iGEM 2020 — Team CCU_Taiwan

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# Team:CCU Taiwan/Notebook

[![](https://static.igem.org/mediawiki/2020/7/7e/T--CCU_Taiwan--logo.png)](https://2020.igem.org/Team%3ACCU_Taiwan)

* Team

  [Team Members](https://2020.igem.org/Team%3ACCU_Taiwan/Team)
  [Attributions](https://2020.igem.org/Team%3ACCU_Taiwan/Attributions)
  [Contribution](https://2020.igem.org/Team%3ACCU_Taiwan/Contribution)
  [Sponsors](https://2020.igem.org/Team%3ACCU_Taiwan/Sponsors)
* Project

  [Description](https://2020.igem.org/Team%3ACCU_Taiwan/Description)
  [Implementation](https://2020.igem.org/Team%3ACCU_Taiwan/Implementation)
  [Design](https://2020.igem.org/Team%3ACCU_Taiwan/Design)
  [Engineering](https://2020.igem.org/Team%3ACCU_Taiwan/Engineering)
  [Model](https://2020.igem.org/Team%3ACCU_Taiwan/Model)
  [Safety](https://2020.igem.org/Team%3ACCU_Taiwan/Safety)
* Results

  [Results](https://2020.igem.org/Team%3ACCU_Taiwan/Results)
  [Improvements](https://2020.igem.org/Team%3ACCU_Taiwan/Improvement)
  [Prospects](https://2020.igem.org/Team%3ACCU_Taiwan/Future)
* Parts

  [Parts](https://2020.igem.org/Team%3ACCU_Taiwan/Parts)
* Human Practices

  [IHP](https://2020.igem.org/Team%3ACCU_Taiwan/Human_Practices)
  [Science Communication](https://2020.igem.org/Team%3ACCU_Taiwan/Education)
  [Inclusivity](https://2020.igem.org/Team%3ACCU_Taiwan/Inclusion)
  [Collaborations](https://2020.igem.org/Team%3ACCU_Taiwan/Collaborations)
* Notebook

  [Notebook](https://2020.igem.org/Team%3ACCU_Taiwan/Notebook)
  [Protocols](https://2020.igem.org/Team%3ACCU_Taiwan/Protocols)
* Awards

  [Awards](https://2020.igem.org/Team%3ACCU_Taiwan/Awards)
  [Criteria](https://2020.igem.org/Team%3ACCU_Taiwan/Criteria)

# Notebook

Week1 (04/05-04/11)

#### WL

---

Understand the project and discuss the procedures of experiment.

#### DL

---

Search for the principles and materials of the NS1 detection kits on the market.

#### HP

---

Schedule the progress of HP.

Week2 (04/12-04/18)

#### WL

---

1. Find the principle of LAE and experimental procedures.
2. Confirm the interaction between the CLEC5A and E protein. (in progress)

#### DL

---

1. Search for the structures and binding regions of the envelope protein (E protein) and CLEC5A.
2. Learn how to use Chimera.

#### HP

---

Search for the current products on market.

Week3 (04/19-04/25)

#### WL

---

1. Optimize the experimental procedure of LAE.
2. Confirm the sequence and structure of E protein and design the procedure of CLEC5A expression.

#### DL

---

Use Chimera to simulate the docking of E protein and CLEC5A.

#### HP

---

Analyze the pros and cons between our product and the current products.

Week4 (04/26-05/02)

#### WL

---

1. Design the TR-PCR primers. (in progress)
2. Confirm the interaction between the CLEC5A and E protein. (done)

#### DL

---

1. Analyze the results of Chimera.
2. Learn how to use Rosetta.

#### HP

---

Virtual meetup with NCTU\_Formosa iGEM.

Week5 (05/03-05/09)

#### WL

---

1. Design the TR-PCR primers and choose the plasmid for expression.
2. Design the procedure of E protein expression, E protein and CELC5A’s primers and choose the plasmid for expression. (in progress)

#### DL

---

1. Search for the principles of the NS1 antigen test and pregnancy test and design our detection kit.
2. Try to use the *ab initio* method to produce the peptides and the FlexPepDock method to simulate peptide and protein docking by using Rosetta.

#### HP

---

Fund raising for project.

Week6 (05/10-05/16)

#### WL

---

1. Design the TR-PCR, E protein and CLEC5A primers. (done)
2. Choose the plasmid for E protein and CLEC5A expression. (E protein-pET-29a (+), CLEC5A-pET-29b (+))

#### DL

---

1. Design each component of the detection pads.
2. Practice the FlexPepDock method to simulate peptide and protein docking.
3. Generate fragments which is the input of producing the peptides.

#### HP

---

Conduct the survey on dengue.

Week7 (05/17-05/23)

#### WL

---

1. Practice the instrument operating.
2. Culture the DH5α-pET29b, DH5α-pCMV6-CLEC5A and DH5α-pKz-V5 tag-C-CI-prM-E-(HA tag)2

#### DL

---

1. Search for the concentration of each stage of dengue virus viremia.
2. Use the *ab initio* method to produce the peptides.

#### HP

---

Conduct the survey on dengue.

Week8 (05/24-05/30)

#### WL

---

1. Culture DH5A-pCMV6-CLEC5A.
2. Extract the pET-29b (+) & pCMV6-CLEC5A & pKz-V5 tag-C-CI-prM-E-(HA tag)2.

#### DL

---

1. Discuss the production of the detection kit with Professor Chau, Lai-Kwan.
2. Search for the nature of gold nanoparticles and how to attach gold nanoparticles to the peptides.
3. Use the *ab initio* method to produce the peptides.
4. Utilize the Mutation method to produce E protein (PL046).

#### HP

---

1. Build the lesson plan of synthetic biology on podcast.
2. Contact department of health in Kaohsiung city government.

Week9 (05/31-06/06)

#### WL

---

1. TR-PCR 110 & design the AD-primers.
2. Plasmid extraction of pCMV6-CLEC5A.

#### DL

---

1. Discuss how to attach gold nanoparticles to the peptides, the size of gold nanoparticles and nonspecific adsorption of the reagent with Postdoctor Tseng, Yen-Ta.
2. Fill in the biosafety form.
3. Use the *ab initio* method to produce the peptides.
4. Utilize the Mutation method to produce E protein (PL046).

#### HP

---

1. Contact Dr.Jiang, Yi-huah.
2. Contact Center for Dengue Prevention and Control, Tainan City Government.

Week10 (06/07-06/13)

#### WL

---

Design the AD-primers. (done)

#### DL

---

1. Use the *ab initio* method to produce the peptides.
2. Utilize the Mutation method to produce E protein (PL046).

#### HP

---

Visit Dr.Jiang, Yi-huah.

Week11 (06/14-06/20)

#### WL

---

Optimize the conditions of TR-PCR.

#### DL

---

1. Use the *ab initio* method to produce the peptides.
2. Utilize the Mutation method to produce E protein (PL046).

#### HP

---

Contact Department of Health, Kaohsiung City Government.

Week12 (06/21-06/27)

#### WL

---

Optimize the conditions of TR-PCR.

#### DL

---

1. Use the *ab initio* method to produce the peptides.
2. Utilize the Mutation method to produce E protein (PL046).

#### HP

---

1. Contact Center for Dengue Fever Control and Research, Kaohsiung Medical University Chung-Ho Memorial Hospital.
2. Contact National Mosquito-Borne Diseases Control Research Center.

Week13 (06/28-07/04)

#### WL

---

1. TR-PCR 151(done) & TR-PCR 110.
2. Amplify the CLEC5A and E protein.

#### DL

---

1. Confirm the source of the materials of the detection kit.
2. Design the home page of wiki.
3. Use the *ab initio* method to produce the peptides.
4. Utilize the RosettaCM method to produce E protein (PL046).
5. Deal with the carbohydrate on the E protein.

#### HP

---

1. Record the first episode of podcast.
2. Build the lesson plan of education on Mingyang High School.

Week14 (07/05-07/11)

#### WL

---

1. TR-PCR 151(done), TR-PCR 110. (done) and AD-PCR 151 and AD-PCR 110 (done).
2. TA-CLEC5A cloning.

#### DL

---

1. Modify the component of the control line.
2. Use the *ab initio* method to produce the peptides.
3. Utilize the RosettaCM method to produce E protein (PL046).
4. Deal with the carbohydrate on the E protein.

#### HP

---

Virtual meetup with NCTU \_Formosa iGEM.

Week15 (07/12-07/18)

#### WL

---

1. TA-110, TA-151, TA-151R cloning and TA-110, TA-151, TA-151R colony PCR.
2. TA-CLEC5A colony PCR & plasmid extraction (TA-CLEC5A) & DNA sequencing. (M13F(R)-TA-CLEC5A)

#### DL

---

1. Search for the ways of modeling by 3D printer.
2. Confirm the component of the control line.
3. Use the *ab initio* method to produce the peptides.
4. Utilize the RosettaCM method to produce E protein (PL046).
5. Deal with the carbohydrate on the E protein.

#### HP

---

1. Visit TFDA.
2. Visit Center for Dengue Prevention and Control, Tainan City Government.
3. Visit National Mosquito-Borne Diseases Control Research Center.
4. Education at Cheng-He Confucian Academic Center.

Week16 (07/19-07/25)

#### WL

---

1. TR-PCR 110R. (done)
2. Sequencing M13F(R)TA-151 and M13F(R)TA-151R.
3. Digest TA-CLEC5A, ligate CLEC5A to pET-29b (+) and transform pET-29b (+)-CLEC5A into DH5α.
4. pET-29b (+)-CLEC5A colony PCR, TA-E protein cloning and cultivatepET-29a (+).

#### DL

---

1. Design the layout of wiki.
2. Design the project promotion video.
3. Arrange the content of the poster.
4. Use the *ab initio* method to produce the peptides.
5. Utilize the RosettaCM method to produce E protein (PL046).
6. Use the Protein-protein Docking method to simulate peptide and E protein docking.

#### HP

---

1. Visit Department of Health, Kaohsiung City Government.
2. Visit Center for Dengue Fever Control and Research, Kaohsiung Medical University Chung-Ho Memorial Hospital.
3. Virtual meetup with iGEM CSMU Taiwan.
4. Virtual meetup with Team iGEM IISER Berhampur

Week17 (07/26-08/01)

#### WL

---

1. AD-PCR 110R& AD-PCR TA-151R (done), TA-110R cloning & TA-110R colony PCR.
2. Transform pET-29b (+)-CLEC5A into BL21(DE3) and sequencing (M13F(R)-TA-E protein).

#### DL

---

1. Design the home page of wiki.
2. Design the project promotion video.
3. Arrange the content of the poster.
4. Use the *ab initio* method to produce the peptides.
5. Utilize the RosettaCM method produce E protein (PL046).
6. Use the Protein-protein Docking method to simulate peptide and E protein docking.

#### HP

---

1. Design the project logo.
2. Record the second episode of podcast.
3. Compose the children storybook.

Week18 (08/02-08/08)

#### WL

---

1. Digest TA-110 and TA-151 & ligate 110 & 151 to pET-29b (+).
2. Sequencing (M13F(R)TA-110R) and AD-PCR TA-151R (done).
3. Express CLEC5A with 1μM IPTG and run SDS-PAGE.

#### DL

---

1. Design the layout of the poster.
2. Shoot the member introduction of the project promotion video.
3. Calculate the force between gold nanoparticles.
4. Use the *ab initio* method to produce the peptides (110-3 and 151-8).
5. Use the Protein-protein Docking method to simulate peptide (110-3) and E protein (PL046) docking.

#### HP

---

1. Virtual meetup with Dr.Liang, Shih-Yuan.
2. Taiwan National Education Radio interview.
3. Visit senior citizen service center for dengue fever informational audio recording in Taiwanese Hokkien.

Week19 (08/09-08/15)

#### WL

---

1. Culture DH5A-pET-29b-110 & DH5A-pET-29b (+)-151.
2. Run the CLEC5A western blot.

#### DL

---

1. Make the poster.
2. Shoot and edit the member introduction of the project promotion video.
3. Print the model for NCTU and the detection kit by using a 3D printer.
4. Calculate the force between gold nanoparticles.
5. Use the Protein-protein Docking method to simulate peptide (110-3) and E protein (PL046) docking.

#### HP

---

Education at Cheng-He Confucian Academic Centre.

Week20 (08/16-08/22)

#### WL

---

1. pET-29b (+)-110 colony PCR & pET-29b-151 colony PCR.
2. Ligate E protein into pET-29a (+) & transform pET-29a (+)-E protein into DH5α and pET-29a (+)-E protein colony PCR.

#### DL

---

1. Shoot and edit the member introduction and the beginning of the project promotion video.
2. Calculate the force between gold nanoparticles.
3. Done the template of wiki.
4. Print the team name by using a 3D printer.
5. Use the Clustering method to deal with the result of the peptide.
6. Use the Protein-protein Docking method to simulate peptide (151-8) and E protein (PL046) docking.

#### HP

---

1. Record the third episode of podcast.
2. Taiwan iGEM conference.

Week21 (08/23-08/29)

#### WL

---

1. Optimize the procedure from digestion of TA-110 to pET-29b(+)-110 colony PCR.
2. Express the CLEC5A in large scale and find that protein was expressed in the pellet.

#### DL

---

1. Done the member introduction and the beginning of the project promotion video.
2. Code the home page.
3. Use the Clustering method to deal with the result of the peptide.
4. Use the Protein-protein Docking method to simulate protein and protein docking for NYMU.

#### HP

---

1. Contact with Mingyang High School.
2. Virtual meetup with Team iGEM IISER Berhampur.
3. Virtual meetup with NYMU Taipei.

Week22 (08/30-09/05)

#### WL

---

1. Ligate 151R to pET-29b (+).
2. Optimize the procedure of TA-110 with pET-29b ligation and the procedure from transformation pET-29a (+)-E protein into DH5A to protein colony PCR.

#### DL

---

1. Code the wiki page of safety and sponsors.
2. Use the Clustering method to deal with the result of the peptide.
3. Use the Protein-protein Docking method to simulate peptides and E protein (PL046) docking.
4. Shoot and edit the project promotion video.
5. Do the gold nanoparticles liquidity experiment.

#### HP

---

Contact with glove puppetry show troupe.

Week23 (09/06-09/12)

#### WL

---

Optimize the condition of IPTG induction in CLEC5A expression.

#### DL

---

1. Shoot and edit the project promotion video.
2. Code the wiki page of description, notebook and team members.
3. Design and do the experiment of modifying gold nanoparticles and glass fiber.
4. Analysis the result of the clustering result.

#### HP

---

1. Record the fourth episode of podcast.
2. Mentoring Mingdao High School.

Week24 (09/13-09/19)

#### WL

---

1. Culture DH5α-pET-29b(+)-110 & DH5A-pET-29b (+)-151.
2. pET-29b (+)-110 colony PCR & pET-29b (+)-151 colony PCR.

#### DL

---

1. Shoot and edit the project promotion video.
2. Code the wiki page of notebook and team members.
3. Design and do the experiment of modifying gold nanoparticles and glass fiber.

#### HP

---

1. Record the fifth episode of podcast.
2. Mentoring Mingdao High School.

Week25 (09/20-09/26)

#### WL

---

1. Culture DH5α-pET-29b(+)-110 & DH5A-pET-29b (+)-151.
2. pET-29b (+)-110 colony PCR & pET-29b (+)-151 colony PCR.
3. Do the digest and ligation again, transform to DH5α and do the colony PCR.

#### DL

---

1. Edit the project promotion video.
2. Do the experiment of modifying gold nanoparticles and glass fiber.

#### HP

---

1. Contact with glove puppetry show troupe.
2. Glove puppetry show filming.

Week26 (09/27-10/03)

#### WL

---

1. Optimize the procedure from digestion of TA-151 to pET-29b(+)-151 colony PCR.
2. Do the digest and ligation again, transform to DH5α and do the colony PCR.

#### DL

---

1. Code the wiki page of contribution, parts and collaborations.
2. Changing from MUA/MCH to MHA/SBSH & EDC to DCC.

#### HP

---

1. SDG panel discussion.
2. Education at Mingyang High School.
3. Record the sixth episode of podcast.

Week27 (10/04-10/10)

#### WL

---

1. Optimize the procedure from digestion of TA-151 to pET-29b(+)-151 colony PCR.
2. Optimize the procedure from digestion of TA-110 to pET-29b(+)-110 colony PCR.
3. Culture DH5α-pET-29b(+) and optimize the quality of plasmid pET-29b(+).
4. Do the digest and ligation again, transform to DH5α and do the colony PCR.

#### DL

---

1. Code the wiki page of design and model.
2. Design the narrative thread of the presentation video.

#### HP

---

1. Education at Mingyang High School.
2. Record the seventh episode of podcast.

Week28 (10/11-10/17)

#### WL

---

Do the digest and ligation again, transform to DH5α and do the colony PCR.

#### DL

---

1. Code the wiki page of education, implementation and protocols.
2. Revise the script of the presentation video.
3. Design the storyboard of the presentation video.

#### HP

---

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# Team:CCU Taiwan/Engineering

[![](https://static.igem.org/mediawiki/2020/7/7e/T--CCU_Taiwan--logo.png)](https://2020.igem.org/Team%3ACCU_Taiwan)

* Team

  [Team Members](https://2020.igem.org/Team%3ACCU_Taiwan/Team)
  [Attributions](https://2020.igem.org/Team%3ACCU_Taiwan/Attributions)
  [Contribution](https://2020.igem.org/Team%3ACCU_Taiwan/Contribution)
  [Sponsors](https://2020.igem.org/Team%3ACCU_Taiwan/Sponsors)
* Project

  [Description](https://2020.igem.org/Team%3ACCU_Taiwan/Description)
  [Implementation](https://2020.igem.org/Team%3ACCU_Taiwan/Implementation)
  [Design](https://2020.igem.org/Team%3ACCU_Taiwan/Design)
  [Engineering](https://2020.igem.org/Team%3ACCU_Taiwan/Engineering)
  [Model](https://2020.igem.org/Team%3ACCU_Taiwan/Model)
  [Safety](https://2020.igem.org/Team%3ACCU_Taiwan/Safety)
* Results

  [Results](https://2020.igem.org/Team%3ACCU_Taiwan/Results)
  [Improvements](https://2020.igem.org/Team%3ACCU_Taiwan/Improvement)
  [Prospects](https://2020.igem.org/Team%3ACCU_Taiwan/Future)
* Parts

  [Parts](https://2020.igem.org/Team%3ACCU_Taiwan/Parts)
* Human Practices

  [IHP](https://2020.igem.org/Team%3ACCU_Taiwan/Human_Practices)
  [Science Communication](https://2020.igem.org/Team%3ACCU_Taiwan/Education)
  [Inclusivity](https://2020.igem.org/Team%3ACCU_Taiwan/Inclusion)
  [Collaborations](https://2020.igem.org/Team%3ACCU_Taiwan/Collaborations)
* Notebook

  [Notebook](https://2020.igem.org/Team%3ACCU_Taiwan/Notebook)
  [Protocols](https://2020.igem.org/Team%3ACCU_Taiwan/Protocols)
* Awards

  [Awards](https://2020.igem.org/Team%3ACCU_Taiwan/Awards)
  [Criteria](https://2020.igem.org/Team%3ACCU_Taiwan/Criteria)

# Engineering

* [Inspiration](#ins)
* [Highlights](#high)
* [The Core Design](#des)
* [The Lateral-Flow Based Kit](#kit)

* **Abbreviations**
* CLEC5A: C-type lectin domain, family 5, member A
* E protein: dengue virus envelope protein
* TRS: tandem-repeated sequence
* PTRS: peptide of tandem-repeated sequence
* TR-PCR: template-repeated PCR

## Inspiration

We noticed that dengue fever is a severe disease, which could affect almost half the world. We learned through literature and reports that dengue hemorrhagic fever and dengue shock syndrome have high mortality rates. These symptoms result from the interaction between C-type lectin domain, family 5, member A (CLEC5A) on macrophages and the envelope protein
(E proteins) of dengue virions.

Imagine if there are enough proteins or antibodies to “disturb” the CLEC5A – dengue interaction, just like “smoke grenades” in our body? That would be a fantastic treatment option. It would take a long time to achieve, though.

Instead, we thought about using peptides as “peptides cloud” to detect virions outside the body since they are small and can be produced much faster than antibodies. The goal is to implement this mechanism on a detection kit. We summarize the accomplishments to achieve our idea.

## Engineering Success

1. **We obtained the TRSs using LAE.**
2. **We expressed E protein and CLEC5A.**
3. **We conjugated primary amines to the glass fiber membranes and AuNPs.**

## The Core Design

We designed two parts, which we called TRS-1 and TRS-2, using the binding site of CLEC5A as a reference. We use TR-PCR as a method of producing these the PTRSs, which takes primers as PCR templates and produces a long chain of repeating sequences of peptide DNA. There is a problem that the primer concentration is highly sensitive to the TRS chain length (repeat times). We found the lower the concentration the longer the chain that will be produced.

![](https://static.igem.org/mediawiki/2020/c/cb/T--CCU_Taiwan--Engineering1.png)

Tandem-repeated sequence (TRS)

In the next stage of the experiment, we use pET-29b(+) as a vector of TRS-1 and TRS-2 to transform *E.coli* DH5α to clone more TRS. After that, we then transform the chosen TRS into *E.coli* BL21 (DE3) to express PTRS. *E.coli* DH5α has an property of high transformation efficiency, and *E.coli* BL21 (DE3) can express protein or peptide effectively. So, we choose these two different strains to do different parts.

To ensure that our PTRS can bind to E protein, we design the experiment to check it. Therefore, we also do the experiment to express the E protein and CLEC5A extracellular domain as the positive control in this experiment. We ended up expressing CLEC5A extracellular domain and E protein.

![](https://static.igem.org/mediawiki/2020/9/9b/T--CCU_Taiwan--Engineering2.png)

C-type lectin domain, family 5, member A (CLEC5A)

![](https://static.igem.org/mediawiki/2020/f/fc/T--CCU_Taiwan--Engineering3.png)

Dengue virus envelope protein (E protein)

In the initial design, we planned to add an arginine sequence to the end of the peptide sequence, which could be cleaved by trypsin at the arginine C-terminal of the peptide. Unfortunately, based on a calculation of the electrostatic force of gold nanoparticles, we decided we should use a longer PTRS instead of the PTRS monomer because there is an electrostatic force between the gold nanoparticles, so if one gold nanoparticle with TRS interacts with an E protein on the virion, the other gold nanoparticles might find it difficult to interact with the E protein. Thus, we designed the TR-PCR primer without an arginine sequence to produce the PTRS and chose the size that is most likely to interact with dengue E proteins, according to Rosetta simulations.

## The Lateral-Flow Based Kit

Our rapid diagnostic test kit, **DENDETX**, is referred to the pregnancy test. DENDETX consists of the sample pad, conjugate pad, test line, control line, and absorbent pad. The sample pad and the absorbent pad are made of cellulose fiber membranes, and the conjugate pad, test line, and control line are made of glass fiber membranes.

The difference between DENDETX and the pregnancy test kit is that we replaced the antibodies by PTRSs. Conventionally, the substrate for the test and control lines is nitrocellulose, it forms the dipole-dipole interaction with the antibodies. In order to have a strong conjugation of PTRSs to the test and control lines, we used glass fiber membranes instead of nitrocellulose membranes. We can chemically modify the glass membranes, which forms the strong covalent bonds with PTRSs.

For the modification of the glass fiber membranes, we first utilized the oxygen plasma treatment to remove the impurities and contaminants from the surfaces of membranes. The treatment generates silanol groups (−SiOH) fully covers the surface of the glass fiber membranes. In the beginning, we planned to use APTES to modify the glass fiber membranes, which have been processed with oxygen plasma treatment, so that the silanols on the surface of the glass fiber membranes will be replaced by primary amines. Then, we can use the EDC/NHS approach to conjugate the carboxylic groups of CM-Dextran to the membrane surface with amines. Finally, the PTRSs could be conjugated to the available carboxylic groups of CM-Dextran again use the EDC/NHS approach again.

However, we realized the hydroxyls CM-Dextran would have non-specific interactions to the compositions in the liquid sample, which would mislead the results. In order to reduce the uncertainty of the experiment, we decided to use CES to replace CM-Dextran. To the best of our knowledge, there is no record of modifying glass fiber membranes using CES. We have successfully conjugated CES to the glass fiber membrane and PTRSs to CES using the EDC/NHS approach.

![](https://static.igem.org/mediawiki/2020/e/e4/T--CCU_Taiwan--Engineering4.png)

**Abbreviations**: APTES: (3-aminopropyl)tiethoxysilane; EDC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; NHS: N-hydroxysuccinimide; CM-Dextran: carboxymethyl-dextran; CES: carboxyethylsilanetriol

---

#### References

Tung YT, Wu MF, Wang GJ, Hsieh SL. Nanostructured electrochemical biosensor for th0065 detection of the weak binding between the dengue virus and the CLEC5A receptor. Nanomedicine. 2014 Aug;10(6):1335-41. doi: 10.1016/j.nano.2014.03.009. Epub 2014 Mar 24. PMID: 24674971.
Alen, M. M. F., & Schols, D. (2012). Dengue Virus Entry as Target for Antiviral Therapy. Journal of Tropical Medicine, 2012, 1–13. doi:10.1155/2012/628475
Fahimi, H., Mohammadipour, M., Haddad Kashani, H. et al. Dengue viruses and promising envelope protein domain III-based vaccines. Appl Microbiol Biotechnol 102, 2977–2996 (2018). https://doi.org/10.1007/s00253-018-8822-y
Mondotte JA, Lozach PY, Amara A, Gamarnik AV. Essential role of dengue virus envelope protein N glycosylation at asparagine-67 during viral propagation. J Virol. 2007 Jul;81(13):7136-48. doi: 10.1128/JVI.00116-07. Epub 2007 Apr 25. PMID: 17459925; PMCID: PMC1933273.
Sprokholt, J. K. (2019). Dengue virus infection of dendritic cells: Sensing viral replication during inflammation and immunity.
Sung PS., Chang WC., Hsieh SL. (2020) CLEC5A: A Promiscuous Pattern Recognition Receptor to Microbes and Beyond. In: Hsieh SL. (eds) Lectin in Host Defense Against Microbial Infections. Advances in Experimental Medicine and Biology, vol 1204. Springer, Singapore. https://doi.org/10.1007/978-981-15-1580-4\_3
del Fresno C, Iborra S, Saz-Leal P, Martínez-López M and Sancho D (2018) Flexible Signaling of Myeloid C-Type Lectin Receptors in Immunity and Inflammation. Front. Immunol. 9:804. doi: 10.3389/fimmu.2018.00804
Huang, Y., Chen, S., Liu, R. et al. CLEC5A is critical for dengue virus-induced osteoclast activation and bone homeostasis. J Mol Med 94, 1025–1037 (2016). https://doi.org/10.1007/s00109-016-1409-0
Teng, O., Chen, S. T., Hsu, T. L., Sia, S. F., Cole, S., Valkenburg, S. A., Hsu, T. Y., Zheng, J. T., Tu, W., Bruzzone, R., Peiris, J., Hsieh, S. L., & Yen, H. L. (2016). CLEC5A-Mediated Enhancement of the Inflammatory Response in Myeloid Cells Contributes to Influenza Virus Pathogenicity In Vivo. Journal of virology, 91(1), e01813-16. https://doi.org/10.1128/JVI.01813-16
Miller, J. L., de Wet, B. J., Martinez-Pomares, L., Radcliffe, C. M., Dwek, R. A., Rudd, P. M., & Gordon, S. (2008). The mannose receptor mediates dengue virus infection of macrophages. PLoS pathogens, 4(2), e17. https://doi.org/10.1371/journal.ppat.0040017
Ishak, H., Takegami, T., Kamimura, K. and Funada, H. (2001), Comparative Sequences of Two Type 1 Dengue Virus Strains Possessing Different Growth Characteristics In Vitro. Microbiology and Immunology, 45: 327-331. doi:10.1111/j.1348-0421.2001.tb02627.x
Fuchs, A., Lin, T.-Y., Beasley, D. W., Stover, C. M., Schwaeble, W. J., Pierson, T. C., & Diamond, M. S. (2010). Direct Complement Restriction of Flavivirus Infection Requires Glycan Recognition by Mannose-Binding Lectin. Cell Host & Microbe, 8(2), 186–195. doi:10.1016/j.chom.2010.07.007
Lee, E., Weir, R. C., & Dalgarno, L. (1997). Changes in the Dengue Virus Major Envelope Protein on Passaging and Their Localization on the Three-Dimensional Structure of the Protein. Virology, 232(2), 281–290. doi:10.1006/viro.1997.8570
Mason, P. W., McAda, P. C., Mason, T. L., & Fournier, M. J. (1987). Sequence of the dengue-1 virus genome in the region encoding the three structural proteins and the major nonstructural protein NS1. Virology, 161(1), 262–267. doi:10.1016/0042-6822(87)90196-6
Osatomi, K., & Sumiyoshi, H. (1990). Complete nucleotide sequence of dengue type 3 virus genome RNA. Virology, 176(2), 643–647. doi:10.1016/0042-6822(90)90037-r
Crill, W. D., & Roehrig, J. T. (2001). Monoclonal Antibodies That Bind to Domain III of Dengue Virus E Glycoprotein Are the Most Efficient Blockers of Virus Adsorption to Vero Cells. Journal of Virology, 75(16), 7769–7773. doi:10.1128/jvi.75.16.7769-7773.2001
Johnson, A. J., Guirakhoo, F., & Roehrig, J. T. (1994). The Envelope Glycoproteins of Dengue 1 and Dengue 2 Viruses Grown in Mosquito Cells Differ in Their Utilization of Potential Glycosylation Sites. Virology, 203(2), 241–249. doi:10.1006/viro.1994.1481
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ROGERO, Celia, et al. Silicon surface nanostructuring for covalent immobilization of biomolecules. The Journal of Physical Chemistry C, 2008, 112.25: 9308-9314.
S. Bhattacharya, A. Datta, J. M. Berg and S. Gangopadhyay, "Studies on surface wettability of poly(dimethyl) siloxane (PDMS) and glass under oxygen-plasma treatment and correlation with bond strength," in Journal of Microelectromechanical Systems, vol. 14, no. 3, pp. 590-597, June 2005, doi: 10.1109/JMEMS.2005.844746.
CHEANG, Tuck-yun, et al. Promising plasmid DNA vector based on APTES-modified silica nanoparticles. International journal of nanomedicine, 2012, 7: 1061.
FISCHER, Marcel JE. Amine coupling through EDC/NHS: a practical approach. In: Surface plasmon resonance. Humana Press, 2010. p. 55-73.

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# Team:CCU Taiwan/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)

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# Team:CCU Taiwan/Results

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* Team

  [Team Members](https://2020.igem.org/Team%3ACCU_Taiwan/Team)
  [Attributions](https://2020.igem.org/Team%3ACCU_Taiwan/Attributions)
  [Contribution](https://2020.igem.org/Team%3ACCU_Taiwan/Contribution)
  [Sponsors](https://2020.igem.org/Team%3ACCU_Taiwan/Sponsors)
* Project

  [Description](https://2020.igem.org/Team%3ACCU_Taiwan/Description)
  [Implementation](https://2020.igem.org/Team%3ACCU_Taiwan/Implementation)
  [Design](https://2020.igem.org/Team%3ACCU_Taiwan/Design)
  [Engineering](https://2020.igem.org/Team%3ACCU_Taiwan/Engineering)
  [Model](https://2020.igem.org/Team%3ACCU_Taiwan/Model)
  [Safety](https://2020.igem.org/Team%3ACCU_Taiwan/Safety)
* Results

  [Results](https://2020.igem.org/Team%3ACCU_Taiwan/Results)
  [Improvements](https://2020.igem.org/Team%3ACCU_Taiwan/Improvement)
  [Prospects](https://2020.igem.org/Team%3ACCU_Taiwan/Future)
* Parts

  [Parts](https://2020.igem.org/Team%3ACCU_Taiwan/Parts)
* Human Practices

  [IHP](https://2020.igem.org/Team%3ACCU_Taiwan/Human_Practices)
  [Science Communication](https://2020.igem.org/Team%3ACCU_Taiwan/Education)
  [Inclusivity](https://2020.igem.org/Team%3ACCU_Taiwan/Inclusion)
  [Collaborations](https://2020.igem.org/Team%3ACCU_Taiwan/Collaborations)
* Notebook

  [Notebook](https://2020.igem.org/Team%3ACCU_Taiwan/Notebook)
  [Protocols](https://2020.igem.org/Team%3ACCU_Taiwan/Protocols)
* Awards

  [Awards](https://2020.igem.org/Team%3ACCU_Taiwan/Awards)
  [Criteria](https://2020.igem.org/Team%3ACCU_Taiwan/Criteria)

# Results

[![](https://static.igem.org/mediawiki/2020/3/3b/T--CCU_Taiwan--Results01.png)](#lae)

[![](https://static.igem.org/mediawiki/2020/3/35/T--CCU_Taiwan--Results02.jpg)](#epro)

[![](https://static.igem.org/mediawiki/2020/2/24/T--CCU_Taiwan--Results03.jpg)](#cle)

[![](https://static.igem.org/mediawiki/2020/d/d9/T--CCU_Taiwan--Results04.jpg)](#con)

[![](https://static.igem.org/mediawiki/2020/a/ae/T--CCU_Taiwan--Results05.jpg)](#avo)

* [Linear Array Epitope](#lae)
* [Envelope Protein](#epro)
* [CLEC5A](#cle)
* [Conjugations of Primary Amines to the Glass Fiber Membranes](#con)
* [Gold Nanoparticles](#avo)

## Linear Array Epitope

The results of template-repeated PCR (TR-PCR), shown in Figure 1, indicate that the length of the tandem-repeated sequence (TRS) varies with the concentration of the primer. We found the typical trend that the lower the concentration of primer, the higher the number of repeats of the TR-PCR products. However, the product concentration decreases when the primer concentration increases. In our project, the optimal primer concentrations are 0.2 μM for TRS-151\*7 and 0.08 μM for TRS-110\*3.

![](https://static.igem.org/mediawiki/2020/3/39/T--CCU_Taiwan--Results1a.png)

(a)

![](https://static.igem.org/mediawiki/2020/a/a4/T--CCU_Taiwan--Results1b.png)

(b)

Figure 1. The TR-PCR results of (a) TRS-151\*7 and (b) TRS-110\*3. The values listed on each represent the concentration of primer (in μM). The yellow arrow shows the typical ladder pattern of TR-PCR. M = DNA marker.

TRS-110\*3 and TRS-151\*7 obtained from TR-PCR were transferred to adaptor PCR (AD-PCR). The ladders shown in Figure 2 are the products obtained from AD-PCR. The sequences ranging in size from 200-400 bp and 50-200 bp, identified as TA-151 and TA-110, respectively, were extracted and ligated to TA vectors. Mutiple bands found in the results of AD-PCR due to the fact that there were several sites available for ligation of the adapter. Therefore, various lengths of sequences were generated during AD-PCR. These ligated sequences were then successfully transformed into DH5α competent cells. The colonies shown in Figure 3 suggest the bacteria survives due to the antimicrobial peptides (AMP) in the TA vector.

![](https://static.igem.org/mediawiki/2020/c/c6/T--CCU_Taiwan--Results2a.png)

(a)

![](https://static.igem.org/mediawiki/2020/b/bd/T--CCU_Taiwan--Results2b.png)

(b)

Figure 2. The results of AD-PCR of (a) TRS-151\*7 and (b) TRS-110\*3. The value listed on each lane represents the concentration of primer (in μM). M = DNA marker.

![](https://static.igem.org/mediawiki/2020/a/a8/T--CCU_Taiwan--Results3a.png)

(a)

![](https://static.igem.org/mediawiki/2020/6/62/T--CCU_Taiwan--Results3b.png)

(b)

Figure 3. The culture plates suggest that TA-151 (a) and and TA-110 (b) were successfully transformed into DH5α competent cells.

The results of colony PCR show that the sizes of TRS-151\*7 (Figure 4(a)) and TRS-110\*3 (Figure 4(b)) are estimated to be 420 bp and 300 bp, respectively.

![](https://static.igem.org/mediawiki/2020/7/7a/T--CCU_Taiwan--Results4a.png)

(a)

![](https://static.igem.org/mediawiki/2020/5/50/T--CCU_Taiwan--Results4b.png)

(b)

Figure 4. Colony PCR of (a) TRS151\*7 and (b) TRS110\*3. The sizes of TRS-151\*7 and TRS-110\*3 are estimated to be 420 bp and 300 bp (including the M13 primers and some sequences on the TA-vector), respectively. The numbers above the lanes indicate colony number. M = DNA marker.

Plasmids from these colonies were extracted, and TRS were obtained using the restriction sites of Ncol and Hindlll on the TA vector. TRS-151\*7 and TRS-110\*3 have sizes of 200-300 bp (Figure 5(a)) and 90-100 bp (Figure 5(b)), respectively, which fits our expectations.

![](https://static.igem.org/mediawiki/2020/a/aa/T--CCU_Taiwan--Results5a.png)

(a)

![](https://static.igem.org/mediawiki/2020/c/ce/T--CCU_Taiwan--Results5b.png)

(b)

Figure 5. The result of digestion of (a) TA-151 and (b) TA-110, which have sizes of 200-300 bp and 90-100 bp, respectively. M = DNA marker.

Finally, these TRSs were extracted and ligated to pET-29b(+). However, we ran into a problem during transformation of pET-29b(+) into DH5α. Currently, we are still seeking to optimize the transformation conditions.

## Envelope Protein (E protein)

A plasmid containing the whole structural protein of the dengue virus was obtained from National Health Research Institute. The Envelope protein (E protein) with two HA tags from the plasmid were amplified using PCR. Figure 6 shows that the sequence of the E protein with the HA tags were amplified, matching the expected size of 1,551 bp.

![](https://static.igem.org/mediawiki/2020/f/f9/T--CCU_Taiwan--Results6.jpg)

Figure 6. E protein with two HA tags, matching the expected size of 1,551 bp. M = DNA marker.

We prepared pET-29a(+) as the vector for expressing E protein and confirmed in Figure 7 that plasmid extraction shows the size of pET-29a(+) is about 5,000 bp, which is close to the theoretical size, 5,371 bp. Also, we transformed the pET-29a(+)\_E protein into DH5α and check the transformation using colony PCR. Figure 8 shows the size of E protein with two HA tags, and T7 promoter and terminator is about 2,000 bp, close to the theoretical size of 1,903 bp.

![](https://static.igem.org/mediawiki/2020/5/5b/T--CCU_Taiwan--Results7.jpg)

Figure 7. Plasmid extraction of pET-29a(+), matching the expected size of about 5,000 bp. M = DNA marker.

![](https://static.igem.org/mediawiki/2020/c/c9/T--CCU_Taiwan--Results8.jpg)

Figure 8. Colony PCR of pET-29a(+)\_E protein, which has a size of about 2,000 bp, containing E protein with two HA tags, and T7 promoter and terminator. M = DNA marker.

pET-29a(+)\_E protein was then transformed to BL21(DE3), cultured with 3 ml medium at 37 °C, and induced with 1.8 ml bacteria and 1 mM IPTG for 2 hours. Figure 9 shows the results from four different colonies with and without induction. The weak bands from 63 to 48 kDa indicate the E protein expression with the HA tag, which has a size of 61.2 kDa. However, it is too hard to see, and we are trying to improve the expression. Also, the bacterial culture was lysed with sonication, then separated with a high-speed centrifuge. It indicates that E protein found in the supernatant in Figure 10. The results were also confirmed using Western blot based on the HA tag, which is part of pET-29a(+). Figure 11 shows that the anti-His tag binds to the His tag, suggesting that the protein expression was successful.

![](https://static.igem.org/mediawiki/2020/e/e8/T--CCU_Taiwan--Results15.jpg)

Figure 9. SDS-PAGE of E protein from a small-scale culture. After induction with IPTG, the strong bands at about 61.270 kDa indicate the expressing of E protein. M = protein marker. NI = Non-Induction. I = Induction.

![](https://static.igem.org/mediawiki/2020/9/90/T--CCU_Taiwan--Results16.png)

Figure 10. SDS-PAGE of E protein from a small-scale expression. The bacterial solution was lysed with sonication and separated with a high-speed centrifuge. The results indicate E protein is expressed in the supernatant. M = protein marker. Sup. = supernatant.

![](https://static.igem.org/mediawiki/2020/e/ed/T--CCU_Taiwan--Results11new.jpg)

Figure 11. Western blot of E protein. An anti-His tag was used to bind to the His tag on the E protein. The stronger band on the both non-induction and induction lanes suggests the experiments were successful. NI = Non-Induction. I = Induction.

## CLEC5A

A plasmid containing the CLEC5A extracellular domain with a Myc tag was obtained from OriGene Technologies. The CLEC5A extracellular domain and Myc tag were amplified using PCR. Figure 12 shows that the sequence of the CLEC5A extracellular domain with a Myc tag were amplified successfully, with a size of 564 bp.
pET-29b(+) was prepared successfully as a vector, as shown in Figure 13, with a theoretical size of 5,370 bp. pET-29b(+)\_CLEC5A was obtained by inserting the CLEC5A into pET-29b(+) and then transforming it into DH5α which was confirmed using colony PCR.

![](https://static.igem.org/mediawiki/2020/7/76/T--CCU_Taiwan--Results9.jpg)

Figure 12. CLEC5A with a Myc tag, matching the expected size of about 600 bp. M = DNA marker.

![](https://static.igem.org/mediawiki/2020/8/8c/T--CCU_Taiwan--Results10.jpg)

Figure 13. Plasmid extraction of pET-29b(+), matching the expected size of about 5,000 bp. M = DNA marker.

Figure 14 shows the size of CLEC5A extracellular domain with Myc tag, and T7 promoter and terminator is about 900 bp, close to the theoretical size of 904 bp.

![](https://static.igem.org/mediawiki/2020/c/c1/T--CCU_Taiwan--Results11.jpg)

Figure 14. Colony PCR of pET-29b(+)\_CLEC5A, which has an expected size of about 900 bp including the CLEC5A extracellular domain and Myc tag and T7 promoter and terminator. M = DNA marker.

pET-29b(+)\_CLEC5A was then transformed to BL21(DE3) and induced to express protein with 1.8 ml bacteria and 1 mM IPTG for 2 hours. Figure 15 shows the results from four different colonies with and without induction. The strong bands near 28 kDa indicate expression of the CLEC5A extracellular domain with the Myc tag, which has a size of 26.6 kDa. The results were also confirmed using Western blot based on the HA tag, which is part of pET-29b(+). Figure 16 shows that the anti-His tag binds to the His tag, suggesting that the protein expression was successful.

![](https://static.igem.org/mediawiki/2020/0/02/T--CCU_Taiwan--Results12.jpg)

Figure 15. SDS-PAGE of CLEC5A from a small-scale culture. After induction with IPTG, the strong bands at about 26.6 kDa indicate the expressing of CLEC5A. M = protein marker. NI = Non-Induction. I = Induction.

![](https://static.igem.org/mediawiki/2020/5/56/T--CCU_Taiwan--Results13.jpg)

Figure 16. Western blot of CLEC5A. An anti-His tag was used to bind to the His tag on the CLEC5A protein. The stronger band on the induction lanes suggests the experiments were successful. NI = Non-Induction. I = Induction.

Finally, we expressed CLEC5A on a large scale. The bacterial culture was lysed using a French press, then separated with a high-speed centrifuge. Figure 17 indicates CLEC5A is always found in the pellets no matter how long they were induced.

![](https://static.igem.org/mediawiki/2020/7/79/T--CCU_Taiwan--Results14.jpg)

Figure 17. SDS-PAGE of CLEC5A large-scale expression. The protein was induced for 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours, separately. The bacterial solution was lysed with a French press and separated with a high-speed centrifuge. The results indicate CLEC5A is always expressed in the pellet under these conditions. M = protein marker. Sup. = supernatant.

## Conjugations of Primary Amines to the Glass Fiber Membranes

We expressed green fluorescent protein (GFP) as a mock E protein to show that that after modification, the glass fiber membrane can bind to the primary amines (side-chain amines) from a peptide or protein. We appreciate Mingdao iGEM 2020, who generously provided us with a plasmid containing the green fluorescent protein gene (GFPmut1, BBa\_K1159311). The modified glass fiber membranes were immersed in GFP supernatant from the culture medium for 30 mins to form amide bonds. To verify that the experiments work, we measured the intensity of the fluorescence from GFP using a Synergy H1 Hybrid Multi-Mode Microplate Reader (also provided by Mingdao iGEM 2020). The intensity of membrane reaction with 0.5x and 1x GFP supernatant are shown in Figure 18. We carefully washed the membranes with double-distilled water after the reaction. The intensity was about twice as strong in the 1x supernatant as the 0.5x one, suggesting the conjugation experiments were successful. We also performed the same experiment with non-modified glass fiber membranes as a control, and the intensity of fluorescence is within the range of measurement error.

![](https://static.igem.org/mediawiki/2020/c/c6/T--CCU_Taiwan--Results_dl1.png)

Figure 18. Fluorescence from modified glass fiber membranes reacting with 1x GFP stock, modified glass fiber membranes reacting with 0.5x GFP stock, and non-modified glass fiber membranes (control).

We also confirmed the reactivity of glass fiber membranes to amines using DNA primers (TRS-110R\*7, BBa\_K3648007) by measuring the fluorescence spectra with a plate reader (provided by Mingdao iGEM 2020). We used the same coupling conditions for GFP. Figure 19 shows that with the modification, the emission has a 0.5-fold enhancement in DNA conjugation.

![](https://static.igem.org/mediawiki/2020/3/38/T--CCU_Taiwan--Results_dl2.png)

Figure 19. The fluorescence spectra of DNA conjugated glass fiber membrane (blue), the control experiment (red), in which DNAs react with non-modified membranes, and the empty well (yellow). The spectra were obtained at an excitation of 320 nm.

## Gold Nanoparticles (AuNPs)

### Avoiding the aggregation of AuNPs

To conjugate the peptides to the AuNPs, pre-treatment of the AuNPs is necessary. However, during this process, aggregation of AuNPs could happen, which would result in failure of the experiment. We measured the sizes of AuNPs using dynamic light scattering shown in Figure 20.

First, we used MUA/MCH to modify 13-nm AuNPs by adding carboxylic groups to the surface. However, the AuNPs aggregated to a size of about 1,800 nm after we applied the EDC/NHS treatment. We found that if we replaced EDC with DCC, the size of AuNPs aggregation was reduced to about 350 nm. But 350 nm is not small enough, so we further tried replacing MUA/MCH with MHA /SB thiol, which was then treated with EDC/NHS. We obtained a significantly smaller size of 90 nm. We used these particles for the following experiments.

![](https://static.igem.org/mediawiki/2020/d/dd/T--CCU_Taiwan--Results_dl3.png.png)

Figure 20. The size distributions of AuNPs modified using MUA/MCH + EDC/NHS (blue), modified using MUA/MCH + DCC/NHS (red), and modified using MHA/SB thiol + EDC/NHS (yellow)

**Abbreviations:** MUA: 11-mercaptoundecanoic acid; MCH: 6-hydroxy-1-hexanethiol; EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; NHS: N-hydroxysuccinimide; DCC: N,N’-dicyclohexylcarbodiimide; MHA: 16-mercaptohexadecanoic acid; SB thiol: 1-(2-sulfosulfanylethylamino)tetradecane

### Conjugations of primary amines to AuNPs

As a proof of concept that we are able to form the covalent bonds between the primary amines (from the PTRS) and AuNPs, we tried to conjugate the DNA primers (TRS-110R\*7, BBa\_K3648007) with the modified AuNPs.

We used Raman spectrometer verify if the experiments worked. We found that with the DNA conjugation, the Raman signals have a significant decrease (Figure 21). Although we have no model to explain this effect, we believe it resulted from interactions with the DNA, suggesting DNA can bind to AuNPs.

![](https://static.igem.org/mediawiki/2020/a/aa/T--CCU_Taiwan--Results_dl4.png)

Figure 21. The Raman spectra of DCC/NHS modified AuNPs (yellow), and DCC/NHS modified AuNPs conjugated with 1 μM (blue) and 0.1 μM (red) DNA primers.

**Abbreviations:** DCC: N,N’-dicyclohexylcarbodiimide; NHS: N-hydroxysuccinimide

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