Difference between revisions of "Team:CCU Taiwan/Poster"

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We took a step back to seek an alternative. We aimed to find something smaller and easier to produce to detect dengue virus instead of using antibodies. Symptoms of DHF and DSS result from interaction between C-type lectin domain, family 5, member A (CLEC5A) on macrophages and the envelope protein (E proteins) of dengue virus.
 
We took a step back to seek an alternative. We aimed to find something smaller and easier to produce to detect dengue virus instead of using antibodies. Symptoms of DHF and DSS result from interaction between C-type lectin domain, family 5, member A (CLEC5A) on macrophages and the envelope protein (E proteins) of dengue virus.
 
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We found the sequences from CLEC5A most likely to bind the dengue virus and confirmed this using Rosetta simulations. We believe these peptides have potential to detect the dengue virus.
 
We found the sequences from CLEC5A most likely to bind the dengue virus and confirmed this using Rosetta simulations. We believe these peptides have potential to detect the dengue virus.
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Figure 2. 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).
 
Figure 2. 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).
 
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<b>Current Progress of DENDETX:</b>
 
<b>Current Progress of DENDETX:</b>
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We consulted with government officials, experts, and academic specialists to consider the application of our project in society (Figure 1, Figure 2).
 
We consulted with government officials, experts, and academic specialists to consider the application of our project in society (Figure 1, Figure 2).
 
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Figure 1. Consulting with Dr. De-Syuan Chen and Dr. Jin-Yu Lee from TFDA.
 
Figure 1. Consulting with Dr. De-Syuan Chen and Dr. Jin-Yu Lee from TFDA.
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This shows that the public in Taiwan lack common understanding about dengue, which leads to low awareness about the severity of this disease in Taiwan. This urged us to conduct outreach and education programs to popularize knowledge of dengue fever.
 
This shows that the public in Taiwan lack common understanding about dengue, which leads to low awareness about the severity of this disease in Taiwan. This urged us to conduct outreach and education programs to popularize knowledge of dengue fever.
 
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Figure 1. Public knowledge of dengue fever.
 
Figure 1. Public knowledge of dengue fever.
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we created a children’s storybook to spark children’s interest in science. To reach to more children and increase the impact of our storybook, we translated it into English, Chinese, and Hindi.
 
we created a children’s storybook to spark children’s interest in science. To reach to more children and increase the impact of our storybook, we translated it into English, Chinese, and Hindi.
 
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Revision as of 11:56, 9 November 2020

DENDETX
  • Team member:
    Chia-Wei Shen, Jyue-Han Chen, Xiang-Yen Wong, Chiau-Shu Pan, Hong-Fu Liao, Yuan-Zhi Li, Young-Cheng Lin, Chen-Shing Tseng, Li-Yun Cheng, Ching-Chia Kuo, Yu-Shiuan Chang, Wen-Han Chang, Xin-Yu Chang, En-Chi Chang

  • Instructors:
    Eugene C. Lin, Hau-Ren Chen, Chun-Ying Yu, Cheng-I Lee, Victoria Rau, Gerald Rau, Yen-Ta Tseng

  • Advisors:
    Cheng-Yu Lee, Wei-Cheng Wang, Hooi-Hui Khor, Tzu-Cheng Chuang, Meng-Jing Lee, Yu-Xian Cheng

  • Sponsors:
    National Chung Cheng University, Academic Foundation Chung Cheng University, Association of CCU Alumni, 國立中正大學之友會, Integrated DNA Technologies, Software for molecular biology, Benching, MathWorks
Project Goals
  1. Develop a rapid test for dengue fever to improve treatment outcomes.
  2. Develop a method to mass-produce peptides for virus recognition, reducing cost.
  3. Raise awareness of dengue fever in scientifically illiterate and high-risk groups.
Introduction
Dengue fever occurs in tropical and subtropical regions. Half of the world population is now at risk, with 390 million infections every year, causing dengue fever to be listed by WHO as a top 10 health threat.

Dengue fever can progress into more serious conditions like dengue hemorrhagic fever (DHF), which has a 20% mortality rate. If conditions continue to worsen, patients experience dengue shock syndrome (DSS), which has a mortality rate up to 40%. An estimated 500,000 patients develop DHF or DSS, resulting in about 25,000 deaths every year.

Taiwan is in the area under the threat of dengue virus. From 2014 to 2015, there were over 44,000 confirmed cases during a dengue fever outbreak that spread from Tainan to other cities, causing 228 deaths. Recently, there was local transmission of dengue fever in Taoyuan, showing that the potentially affected population keeps increasing.

There is no specific treatment or vaccine for dengue, and health care systems are still facing a challenge from dengue fever.
Inspiration: Interaction between the CLEC5A and E protein suggest proteins might be effective in detection
After infection by the dengue virus, a nonstructural protein, NS1, appears in blood serum early in the infection. The current method of detecting dengue fever uses antibodies to recognize NS1 antigens. Nevertheless, the cost of the antibodies is high, the antibodies are hard to purify, and the procedure of making antibodies is time-consuming.

We took a step back to seek an alternative. We aimed to find something smaller and easier to produce to detect dengue virus instead of using antibodies. Symptoms of DHF and DSS result from interaction between C-type lectin domain, family 5, member A (CLEC5A) on macrophages and the envelope protein (E proteins) of dengue virus.

We found the sequences from CLEC5A most likely to bind the dengue virus and confirmed this using Rosetta simulations. We believe these peptides have potential to detect the dengue virus.

We designed a detection kit with these peptides that can bind to the E protein of dengue virus. This would allow people who may have dengue fever or live in an outbreak region of dengue fever to get treatment or protection and control the spread early.
Linear Array Epitope
Linear array epitope (LAE) is a technique to produce tandem-repeated sequences (TRSs). After the procedure, the TRSs would be expressed as a long-chain peptide which can be cleaved by an enzyme, resulting in many identical peptides.

The Procedure of LAE:
  1. Design DNA oligo primers.
  2. Template-repeated PCR (TR-PCR).
  3. Adaptor PCR (AD-PCR).

For the primer design, TR-PCR primers need to be partially complementary to each other and AD-PCR primers need to have restriction sites.

In TR-PCR, those primers act as both primer and template and they are partially complementary. The TRSs can be produced during the PCR cycling process. In AD-PCR, restriction sites need be introduced into the products of TR-PCR by adding the AD-PCR primers in PCR.
Parts
The BioBrick of TRSs is composed of T7 promoter, lac operator, thrombin site, TRSs, and His tag.


The BioBrick of E protein is composed of a T7 promoter, lac operator, thrombin site, E protein gene, HA tag, and His tag.


The BioBrick of CLEC5A is composed of a T7 promoter, lac operator, thrombin site, CLEC5A gene, Myc tag, and His tag.


Our peptides and proteins were expressed in E. Coli with T7 promotor and lac operator, which can be induced by IPTG. The thrombin site was included to remove unnecessary sections. The His tag was used in Western blot to confirm the expression of protein, and the HA tag and myc tag were utilized for purification.
Modeling: Interaction between the tandem-repeated sequence peptide and E protein
Purpose:
Our goal is to design a peptide of tandem-repeated sequence (PTRSs) to imitate the binding of CLEC5A to the E protein from the dengue virus. In order to ensure the PTRSs and the E protein interact, all the structure of PTRSs and proteins and their interactions were modeled using Rosetta.

Method and Results:
  1. The structure of E protein (Figure 1) from a local strain (PL046) was generated using RosettaCM (comparative modeling with Rosetta) based on the crystal structure (PDB: 1OAN).
  2. The structures of PTRSs were predicted purely based on their sequences using the ab initio method in Rosetta, and the representative structures are shown in Figure 2.
  3. The most probable structure of the PTRS were identified by clustering.
  4. The interactions between the PTRSs and the E protein were evaluated using global protein-protein docking. The 100 most frequent docking sites are shown in Figure 3.


Figure 1. The homologous structure of PL046 E protein (cyan) based on the deposited structure (PDB: 1OAN) magenta.


Figure 2. The representative structures of a PTRS.


Figure 3. The best 100 results (based on the Rosetta score) of PTRS docking to PL046 E protein (red). The space above the plane (grey) indicates the external surface of the virion, where the interactions occur.

Modeling: Interaction between Gold Nanoparticles
Purpose:
A potential weakness of our design is that peptides on the gold nanoparticles and on the glass fiber compete for the same binding sites on the E protein. If the E proteins on the virus particles are fully covered by gold nanoparticles, there would be no sites available to interact with the peptides attached to the glass fiber. Hence, we need to understand if this situation would hinder development of our kit.

Method:
We used DLVO theory to calculate the repulsion between gold nanoparticles to estimate the number of gold nanoparticles that would bind to a virus particle. The structure of dengue virus is icosahedral, and there are nine E proteins on each surface. The distances between the potential binding sites can be obtained from the structure in the protein data bank (1K4R).
DLVO theory can be described as follows,

Wtotal(D) = Wa(D) + Wr(D) = -AR/12D + 2πεε0RΨδ2exp(-κD)

Wtotal(D): total energy
Wa(D): van der Waals interaction energy
Wr(D): electrostatic interaction energy
A: Hamaker constant = 2.5×10-19 J
R: radius = 1.3x10-8 m (based on the size of gold nanoparticles)
C: center to center distance
D: C-R
ε: permittivity of vacuum = 8.854x10-12 F/m
ε0: dielectric of solution = 80.4 (at 20°C)
Ψδ2: Stern layer potential (zeta potential) = 1.794 mV
κ-1: diffuse layer thickness = 7.95x10-10 m

We took several representative positions on adjoining faces of the icosahedron to calculate the interactions between the gold nanoparticles based on DLVO theory.

Results:
The total energies are all positive, indicating repulsive forces, (Table 1) and these total energies are also larger than the typical biological interactions (~0.5 kcal/mol or 3.49 x10-21 J). The results suggest that there will always be free faces on the virus particles to interact with the peptides conjugated on the test line.

Table 1. Energies of the gold nanoparticles between representative positions.
The Detection Kit - DENDETX
Content of DENDETX and Materials Used:
  1. Sample pad (cellulose fibers)
  2. Conjugate pad (glass fibers)
  3. Test line (glass fibers)
  4. Control line (glass fibers)
  5. Absorbent pad (cellulose fibers)
The conjugate pad contains the gold nanoparticles (AuNPs) coupled with the first PTRS (PTRS-1), in which PTRS-1 is designed to interact with the virions. The second PTRS (PTRS-2) is mounted on the test line to capture the virion. E proteins are conjugated on the control line, which should always interact with PTRS-1, to ensure the kit functions normally.

Sample without Dengue Virions:
The AuNPs will not accumulate on the test line, and no color change will be found. The AuNPs will aggregate on control line via the interaction of PTRS-1 and E protein, so the control line turns red.

Sample with Dengue Virions:
The AuNPs bound to the virions via PTRS-1 will accumulate on the test line, and the residual AuNPs will aggregate on the control line. Both the test and control lines will be red.

Method:
The AuNPs were first modified with MUA/MCH so that carboxyl groups are exposed on the surface of the particles. Then, peptides with the amine sidechains could be conjugated to the AuNPs using the EDC/NHS approach. Similarly, the glass fiber membranes, the substrate of the test and control lines, were modified with CES to form a carboxyl surface, which allows us to employ the EDC/NHS approach to conjugate with peptides or proteins.

Results:
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 with the modified AuNPs. We found that with the DNA conjugation, the Raman signals have a significant decrease (Figure 1). Although we have no model to explain this effect, we believe it resulted from interactions with the DNA, suggesting DNA can bind to AuNPs.

Figure 1. 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.

We expressed green fluorescent protein (GFP) as a mock E protein to show that after modification the glass fiber membrane can bind to the primary amines (side-chain amines) from a peptide or protein. The intensity of membrane reaction with 0.5x and 1x GFP supernatant are shown in Figure 2. The intensity was about twice as strong in the 1x supernatant as the 0.5x one, suggesting the conjugation experiments were successful.

Figure 2. 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).

Current Progress of DENDETX:
We have shown that we are able to conjugate peptides/proteins to the AuNP, control line, and test line.
Results
LAE
The results of TR-PCR (Figure 1) indicate the length of TRS varies with the concentration of the primers. We found the typical trend that the lower the concentration, the higher the repeats of the TR-PCR products. However, the product concentration decreases when the primer concentration increases. TRS-110*3 and TRS-151*7, with sizes ranging from 200-500 bp, were taken to AD-PCR. The ladders shown in Figure 2 are the products obtained from AD-PCR. The AD-PCR product of TRS-110*3 TRS-151*7 with sizes ranging from 200-500 bp were extracted and ligated to TA vectors.
(a)
(b)
Figure 1. The TR-PCR results of TRS-151*7 (a) and TRS-110*3 (b). The value listed on each represents the concentration of primer (in μM). M = DNA marker.
(a)
(b)
Figure 2. The results of AD-PCR of TRS-151*7 (a) and TRS-110*3 (b). The value listed on each lane represents the concentration of primer (in μM). M = DNA marker.

E Protein
We expressed E protein to use it in the control line.

Figure 3 shows the size of E protein we induced is 61.2 kDa. The weak bands from 63 to 48 kDa indicate expression of the E protein with the HA tag, which has a size of 61.2 kDa. However, the concentration is so weak that it is hard to see.

Figure 3. SDS-PAGE of E protein from a small-scale culture. After induction with IPTG, the strong bands at about 61.2 kDa indicate the expression of E protein. M = protein marker. NI = Non-Induction. I = Induction.

Figure 4 shows the result confirmed with Western blot. It shows that anti-His tag binds to the His tag, suggesting that the protein expression was successful.

Figure 4. 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 both non-induction and induction lanes suggests the experiments were successful. NI = Non-Induction. I = Induction.

CLEC5A
In addition to the required E protein used in our kit, we attempted to express CLEC5A as a model protein to interact with E protein.

Figure 5 shows the results from four different colonies with and without induction. The strong bands on 26.6 kDa indicate expression of the CLEC5A extracellular domain with the Myc tag, which has a size of 26.6 kDa.

Figure 5. SDS-PAGE of CLEC5A from a small-scale culture. After induction with IPTG, the strong bands at about 26.6 kDa indicate the expression of CLEC5A. M = protein marker. NI = Non-Induction. I = Induction.

Figure 6 is the result confirmed with western blot. It shows that anti-His tag binds to the His tag, suggesting that the protein expression was successful.

Figure 6. 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.
Human Practices
Interviews
Purpose:
  1. We wish to understand public views on dengue and our project.
  2. We want to shape our project from different perspectives to achieve socially responsible research.
Process:
We consulted with government officials, experts, and academic specialists to consider the application of our project in society (Figure 1, Figure 2).

Figure 1. Consulting with Dr. De-Syuan Chen and Dr. Jin-Yu Lee from TFDA.

Figure 2. Learning the way on modification of gold nanoparticles from Dr. Yen-Ta Tseng.

Achievement:
  1. We understood the process and the standards for verification for medical devices from the Taiwan Food and Drug Administration (TFDA).
  2. We learned about pretreatment of blood sample from The Dengue Prevention and Control Center.
  3. We comprehended the modification of gold nanoparticles after consulting with experts in the Department of Chemistry and Biochemistry at National Chung Cheng University.
Public Survey
Purpose:
Find out how much the public knows about dengue and their demands for a dengue virus detection kit.
Process:
We created a questionnaire, and we received 248 questionnaires by sharing the questionnaire online and distributing it in the community.
Achievement:
The results showed nearly 70% of people did not know about severe dengue and nearly 70% did not know what kind of treatment is required for dengue fever (Figure 1).

This shows that the public in Taiwan lack common understanding about dengue, which leads to low awareness about the severity of this disease in Taiwan. This urged us to conduct outreach and education programs to popularize knowledge of dengue fever.


Figure 1. Public knowledge of dengue fever.
We determined that our detection kit meets their needs, as it should have high sensitivity during the first five days of symptoms, which is when people would consult a doctor.

Education

Podcast
Podcast has become a popular platform to convey knowledge in Taiwan. In order to reach the general public, we created a podcast program with 7 episodes talking about synthetic biology.

Dengue Fever Information in Taiwanese Hokkien
Taiwan is considered an aged society, as the aged population reached 16% in 2018. We invited the elderly in our community help us record a dengue fever information audio in Taiwanese Hokkien, targeting senior citizens, who mostly use this local dialect.

Children’s Storybook
we created a children’s storybook to spark children’s interest in science. To reach to more children and increase the impact of our storybook, we translated it into English, Chinese, and Hindi.


Educational Activity
We went to the Cheng-He Confucian academic center, a religious center where children study Confucianism, teaching them basic ideas about biology.

Radio Interview
We were invited by National Education Radio to introduce our project and the iGEM competition.
Inclusion
Inmates

Purpose:
We wanted to reach out to communities that are underrepresented in science to allow them to participate in science.
Process:
We went to a juvenile correction school to popularize science, as juvenile inmates lack opportunities to learn science (Figure 1).

Figure 1. Science instruction at a juvenile correction school.

Achievement:
We found that inclusion in science communication is a two-way street. Science should include communities who lack access to science by eliminating the access barriers with diverse and engaging ways to bring science closer to them. In order the experience the beauty of science, underrepresented communities must overcome conventional thinking that science is too difficult.

Glove Puppetry

Purpose:
Glove puppetry is a traditional performing art in Taiwan with its own unique elements such as using the language most spoken by Taiwanese elderly, Taiwanese Hokkien, for narration. This inspired us to integrate this folk art into science communication. We thought that a non-scientific artist could also provide a different perspective on science.

Process:
We collaborated with a glove puppetry troupe to transmit knowledge of dengue by narrating and filming a story as a 13-minute video (Figure 2).

Figure 2. Glove puppetry presentation of dengue fever.

Achievement:
We realized that continuous development of science requires adding diverse fields and perspective to make it more widely recognized, just as glove puppetry integrates modern elements to keep up with the times.
References
Introduction
  1. Taiwan National Infectious Disease Statistics System - Taiwan Centers of Disease Control (TCDC), https://nidss.cdc.gov.tw/old/en/
  2. Dengue and severe dengue - World Health Organization, https://www.who.int/news-room/fact-sheets/detail/dengue-and-severe-dengue
  3. Dengue Fever – Taiwan Centers of Disease Control (TCDC), https://www.cdc.gov.tw/Category/ListContent/bg0g_VU_Ysrgkes_KRUDgQ?uaid=9_Oq7OYHa-l8B05iUwyVvQ

Inspiration
  1. Chen, S., Li, F., Hsu, T. et al. CLEC5A is a critical receptor in innate immunity against Listeria infection. Nat Commun 8, 299 (2017). https://doi.org/10.1038/s41467-017-00356-3
  2. Watson AA, Lebedev AA, Hall BA, Fenton-May AE, Vagin AA, Dejnirattisai W, Felce J, Mongkolsapaya J, Palma AS, Liu Y, Feizi T, Screaton GR, Murshudov GN, O'Callaghan CA. Structural flexibility of the macrophage dengue virus receptor CLEC5A: implications for ligand binding and signaling. J Biol Chem. 2011 Jul 8;286(27):24208-18. doi: 10.1074/jbc.M111.226142. Epub 2011 May 12. PMID: 21566123; PMCID: PMC3129202.

LAE
Peng-Yeh Lai, Chia-Tse Hsu, Shao-Hung Wang, Jin-Ching Lee, Min-Jen Tseng, Jaulang Hwang, Wen-Tsai Ji, Hau-Ren Chen. Production of a neutralizing antibody against envelope protein of dengue virus type 2 using the linear array epitope technique. Journal of General Virology (2014), 95, 2155–2165

Modelling: Interaction between the tandem-repeated sequence peptide and E protein
  1. Elena Pokidysheva, Ying Zhang, Anthony J. Battisti, Carol M. Bator-Kelly, Paul R. Chipman, Chuan Xiao, G. Glenn Gregorio, Wayne A. Hendrickson, Richard J. Kuhn, Michael G. Rossmann. Cryo-EM Reconstruction of Dengue Virus in Complex with the Carbohydrate Recognition Domain of DC-SIGN. Cell Press, 124(3). doi:10.1016
  2. Jörg Polte. Fundamental growth principles of colloidal metal nanoparticles – a new perspective. CrystEngComm, 36. doi:10.1039
  3. Phillip E. Mason, Adrien Lerbret, Marie-Louise Saboungi, George W. Neilson, Christopher E. Dempsey, John W. Brady. Glucose Interactions with a Model Peptide. NCBI, 79(7). doi:10.1002
  4. Taehoon Kim, Kangtaek Lee, Myoung-seon Gong, Sang-Woo Joo. Control of Gold Nanoparticle Aggregates by Manipulation of Interparticle Interaction. ACS Publications. doi:10.1021