iGEM 2021 — Team CCU_Taiwan

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# Team:CCU Taiwan/Notebook

Team:CCU Taiwan/Notebook - 2021.igem.org

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# Team:CCU Taiwan/Poster

![](https://static.igem.org/mediawiki/2021/0/0c/T--CCU_Taiwan--loading.gif)

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* Team
  + [Home](https://2021.igem.org/Team%3ACCU_Taiwan)
  + [Members](https://2021.igem.org/Team%3ACCU_Taiwan/Member)
  + [Attributions](https://2021.igem.org/Team%3ACCU_Taiwan/Attributions)
  + [Sponsors](https://2021.igem.org/Team%3ACCU_Taiwan/Sponsors)
* Project
  + [Description](https://2021.igem.org/Team%3ACCU_Taiwan/Description)
  + [Design](https://2021.igem.org/Team%3ACCU_Taiwan/Design)
  + [Implementation](https://2021.igem.org/Team%3ACCU_Taiwan/Implementation)
  + [Entrepreneurship](https://2021.igem.org/Team%3ACCU_Taiwan/Entrepreneurship)
  + [Contribution](https://2021.igem.org/Team%3ACCU_Taiwan/Contribution)
  + [Improvement](https://2021.igem.org/Team%3ACCU_Taiwan/Improvement)
  + [Proof of Concept](https://2021.igem.org/Team%3ACCU_Taiwan/Proof_Of_Concept)
* Labwork
  + [Protocol](https://2021.igem.org/Team%3ACCU_Taiwan/Protocol)
  + [Engineering](https://2021.igem.org/Team%3ACCU_Taiwan/Engineering)
  + [Measurement](https://2021.igem.org/Team%3ACCU_Taiwan/Measurement)
  + [Model](https://2021.igem.org/Team%3ACCU_Taiwan/Model)
  + [Result](https://2021.igem.org/Team%3ACCU_Taiwan/Result)
  + [Parts](https://2021.igem.org/Team%3ACCU_Taiwan/Parts)
* Human Practice
  + [Integrated Human Practice](https://2021.igem.org/Team%3ACCU_Taiwan/Human_Practices)
  + [Education](https://2021.igem.org/Team%3ACCU_Taiwan/Education)
  + [Science Communication](https://2021.igem.org/wiki/index.php?title=Team:CCU_Taiwan/Communication)
  + [Collaboration](https://2021.igem.org/Team%3ACCU_Taiwan/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3ACCU_Taiwan/Partnership)
  + [Inclusivity](https://2021.igem.org/Team%3ACCU_Taiwan/Inclusivity)
* [Notebook](https://2021.igem.org/Team%3ACCU_Taiwan/Notebook)
* [Safety](https://2021.igem.org/Team%3ACCU_Taiwan/Safety)
* [Awards](https://2021.igem.org/Team%3ACCU_Taiwan/Awards)
  + [Medals](https://2021.igem.org/Team%3ACCU_Taiwan/Medals)
  + [Judging Form](https://igem.org/2021_Judging_Form?id=3778)

![](https://static.igem.org/mediawiki/2021/6/69/T--CCU_Taiwan--Notebook_Top.png)

### This Notebook page will help you understand what we have suffered done in these days.

![](https://static.igem.org/mediawiki/2021/archive/2/26/20210810070247%21T--CCU_Taiwan--Notebook-0.png)

May

# May

## Week01: 05/03-05/09

### WL: * Explore the mechanism how TAT and penetratin function * Explore the mechanism how S. aureus resist antimicrobial peptides * Find out whether antimicrobial peptide harm the organelles * Confirm the latency of S.aureus in professional phagocytes DL: * Determine the materials in each dressing layer * Integrate the preparation of dressing, such as protocol, equipment, etc. * Design the thrombin cleavage assay HP: * Complete the parent-child picture book’s outline * Prepare the teaching plan for high school and elementary school students * Operate our Instagram and Facebook * Seek for collaboration opportunities among iGEMers * Write a promotion video script * Start planning a fundraising plan Week02: 05/10-05/16WL: * Search the lysosomal enzyme candidates for releasing intracellular AMPs * Cloning eGFP-TAT and eGFP into expression vectors-PCR, TA, and sequencing confirmation DL: * Order the material of dressing * Confirm the connection of each layer of dressing * Design the thrombin cleavage assay HP: * Rehearsal the experiment in the high school teaching plan * Shoot promotion video * Start to make our parent-child picture book * Prepare for interviews with domestic experts * Design the flyer for the teaching plans Week03: 05/17-05/23WL: * Design the construct of biobrick * Search the the activity of AMP in different pH level * Search the cleavage preference of lysosomal legumain * Cloning eGFP-TAT and eGFP into expression vectors- ligation into expression vectors DL: * Determine the protocol and equipment of thrombin cleavage assay * Thrombin cleavage site prediction * Determine absorptive layer and collagen layer protocol * Determine the container and size of the dressing HP: * Put our teaching plan on record, responding to the epidemic Week04: 05/24-05/30WL: * Explore how antimicrobial peptide functions * Explore the cleavage preference of lysosomal enzymes-cathepsin H, F, D, S, L * Explore the biosafety of cell penetrating peptides * Cloning eGFP-TAT and eGFP into expression vectors- ligation into expression vectors DL: * Design the thrombin cleavage assay * Preparation of absorptive layer HP: * Make PowerPoint for the teaching plan * Finalize the parent-child picture book * Design our team logo * Design team’s peripheral product * Design postcard(the cooperation with iGEM-Duesseldorf) * Start to cooperate with the other iGEMer for translating our parent-child picture book

![](https://static.igem.org/mediawiki/2021/archive/2/26/20210810070302%21T--CCU_Taiwan--Notebook-0.png)

June

# June

## Week05: 05/31-06/06

### WL: * Explore how antimicrobial peptide functions * Find the the macrophage culture and stimulation protocol * Explore the biosafety of cell penetrating peptide and antimicrobial peptide * Find evidence supporting the severity of MRSA latency * Write the safety form.

### DL: * Design the thrombin cleavage assay and confirm protocol and equipment * Confirm and modify the preparation principle of absorptive layer

### HP: * Online meeting with iGEM Ioannina * Prepare for the parent-child online picture books * Design team uniform

## Week06: 06/07-06/13

### WL: * Search the mechanism of antimicrobial peptide-finished * Find the protocol of hemolysis * Explore the biosafety of cell penetrating peptide and antimicrobial peptide * Find evidence supporting the severity of MRSA latency * Write safety form * Ligate the eGFP-TAT to pET15b * Perform the PCR for CBD-linker, thrombin cleavage site for AMP/TAT mimic release, and thrombin releasable AMP * Perform the colony PCR of TA-CBD-linker, TA-Thrombin cleavage site for AMP/TAT mimic release, TA-thrombin releasable AMP

### DL: * Confirm the human thrombin concentration * Preparation of absorptive layer * Order collagen solution

### HP: * Trim the promotion video * Draw a comic about our project(the cooperation with iGEM IISER Berhampur)

## Week07: 06/14-06/20

### WL: * Explore the biosafety of cell penetrating peptide and antimicrobial peptide * Find evidence supporting the severity of MRSA latency * Write safety form * Perform the colony PCR for TA-CBD-linker, TA-Thrombin cleavage site for AMP/TAT mimic release-eGFP, TA-thrombin releasable AMP * Perform the digestion for TA-CBD-linker, TA-Thrombin cleavage site for AMP/TAT mimic release-eGFP, TA-thrombin releasable AMP

### DL: * Prepare different mixing ratios of the absorptive layer to find the suitable group * Wiki team member page design

### HP: * Prepare a collaboration proposal for Taiwan iGEM Conference * Dub the promotion video * Invite Hsin-Yi Foundation as our interviewee * Hsin-Yi Foundation regards the promotion of early childhood education and parenting education as their mission

## Week08: 06/21-06/27

### WL: * Fighting with midterm examination!!!!!!! XD

### DL: * Fighting with midterm examination!!!!!!! XD

### HP: * Prepare for a meeting with iGEM Team KOR\_HS * Contact Adien Lo, a 17 years old podcaster, to see if we can collaborate * Send out postcards to Germany for worldwide iGEMer’s postcard exchange activity launched by iGEM Team Duesseldorf

![](https://static.igem.org/mediawiki/2021/e/eb/T--CCU_Taiwan--notebook-7.png)

July

# July

## Week09: 06/28-07/04

### WL: * Explore the cleavage preference of lysosomal enzymes-cathepsin B, D. * Explore the biosafety of cell penetrating peptide * Find evidence supporting the severity of MRSA latency * Cloning eGFP-TAT and eGFP into expression vectors- ligation into expression vectors * Cloning CBD-linker, thrombin cleavage site for AMP/TAT mimic release-GFP, and thrombin releasable AMP into expression vector- ligation into expression vectors

### DL: * Find the suitable mixing ratio of alginate and chitosan solution to prepare our absorptive layer * Confirm Swelling test protocol * Confirm thrombin unit conversion * Wiki team member page and homepage design

### HP: * Finish the parent-child picture E-book, and start to translate it into an English version * Participate in Taiwan iGEM Conference hosted by iGEM Team CSMU\_Taiwan * Communicate with Kinmen Hospital to seek professional advice about infection data * Invite Junyi Academy as our interviewee * Junyi Academy creates an online learning platform for students from elementary school to college

## Week10: 07/05-07/11

### WL: * Explore the cleavage preference of lysosomal enzymes-cathepsin B, D * Explore the biosafety of cell penetrating peptide * Find evidence supporting the severity of MRSA latency * Cloning eGFP-TAT and eGFP into expression vectors- ligation into expression vectors * Cloning CBD-linker, thrombin cleavage site for AMP/TAT mimic release-GFP, and thrombin releasable AMP into expression vector- ligation into expression vectors

### DL: * Prepare absorptive layer by different concentrations of alginate and chitosan solution * Conduct the swelling test to compare water uptake ability of the absorptive layer * Collagen layer protocol * Convert the unit of commercial thrombin to human thrombin and confirm its usage in our experiment * Order thrombin * Design Wiki style

### HP: * Have an online meeting with the iGEM Team KOR\_HS * Dub the online teaching video * Do the market research of our parent-child picture book * Turn our parent-child picture E-book into the paper one * Finish the promotion video * Translate parent-child picture book into a Cantonese version, thanks to iGEM Team HKUST

## Week11: 07/12-07/18

### WL: * Explore the mechanism of antibiotic-resistance of S. aureus * Explore the biosafety of cell penetrating peptide * Organize evidence supporting the severity of MRSA latency * Cloning eGFP-TAT and eGFP into expression vectors- ligation into expression vectors * Cloning CBD-linker, thrombin cleavage site for AMP/TAT mimic release-GFP, and thrombin releasable AMP into expression vector- ligation into expression vectors * Practice the procedure of MIC test

### DL: * Prepare absorptive layer by different concentrations of alginate and chitosan solution * Improve steps in the preparation process of the absorptive layer to make its structure more stable, such as pH neutralization * Swelling test * Design Wiki style

### HP: * Co-product online teaching video with iGEM Team NTCU\_Formosa * Pop up with an idea of the iASK symposium with iGEM Team NCKU\_Tainan * Translate parent-child picture book into Kazakh and French version, thanks to iGEM Team NU\_Kazakhstan and iGEM Team Aix-Marseille * Rehearsal online courses for Consistent Children Care organization collaboration

## Week12: 07/19-07/25

### WL: * Organize the protocol of cloning * Organize evidence supporting the severity of MRSA latency * Design the sequence of cathepsin S site * Cloning eGFP-TAT and eGFP into expression vectors- ligation into expression vectors * Cloning CBD-linker, thrombin cleavage site for AMP/TAT mimic release-GFP, and thrombin releasable AMP into expression vector- ligation into expression vectors * Practice the procedure of MIC test

### DL: * Prepare absorptive layer by different concentrations of alginate and chitosan solution * Swelling test * Design Wiki style

### HP: * Typeset our picture book * Prepare the iASK symposium with iGEM Team NCKU\_Tainan * Contect St. Viator Catholic High School

## Week13: 07/26-08/01

### WL: * Explore the stability of antimicrobial peptides in a low pH value environment * Explore the mechanism that S. aureus resistant lysosomal enzyme * Organize evidence supporting the severity of MRSA latency * Cloning eGFP-TAT and eGFP into expression vectors- ligation into expression vectors * Cloning CBD-linker, thrombin cleavage site for AMP/TAT mimic release-GFP, and thrombin releasable AMP into expression vector- ligation into expression vectors * Practice the procedure of MIC test

### DL: * Prepare absorptive layer by different concentrations of alginate and chitosan solution * Swelling test * Protein structure prediction * Build the network structure of the simulation model

### HP: * Have an online meeting with iGEM Team IISER Kolkata * Translate iGEM Team Ioannina’s promotion video into Mandarin Chinese * Upload 1st episode of our soap opera “We Don’t Like Germs.”

![](https://static.igem.org/mediawiki/2021/archive/2/26/20210810070329%21T--CCU_Taiwan--Notebook-0.png)

August

# August

## Week14: 08/02-08/08

### WL: * Explore the stability of antimicrobial peptides in low pH value environment * Explore the mechanism that S. aureus resistant lysosomal enzyme * Organize evidence supporting the severity of MRSA latency * Cloning eGFP-TAT into expression vectors-ligation into expression vector * Cloning eGFP into expression vectors-protein induction * Cloning CBD-linker, thrombin cleavage site for AMP/TAT mimic release-GFP, and thrombin releasable AMP into expression vector- ligation into expression vectors * Practice the procedure of MIC test

### DL: * Analyze the result of swelling test * Combination of collagen layer and absorptive layer * Confirm the experiment information of thrombin cleavage assay * Infer the differential equation from reaction rate equation

### HP: * Operate the picture book readers survey * Participate in a campaign with iGEM Team Aix-Marseille * Collaborate with iGEM Team USP-BR

## Week15: 08/09-08/15

### WL: * Explore the mechanism that S. aureus resistant lysosomal enzyme * Cloning eGFP-TAT into expression vectors-ligation into expression vector * Cloning eGFP into expression vectors-protein induction * Cloning CBD-linker, thrombin cleavage site for AMP/TAT mimic release-GFP, and thrombin releasable AMP into expression vector- ligation into expression vectors * Practice the procedure of MIC test * Stimulate the monocyte to macrophage

### DL: * Combination of dressing * Use differential equation and law of conservation of mass to built the framework simulation model * Cathepsin S cleavage preference prediction

### HP: * Have an online meeting with iGEM Team Tübingen * Launch a picture book promotion collaborate with Teach For Taiwan * Collaborate with Consistent Child Care * Upload online science courses to LearnMode and YouTube * Interview with Yien Home Care Center

## Week16: 08/16-08/22

### WL: * Find out that whether the mechanism of S. aureus resistant lysosomal enzymes impacts Cathepsin S * Cloning TAT-GFP and eGFP into expression vectors-Protein induction * Cloning CBD-linker, thrombin cleavage site for AMP/TAT mimic release-GFP, and thrombin releasable AMP into expression vector- ligation into expression vectors * Practice the procedure of MIC test and hemolysis

### DL: * Use MATLAB to built a simulation model * Confirm the active site of Cathepsin S * Conduct the docking through AutoDock Vina

### HP: * Interview with Associate Professor Chien-Chi Liu * Cohold iASK symposium with iGEM Team NCKU\_Tainan * Record Podcast with Aiden Lo * Conduct a dressing market survey * Prepare for the final video * Join in the SDGs impact challenge launched by iGEM Team TAS\_Taipei * Upload the online science course video

## Week17: 08/23-08/29

### WL: * Explore the Interaction between AMP and antibiotics * Ensure our antimicrobial agent won’t irritate the wound * Cloning TAT-GFP and eGFP into expression vectors-Protein purification * Cloning CBD-linker, thrombin cleavage site for AMP/TAT mimic release-GFP, and thrombin releasable AMP into expression vector- ligation into expression vectors> * Cloning CBD-linker-thrombin cleavage site for buforin II-C mimic release-GFP into expression vector- protein induction> * Practice the procedure of MIC test, hemolysis, and cytotoxicity>

### DL: * Improve the dressing combination method by coating the collagen layer on the absorptive layer * Use MATLAB to built a simulation model * Confirm the substrate binding site on Cathepsin S via crystal structure

### HP: * Upload the third episode of the soap opera * Interview with Dr. Chi, Chia-Yu * Have an online meeting with iGEM Team Korea\_HS * Have an online meeting with iGEM Team IISER Kolkata * Have a picture book promotion collaborate with Concordia Kindergarten * Complete the online science video with iGEM Team NCTU\_Formosa

![](https://static.igem.org/mediawiki/2021/2/26/T--CCU_Taiwan--Notebook-0.png)

September

# September

## Week18: 08/30-09/05

### WL: * Explore the pH-activate intein * Cloning eGFP-D2A21 into expression vectors-Colony PCR and restriction enzyme digestion * Cloning CBD-linker-thrombin releasable AMP into expression vectors-Restriction enzyme digestion and protein induction * Cloning CBD-linker-thrombin releasable TAT-DPK-060 into expression vectors-protein induction * Cloning CBD-linker, thrombin cleavage site for AMP/TAT mimic release-GFP into expression vector- Ligate into pRSET vector, Colony PCR, protein induction * Cloning CBD-linker-thrombin cleavage site for buforin II-C mimic release-GFP into expression vector- protein induction- Time course, cell disruption * Perform of MIC and hemolysis

### DL: * Ligand preparation * Confirm the specificity pocket of CTSS * Combination of three-layer dressing * Dressing & target population analysis * Data analysis via ImageJ

### HP: * Prepare for the interview with the Industrial Technology Research Institute (ITRI) * Prepare for the interview with Dr. Lee, Nan-Yao

## Week19: 09/06-09/12

### WL: * Explore the Anti-biofilm ability of AMP * Perform cell penetration ability test * Cloning CBD-linker-thrombin releasable D2A21 into expression vectors-protein induction * Cloning CBD-linker-thrombin releasable TAT-DPK-060 into expression vectors-protein induction, Time course * Cloning CBD-linker, thrombin cleavage site for AMP/TAT mimic release-GFP into expression vector-protein induction, cell disruption * Perform of MIC, hemolysis, and cytotoxicity

### DL: * Ligand preparation * Protein-ligand docking through AutoDock * Analyze the pros and cons of dressing combination method

### HP: * Interview with experts from ITRI * Interview with Dr. Lee, Nan-Yao * Visit Trade Wind Biotech Co., Ltd * Record Podcast with iGEM Tübingen * Collaborate with Dong Rong Elementary School * Upload the fourth episode of the soap opera

## Week20: 09/13-09/19

### WL: * Perform cell penetration ability test * Cloning CBD-linker-thrombin releasable D2A21 into expression vectors-protein induction, Time course, cell disruption * Cloning CBD-linker-thrombin releasable TAT-DPK-060 into expression vectors-protein induction, watson blot * Cloning CBD-linker, thrombin cleavage site for AMP/TAT mimic release-GFP into expression vector-protein induction, cell disruption in various condition, watson blot * Perform of MIC, hemolysis, and cytotoxicity

### DL: * Practice the procedure of thrombin cleavage assay * Data analysis via ImageJ to get the release curve * Use MATLAB to built a simulation model * Protein-ligand docking through AutoDock

### HP: * Shoot our team presentation video * Interview with Associate Professor Jong-Yuh Cherng * Participate in iGEM TAS\_Taipei SDGs Challenge Conference * Online program with St. Viator Catholic High School

## Week21: 09/20-09/26

### WL: * Cloning CBD-linker-thrombin releasable AMP into expression vectors-Colony PCR, protein induction * Cloning CBD-linker-thrombin releasable TAT-DPK-060 into expression vectors-cell disruption * Cloning CBD-linker, thrombin cleavage site for AMP mimic release-GFP into expression vector-cell disruption, small scale protein purification * Cloning CBD-linker, thrombin cleavage site for TAT mimic release-GFP into expression vector-Colony PCR, Restriction enzyme digestion * Perform cytotoxicity

### DL: * Protein-ligand docking through AutoDock * Practice the procedure of thrombin cleavage assay * Data analysis & fitting (MATLAB)

### HP: * Participate in Innovation and Entrepreneurship Competition * Design a flyer on using antibiotics accurately * Have an interview with the iGEM Guelph team

## Week22: 09/27-10/03

### WL: * Perform cell penetration test * Cloning CBD-linker-thrombin releasable AMP into expression vectors-Colony PCR, small scale protein purification * Cloning CBD-linker-thrombin releasable TAT-DPK-060 into expression vectors-small scale protein purification * Cloning CBD-linker, thrombin cleavage site for AMP mimic release-GFP into expression vector-time course, small scale protein purification * Cloning CBD-linker, thrombin cleavage site for TAT mimic release-GFP into expression vector-Colony PCR * Perform hemolysis and cytotoxicity

### DL: * Normalize the picture of PAGE via MATLAB * Data analysis & fitting via MATLAB * Protein-ligand docking through AutoDock

### HP: * Organize silver dressing information based on Dr. Lee’s feedback * Interview with Dr. Tak-Wah Wong * Contact with local dermatology clinics * Write subtitles for two videos * Edit the video interviewing foreign students in CCU * Design the slides of the presentation video * Edit the presentation video

![](https://static.igem.org/mediawiki/2021/6/67/T--CCU_Taiwan--Notebook-10.png)

October

# October

## Week23: 10/4-10/10

### WL: * Cloning CBD-linker-thrombin releasable TAT-DPK-060 into expression vectors-Cell disruption, FPLC * Cloning CBD-linker, thrombin cleavage site for AMP/TAT mimic release-GFP into expression vector-protein induction, cell disruption, small scale protein purification * Perform MIC, hemolysis, and cytotoxicity

### DL: * Integrate the data and put it on the wiki * Use MATLAB to built a simulation model * Protein-ligand docking through AutoDock * Analyze gel of SDS-PAGE via normalization between different gel images and signal integration of each band * Conduct thrombin cleavage assay * Data analysis & fitting via MATLAB

### HP: * Upload the last episode of the soap opera * Film and edit the presentation video * Subtitle the video interviewing foreign students in CCU

## Week24: 10/11-10/17

### WL: * Perform cell penetration test * Cloning CBD-linker-thrombin releasable TAT-DPK-060 into expression vectors-FPLC * Cloning CBD-linker, thrombin cleavage site for AMP/TAT mimic release-GFP into expression vector-ligate to pET32a vector, Colony PCR, FPLC * Perform MIC and cytotoxicity

### DL: * Integrate the data and put it on the wiki * Use MATLAB to built a simulation model * Protein-ligand docking through AutoDock * Analyze gel of SDS-PAGE via normalization between different gel images and signal integration of each band * Conduct thrombin cleavage assay * Data analysis & fitting via MATLAB

### HP: * Contact Dr. Wu Tingzyen * Edit presentation video

###

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# Team:CCU Taiwan/Engineering

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![](https://static.igem.org/mediawiki/2021/0/0c/T--CCU_Taiwan--loading.gif)

[![tmp](https://static.igem.org/mediawiki/2021/a/af/T--CCU_Taiwan--mrsa.png)](https://2021.igem.org/Team%3ACCU_Taiwan)

* Team
  + [Home](https://2021.igem.org/Team%3ACCU_Taiwan)
  + [Members](https://2021.igem.org/Team%3ACCU_Taiwan/Member)
  + [Attributions](https://2021.igem.org/Team%3ACCU_Taiwan/Attributions)
  + [Sponsors](https://2021.igem.org/Team%3ACCU_Taiwan/Sponsors)
* Project
  + [Description](https://2021.igem.org/Team%3ACCU_Taiwan/Description)
  + [Design](https://2021.igem.org/Team%3ACCU_Taiwan/Design)
  + [Implementation](https://2021.igem.org/Team%3ACCU_Taiwan/Implementation)
  + [Entrepreneurship](https://2021.igem.org/Team%3ACCU_Taiwan/Entrepreneurship)
  + [Contribution](https://2021.igem.org/Team%3ACCU_Taiwan/Contribution)
  + [Improvement](https://2021.igem.org/Team%3ACCU_Taiwan/Improvement)
  + [Proof of Concept](https://2021.igem.org/Team%3ACCU_Taiwan/Proof_Of_Concept)
* Labwork
  + [Protocol](https://2021.igem.org/Team%3ACCU_Taiwan/Protocol)
  + [Engineering](https://2021.igem.org/Team%3ACCU_Taiwan/Engineering)
  + [Measurement](https://2021.igem.org/Team%3ACCU_Taiwan/Measurement)
  + [Model](https://2021.igem.org/Team%3ACCU_Taiwan/Model)
  + [Result](https://2021.igem.org/Team%3ACCU_Taiwan/Result)
  + [Parts](https://2021.igem.org/Team%3ACCU_Taiwan/Parts)
* Human Practice
  + [Integrated Human Practice](https://2021.igem.org/Team%3ACCU_Taiwan/Human_Practices)
  + [Education](https://2021.igem.org/Team%3ACCU_Taiwan/Education)
  + [Science Communication](https://2021.igem.org/wiki/index.php?title=Team:CCU_Taiwan/Communication)
  + [Collaboration](https://2021.igem.org/Team%3ACCU_Taiwan/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3ACCU_Taiwan/Partnership)
  + [Inclusivity](https://2021.igem.org/Team%3ACCU_Taiwan/Inclusivity)
* [Notebook](https://2021.igem.org/Team%3ACCU_Taiwan/Notebook)
* [Safety](https://2021.igem.org/Team%3ACCU_Taiwan/Safety)
* [Awards](https://2021.igem.org/Team%3ACCU_Taiwan/Awards)
  + [Medals](https://2021.igem.org/Team%3ACCU_Taiwan/Medals)
  + [Judging Form](https://igem.org/2021_Judging_Form?id=3778)

![](https://static.igem.org/mediawiki/2021/f/f0/T--CCU_Taiwan--Engineering.png)

* [Thrombin-releasable
  AMPs or TAT-AMPs](#Thrombin-releasable AMPs or TAT-AMPs)
* [The CTSS-releasable
  AMPs](#The CTSS-releasable AMPs)
* [References](#Reference)

123

**Thrombin-releasable AMPs or TAT-AMPs**

**Introduction**

We apply AMPs or TAT-AMPs in our dressing to kill both extracellular and intracellular MRSA on the wounds, but constitutively activated AMPs may induce unexpected immune responses. In addition, the AMPs or TAT-AMPs need to be stably anchored on the collagen layer. Therefore, we tried to design AMPs with collagen anchors that can be released only when necessary.

## Design

For the collagen anchors, we cloned the collagen binding domain (CBD) from the FN1 gene (see [Design](https://2021.igem.org/Team%3ACCU_Taiwan/Design) for details). Inspired by the [2019 iGEM team Linkoping\_Sweden](https://2019.igem.org/Team%3ALinkoping_Sweden), we designed a linker containing thrombin cleavage site to link AMP/TAT-AMPs and CBD. When the dressing is on the wound, the secreted thrombin will cut the thrombin cleavage site to release the AMP.

## Learn from lectures and re-design

Notably, the cleavage of the canonical thrombin cleavage site will leave two residues at the N-terminal of released AMPs, impacting the activity of AMPs or TAT [[1]](#123)[[2]](#123). Petrassi *et al*. showed that the P1’ and P2’ positions of the thrombin cleavage site could endure the amino acid substitution to some extent without seriously hampering the cleavage ability [[3]](#123). Therefore, it is possible to merge the N-terminal of AMP to the P1’ and P2’ positions of the thrombin cleavage site and generate thrombin-released AMPs without extra residues. Therefore, we substituted the amino acids at P1’ and P2’ positions of the thrombin cleavage site with the first two amino acids at the N-terminal of AMPs.

![](https://static.igem.org/mediawiki/2021/archive/9/96/20211010095459%21T--CCU_Taiwan--子揚.png)

#### ▲ Figure 1: The preferred amino acids at P1’ and P2’ positions of the thrombin cleavage site from the reference [[3]](#123).

## Build

To prove our design, we constructed four vectors expressing fusion proteins to mimic the thrombin cleavage site linked AMPs (Table 1). To easily monitor the result of thrombin cleavage, we replaced the AMP with eGFP and saved the first two amino acids from the N-terminal of AMPs. The expressed fusion proteins are subjected into the thrombin cleavage experiment and the result is demonstrated by SDS-PAGE analysis.

Fusion protein | Mimic targets || CBD-linker-LVPRGK-eGFP | CBD-linker- LVPR-DPK-060 |
| CBD-linker-LVPRFA-eGFP | CBD-linker- LVPR-D2A21 |
| CBD-linker-LVPRGR-eGFP | CBD-linker- LVPR-TAT-DPK-060 |

#### ▲ Table 1: Fusion proteins for thrombin activity test.

## Test- hold by virus outbreak

After constructing the vector expressing the CBD-AMP mimic proteins, we start to express the CBD-AMP mimic proteins, and prepare for thrombin cleavage experiment. Unfortunately, the COVID-19 pandemic has come back to Taiwan, and we need to follow the rules and to slow down the experiment.

## Learn

Although we could not finish the thrombin cleavage experiment under COVID-19 pandemic now, we have prepared to restart the experiment as soon as possible. We believe that, through the experimental design, we can clearly show the ability of our AMPs released by thrombin.

# The CTSS-releasable AMPs

## Introduction

MRSA can remain latent in human cells, such as macrophage, to escape from antibodies, antibiotics or AMPs [[4]](#123)[[5]](#123). To tackle the problem of MRSA latency, we designed a TAT linked AMP (TAT-AMP) to deliver the AMP into the macrophage.

## Design

The TAT-AMP will enter into macrophages through macropinosomes and fuse with phagolysosomes. In phagolysosomes, endopeptidases cathepsin D, F, O, L, and S are responsible for degrading the foreign objects [[6]](#123)[[7]](#123). The survey of preference cleavage sequence of the cathepsin family members indicated that DPK-060 and D2A21 would not be attacked by cathepsins in the lysosome (See Table 3 in [Design](https://2021.igem.org/Team%3ACCU_Taiwan/Design)). This result also suggested that cathepsins are good candidates to release the AMPs from TAT in phagolysosomes. Since cathepsin S (CTSS) is specifically expressed in antigen presenting cells (e.g., macrophages), we selected DPK-060 as intracellular AMPs and added a CTSS cleavage site to release DPK-060 in lysosomes in macrophage.

![](https://static.igem.org/mediawiki/2021/archive/9/96/20211021101624%21T--CCU_Taiwan--子揚.png)

#### ▲ Figure 2: Biobrick for intracellular bacteria.

## Build & Test

Because it is too expensive to get commercial CTSS enzymes, we decided to perform molecular docking to examine the preference CTSS cleavage site. Click [Model](https://2021.igem.org/Team%3ACCU_Taiwan/Model) for more details.

## Results

The modeling results indicated that the CTSS cleavage sites could efficiently dock onto CTSS, suggesting successful cleavage. We may extend this result to model different combinations of amino acids around the CTSS cleavage site, and predict the CTSS cleavage sequence in silico. Furthermore, we can apply the same process to other cathepsin family members whose cleavage sequence remains unclear.

![](https://static.igem.org/mediawiki/2021/archive/9/96/20211017141436%21T--CCU_Taiwan--子揚.png)

#### ▲ Figure 3: The tripeptide (VGG shown in orange; LGG shown in purple) docked to cathepsin S.

References:

[1. Muchintala, D., Suresh, V., Raju, D., & Sashidhar, R. B. (2020). Synthesis and characterization of cecropin peptide-based silver nanocomposites: Its antibacterial activity and mode of action. *Materials science & engineering. C, Materials for biological applications, 110*, 110712.](https://doi.org/10.1016/j.msec.2020.110712)

[2. Schmidtchen, A., Pasupuleti, M., Mörgelin, M., Davoudi, M., Alenfall, J., Chalupka, A., & Malmsten, M. (2009). Boosting antimicrobial peptides by hydrophobic oligopeptide end tags. *The Journal of biological chemistry, 284*(26), 17584–17594.](https://doi.org/10.1074/jbc.M109.011650)

[3. Petrassi, H. M., Williams, J. A., Li, J., Tumanut, C., Ek, J., Nakai, T., Masick, B., Backes, B. J., & Harris, J. L. (2005). A strategy to profile prime and non-prime proteolytic substrate specificity. *Bioorganic & medicinal chemistry letters, 15*(12), 3162–3166.](https://doi.org/10.1016/j.bmcl.2005.04.019)

[4. Huo, S., Chen, C., Lyu, Z., Zhang, S., Wang, Y., Nie, B., & Yue, B. (2020). Overcoming Planktonic and Intracellular *Staphylococcus aureus*-Associated Infection with a Cell-Penetrating Peptide-Conjugated Antimicrobial Peptide. *ACS infectious diseases, 6*(12), 3147–3162.](https://doi.org/10.1021/acsinfecdis.0c00264)

[5. Flannagan, R. S., Heit, B., & Heinrichs, D. E. (2016). Intracellular replication of *Staphylococcus aureus* in mature phagolysosomes in macrophages precedes host cell death, and bacterial escape and dissemination. *Cellular microbiology, 18*(4), 514–535.](https://doi.org/10.1111/cmi.12527)

[6. Biniossek, M. L., Nägler, D. K., Becker-Pauly, C., & Schilling, O. (2011). Proteomic identification of protease cleavage sites characterizes prime and non-prime specificity of cysteine cathepsins B, L, and S. *Journal of proteome research, 10*(12), 5363–5373.](https://doi.org/10.1021/pr200621z)

[7. Vidmar, R., Vizovišek, M., Turk, D., Turk, B., & Fonović, M. (2017). Protease cleavage site fingerprinting by label-free in-gel degradomics reveals pH-dependent specificity switch of legumain. *The EMBO journal, 36*(16), 2455–2465.](https://doi.org/10.15252/embj.201796750)

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# Team:CCU Taiwan/Experiments

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![tmp](https://static.igem.org/mediawiki/2021/a/af/T--CCU_Taiwan--mrsa.png)

* Team
  + [Home](https://2021.igem.org/Team%3ACCU_Taiwan)
  + [Members](https://2021.igem.org/Team%3ACCU_Taiwan/Member)
  + [Attributions](https://2021.igem.org/Team%3ACCU_Taiwan/Attributions)
  + [Sponsors](https://2021.igem.org/Team%3ACCU_Taiwan/Sponsors)
* Project
  + [Description](https://2021.igem.org/Team%3ACCU_Taiwan/Description)
  + [Design](https://2021.igem.org/Team%3ACCU_Taiwan/Design)
  + [Implementation](https://2021.igem.org/Team%3ACCU_Taiwan/Implementation)
  + [Entrepreneurship](https://2021.igem.org/Team%3ACCU_Taiwan/Entrepreneurship)
  + [Contribution](https://2021.igem.org/Team%3ACCU_Taiwan/Contribution)
  + [Improvement](https://2021.igem.org/Team%3ACCU_Taiwan/Improvement)
  + [Proof of concept](https://2021.igem.org/Team%3ACCU_Taiwan/Proof_Of_Concept)
* Labwork
  + [Experiments](https://2021.igem.org/Team%3ACCU_Taiwan/Experiments)
  + [Protocol](https://2021.igem.org/Team%3ACCU_Taiwan/Protocol)
  + [Engineering](https://2021.igem.org/Team%3ACCU_Taiwan/Engineering)
  + [Product](https://2021.igem.org/Team%3ACCU_Taiwan/Product)
  + [Model](https://2021.igem.org/Team%3ACCU_Taiwan/Model)
  + [Measurement](https://2021.igem.org/Team%3ACCU_Taiwan/Measurement)
  + [Result](https://2021.igem.org/Team%3ACCU_Taiwan/Result)
  + [Parts](https://2021.igem.org/Team%3ACCU_Taiwan/Parts)
* Human Practice
  + [Integrated human practice](https://2021.igem.org/Team%3ACCU_Taiwan/Human_Practices)
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  + [Inclusivity](https://2021.igem.org/Team%3ACCU_Taiwan/Inclusivity)
* [Notebook](https://2021.igem.org/Team%3ACCU_Taiwan/Notebook)
* [Safety](https://2021.igem.org/Team%3ACCU_Taiwan/Safety)
* [Awards](https://2021.igem.org/Team%3ACCU_Taiwan/Awards)
  + [Medals](https://2021.igem.org/Team%3ACCU_Taiwan/Medals)
  + [Judging form](https://igem.org/2021_Judging_Form?id=3778)

![](https://static.igem.org/mediawiki/2021/b/b2/T--CCU_Taiwan--bigpic-experiment.png)

* [Swelling test](#Swelling test)
* [Trombin test](#Trombin test)

# Swelling test

## Overview

The absorptive layer is the middle layer of our antimicrobial dressing, which is responsible for absorbing exudate secreted from the wounds. Therefore, the absorptive layer should fullfit two basic characteristics, the high integrity and good absorbability.

After the lecture reviewing, we found that alginate and chitosan are famous for their biocompatible, biodegradable, and bioactive properties. Moreover, the mixture of alginate and chitosan has high absorbability and non-toxic properties, which make them ideal materials for constructing an absorptive layer.

Accordingly, we decided to crosslink alginate with chitosan as our material of absorptive layer. The integrity of alginate and chitosan mixture is dependent on the mixture ratio. Additionally, the mixture ratio of alginate and chitosan also affects the absorbability. Therefore, we conduct a swelling test to optimize the mixing ratio of alginate and chitosan to generate an absorptive layer with high integrity and absorbability.

## Definition of Integrity

To unbiasedly monitor the integrity of alginate and chitosan formed sponge (CA-sponge), we record the light image of the CA-sponge using black paper as background. The images are recorded with fixed angle, light, and size (the left panel in Figure 1). Because the black background will show in the region with low integrity, we then analyze the black region of each image by Image J (the middle panel of Figure 1). The integrity is defined as the ratio of white area to all area. For example, if the ratio of withe area is more than 95%, the integrity is higher than 95%.

## Definition of absorbability

To determine the absorbability, we incubate the dry CA-sponge with the same weight in pH 7.4 of phosphate buffer saline (PBS) at room temperature for 1 hour, and the swelling rate of CA-sponges was determined and calculated as follows (Figure 2).

![](https://static.igem.org/mediawiki/2021/archive/9/96/20210912122412%21T--CCU_Taiwan--子揚.png)

![](https://static.igem.org/mediawiki/2021/archive/9/96/20210915124658%21T--CCU_Taiwan--子揚.png)

#### ▲ Figure 1. The first standard---We used imagrJ to divide white/black area, and make sure white area is over 95%.

![](https://static.igem.org/mediawiki/2021/archive/9/96/20210915125213%21T--CCU_Taiwan--子揚.png)

#### ▲ Figure 2. The second standard---We went through swelling test to get swelling rate, and the above is process.

# Trombin test

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# Team:CCU Taiwan/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)