iGEM 2021 — Team GreatBay_United

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:GreatBay United/Notebook

# 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:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:GreatBay United/Engineering

GreatBay\_United

![](https://static.igem.org/mediawiki/2021/9/96/T--GreatBay_United--logo.png)

* [Home](https://2021.igem.org/Team%3AGreatBay_United)
* [Project](https://2021.igem.org/Team%3AGreatBay_United/Background)

  1. [Background](https://2021.igem.org/Team%3AGreatBay_United/Background)
  2. [Description](https://2021.igem.org/Team%3AGreatBay_United/Description)
  3. [Design](https://2021.igem.org/Team%3AGreatBay_United/design)
  4. [Contribution](https://2021.igem.org/Team%3AGreatBay_United/Contribution)
* [Wetlab](https://2021.igem.org/Team%3AGreatBay_United/Proof_Of_Concept)

  1. [Proof of Concepts](https://2021.igem.org/Team%3AGreatBay_United/Proof_Of_Concept)
  2. [Engineering](https://2021.igem.org/Team%3AGreatBay_United/Engineering)
  3. [Result](https://2021.igem.org/Team%3AGreatBay_United/result)
  4. [Measurements](https://2021.igem.org/Team%3AGreatBay_United/Measurement)
  5. [Parts](https://2021.igem.org/Team%3AGreatBay_United/parts)
  6. [Safety](https://2021.igem.org/Team%3AGreatBay_United/Safety)
  7. [Lab Notebook](https://2021.igem.org/Team%3AGreatBay_United/LabNotebook)
* [Drylab](https://2021.igem.org/Team%3AGreatBay_United/Hardware)

  1. [Hardware](https://2021.igem.org/Team%3AGreatBay_United/Hardware)
  2. [Modeling](https://2021.igem.org/Team%3AGreatBay_United/Model)
  3. [Proposed Implementation](https://2021.igem.org/Team%3AGreatBay_United/Implementation)
  4. [Supporting Entrepreneurship](https://2021.igem.org/Team%3AGreatBay_United/Entrepreneurship)
  5. [Sustainability](https://2021.igem.org/Team%3AGreatBay_United/Sustainable)
* [Human practices](https://2021.igem.org/Team%3AGreatBay_United/Human_Practices)

  1. [Integrated Human Practice](https://2021.igem.org/Team%3AGreatBay_United/Human_Practices)
  2. [Education](https://2021.igem.org/Team%3AGreatBay_United/Education)
  3. [Education and Communication](https://2021.igem.org/Team%3AGreatBay_United/Communication)
  4. [Excellence in Other Area](https://2021.igem.org/Team%3AGreatBay_United/Excellence)
* [Team](https://2021.igem.org/Team%3AGreatBay_United/Team)

  1. [Team](https://2021.igem.org/Team%3AGreatBay_United/Team)* [Attibution](https://2021.igem.org/Team%3AGreatBay_United/Attributions)* [Collaboration](https://2021.igem.org/Team%3AGreatBay_United/Collaborations)* [Partnership](https://2021.igem.org/Team%3AGreatBay_United/Partnership)

![](https://static.igem.org/mediawiki/2021/3/3d/T--GreatBay_United--sidebar2.gif)

# Engineering success

The whole system include three parts: detection system, cascade system and agglutination system.Agglutination system plays a role in showing the detection result. In consideration of the complexity of result presentation form in the existing endotoxin testing methods(recombinant C factor), we decided to use agglutination reaction to visualize the detection result, inspired by blood group test paper and limulus amebocyte lysate.

![](https://static.igem.org/mediawiki/2021/c/cf/T--Greatbay_United--凝集对比.jpg)

## Design

In order to achieve this，we decided to use yeasts to build our own agglutination system. The first reason is the size of yeast. Compare to other synthetic biological tools, yeast has a bigger size so the agglutination can be more obvious to observe. The second reason is the maturity of the yeast surface display. We can easily use yeast to express the proteins we need to build the system.

![](https://static.igem.org/mediawiki/2021/3/3b/T--Greatbay_United--凝集gif1.gif)
![](https://static.igem.org/mediawiki/2021/0/0b/T--Greatbay_United--003.png)
![](https://static.igem.org/mediawiki/2021/d/db/T--Greatbay_United--凝集gif2.gif)

## Build

We constructed the yeast surface display plasmid based on Vector pYD1.pYD1 is a 5.0 kb expression vector designed for expression, secretion, and display of proteins on the extracellular surface of Saccharomyces cerevisiae cells. Features of this vector allow regulated expression, secretion, and detection of expressed proteins。

![](https://static.igem.org/mediawiki/2021/b/b6/T--Greatbay_United--009.png)
![](https://static.igem.org/mediawiki/2021/thumb/4/4f/T--Greatbay_United--010.png/1600px-T--Greatbay_United--010.png)

The plasmid A (plasmid of Yeast A) is inserted with sequences that code for GFP, HCV substrate and anti GFP(explained below) and the plasmid B(plasmid of Yeast B) is inserted with sequences that code for anti-GFP. Plamid A and plasmid B is transformed into Yeast EBY100 separately. In this way Yeast A and Yeast B are obtained.

![](https://static.igem.org/mediawiki/2021/thumb/3/33/T--Greatbay_United--015.jpg/1599px-T--Greatbay_United--015.jpg.png)

## Test

In order to verify our assumption in design, we carried out following tests.

## 1. Verification of the construction of Yeast A&B

We cultured the yeast with YNB-CAA containing 2% glucose at 30℃ and YNB-CAA medium containing 2% galactose at 20℃ to induce protein expression. In order to determine whether the target protein is expressed or not, we add anti-mouse IgG conjugated with fluorescein isothiocyanate (FITC) to stain the displayed protein. The fluorescence observation result showed that the proteins are successfully expressed.

![](https://static.igem.org/mediawiki/2021/a/ae/T--Greatbay_United--酵母荧光001.gif)

## 2. Verification of the agglutination system design

In our design, instead of expressing GFP alone, Yeast A also expresses HCV substrate and anti-GFP. These three parts are connected together with linkers, as GFP and anti-GFP will bind together on the surface of Yeast A before they meet Yeast B. In this way, Yeast A will not agglutinate with Yeast B as the GFP on Yeast A is inhibited by self-expressed anti-GFP. By adding HCV protease into the agglutination system, the substrate of HCV between GFP and anti-GFP on Yeast A will be cut and the self-expressed anti-GFP on Yeast A will be separate from the GFP and leaving the yeast surface, making GFP be able to bind with anti-GFP on Yeast B and forming agglutination. This is how we use HCV to control the agglutination system, positive and negative results of detection can be present by presenting HCV or absenting HCV in the system.

![](https://static.igem.org/mediawiki/2021/e/ef/T--Greatbay_United--004.png)

In the verification test, we mixed same amount of Yeast A and Yeast B together in the buffer, and adding HCV protease into this system.The results showed that the adding of HCV trigger the agglutination between yeasts.

## Learn

By testing our design, we think that our design of using yeast surface display to simulate agglutination and using HCV to control the agglutination is workable.

## Redesign

In our first design, we used anti-GFP-low (anti-GFP with lower affinity) :
[Part:BBa\_K3858013](http://parts.igem.org/Part%3ABBa_K3858013)
on Yeast A and anti-GFP-high (anti-GFP with higher affinity) :
[Part:BBa\_K3858014](http://parts.igem.org/Part%3ABBa_K3858014)
on Yeast B to ensure our phenomenon of agglutination is obvious. After verifying the agglutination phenomenon can be observed by using this combination, we decided to test the relationship betwwen the agglutination phenomenon and anti-GFP with different affinities.

By testing our design, we think that our design of using yeast surface display to simulate agglutination and using HCV to control the agglutination is workable.

![](https://static.igem.org/mediawiki/2021/thumb/d/dc/T--Greatbay_United--007.png/1599px-T--Greatbay_United--007.png)

![](https://static.igem.org/mediawiki/2021/thumb/c/cd/T--Greatbay_United--008.png/1600px-T--Greatbay_United--008.png)

## Rebuild

Base on Yeast A and Yeast B, we designed 4 yeasts by using anti-GFP with different affinity.

![](https://static.igem.org/mediawiki/2021/thumb/0/0b/T--Greatbay_United--012.png/1600px-T--Greatbay_United--012.png)

![](https://static.igem.org/mediawiki/2021/b/b4/T--Greatbay_United--013.jpg)

By using the AcLi transformation method, we successfully obtained yeast positive transformers.

![](https://static.igem.org/mediawiki/2021/b/bc/T--Greatbay_United--菌株005.jpg)

By testing our design, we think that our design of using yeast surface display to simulate agglutination and using HCV to control the agglutination is workable.

![](https://static.igem.org/mediawiki/2021/d/d7/T--Greatbay_United--011.png)

## Retest

For the above four combinations of yeast, we did a comparative test on whether to add HCV crude enzyme solution to the system. The results are shown in the following figure. Samples No. 7-10 are samples without addition of HCV crude enzyme solution, showing homogeneous bacterial liquid morphology. Samples No. 11-14 are samples with addition of HCV crude enzyme solution. Compared with the treatment group without enzyme, an obvious coagulation phenomenon appeared in samples No.11-14 after the same time of culture.

![](https://static.igem.org/mediawiki/2021/thumb/9/93/T--Greatbay_United--7-10.jpg/1600px-T--Greatbay_United--7-10.jpg)
![](https://static.igem.org/mediawiki/2021/thumb/f/f7/T--Greatbay_United--11-14.jpg/1600px-T--Greatbay_United--11-14.jpg)

The results of the above tests verified that our agglutination system design is reasonable and feasible. These tests had confirmed that the agglutination system can work successfully. When HCV enzymes appear in the reaction system, agglutination will occur, which can be used to construct an artificial Limulus reagent system.

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:GreatBay United/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

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:GreatBay United/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)