%3ASHSBNU_China/Collaborations)
  + [Partnership](https://2020.igem.org/Team%3ASHSBNU_China/Partnership)
* [Parts](https://2020.igem.org/Team%3ASHSBNU_China/Part%20Overview)

  + [Part Overview](https://2020.igem.org/Team%3ASHSBNU_China/Part%20Overview)
  + [Contribution](https://2020.igem.org/Team%3ASHSBNU_China/Contribution)
  + [Engineering Success](https://2020.igem.org/Team%3ASHSBNU_China/Engineering)

  * [HP](https://2020.igem.org/Team%3ASHSBNU_China/Human%20Practice)

    + [Human Practices](https://2020.igem.org/Team%3ASHSBNU_China/Human_Practices)
    + [Science Communication](https://2020.igem.org/Team%3ASHSBNU_China/Education)
  * [Model](https://2020.igem.org/Team%3ASHSBNU_China/Model)
  * [Judging](https://2020.igem.org/Team%3ASHSBNU_China/Judging)
  * [Safety](https://2020.igem.org/Team%3ASHSBNU_China/Safety)

# Engineering

In an article from AMB Express in 2019, two key components of degrading guaiacol are mentioned, which are cytochrome P450 (CYP) and Ferredoxin Reductase (FR). We then discovered that plasmids contain genes that express these two substances are respectively WP\_085469912 from R.rhodochrous J3 and WP\_085469913 from R.rhodochrous J3. Next, we searched for the sequence of these two genes and synthesize them from genetic company.

![](https://static.igem.org/mediawiki/2020/7/71/T--SHSBNU_China--123123.jpeg)

Figure 1. Linear map of pSEVA424 plasmid and its relevant features. b Structure of the polycistronic operons constructed in pSEVA424. Strains GI, GII, and GIII contain all three gene elements, strain G0 only carries the first gene (CYP gene), and strain GIV contains the first two genes only. Strains GI–III differ by the origin of the ferredoxin gene (WP\_085470952 from R. rhodochrous J3 in GI, WP\_085469096 from R. rhodochrous J3 in GII and WP\_020416430 from Amycolatopsis ATCC 39116 in GIII)

### ·Problems that we encountered

This plasmid was mainly constructed by Gongze He. According to his response, the construction of this plasmid is relatively successful. We did not encounter any severe problems, and all the works were completed for only one time.

### ·Experiments & Results

we constructed two plasmids PSB4K5\_pTac\_CYP\_FR (PSHS4) and PSB4K5\_pTac\_MAO\_ALDR (PSHS5) by molecular cloning. We transferred these two plasmids into E. coli BL21, separately. After IPTG induction, we found the target protein expressed, except MAO, by SDS-PAGE.

![](https://static.igem.org/mediawiki/2020/e/eb/T--SHSBNU_China--444.jpeg)

Figure 4. The process we do in order to determine the expressing of protein.

![](https://static.igem.org/mediawiki/2020/9/91/T--SHSBNU_China--555.jpeg)

Figure 5. The analysis of SDS-PAGE of PSHS4 and PSHS5. Lane 1 and 2 are samples of PSHS4 and 5 by overnight LB culture. Lane 3 and 4 are PSHS4 and 5 after overnight culture, and then diluted to M9 and cultured to an OD approximately equal to 0.6. Lane 5 and 6 are PSHS4 and 5 adding IPTG for 3h under 37 degrees centigrade.

We can see the result obviously lane 5 and 6, which can prove that the systems we constructed are successful. However, we found that there are also some protein expressions before induction, which we thought was caused by the leakage of promoter. Moreover, we did not see the obvious expression of MAO, and we will find out the cause and optimize our system in future experiments. In the future, we would determine the activity of enzymes.

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:SHSBNU China/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

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:SHSBNU China/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.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)