iGEM 2018 — Team Tokyo_Tech

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# Team:Tokyo Tech/Notebook

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[HOME](https://2018.igem.org/Team%3ATokyo_Tech)

TEAM

[Team Members](https://2018.igem.org/Team%3ATokyo_Tech/Team)
[Collaborations](https://2018.igem.org/Team%3ATokyo_Tech/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3ATokyo_Tech/Description)
[Design](https://2018.igem.org/Team%3ATokyo_Tech/Design)
[Experiments](https://2018.igem.org/Team%3ATokyo_Tech/Experiments)
[Notebook](https://2018.igem.org/Team%3ATokyo_Tech/Notebook)
[InterLab](https://2018.igem.org/Team%3ATokyo_Tech/InterLab)
[Model](https://2018.igem.org/Team%3ATokyo_Tech/Model)
[Results](https://2018.igem.org/Team%3ATokyo_Tech/Results)
[Demonstrate](https://2018.igem.org/Team%3ATokyo_Tech/Demonstrate)
[Improve](https://2018.igem.org/Team%3ATokyo_Tech/Improve)
[Attributions](https://2018.igem.org/Team%3ATokyo_Tech/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3ATokyo_Tech/Parts)
[Basic Parts](https://2018.igem.org/Team%3ATokyo_Tech/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3ATokyo_Tech/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3ATokyo_Tech/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3ATokyo_Tech/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3ATokyo_Tech/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3ATokyo_Tech/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3ATokyo_Tech/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3ATokyo_Tech/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3ATokyo_Tech/Hardware)
[Measurement](https://2018.igem.org/Team%3ATokyo_Tech/Measurement)
[Model](https://2018.igem.org/Team%3ATokyo_Tech/Model)
[Plant](https://2018.igem.org/Team%3ATokyo_Tech/Plant)
[Software](https://2018.igem.org/Team%3ATokyo_Tech/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=Tokyo_Tech)

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Tokyo Tech/Experiments

<!DOCTYPE html>

[![Tokyo Tech](https://static.igem.org/mediawiki/2018/1/14/T--Tokyo_Tech--logo13.png)](https://2018.igem.org/Team%3ATokyo_Tech/)
[Tokyo Tech](https://2018.igem.org/Team%3ATokyo_Tech/)

* [Project](https://2018.igem.org/Team%3ATokyo_Tech/Description)

  [Description](https://2018.igem.org/Team%3ATokyo_Tech/Description)
  [Parts](https://2018.igem.org/Team%3ATokyo_Tech/Parts)
  [Collaboration](https://2018.igem.org/Team%3ATokyo_Tech/Collaborations)
  [Achievement](https://2018.igem.org/Team%3ATokyo_Tech/Achievement)
* [Human Practice](https://2018.igem.org/Team%3ATokyo_Tech/Human_Practices)
* [Modelling](https://2018.igem.org/Team%3ATokyo_Tech/Model)
* [Experiments](https://2018.igem.org/Team%3ATokyo_Tech/Experiments)

  [Experiments](https://2018.igem.org/Team%3ATokyo_Tech/Experiments)
  [InterLab](https://2018.igem.org/Team%3ATokyo_Tech/InterLab)
* [About Us](https://2018.igem.org/Team%3ATokyo_Tech/Team)

  [Team](https://2018.igem.org/Team%3ATokyo_Tech/Team)
  [Attribution](https://2018.igem.org/Team%3ATokyo_Tech/Attributions)
  [Sponsor](https://2018.igem.org/Team%3ATokyo_Tech/Sponsor)

Experiments

[develop your own website](https://mobiri.se/k)

# Experiment

### Establishment of dengue virus serotype detection system

# Abstract

To identify which serotype of dengue virus your target patient is infected with, many methods have been explored and established. Since our prediction model requires the certain number of consensus time-scale data, it has been urgent to develop a high-throughput and cost-efficient tool for the serotype identification.

With the assistance of Dr. Suzuki at National Institute of Infectious Diseases, our team designed a new detection system with fluorescence protein. Three types of constructs (Genetic feactures: 1. C, 2. prM-E and 3. Fluorescence protein-FMDV2a-NS) should be prepared first. After that, they are introduced into HEK293T cells and pseudo-viruses are produced after a certain interval. They are added to cultured Vero cells to check the infectivity by measuring the intensity of fluorescence emitted from fluorescence protein that works as mono-cistron.

Since each one of the constructs contains either structural or non-structural region, the pseudo-virus just causes single-round infection. In terms of biosafety, our system and products that apply this system can be demonstrated even in the real world.

![Mobirise](https://static.igem.org/mediawiki/2018/0/07/T--Tokyo_Tech--presentation12.png)
![Mobirise](https://static.igem.org/mediawiki/2018/8/81/T--Tokyo_Tech--presentation13.png)

Figure 0: How Our Type Detection System Works

# Background

RT-PCR, ELISA and neutrilization assay - these methods have been widely used to identify infection experience and its serotype. However, RT-PCR is valid only for 0 to 5 days after infection and ELISA and neutralization assay have problem with cost-efficiency.

![Mobirise](https://static.igem.org/mediawiki/2018/a/a5/T--Tokyo_Tech--response_graphic.jpg)

Figure 1: Change in viral load and antibodies (Source: [Guzman, M. G. et al., 2010](https://www.nature.com/scitable/topicpage/host-response-to-the-dengue-virus-22402106))

![Mobirise](https://static.igem.org/mediawiki/2018/b/b0/T--Tokyo_Tech--presentation30.png)

Figure 2: Comparison of dengue detection system with existing ones

# Experiment Overview

Our experiment consists of three parts: 1. Construction, 2. Pseudo-virus Production and 3. Pseudo-virus Infection. After three types of constructs are introduced into HEK293T cells, pseudo-viruses are produced. They are added to cultured Vero cells, and you can check the infection after a certain interval.

![Mobirise](https://static.igem.org/mediawiki/2018/9/9e/T--Tokyo_Tech--presentation5.png)

Figure 3: Experiment Overview (iGEM Tokyo Tech 2018)

# 1. Construction

For the pseudo-virus production, we prepared structural gene, and non-structural gene with fluorescence protein gene.

About structural gene, capsid (C), membrane (prM) and envelope (E) are necessary for the formation of viral structure. We prepared pCAG-C and pCAG-prM-E for Serotype I to IV.

About non-structural gene with fluorescence protein gene, we prepared EGFP-FMDV2a, DsRed-Express-FMDV2a, ZsYellow-FMDV2a and AmCyan-FMDV2a, and inserted each of them to non-structural genes of dengue virus.

[Protocol](https://static.igem.org/mediawiki/2018/3/3a/T--Tokyo_Tech--protocol_construction.pdf)

![Mobirise](https://static.igem.org/mediawiki/2018/9/95/T--Tokyo_Tech--presentation40.png)

Figure 4: Construct line-up and original genes derived from dengue virus

# 2. Pseudo-virus Production

We introduced pCAG-C, pCAG-prM-E and FP-FMDV2a (FP: Fluorescence Protein) into HEK293T cells. After cell culture for about 2 days, we collect the supernatant that contains pseudo-viruses produced from HEK293T cells.

[Protocol](https://static.igem.org/mediawiki/2018/b/b3/T--Tokyo_Tech--protocol_transfection.pdf)

![Mobirise](https://static.igem.org/mediawiki/2018/9/91/T--Tokyo_Tech--presentation9.png)

Figure 5: Basic flow of transfection and pseudo-virus production

# 3. Pseudo-virus Infection and Functional Evaluation of Pseudo-virus

After collecting pseudo-viruses from HEK293T cells, we added them to Vero cells. When they can successfully infect the cells, the enclosed fluorescence protein gene is coded and we tried to estimate the amount by measuring the fluorescence intensity.
Since we split up the structural region and non-structural region, the pseudo-viruses produced in our system can infect host cells only once. Thus, it will not spread out of the system and will not be harmful.

[Protocol](https://static.igem.org/mediawiki/2018/6/63/T--Tokyo_Tech--protocol_infection.pdf)

![Mobirise](https://static.igem.org/mediawiki/2018/3/34/T--Tokyo_Tech--presentation10.png)

Figure 6: Basic flow of pseudo-virus infection and how fluorescence protein works

# Note: Validated Parts (Silver Medal Criteria)

DENV2 C, EGFP-FMDV2a and DsRed-Express-FMDV2a are essential for pseudo-virus production. As one of the structural genes, DENV2 C plays an important role in virus structural formation. EGFP-FMDV2a and DsRed-Express-FMDV2a are enclosed in pseudo-virus and programmed to code functional fluorescence proteins after infecting the host cells.
To get specific sequence and charanterization information, please see the following Parts Pages.

![Mobirise](https://static.igem.org/mediawiki/2018/9/9c/T--Tokyo_Tech--silver_val_parts.png)

Figure 7: List of validated parts and the features

# Experiment Progress

## Report on Oct. 17th, 2018

* Our team successfully validated that all the necessary constructs are produced by using electrophoresis (please refer to our Parts Pages).
* After completing the construction, our team shifted to the transfection into HEK293T cells for pseudo-virus production. Using lipofection, the constructs have been already introduced into the cells and the pseudo-viruses will be collected in a few days.
* After collecting the pseudo-viruses, they will be added to cultured Vero cells. Our team will test the infectivity by measuring the fluorescence intensity at the end.
* The whole results will be announced in Giant Jamboree, so please do not miss it!

# Our Goal

We try to validate that the pseudo-viruses are produced, and they can infect the host cells. Making the most of our system and lab experience, our team will make a kit to identify which type your target patient was infected with before.
As you can see in Fig. 8, if the patient was infected with type II virus before, type II-specific antibodies in blood serum decrease the infectivity of type II pseudo-virus. As a result, fluorescence protein programmed in the pseudo-virus would not be coded so much due to the decrease in the number of infectious viruses.
Thus, you can identify the infection experience and its type by measuring or observing the fluorescence intensity for each type/color.

![Mobirise](https://static.igem.org/mediawiki/2018/8/8a/T--Tokyo_Tech--infection_four_blocked.png)

Figure 8: Mechanism of colorimetric type detection system with different fluorescence proteins

# Feedback to Modeling

Since our detection system enables us to collect more samling data in many areas quickly and with low cost, our prediction model can be more precise thanks to the increase in the consensus time-scale data of dengue-affected patients and serotype ratio.

# Biosafety

As you can see in Fig. 9, on native virus genome, structural genes and non-structural genes are integrated on the same sequence. Thus, the whole genome are replicated and that make viruses possible to replicate themselves in host cells.
However, as you can see our construct design in Fig. 10, structural and non-structural regions are splitted so that the pseudo-virus cannot replicate themselves in host cells because RNA polymerase from non-structural V gene cannot replicate structural genes.
To sum up, our system starts and ends in one place and doesn't harm environment.

![Mobirise](https://static.igem.org/mediawiki/2018/7/76/T--Tokyo_Tech--infectious_particle_ex.png)

Figure 9: Infection process of native virus and production of infectious pseudo-virus

![Mobirise](https://static.igem.org/mediawiki/2018/9/91/T--Tokyo_Tech--presentation9.png)

Figure 10: Infection process of our pseudo-virus for validating biosafety

# Reference

### [National Institute of Infectious Diseases HP](https://www0.niid.go.jp/)

### [Scitable: Host Response to the Dengue Virus](https://www.nature.com/scitable/topicpage/host-response-to-the-dengue-virus-22402106)

### [Neutralization Assay for Zika and Dengue Viruses by Use of Real-Time-PCR-Based Endpoint Assessment.](https://www.ncbi.nlm.nih.gov/pubmed/28794181)

### [WHO: Dengue control](http://www.who.int/denguecontrol/en/)

[InterLab Page](https://2018.igem.org/Team%3ATokyo_Tech/InterLab)
[Modeling](https://2018.igem.org/Team%3ATokyo_Tech/Model)

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##### Address

2 Chome-12-1

Ookayama, Meguro, Tokyo

##### Contacts

Email: igem2018tokyotech@gmail.com

##### Links

[Facebook](https://www.facebook.com/igem.tokyo.tech/)

[Twitter](https://twitter.com/igem2017titech?lang=en)

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# Team:Tokyo Tech/Results

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[HOME](https://2018.igem.org/Team%3ATokyo_Tech)

TEAM

[Team Members](https://2018.igem.org/Team%3ATokyo_Tech/Team)
[Collaborations](https://2018.igem.org/Team%3ATokyo_Tech/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3ATokyo_Tech/Description)
[Design](https://2018.igem.org/Team%3ATokyo_Tech/Design)
[Experiments](https://2018.igem.org/Team%3ATokyo_Tech/Experiments)
[Notebook](https://2018.igem.org/Team%3ATokyo_Tech/Notebook)
[InterLab](https://2018.igem.org/Team%3ATokyo_Tech/InterLab)
[Model](https://2018.igem.org/Team%3ATokyo_Tech/Model)
[Results](https://2018.igem.org/Team%3ATokyo_Tech/Results)
[Demonstrate](https://2018.igem.org/Team%3ATokyo_Tech/Demonstrate)
[Improve](https://2018.igem.org/Team%3ATokyo_Tech/Improve)
[Attributions](https://2018.igem.org/Team%3ATokyo_Tech/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3ATokyo_Tech/Parts)
[Basic Parts](https://2018.igem.org/Team%3ATokyo_Tech/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3ATokyo_Tech/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3ATokyo_Tech/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3ATokyo_Tech/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3ATokyo_Tech/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3ATokyo_Tech/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3ATokyo_Tech/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3ATokyo_Tech/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3ATokyo_Tech/Hardware)
[Measurement](https://2018.igem.org/Team%3ATokyo_Tech/Measurement)
[Model](https://2018.igem.org/Team%3ATokyo_Tech/Model)
[Plant](https://2018.igem.org/Team%3ATokyo_Tech/Plant)
[Software](https://2018.igem.org/Team%3ATokyo_Tech/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=Tokyo_Tech)

# Results

Here you can describe the results of your project and your future plans.

### 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 be on part pages in the Registry.
* 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.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)