iGEM 2021 — Team DKU

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

# Team:DKU/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:DKU/Engineering

# Engineering Success

### Synthesis of the peptide

**1) Design:** In our project, we decided to utilize a peptide named HPA3NT3-A2. Previous records suggested that such a peptide presents low hemolytic and cytotoxic effects on mammalian cells at a relatively high concentration up to 250 μM (Lee & Park, 2020), but a significant and selective algicidal effect toward Microcystits aeruginosa even at a low concentration down to 10 μM (Han et al., 2019). We, therefore, acknowledged it as a wonderful tool to help us achieve the goal of eliminating water blooms in the environment.

**2) Hypothesis:** Different from the chemical methods of synthesis of HPA3NT3-A2 in previous studies, we wanted to perform biosynthesis, which theoretically saves energy but gives a higher yield. We noticed that the peptide might be able to present significant toxicity towards E. coli so that direct synthesis of the peptide itself might not be feasible.

**3) Testing:** To test our hypothesis, we first design the original DNA sequence to be a 6xHis-tag at the 5’ end with a single peptide sequence directly attached to at the 3’ end. The DNA sequence was then cloned onto plasmid pET-30a (+) downstream of a T7 promoter. The plasmid was first amplified in the DH5α strain of E. coli and uptaken by the competent BL21(DE3) strain of E. coli which expresses T7 RNA polymerase allowing rapid expression of the peptide. The expression of the peptide is induced at 37 degrees Celcius and 20 degrees Celcius with IPTG of 0.5 mM right at the exponential growth stage of E. coli. No significant expression was detected in either common SDS-page gel electrophoresis or tricine-SDS-page gel electrophoresis for small peptides. Recognizable cell precipitation can be observed after the IPTG induction or even after 12 hours of incubation without IPTG, implying that the peptide cannot be directly synthesized either because of its extremely small size (2 kDa) or because of its strong toxicity towards bacterial cells.

**4)Learn and modification:** The DNA sequence clearly required further modification. To shield the toxicity of the peptide, a SUMO-tag with a linker sequence of GGGGS at its 5’ end is added to the 3’end of the 6x His-tag, which can be cut away altogether by SUMO protease in later processes. To increase the algicidal activity, following the “His-tag, linker sequence, SUMO-tag” sequence, a triple-tandem peptide sequence with GGGGS between each complete peptide is added to its 3’ end. The linker sequence (GGGGS) maintains the flexibility of rotation or movement of every single peptide sequence, allowing the tight contact of each peptide sequence to the surface of algae without steric hindrance (Chen et al., 2013). This plasmid is named SUMO-H3, while another SUMO-H1 plasmid with only one single peptide sequence is built up to examine the potentially increased algicidal effect of the triple-tandem peptide. For more information, please refer to the PARTS section of our wiki.

### Efficient attack on algae

**1) Design:** To conduct an efficient attack on cyanobacteria, we choose to load the peptide onto and into the exosomes so that when it targets the cell membrane, the local concentration of the peptide can be higher than simply diffusing the drug evenly in the water. In this project, the nano-scale exosomes were produced and separated from the engineering E. coli DH5a strain. To promote the production of the exosome, the degP gene in the bacteria genome was knocked out (Schwechheimer & Kuehn, 2013). We applied a two plasmid CRISPR-cas9 system invented by Jiang et al. (2015).

**2) Build:** First, the pCAS plasmid was transformed into the bacteria through electroporation. Then, positive transformants were selected with kanamycin and made into competent cells. Then, pTagetF-degP and homologous sequences were transformed into the bacteria through electroporation. The positive transformants were selected with Streptomycin. After recovering at 37˚C, two plasmids were cured.

**3) Test:** Sanger sequencing targeting degP was performed to test either gene knock-out was successful. For the gene knock-out strains, they were cultured in M9 media with 2% glucose overnight before harvesting. The bacteria were filtered from the media and then the exosome was purified via ultracentrifugation. To examine the quality and the yield, flow-cytometry and fluorescent staining were performed.

### Delivery system

**1) Design:** We aim to design a drug delivery system that can slowly release the peptide into the surrounding water. The drug delivery system is composed of small floating units made of biodegradable environmentally friendly plastic called polyhydroxyalkanoates (PHA) whose biodegradation in marine water and the freshwater system have been confirmed (Morohoshi et al., 2018; Sun et al., 2021). Each floating unit consists of two horizontal hollow capsule-like structures to maintain buoyancy and a short vertical hollow cylinder in between to control the sustained release of the peptide. Inside the vertical cylinder, a drug pump is set up in which the peptide and the water-absorbing material are separately wrapped by semi-permeable membrane sacs. Once adequate water flows into the cylinder through a small hole at its bottom, the water-absorbing material starts to expand and squeeze the peptide out and release it into the water system. Multiple floating units can be connected with PHA threads into a bigger net-like unit to flow with the current along with water bloom cyanobacterial species. In this way, the drug is enriched at the water surface to better target the Microcystis aeruginosa.

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

# Team:DKU/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:DKU/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)