iGEM 2021 — Team SHSBNU_China

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

# Team:SHSBNU China/Notebook

BootStrap

![](https://static.igem.org/mediawiki/2021/4/42/T--SHSBNU_China--loading.jpg)

* Team
  + Teammembers
  + Attribution
  + Collaboration
  + Partnership
* Project
  + Description
  + Design
  + Proof of concept
  + Proposed Implementation
  + Hardware
* Model
* Part
  + Part overview
  + Contribution
  + Engineering success* HP
    + Human Practice
    + Science Communication
    + Education
  * Lab
    + Notebook
  * Judge
  * Safty
  * Acknowledgement

![](https://static.igem.org/mediawiki/2021/4/49/T--SHSBNU_china--505.jpg)

![](https://static.igem.org/mediawiki/2021/f/ff/T--SHSBNU_China--below1.jpg)

![](https://static.igem.org/mediawiki/2021/8/85/T--SHSBNU_China--navbar4.jpg)

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

# Team:SHSBNU China/Engineering

![](https://static.igem.org/mediawiki/2021/4/42/T--SHSBNU_China--loading.jpg)

* Team
  + Teammembers
  + Attribution
  + Collaboration
  + Partnership
* Project
  + Description
  + Design
  + Proof of concept
  + Proposed Implementation
  + Hardware
* Model
* Part
  + Part overview
  + Contribution
  + Engineering success* HP
    + Human Practice
    + Science Communication
    + Education
  * Lab
    + Notebook
  * Judge
  * Safty
  * Acknowledgement

* [Engineering](#text1)
* [Introduction](#intro)
* [Failed attempt](#failed)
* [Learn and redesign](#finding)
* [Antibiotics](#antibiotics)
* [Optimization of experiment condition](#temperature)
* [Modeling](#modeling)

Engineering

**Intro**
In the experiments about the cultivation of fusion protein(CBD-SA), SHS1-pET28b-CBD-EGFP-CBD, SHS2-pET28b-CBD-SA-CBD and CBD-SA-CBD-BS- pUC were inoculated into culture medium. ThenSHS1&2 were extracted and transformed them into E.coil BL21 separately, and were induced with IPTG. After purified the protein by using nickel column, we tested if these proteins have expressed by SDS-PAGE.

**Failed attempt**
At the beginning of the experiments, we cultivated the proteins with carbon source of glucose and 0.3mM of IPTG to induce those proteins in 16℃.

![](https://static.igem.org/mediawiki/2021/4/46/T--SHSBNU_china--300.jpg)

Figure 1 The result of SDS-PAGE of CBD-eGFP

![](https://static.igem.org/mediawiki/2021/8/89/T--SHSBNU_china--301.jpg)

Figure 2 The result of SDS-PAGE. Unfortunately, both proteins didn't express.

Although the lysed cell is green indicating some protein had been expressed, the SDS-PAGE results of the purification does not yield satisfying results. As shown in the SDS gel above, no clear band of 51.4kDa is visible. And can be seen from figure 2, no band of 38.7kDa is visible as well.

**Finding the cause**

To verify that it was because of inclusion body but not the not fully lysed cells, we sonicated the remaining sediments of previous experiment with 300watts, which is as high as this machine can get that should be able to fully lysis any cells. However, after sonication, the supernatant of the sonicated cells still lack fluorescence, indicating inclusion body formation is responsible for the failure of the purification. Therefore, the following experiments are meanly focused on improving protein production by reducing the inducing intensity of the bacteria.

We then tried to find out whether IPTG concentration can have effects on the expression. We used different concentrations of IPTG to induce the expression of CBD-eGFP protein. From Figure3, it is obvious that the concentration of IPTG can have profound influences on the expression of the protein. Although it seems that in the picture IPTG concentration of 0.05mM and 0.1mM have the best expression result, we continued to proceed experiments to confirm the best IPTG concentration.

![](https://static.igem.org/mediawiki/2021/d/d0/T--SHSBNU_china--302.jpg)

Figure 3 Numbers on the picture are different IPTG concentration in each system.

The results didn’t accord with our expectation due to the lack of fluorescence.
Concluded from the results of Figure 2, we hypothesized that the reason why our first attempt of expression failed was because of the IPTG concentration was too high.

**Antibiotics**
We used different concentration of IPTG to induce the protein under 16 ℃. The samples were divided into three groups based on the concentration of IPTG(0.01mM/0.02mM/0.03mM), and each group contains six samples which three of them were added kana while others were not. We tested the fluorescent value of all eighteen samples. The results were the samples which have no kana have higher fluorescent value, which means more protein are remained.

![](https://static.igem.org/mediawiki/2021/0/06/T--SHSBNU_china--303.jpg)

From this result, we decided to not use kanamycin in further experiments.

**Temperature**
Then, we induced protein with different concentration of IPTG under 16 and 24 Celsius and both samples have no kana. Other conditions were same.

![](https://static.igem.org/mediawiki/2021/5/59/T--SHSBNU_china--304.jpg)![](https://static.igem.org/mediawiki/2021/b/bf/T--SHSBNU_china--305.jpg)

Figure 5 Fluorescence production comparison in different IPTG concentration under 16℃ and 24℃.

As can see in figure 5, the fluorescent value of the supernatant of sonicated cells under 16℃ of induction did not exceed 20000 in its best attempt of 0.03mM IPTG. If the fluorescent value to IPTG concentration is to increase linearly, then we can well conclude that the production of protein is better under 24℃ since the 24℃ induced cells can yield a higher fluorescent value than that of the 16℃’s. Therefore, all the production attempts after this experiment utilize inducing temperature of 24℃.
According to the fluorescent value, when the concentration of IPTG is 0.05mM and 0.2mM under 24 Celsius, more protein was produced.

**Modeling**
After all other factors except IPTG concentration are determined, we now aim to estimate the best IPTG concentration by mathematical simulation because it can provide a more accurate and rigorous concentration.
We figured it out, through modeling, that when the IPTG concentration is 0.05mM, the protein expression can be maximized. For more information about our model: Click here!

![](https://static.igem.org/mediawiki/2021/5/5f/T--SHSBNU_china--306.jpg)

Figure 6 Maximum (approximately 20000) responds to concentration of 0.05mM

After we got the results from modeling, we constructed a concentration gradient around 0.05mM and tried to find out the best condition for the induction of the protein.

![](https://static.igem.org/mediawiki/2021/a/a2/T--SHSBNU_china--307.jpg)

Figure 7 Fluorescence production under different IPTG concentrations

From the result above, it is evidential that with 0.05mM of IPTG, the bacteria can produce the greatest amount of the fusion protein. To our surprise, the best concentration was actually exactly 0.05mM, very consistent with our model.

According to the result, the best condition to cultivated protein is 0.05mM IPTG in 24℃ without kanamycin. Thus, that condition was going to be the condition when we induced our protein.

![](https://static.igem.org/mediawiki/2021/d/d6/T--SHSBNU_china--308.jpg)

Figure 8 The result of SDS-PAGE for CBD-eGFP

We can obviously see that the expression of protein, which means our experiment was successful.

**For more information about our proof\_Of\_Concept:** [Click here!](https://2021.igem.org/Team%3ASHSBNU_China/Proof_Of_Concept)

![](https://static.igem.org/mediawiki/2021/f/ff/T--SHSBNU_China--below1.jpg)

![](https://static.igem.org/mediawiki/2021/8/85/T--SHSBNU_China--navbar4.jpg)![](https://static.igem.org/mediawiki/2021/4/4c/T--SHSBNU_china--503.jpg)

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

* [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: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.

* [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)