Team:NYMU-Taipei/Poster

Off the Crown: A Quest to Dethrone the Notorious Coronavirus
Presented by Team NYMU Taipei 2020

You-Cheng Lee¹, Yu-Te Lan¹, Jo-An Hsiung¹, Terrance Yu-Hao Chen¹, Yu-Chuan Yang¹, Huang-Yen Hsun¹, Jia-Yin Chen¹, Tao-Wei Chang¹, Chun-Yi Wang¹, Chieh-Yun Peng¹, You-Sheng Huang¹, Yu-Fan Lin¹, Hsin-Pei Yang¹, Yi-Lin Liao², Bo-Han Li², Ching-Fen Chang³, Chuan-Hsiung Chang³, and National Yang-Ming University, Taipei, Taiwan§

¹iGEM Student Team Member, ²iGEM Team Advisor, ³iGEM Team Instructor, §Faculty Sponsor

Abstract

The “crown” symbolizes the novel coronavirus that has wreaked havoc in 2020. It is a symbol of the virus in biological terms and a symbol of its dominance in societal terms. As of today, the number of total global cases is around 50 million, and the number of global deaths passing 1 million. The prevalence of COVID-19 is mainly through its transmission mechanics. Close interactions such as direct or indirect contact of respiratory droplets and aerosols with human mucous membranes are the major routes of transmission. Infections through fecal-oral routes or contaminated routes are also possible.

Of the structural proteins of SARS-CoV-2, spike protein is the main focus of our project studies. Spike is composed of a transmembrane trimetric glycoprotein protruding from the viral surface, which determines the diversity of coronaviruses and host tropism. SARAS-CoV-2 enters human cells via spiking binding to human angiotensin-converting 2 (hACE2). This property of the virus is our project inspiration.

Off the Crown wants to capture and even destroy virus particles that may come in close contact with individuals. Our plan is to implement our designed product on surgical masks, contact lenses, and even filtration devices to reduce the possibility of infection.


What we expect to achieve:
  1. Lower infection chance
    Lower the virus’s infection rate on humans and the disease occurence.
  2. Protect
    Improve products that can enhance human protection from the virus such as surgical masks and contact lenses.
  3. Alert
    Alert humans of the virus’s presence through a visual signal.


Project Goals
Capture and diminish the amount of viruses that comes into contact with human beings, and, thus, provide first-line protection for the world in this fatal pandemic.

Implement our design on surgical masks, contact lenses, and our detection box (debux) as to create a positive impact on the public’s everyday life.

Screen the potential virus carriers in the near future. Provide measures to retaliate against pandemics that might happen in the future.

Inspiration

We were inspired from the inconvenience brought by COVID-19 from the start of the year. All meetings, lab activity and even the trip to Boston have been affected by this prevailing pandemic. In order to overcome this difficulty, we started to do research on COVID-19’s characteristics, which brought us to the following odyssey of synthetic biology.



Design and Experiment Results

To fight against the spreading of COVID-19, most scientists focus on either developing antibodies that deactivate the SARS-CoV-2 virus or vaccines that trigger immune responses of the human body. However, NYMU Taipei aims to tackle the problem in a more fundamental way via the construction of the following parts, which highlights our contribution to the iGEM community.


  1. hACE2 receptor binding domain (part BBa_K3682002)

    One of our aims is to capture the SARS-CoV-2 virus outside the human body. In order to achieve this goal, we need a construct that may bind to the spike protein attached to the surface of SARS-CoV-2. According to former research findings, the spike protein on the surface of SARS-CoV-2 binds with human angiotensin-converting enzype 2 (hACE2) and enters host cells. This infection process of SARS-CoV-2 serves as a great inspiration for us. Utilizing this characteristic, we chose to use the receptor-binding domain (RBD) of hACE2 as the capturer of SARS-CoV-2. By adding the hACE2 RBD-like construct to our design, SARS-CoV-2 virus particles may be trapped before entering human bodies since it is bound to our specifically designed protein construct.

    2. successful bacterial expression hACE2 RBD plasmid construction
  2. pepP protease (part BBa_K3682001)

    Another goal of our project is to deactivate the virus. To achieve this goal, we chose aminopeptidase P (pepP) as our weapon to deactivate the spike protein on the surface of SARS-CoV-2. According to the data we attained from our MEROPS crawler software, we discovered that there exists four cleavage sites for pepP on the spike protein. Therefore, we are strongly convinced that the spike protein will be destroyed or inactivated after being cleaved by pepP, our protease of choice.

    3. successful bacterial expression pepP plasmid construction
    successful SDS-page result of expressed pepP(51.53kDa)
  3. hACE2-pepP fusion protein construct (part BBa_K3682008)

    In order to capture and deactivate the virus simultaneously, we designed a fusion protein construct consisting of both the hACE2 receptor binding domain and pepP, both of which are connected by a flexible polypeptide linker. With this fusion protein, a spike protein can be successfully cleaved right after being captured by our construct.

    4. illustration of pepP-hACE2 construct cleaving spike protein
    successful bacterial expression pepP-hACE2 plasmid construction
  4. hACE2-RFP fusion protein construct

    Detecting the presence of SARS-CoV-2 is also a major part of our project. After capturing the virus particle with our hACE2 receptor binding domain, we use a construct with hACE2 RBD linked with red fluorescent protein(RFP) to detect the virus by adding it into the detection area. With hACE2 RBD-RFP fusion protein bound to the spike protein on the virus particle, a signal of red fluorescence will be emitted to indicate the presence of the virus.

    5. `
  5. Hydrogel for histag chelation

    To achieve our goal, we need a surface that immobilizes our protein construct on it. Therefore, we refer to Ni-NTA his tag protein purification column and design our gel that chelates and holds his tagged protein on the gel surface.

    To specify, we modify polyHEMA (Polyhydroxyethylmethacrylate) hydrogel with KMnO4 and CuSO4 to create the surface similar to Ni-NTA column. To proof that our gel works as the column, we add his tagged green fluorescent protein to the gel and rinse with wash buffer afterwards. The successful results of green fluorescence existing on our gel is proved by the use of fluorescent microscope. Also, all of the constructs we use to immobolize on the gel are subjoined with a histag on their C terminal and supposely they will also be immobolized on our hydrogel surface.

    6. green fluorescence observed indicating a successful his tag chelation of our hydrogel

Modeling and Software
Software

We have developed an in-house tool ‘MEROPS Crawler’ wrote in python to figure out the protease cleavage site profile of proteins. MEROPS Crawler is deposited on Github( https://github.com/YHTerrance/Merops-Crawler ). With this in house tool, we identified Xaa-Pro aminopeptidase (pepP) for this project.



Docking

The purpose of performing docking simulations is to make sure that the selected proteins will interact as expected before the wet lab experiment. In our design, we conducted three combinations of protein-protein docking. The docking of spike and pepP, the docking of ACE2 and pepP, and the interaction between ACE2 and spike. The following is our protocol:

  1. Load protein and define pockets
  2. Select pocket and load ligand
  3. Dock and calculate affinity


Docking Results

The docking between spike and pepP

There are lots of pockets on a spike protein (Figure 1). After filtering through this sequence, we selected the pocket with the sequence Pro-Phe in order (in the reference, it stated that pepP would cut the sequence Pro-Phe into two pieces), and found the site which is mostly on the exterior of the protein. Then, we load pepP as a ligand and perform docking. As for docking analysis, we found that there are several poses which show high affinity without clashing.

From this outcome, we could conclude that our protease (pepP) construct has a high possibility of binding to the spike protein and destroying it as expected.



The docking between ACE2 and pepP

Though there are 10 pockets on the ACE2 RBD, there is only one pocket that contains a Pro-Phe ordered sequence. Fortunately, that pocket is the only pocket located in the core instead of on the exterior. We then use the pocket to dock ACE2 with pepP. Results show that there is still a chance for ACE2 RBD to bind with pepP. To avoid this, we make a point mutation to turn YAA into AAA, which would stop pepP from cleaving ACE2.



Interaction between ACE2 and spike

Using ClusPro (a web-based protein-protein docking server), we generated different clusters and discovered that one of the clusters, cluster 24, is similar to what previous research have discovered[1], which proves that our experiment is likely to produce the similar binding mechanics between normal ACE2 and spike.



Monitoring Spike Protein

Through monitoring spike protein, we can see how often the spike protein mutates and what mutations are most prevalent. We also visualized the mutation sites through protein structural modeling to see where exactly on the spike protein structure do the mutations potentially occur. In our analysis, two types of mutations were more frequently seen.



D614G Mutation

After collecting, preprocessing, and alignment of SARS-CoV-2 spike sequences, a graph showing incidence record of mutations for site 614 on SARS-CoV-2 Spike Protein sequence before and after March 1st based on location was made. We found that the emergence and spread of D614G was seen in various locations in Taiwan.



T791I Mutation

Incidence record of mutations for site 791 on SARS-CoV-2 Spike protein sequence before and after March 1st based on location showed that T791 mutation only showed up briefly and was only seen in one area of Taiwan.



Structural Visualization of Mutation Sites

We also modeled the protein structure of spike protein to see which regions did the mutations occur.



Reference: [1]Lan, J., Ge, J., Yu, J. et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature 581, 215–220 (2020)
Application
  1. Contact lens
    Goal: To reduce infection through the eye.
    Design: The main material of contact lens is polyHEMA, which is the hydrogel we have chosen to make our gel. To prevent the protein from falling off or blocking our vision when putting on the contact lens, we only made the non-central area undergo functional group conversion. Contrarily, the center area without proteins will be 6mm in diameter.

  2. Mask
    Goal: To inactivate SARS-CoV-2 so that viruses will not transmit through the contact of masks.
    Design: Put a piece of hydrogel with hACE2-pepP fusion protein between layers of the mask. When a virus makes contact with the gel, hACE2 will capture the virus, while the protease, pepP, should be able cut the spike protein on SARS-CoV-2 into pieces to prevent the binding of SARS-CoV-2 with human cells.

  3. Debux: Our in-house detection device
    Goal:Detect the presence of SARS-CoV-2 in a subject’s breathe immediately through a portable and affordable device.
    Design: First, let the subject breathe through the hose connected to the upper box where the ACE2 membrane is at the bottom of the upper box. Then, add ACE2-RFP fusion protein to the upper box. By doing so, viruses would bind with ACE2-RFP. Next, shake the box for 3 minutes. Afterwards, open the water box and wash the membrane twice to make sure unbound ACE2 with RFP construct are removed. The waste liquid would be collected in the lower box. Eventually, if the membrane turns red, it means that the subject’s breath contains the virus.

  4. MEROPS Crawler: Protease cutting site finder
    Goal:Find suitable proteases to inactivate SARS-CoV-2’s spike protein.
    Design: A program that crawls protease data from MEROPS, a well-known protease database. Peptidases will align with target proteins such as SARS-CoV-2 spike protein and ACE2 according to their cleavage sites. Our software sorted peptidases according to their possible cleavage sites on ACE2-RBD, in order to keep hACE2’s binding function. Next, we find the peptidase which has the maximum cleavage sites on spike protein so as to inactivate SARS-CoV-2 to the most extent. After all, pepP was the best choice among all the peptidases.
    Furthermore: This python code is now available on github. We enhanced its power by adjusting it to compare any proteins as long as the user enters its sequence. The results will tell us the cleavage site profile of the protein by different proteases. Therefore, we can select the protease that best fits our need.
    Github:YHTerrance/Merops-Crawler

Human Practices

Human Practices, which includes consulting experts, understanding corporative goals, and collecting field surveys, is an important part when it comes to enabling our project to connect with the real world more tightly. The followings illustrate what we have done.


  1. Our team walked out of the laboratory and took to the streets to discuss COVID-19 with people. Based on the survey we made, we learned that in order to make the public accept our product, our product should not only be convenient but also be able to provide protection against infection.

  2. We went to a hospital and interviewed a doctor working in the frontlines to get some feedback, which gave us the idea to design an equipment that protects us from SARS-CoV-2.

  3. While working on our project, our team discussed with a lot of experts in different fields such as material science and modeling. By doing so, we found both problems and solutions in different parts of our project and was able to it.

  4. What’s more, we also visited a contact lens company and a face mask factory. Through interactions with those companies, we learned about how their products were made, which also gave us inspirations in our own product designs. On the other hand, the companies also gained inspirations from us, such as feedback on issues related with company responsibility to the public and strong passion for research as well.

Future Directions

To capture and inactivate SARS-CoV-2, we focus on the inactivation of the spike proteins on SARS-CoV-2. Thus, we have created a database crawling and searching software - “MEROPS Crawler” with an aim to find the most suitable protein for our experiment. This particular software is not only designed for our project to fight against COVID-19 but can also be used for other pathogens as well. Whenever somebody wants to find a protease to act on a specific protein, our software would be their best resort as we can identify the cleavage sites and compare them with different proteases and sequences simultaneously.

We also designed a 3D-printed detection box which is portable and can attain fast pathogen detection, which holds a great edge over the current mainstream screening method (PCR). This way, we could aid the government in conducting preliminary screening at airports and other occasions where people are in high risk of being infected.

Lastly, our product is expected to protect high-risk groups such as doctors and customs staffs since they are more likely to be exposed to the contagious virus. For example, we wish to combine our gel design with current COVID-19 testing kits to significantly lower the rate of cluster infections for people working at the forefront.


Acknowledgements and Sponsors
Acknowledgements
Doctor Chan Yu-Jiun
Associate Professor Szu-Hao Kung
Associate Professor Yeou-Guang Tsay
Professor Sheh-Yi Sheu
Assistant Professor Chung Te Chang
Associate Professor Hsiao-Hui Lee
Professor Wolfgang Fischer
Associate Professor Yuan-Min Lin
Teacher Assistant Nian Zhong Yang
Teacher Assistant Tzu Han Hsu
Teacher Assistant Chong Rui Zhang
Professor Yung-Chuan Liu
Professor Ma Che Alex


Sponsors