iGEM 2023 — Team h14z-ap

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## Note Book

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## Engineering Success

## Overview

Our project follows the engineering cycle (Design, Build, Test, and Learn) throughout the experiment process. For each experiment we followed Design, Build, Test, and Learn cycle to overcome numerous technical issues.

Experiment process:

[* Introduction](#Introduction)
[* Part 1 Building a UAS expressing plasmid](#part1)
[* Part 2 Testing pUASattB-CASH3-V5 plasmid](#part2)
[* Part 3 Expressing CASH3-V5 protein in Tau expressing flies](#part3)

![Engineering cycle](https://static.igem.wiki/teams/4723/wiki/engineering-cycle/engineering-cycle.png)

#### Introduction

Similar to many neurodegenerative diseases, Alzheimer’s disease (AD) is characterized with protein misfolding and neuronal cell death. CAHS3 protein has been shown to help protein folding and maintain protein activity in the in vitro conditions. It can prevent cell death when it is expressed in mammalian culture cells. We hypothesized that CAHS3 overexpression may help to conquer the diseases conditions in AD animal models. In our project, we chose to use hTau expressing fly models to test if CAHS3 protein can reduce the toxicity of hTau protein.

By using the GAL4-UAS system, Drosophila can express numerous exogeneous proteins. In this system, the GAL4 gene that encodes yeast transcription activator protein Gal4 binds with the upstream activation sequence (UAS) to trigger gene transcription, allowing exogeneous protein expression. External eye morphology can be observed in hTau fly models, which makes it a successful model for AD research. By expressing CAHS3 protein alongside hTau, we can determine whether CAHS3 can help protein folding and thus prevent cell death and improve eye defects caused by hTau expression.

#### Part 1 Building a UAS expressing plasmid

### Design

1. Obtained a plasmid pAcGFP1-N1-CAHS3 that carry the cDNA of CAHS3 from addgene.
2. Using VectorNTI design a pUAS plasmid that could express a V5 tagged CAHS3 protein (pUASattB CAHS3-V5) at the present of Gal4. We design CASH3 protein with a V5 tag to make the expression of the protein easy to follow during the experiment.
![zhili](https://static.igem.wiki/teams/4723/wiki/engineering-cycle/ec2.png)3. Design PCR primers
![](https://static.igem.wiki/teams/4723/wiki/engineering-cycle/ec2-1-pcr.png)

### Build

1. We recovered the ordered plasmid by strip a plate, inoculate in liquid culture, and did DNA miniprep.
2. We did PCR to amplify the DNA fragment of CAHS3-V5
![](https://static.igem.wiki/teams/4723/wiki/engineering-cycle/ec3-cahs3-v5.png)

### Test

1. We did clonal PCR for the liquid culture- and select the positive clone for sequencing.
2. The sequencing results showed that we got the right clone.

### Learn

After failed PCR reactions, we learned to optimize PCR conditions to obtain ideal amount of PCR product for further experiments.

#### Part 2 Testing pUASattB-CASH3-V5 plasmid

### Design

Before generate transgenic flies, we designed experiments to test whether the plasmid could express the protein. We decided to transfect the plasmid to Drosophila S2 cells and examine the protein expression by using immunostaining and Western blot. To ensure our expression system is working, we used pUAS-MitoGFP as a control to express MitoGFP protein in S2 cells as a control.

### Build

pUASattB-CASH3-V5 plasmid and pUAS MitoGFP construct were transfected together with ubiGal4 plasmid into S2 cells. As control, pUAS MitoGFP was transfected with ubiGal4.

### Test

1. The immunostaining was performed to test the expression of CASH3-V5.
![](https://static.igem.wiki/teams/4723/wiki/engineering-cycle/ec4.png)2. The Western blot was performed to test the expression of CASH3-V5 in S2 cells.
![](https://static.igem.wiki/teams/4723/wiki/engineering-cycle/ec5.png)

### Learn

pUASattB CASH3-V5 protein could be effectively expressed in Drosophila cells.

#### Part 3 Expressing CASH3-V5 protein in Tau expressing flies

### Design

Human tau (hTau) protein expressing in fly eyes could cause eye developmental defects and the flies have eye morphology defects. To examine whether CASH3 expression could reduce the toxicity of hTau, we design to express the CASH3 protein in the flies with hTau expression and examine the eye morphology with light microscopy and scanning electro-microscopy. As control, we also express a protein LacZ in flies under the same condition, to exclude the possible dosage effects or other systematic effects.

### Build

1. We obtained the flies with hTau expression in the fly eyes from Bloomington stock center (stock number 93133). Genotype
   (w1118; P{GMR-htau.Ex}1.1 P{GMR-UAS-lacZ}2/CyO).
2. Generate a fly stock with a Gal4 that could drive pUASattB CASH3-V5 expression.
![](https://static.igem.wiki/teams/4723/wiki/engineering-cycle/ec6-1-1.png)3. Generate a transgenic fly with pUASattB CASH3-V5.
4. Generate flies with both hTau and CASH3-V5 expression or flies with hTau and LacZ expression. As control, also generate flies with LacZ or CASH3-V5 expression alone.
![](https://static.igem.wiki/teams/4723/wiki/engineering-cycle/ec6-2-2.png)5. Build a mathematic model to analyze the fly eye morphology
![](https://static.igem.wiki/teams/4723/wiki/engineering-cycle/model.png)

### Test

1. Examine and image the fly eyes with light microscopy and use these image for mathematics analysis.
![](https://static.igem.wiki/teams/4723/wiki/engineering-cycle/ec6.png)2. Examine and image the fly eyes with scanning electo-microscopy.
![](https://static.igem.wiki/teams/4723/wiki/engineering-cycle/ec7.png)

### Learn

It is observed under scan electron microscopy that hTau expressing flies have a significant external eye morphology defect. CAHS3 protein expressing flies have no obvious eye morphology defect. When CAHS3 was overexpressed together with hTau in fly eyes, it did not improve the eye developmental defects caused by hTau. Our mathematical model shows no significant change brought by CAHS3 expression.

It can be concluded that CAHS3 protein does not significantly reduce external eye morphology defects caused by hTau toxicity. Further experimentation can be conducted to examine the internal eye structures of the CAHS3 protein expressing flies during aging process. The CAHS3 fly strain could also be used to test its function in other neurodegeneration disease models. The mathematical model could be used to analyze fly eye morphology in many different scenarios.

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## Experiments

## Our Goal

In our project, we are going to try to make a transgenic
fruit fly strain expressing tardigrade CAHS protein and test
whether its expression could reduce the damage of tau to fly
eyes.

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## Results

![]()

### Conclusion

1. We successfully constructed a plasmid that can express the CAHS3 protein in cells and fruit flies.
2. CAHS3 does not have harmful effect on fruit fly, but it cannot attenuate hTau expression induced eye morphology defects.
![](https://static.igem.wiki/teams/4723/wiki/engineering-cycle/ec7.png)

The picture from the electron microscope and data analyzed using the method of mathematical modeling show that CAHS3 expression alone has no impact to the fly eye morphology. The expression of CAHS3 does not significantly attenuate the eye morphology defects caused by hTau protein expression. Here, LacZ was expressed alone or together with hTau as a control.

![](https://static.igem.wiki/teams/4723/wiki/resultsfinal.png)

The table shows the comparison between the corresponding groups has no statistical significance (p>0.05), suggesting that the expression of CAHS3 has no significant effects to both the wildtype and hTau expressing fly eyes.

3. The Western blot experiments showed that CAHS3 may be partially degraded in Drosophila, which may explain why it was not effective. We observed that there were extra bands under the band with the molecular weight corresponding to CAHS3, which suggested that CAHS3 protein was partially degraded in flies.
![](https://static.igem.wiki/teams/4723/wiki/v5finnalnew.png)

### Future Plan

1. Perform more detailed analysis on the effects of CAHS3 when expressed in fruit flies, such as effects during aging and other stressed conditions.
2. Explore CAHS3's effects on other neurodegenerative disease models.
3. Laverage our genetic statistical model. This model could be used to analyze how genetic and environmental factors influence Drosophila eye health over time. Using this model can reduce human error and improve efficiency for Drosophila experiments.

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