iGEM 2021 — Team Worldshaper-Shanghai

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# Team:Worldshaper-Shanghai/Notebook

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  (current)](/Team%3AWorldshaper-Shanghai)
* Project

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  [Design](/Team%3AWorldshaper-Shanghai/Design)
  [Implementation](/Team%3AWorldshaper-Shanghai/Implementation)
  [Contribution](/Team%3AWorldshaper-Shanghai/Contribution)
* Experiment

  [Engineering](/Team%3AWorldshaper-Shanghai/Engineering)
  [Proof of Concept](/Team%3AWorldshaper-Shanghai/Proof_Of_Concept)
  [Notebook](/Team%3AWorldshaper-Shanghai/Notebook)
  [Protocols](/Team%3AWorldshaper-Shanghai/Protocols)
  [Safety](/Team%3AWorldshaper-Shanghai/Safety)
* Dry Lab

  [Human Practices](/Team%3AWorldshaper-Shanghai/Human_Practices)
  [Education](/Team%3AWorldshaper-Shanghai/Communication)
  [Collaborations](/Team%3AWorldshaper-Shanghai/Collaborations)
* [Parts](/Team%3AWorldshaper-Shanghai/Parts)
* Team

  [Team Members](/Team%3AWorldshaper-Shanghai/Team_Members)
  [Partnership](/Team%3AWorldshaper-Shanghai/Partnership)
  [Attributions](/Team%3AWorldshaper-Shanghai/Attributions)

#### Notebook

### 2021 Timeline

March:

· Team Gathering

· Professional theory training (online): molecular biology, scientific research, media management

April:

· Creating specific groups

· Professional theory training (online): synthetic biology, lab techniques

· Developing topic: focus on Alzheimer’s diseases and using full-length APP to create a drug screening model on drosophila

May:

· Professional theory training: bio-engineering

· iGEM competition rules learning

· project background research

Since June, members were divided into wet team and dry team. Everyone was arranged by different jobs.

June:

Wet Team:

· Getting ready for lab

· Researching relevant topic

Dry Team:

· Initiate a round of fundraising

· Developing online platform

· Start to construct wiki framework

· Planning of publicity propaganda

· Design team logo

· Designing team jersey

July:

Wet team:

· Start Lab:

  oConstrcut APP plasmid

  oDrosophila Crossing

  oDrosophila behavioral experiment

· Writing daily Lab notes

Dry team:

· Interview planning

· Live media platform

· Start PR and raise attention of the project

· Printing, Distributing team jersey

· Initiate a new round of fundraising

· Planning on publicity events (School, Speech…)

August

Wet Team:

· In lab experiment

  oDrosophila behavioral experiment

  oDrosophila drug screening experiment

· Writing Lab Design and protocol

· Analyzing Data, ready for presentation

Dry Team:

· Modify partnership line

· Wiki design

· Publicity events

· Social research and interview with professionals of the field

· Researching and writing implementation.

September & October:

Wet Team:

· Finish Lab write up, design, notebook, protocol

· Uploads all wet team information on wiki

Dry Team:

· Researching and writing implementation

· Manuscript collection of human practice

· Finishing Wiki

  Presentation video

### Summer Journal

2021/0705

Lab crew uses PCR to replicate and create high number of APP 696 genes for further usage

2021/0706

Lab crew performs DNA gel electrophoresis to test for PCR results. DNA was then extracted, and performed Double Digestion using restriction enzyme to cut both APP gene and plasmid for connection. The lab crew left DNA and plasmid together overnight to create recombinant DNA

2021/0707

Lab crew transferred recombinant plasmid into competent E.coli cell. E.Coli was left overnight to reproduce so plenty of Recombinant plasmid was acquired. Then, E. Coli was sent to institution to transfer recombinant plasmid to Drosophila Chromosome.

2021/0708

Western blot

Crossed Drosophila to acquire genotype needed for experiment

2021/0712

Lab crew made culture medium for Drosophila in different concentrations of drug

2021/0713-14

Performed behavioral test: Mating Selection between young and old drosophila

2021/0715

Performed behavioral test: Female and male distinguishing courtship selection

2021/0721

Performed behavioral test: Pupa Height Test

2021/0722

Performed behavioral test: Adult locomotor test

2021/0728

Performed behavioral test: Drosophila learning and memory experiment

2021/0801

Performed behavioral test: Wing expansion defect test

2021/0803

Performed Larva eye disc and VNC disc anatomy with AO and Lysosome dye

2021/0805

Analyzed data from Larva anatomy experiment

2021/0807

Tested Effects of drugs on Drosophila growth

2021/0809

Tested effects of drugs on Drosophila lifespan

### CONTACT US

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Copyright © 2021, Worldshaper\_Shanghai all reserved.

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# Team:Worldshaper-Shanghai/Engineering

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* [Home
  (current)](/Team%3AWorldshaper-Shanghai)
* Project

  [Description](/Team%3AWorldshaper-Shanghai/Description)
  [Design](/Team%3AWorldshaper-Shanghai/Design)
  [Implementation](/Team%3AWorldshaper-Shanghai/Implementation)
  [Contribution](/Team%3AWorldshaper-Shanghai/Contribution)
* Experiment

  [Engineering](/Team%3AWorldshaper-Shanghai/Engineering)
  [Proof of
  Concept](/Team%3AWorldshaper-Shanghai/Proof_Of_Concept)
  [Notebook](/Team%3AWorldshaper-Shanghai/Notebook)
  [Protocols](/Team%3AWorldshaper-Shanghai/Protocols)
  [Safety](/Team%3AWorldshaper-Shanghai/Safety)
* Dry Lab

  [Human
  Practices](/Team%3AWorldshaper-Shanghai/Human_Practices)
  [Education](/Team%3AWorldshaper-Shanghai/Communication)
  [Collaborations](/Team%3AWorldshaper-Shanghai/Collaborations)
* [Parts](/Team%3AWorldshaper-Shanghai/Parts)
* Team

  [Team
  Members](/Team%3AWorldshaper-Shanghai/Team_Members)
  [Partnership](/Team%3AWorldshaper-Shanghai/Partnership)
  [Attributions](/Team%3AWorldshaper-Shanghai/Attributions)

#### Engineering

#### Summary

The goal of our project is to create a drug screen model for Alzheimer’s diseases and do some
potential drug testing. We used the full-length Human APP 596 gene, constructed the UAS-APP-Myc
with the pUAST plasmid. Through UAS/Gal4 binary system, we have successfully obtained fruit
flies expressing human APP. A series of drosophila behavioral experiments were conducted and
chloroquine was selected as a potential medicine to test the model. The results proved the
feasibility of the Drosophila model we constructed for drug screening.

**1 Plasmid construction**

To make the pUAST-APP-Myc plasmid, the first step is to amplify APP (APP695) from the pCAX
APP695 plasmid (Addgene#30137, USA) through Polymerase Chain Reaction (PCR), which loops
Denaturation, Annealing and Extension 35 times.
To check the PCR products, we took an aliquot of DNA sample from each PCR product, mixed it with
purple loading dye, and loaded it into DNA gel for electrophoresis at 120 volts for 40 min. The
figure below shows the results of DNA gel electrophoresis under UV lights (The control group is
on the right). 8/12 of our sample displays a DNA band at the range of 2kb, which corresponds to
the size of APP.

![](https://static.igem.org/mediawiki/2021/6/68/T--Worldshaper-Shanghai--engineerfig1.png)

Figure 1 PCR gel

Once we identified the PCR products with the right size, the remaining DNA of each PCR product
was double digested with two restriction enzymes: SacII and XbaI. The pUAST-Myc vector was also
double digested with the same two enzymes. The digested APP fragment and pUAST-Myc vector were
mixed with ligation buffer and DNA ligase, incubated at 37℃ for 120 min to promote their
recombination.

After ligation, recombinant plasmids were purified, mixed with E. Coli DH5α competent cells
for transformation. After transformation, cells were evenly applied on the surface of LB medium
with antibiotics, incubated at 37℃ overnight.

The results have been shown below, where white dots represent bacterial colonies, shows that our
bacterial are successfully transformed and cultivated.

![](https://static.igem.org/mediawiki/2021/d/dc/T--Worldshaper-Shanghai--engineerfig2.png)

Figure 2 E.coli Flat Figure

To verify the presence of correct plasmids, DNA was extracted from each E. Coli colony, PCR and
DNA gel electrophoresis were perform to check the presence of APP-containing plasmids.

(Figure below shows the result of DNA gel electrophoresis, 7 samples show DNA fragments of
about 2 kb , which correspond to the size of APP)

![](https://static.igem.org/mediawiki/2021/b/bf/T--Worldshaper-Shanghai--engineerfig3.png)

Figure 3 Positive clone identification glue map

To make sure no mutations were introduced into the plasmids, DNA sequencing was performed to
examine the obtained sample. The figures below show the DNA sequencing results (The gray part
indicates that the sequencing result is the same as the APP standard sequence).

![](https://static.igem.org/mediawiki/2021/f/f9/T--Worldshaper-Shanghai--engineerfig4.png)

Figure 4 Graph of segmented sequencing results

After DNA sequencing verification, the pUAST-APP-Myc plasmids were microinjected into Drosophila
early embryos, and transgenic flies carrying the UAS-APP-Myc transgene were obtained. These
flies show orange eyes, as compared with the white eye control flies.

To check whether the APP-Myc protein is expressed in Drosophila, we extracted total proteins
from Appl-Gal4 control flies and Appl-Gal4/UAS-APP-Myc flies, performed Western Blotting (WB)
with anti-Myc antibody. WB can be used to test the expression of protein with combination of gel
electrophoresis and immunochemical analysis techniques. The figure below shows the results of WB
analysis using GE Image Quant LAS4000. The 1st lane show the Appl-Gal4 control flies, the 2nd
and 3rd lanes show APP-Myc is expressed in two Appl-Gal4/UAS-APP-Myc fly lines.

![](https://static.igem.org/mediawiki/2021/6/69/T--Worldshaper-Shanghai--engineerfig5.png)

Figure 5 Results of Western Blotting

**2 Behavioral Experiment**

**Eclosion Defects:**

Chloroquine treatment does not affect the developmental progress of Drosophila, which takes
about 10 days to complete the development from an egg to an adult fly (Figure 6A). 2mg/ml
Chloroquine does not affect the viability in development, while 5mg/ml Chloroquine causes 30%
developmental lethality, suggesting a toxic effect of 5mg/ml Chloroquine on fly development
(Figure 6B).

![](https://static.igem.org/mediawiki/2021/7/79/T--Worldshaper-Shanghai--engineerfig6.png)

Figure 6 (A) Results of eclosion defects, (B) Results of statistical
analysis.

**Pupation Height:**

The height of pupation represents the climbing ability of Drosophila larvae.In the Figure 7,
2mg/ml (2, 5) and 5mg/ml (3, 6) Chloroquine does not affect the pupation height of Appl-Gal4 (1)
and Appl>Dcr2 (4) control animals. Over-expression of APP blocks the climbing ability of larvae,
which dramatically reduces the height of pupation (7). APP-induced larval climbing disability,
as shown by declined pupation height, is significantly rescued by 2mg/ml Chloroquine (8), but
not by 5mg/ml Chloroquine (9), further confirming the toxic effect of 5mg/ml Chloroquine on fly
development. Thus we decided to choose 2mg/ml Chloroquine for future experiments.

![](https://static.igem.org/mediawiki/2021/d/de/T--Worldshaper-Shanghai--engineerfig7.png)

Figure 7 (A)Results of pupation height, (B) Results of statistical
analysis.

**Learning Defects:**

Chloroquine restores APP‐induced learning defects. Figure
8A shows the male learning experiment model. To evaluate the learning ability, we performed
the one‐hour courtship suppression training, measured the CI of the initial (CIinitial) and
final (CIfinal) 10 min, and defined the learning index as

LI = (CIinitial− CIfinal)/CIinitial.

Compared with the controls (+), APP-expressing males
(APP) display a learning impairment, which is improved by 2mg/ml Chloroquine (Figure 8B).

![](https://static.igem.org/mediawiki/2021/d/de/T--Worldshaper-Shanghai--engineerfig8.png)

Figure 8 (A)Male learning experiment model, (B) Results of statistical
analysis.

**Courtship Choice:**

Chloroquine salvages the courtship choice defects caused by APP over-expression. Figure 9A
shows male courtship choice experiments model, in which male flies were provided with both
younger and older virgin females simultaneously. To accurately quantify the extent of males’
preference for younger or older females in courtship choice assay, we measured the preference
index (PI) indicating a relative difference between males’ CI toward younger or older females
(Figure 9A).

PI=CIyoung/CIold

Compared with the control males (+) that preferred to court
younger females, APP-expressing males (APP) were unable to distinguish between younger and older
females, and failed to show the courtship preference for young females. 2mg/ml Chloroquine could
rescue the males’ choice disability induced by APP expression (Figure 9B).

![](https://static.igem.org/mediawiki/2021/2/2c/T--Worldshaper-Shanghai--engineerfig9.png)

Figure 9 (A)Male courtship choice experiments model, (B) Results of
statistical analysis.

**Lyso-tracker positive cell number (VNC):**

Aberrant autophagy is frequently observed in the brains of AD patients. To confirm that APP
could induce autophagy in Drosophila nervous system, and that chloroquine treatment could
effectively suppress APP-induced autophagy in fly, we checked Lyso Tracker staining, which is a
biomarker for autophagy, in Drosophila ventral nerve cord (VNC). Compared with the controls (A),
APP expression resulted in increased autophagy (C), which was significantly suppressed by 2mg/ml
Chloroquine treatment (D). On the other hand, 2mg/ml Chloroquine did not affect autophagy in the
control VNC (B)( Figure 10).

These results indicate that APP expression induces autophagy in fly VNC, which could be
effectively blocked 2mg/ml Chloroquine treatment.

![](https://static.igem.org/mediawiki/2021/d/d5/T--Worldshaper-Shanghai--engineerfig10.png)

Figure 10 (A) Results of Lyso-tracker positive cell number (VNC), (B)
The statistical analysis of data shown in A.

**Lyso-tracker positive cell number (Eye disc):**

Same as above, we also measure autophagy in eye disc to test the effectiveness of
Chloroquine. Compared with the controls (A), APP expression resulted in increased autophagy in
photoreceptor cells in the eye disc (C), which was significantly suppressed by 2mg/ml
Chloroquine treatment (D). On the other hand, 2mg/ml Chloroquine did not affect autophagy in the
control eye disc (B)( Figure 11).

These results indicate that APP expression induces autophagy in the photoreceptor cells of
eye disc, which could be effectively blocked 2mg/ml Chloroquine treatment.

![](https://static.igem.org/mediawiki/2021/2/21/T--Worldshaper-Shanghai--engineerfig11.png)

Figure 11 (A) Results of Lyso-tracker positive cell number (Eye disc),
(B) The statistical analysis of data shown in A.

**AO Positive cell number (VNC):**

To measure the effect of chloroquine on APP-induced autophagy-mediated neuronal cell death,
we performed Acridine Orange (AO) staining, a biomarker for cell death, in Drosophila ventral
nerve cord (VNC) to reflect the degree of neuronal damage. Compared with the controls (A), APP
expression resulted in increased neuronal cell death (C), which was significantly suppressed by
2mg/ml Chloroquine treatment (D). On the other hand, 2mg/ml Chloroquine did not affect neuronal
death in the control VNC (B) (Figure 12).

These results indicate that APP expression induces autophagy-mediated neuronal cell death in
fly VNC, which could be effectively blocked 2mg/ml Chloroquine treatment.

![](https://static.igem.org/mediawiki/2021/e/e6/T--Worldshaper-Shanghai--engineerfig12.png)

Figure 12 (A) Results of AO Positive cell number (VNC), (B) The
statistical analysis of data shown in A.

**AO Positive cell number (Eye disc):**

Same as above, we also measure photoreceptor cell death in eye disc to test the
effectiveness of Chloroquine. Compared with the controls (A), APP expression resulted in
increased cell death (C), which was significantly suppressed by 2mg/ml Chloroquine treatment
(D). On the other hand, 2mg/ml Chloroquine did not affect photoreceptor cell death in the
control eye disc (B) (Figure 13).

These results indicate that APP expression induces autophagy-mediated neuronal cell death in
fly eye discs, which could be effectively blocked 2mg/ml Chloroquine treatment.

![](https://static.igem.org/mediawiki/2021/5/54/T--Worldshaper-Shanghai--engineerfig13.png)

Figure 13 (A) Results of AO Positive cell number (Eye disc), (B) The
statistical analysis of data shown in A.

#### Conclusion

In summary, we successfully constructed pUAST-UAS-APP-Myc plasmid and successfully obtained the
fruit fly expressing human APP. The results of chloroquine as the potential drug to test our
model indicated that chloroquine treatment could suppress APP-induced autophagy and neuronal
cell death, and ameliorate APP-triggered behavioral defects in pupation height, learning, and
memory, and choice. These experiments proved the potential effect of chloroquine as an inhibitor
of AD symptoms, and at the same time proved the feasibility of the Drosophila model we
constructed for drug screening.

#### Future work

Through the use of the APP transgenic Drosophila model, we proved that chloroquine has the
potential to inhibit AD symptoms with experiments. Therefore, for subsequent drug screening for
neurodegenerative diseases such as Alzheimer's disease, we hope our model can be used for
large-scale rapid screening.

### CONTACT US

![](https://static.igem.org/mediawiki/2021/c/cf/T--Worldshaper-Shanghai--wechat.png)

Copyright © 2021, Worldshaper\_Shanghai all reserved.

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

# Team:Worldshaper-Shanghai/Experiments

Template:Worldshaper..

# 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:Worldshaper-Shanghai/Results

Template:Worldshaper..

# 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)