iGEM 2021 — Team Think_Edu_China

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

# Team:Think Edu China/Notebook

# 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:Think Edu China/Engineering

![](https://static.igem.org/mediawiki/2021/b/b8/T--Think_Edu_China--logo.jpg)

Engineering

Step 1: Design the mechanism of degradation

After established a thourough understanding of our project direction, which is the degradation of antibiotics, we selected a specific enzyme named laccase 6 as the major player for the process. According to research that we have done, the laccase enzyme was previously used in enzymatic membrane reactor (EMR) as a mean to remove the antibiotics. Therefore, we cut out a specific gene fragment laccase 6 to be inserted into our constructed bacterial strain.

![](https://static.igem.org/mediawiki/2021/9/9d/T--Think_Edu_China--01.jpg)

Step 2: Construct the whole cell catalyst

After designed our engineered plasmid, in order to determine whether or not the gene lacc 6 was successfully anchored on the extra cellular membrane of the bacteria strain, the study utilized overlap PCR to connect gene gfp and the end of INP-N-Lacc6. Using fluorescence microscopy, the recombinant strain ECN-ILG observed to emit green fluorescence, while the control strain ECN-PSB18A cannot. Thus, came to the conclusion that Lacc6 gene fragment was successfully anchored on the surface of EcN cells surface.

![](https://static.igem.org/mediawiki/2021/e/e2/T--Think_Edu_China--02.jpg)

Step 3: Test the sensitivity of bacterial strain toward antibiotics

In order to ensure that the engineered bacterial strain can be obtain by the antibiotic resistant screening, the sensitivity of the wild strain of EcN toward several types of antibiotics should be determined. In order to test the sensitivity of the bacterial strain, we inoculate the EcN strain into test tubes that each contains Chloramphenicol (30 μg/ mL), Ampicillin (50 μg/ mL) and Kanamycin (50 μg/ mL). Surprisingly, after 12 hours of incubation, if the wild-type EcNs cannot survive in the presence of the above antibiotics that is used for cloning screening, the strain can then be used as host bacteria serves to express the protein.

![](https://static.igem.org/mediawiki/2021/4/4e/T--Think_Edu_China--03.jpg)

Step 4: Advancement and redesigned the EcN-IL

After we have found out that the EcN cannot really survive amount the antibiotics, we were thinking of a way to increase the efficiency of degradation. Instead of directly apply laccase to antibiotics, we construct a new system using the cell surface display technology. Sulfonomide antibiotics is known for not kill bacteria, but it interferes with the ability of bacteria to grow and multiply. Thus, we learn the Nissle 1916 won't be killed by the Sulfonomide. For the advancement of the project, we construct the whole cell catalyst that utilize INP-N protein to anchor the laccase to the extracellular membrane of EcN to form EcN-IL.

Step 5: Test analysis of the sensitivity of the constructed EcN-IL

After EcN-IL was created , we tested its rate of degradation in vitro. We used ABTS as substrates and the tested laccase activity of whole cell catalyst was 1.99 ± 087 U/ cell dry weight when the absorbance value was 420nm (Fig. 3-1). while the enzyme activity was not detected in ECN - p18a and wild-type EcN.

![](https://static.igem.org/mediawiki/2021/9/91/T--Think_Edu_China--04.jpg)

Step 6: Secondary design of the optimal operation conditions

After find out the EcN-IL has comparable effect in degradation of Sulfonomide antibiotics, we attempt to search for the best condition for the enzymes to process. We specifically investigated the effects of pH level and temperature on the degradation rate of sulfadiazine by the engineering strains. As the result shows in Figure 3-2, when the pH level is in the range of 4~7, the degradation rate of ECN on SDZ gradually increases, and reaches the maximum when pH level equals to 7.0. With the gradual increase of pH, the degradation efficiency began to decreases gradually. Moreover, Fig. 3-2 also shows that the degradation rate of SDZ increases gradually with the increase of temperature, and reaches highest around 40 ℃. Therefore, the optimum pH level is 7.0 with temperature of 40 ℃.

![](https://static.igem.org/mediawiki/2021/b/b0/T--Think_Edu_China--05.jpg)![]()

![](https://static.igem.org/mediawiki/2021/b/b8/T--Think_Edu_China--logo.jpg)

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

# Team:Think Edu 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:Think Edu 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)