iGEM 2023 — Team shanghai-sdg

{% extends "layout.html" %} {% block title %}Notebook{% endblock %} {% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your
project.{% endblock %} {% block page\_content %}

# Notebook

## **Summary**

---

Establishment of engineering Drosophila is a time-consuming task. The following is a record of the process of our project experiment. For detailed operation procedures and experimental
results, please refer to the [Protocols](https://2023.igem.wiki/shanghai-sdg/protocols%20) and [Engineering](https://2023.igem.wiki/shanghai-sdg/engineering%20) pages

## **Notebook**

#### [1 January](#Header1)

#### [2 February – March](#Header2)

#### [3 April - June](#Header3)

#### [4 July](#Header4)

#### [5 August](#Header5)

## **1 January**

#### **(weekends & winter vacation):**

#### **Constructions of vectors of pUAST-MTF-1, pMRE-Hid and pMRE-GFP**

1. Primer design and synthesis
2. Total RNA extraction
3. Reverse transcription
4. PCR and product recovery
5. Digestion and purification
6. Overnight ligation
7. Transformation
8. Positive colony identification
9. Sequencing

## **2 February – March**

#### **(winter vacation & weekends):**

### 1.Microinjection of Drosophila embroy

* Plasmid extraction
* Sending plasmid samples for Drosophila embryo injection.

### 2.Cell biologyexperiments

#### 2.1 Co-transfection of plasmids into Drosophila S2 cells

* Recovery of S2 cells
* Co-transfection of plasmids into cells using transient transfection reagent
* Culturing of cells in media with different concentrations of metal ions

#### 2.2 Real-time PCR

* Collecting cells and extracting total RNA
* Reverse transcription
* Real-time PCR

#### 2.3 Western blot detection of MTF-1 and Hid genes transcription and expression

* Collecting and lysing cells after transfection for 48 hours
* Preparation of SDS-PAGE gel
* SDS-PAGE electrophoresis
* Membrane transfer
* Blocking
* Hybridization (overnight)
* Development

## **3 April - June**

#### **(weekends) :**

### 1.Establishment of stable genetic strains of Drosophila

### 2.Pre-experiment of Drosophila hybridization

* Drosophila medium preparation
* Common culture of Drosophila
* Dissection and observation of Drosophila larvae
* Dissection and observation of adult Drosophila
* Cultivation of Drosophila in medium with different concentrations of metal ions

## **4 July**

#### **(summer vacation):**

### 3.Drosophila hybridization

* Hybridizing Drosophila strains GMR-GAL4/ Vg-GAL4/ptc-GAL4 and UAS-MTF-1；MRE-Hid or UAS-MTF-1；MRE-GFP
* Culture hybrid oosperm in media with different concentrations of metal ions

### 4.Phenotypic observations of Drosophila larvae

* Larva dissection and identification of imaginal disks
* AO staining
  1. Staining
  2. make slide and photography
  3. c.Counting of bright spots
* Dcp-1 staining
  1. Fixation of larval tissue
  2. Cleaning of larval tissue
  3. Low-temperature incubation with primary antibody and cleaning
  4. Staining with secondary antibody solution and cleaning
  5. Dissection and make slide
  6. Observation and photography under fluorescence microscope.
  7. Counting of bright spots
* GFP observation

* Literature review

## **5 August**

#### **(summer vacation) :**

### Phenotypic observations of Drosophila adults

* Culture hybrid oosperm in media with different concentrations of metal ions
* Collect adult flies of F1 generation
* Observe the eye and wing phenotypes and take photos
* Calculate eye size and wing size of flies from each metal ion concentration treatment

{% endblock %}

{% extends "layout.html" %} {% block title %}Engineering Success{% endblock %} {% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the
engineering design cycle.{% endblock %} {% block page\_content %}

# Engineering

## **Engineering**

#### [1 Abstract](#Header1)

#### [2 Missions accomplished](#Header2)

[2.1 Build](#Header3)
[2.2 Test](#Header4)

#### [3 Response modeling to heavy matals](#Header5)

#### [4 Conclusion and Future work](#Header6)

## **1 Abstract**

Our team aims to develop a convenient and inexpensive visual monitoring system for heavy metals by constructing transgenic Drosophila. We inserted MTF-1 gene driven by UAS and Hid gene
driven by MRE in Drosophila S2 cells, respectively. These two transgenic Drosophila lines interbreed and the offspring with both MTF-1 and Hid genes cross with the Drosophila line with
genotype GMR-GAL4 or Vg-GAL4 or ptc-GAL4 which express GAL4 during eye/wing development. In this way, we constructed three Drosophila lines with genotype UAS-MTF-1;MRE-Hid/GMR-GAL4,
UAS-MTF-1;MRE-Hid/Vg-GAL4, and UAS-MTF-1;MRE-Hid/ptc-GAL4 which express MTF-1 during eye/wing development due to the GAL4-UAS system. In response to heavy metals in the environment,
MTF-1 is activated, binds to MRE and activates Hid expression, leading to apoptosis in the developing eyes/wings. Thus, in the first generation within 5-10 days, these lines show
abnormally small eye/wing sizes observable by naked eyes. The system is sensitive, cost-effective, and environmentally friendly, requiring minimum equipment and professional personnel,
and is especially of great value to remote and underdeveloped regions.

Our team successfully constructed 9 parts. BBa\_K4890001, BBa\_K4890004, BBa\_K4890011, and BBa\_K4890013 were developed for expressing MTF-1 gene driven by UAS. BBa\_K4890002- BBa\_K4890004,
BBa\_K4890012 and BBa\_K4890014 were developed for expressing Hid gene driven by MRE. Plasmids based on these parts and pAc-GAL4 plasmid were co-transfected into Drosophila S2 cells to
verified the efficiency of this monitor system in response to heavy metals. The cells was assessed by PCR, gene sequencing, real-time PCR and Western blot. Engineering based on these
plasmids and hybridization were used to construct Drosophila lines with genotype UAS-MTF-1;MRE-Hid/GMR-GAL4, UAS-MTF-1;MRE-Hid/Vg-GAL4, and UAS-MTF-1;MRE-Hid/ptc-GAL4. Treated with heavy
metal ions, eye/wing imaginal discs from larvae were evaluated by AO staining and Dcp-1 staining and their adults’ eye/wing areas were analyzed. BBa\_K4890015 was developed for expressing
GFP gene driven by MRE. Engineering based on this part and hybridization were used to construct Drosophila line with genotype UAS-MTF-1;MRE-GFP/GMR-GAL4. Treated with heavy metal ions,
fluorescence intensity in the eye imaginal discs from larvae was analyzed.

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| No. | Part Number | Type | Description | Designer | Length |
| 1 | BBa\_K4890001 | Basic | MTF-1 |  | 2535 |
| 2 | BBa\_K4890002 | Basic | MRE |  | 73 |
| 3 | BBa\_K4890003 | Basic | Hid |  | 1537 |
| 4 | BBa\_K4890004 | Basic | Hsp70 |  | 262 |
| 5 | BBa\_K4890011 | Composite | UAS-Hsp70 |  | 342 |
| 6 | BBa\_K4890012 | Composite | MRE-Hsp70 |  | 320 |
| 7 | BBa\_K4890013 | Composite | UAS-Hsp70-MTF1 |  | 2724 |
| 8 | BBa\_K4890014 | Composite | MRE-Hsp70-Hid |  | 1556 |
| 9 | BBa\_K4890015 | Composite | MRE-Hsp70-GFP |  | 1032 |

## **2 Missions accomplished**

To construct Drosophila lines with genotype of UAS-MTF-1;MRE-Hid/GMR-GAL4, UAS-MTF-1;MRE-Hid/Vg-GAL4, and UAS-MTF-1;MRE-Hid/ptc-GAL4, we accomplished three missions.

### **2.1 Build**

**Mission 1: Construction of pUAST-MTF-1, pMRE-Hid, and pMRE-GFP plasmids**
**(1)Construction of pUAST-MTF-1 plasmid**

We took a commercialized recombinant plasmid pUAST as template, and used restrictive endonuclease (BglII and XhoI) digestion to obtain a linearized pUAST vector. MTF-1 gene fragment was
amplified from the cDNA of wildtype Drosophila melanogaster by PCR. DNA electrophoresis confirmed the length of the PCR product (2376bp). MTF-1 gene fragment was ligated with the pUAST
linearized vector by T4 ligase. pUAST-MTF-1 was transformed into E. coli DH5α strain. Colony PCR and DNA electrophoresis (2376bp) was performed to confirm the positive colonies. These
colonies were transferred and expanded. Plasmid extracted from the colonies was confirmed to be pUAST-MTF-1 plasmid by gene sequencing (Figure 1-2,4).

**(2) Construction of pMRE-Hid and pMRE-GFP plasmids**

We also reformed the pUAST plasmid into pMRE plasmid by restrictive endonuclease (Pst1) digestion to obtain a linearized pUAST vector and then substitute UAS sequence for MRE sequence.
MRE was obtained by DNA synthesis and T4 ligase was used to combine the linearized vector into complete plasmid. pMRE plasmid was transformed into E. coli DH5α strain. Colony PCR and DNA
electrophoresis (600 bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmid extracted from the colonies was confirmed to be pMRE by
gene sequencing (Figure 2-3).

Start from pMRE as template, we used restrictive endonuclease (NotI and XbaI) digestion to obtain a linearized pMRE vector. Hid gene fragment was amplified from the cDNA of wildtype
Drosophila melanogaster by PCR. GFP gene fragment was amplified from the plasmid of pUAST-GFP by PCR. DNA electrophoresis confirmed the lengths of the PCR products (1233bp and 720bp).
Hid and GFP gene fragments were ligated with pMRE linearized vector by T4 ligase, respectively. pMRE-Hid and pMRE-GFP plasmids were transformed into E. coli DH5α strain, respectively.
Colony PCR and DNA electrophoresis (1233bp and 720 bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmids extracted from the colonies
were confirmed to be pMRE-Hid and pMRE-GFP by gene sequencing (Figure 1-2, 4).

![](https://static.igem.wiki/teams/4890/wiki/images/engineering/01.png)

**Fig.1 Genetic circuits of pUAST-MTF-1, pMRE-Hid, and pMRE-GFP plasmids**

![](https://static.igem.wiki/teams/4890/wiki/images/engineering/02.png)

**Figure 2 Gel electrophoresis of PCR products for the construction of pUAST-MTF-1, pMRE-Hid, and pMRE-GFP plasmids**

![](https://static.igem.wiki/teams/4890/wiki/images/engineering/03.png)

**Figure 3 Gene sequencing of pMRE**

![](https://static.igem.wiki/teams/4890/wiki/images/engineering/04.png)

**Figure 4 Gel electrophoresis of pUAST-MTF-1, pMRE-Hid, and pMRE-GFP plasmids (From left to right: marker, pUAST-MTF-1, pMRE-Hid and pMRE-GFP)**

### **2.2 Test**

**Mission 2: Transient co-transfection of Drosophila S2 cells with pUAST-MTF-1, pMRE-Hid, and pAc-GAL4 plasmids**

We cultured Drosophila S2 cells on plates for 24h, and then transiently co-transfected pUAST-MTF-1, pMRE-Hid and pAc-GAL4 (which contains actin-GAL4) plasmids into the S2 cells by using
ROCHE X-treme GENE™ HP DNA Transfection Reagent. pAc-GAL4 plasmid was previous constructed by Genetic Lab, School of Life Science and Technology, Tongji University. The transfected S2
cells were notated as Drosophila UAS-MTF-1/MRE-Hid/Ac-GAL4 cells

Drosophila UAS-MTF-1/MRE-Hid/Ac-GAL4 cells were cultured for 48h and then divided into 5 groups. The control group received no treatment, and the other 4 groups were treated with 10μM
ZnCl2, 100μM ZnCl2, 10μM CdCl2, and 100μM CdCl2, respectively, for 4h.

Real-time PCR and Western Blot results confirmed that both mRNAs and proteins of MTF-1 and Hid were expressed in Drosophila UAS-MTF-1/MRE-Hid/Ac-GAL4 cells treated with 10μM and 100μM
ZnCl2 or CdCl2 (Figure 5-6). The mRNA and protein levels of Hid were concentration-dependent (P<0.05). The mRNA level of MTF-1 was not changed with the addition of metal ions (P>0.05).

![](https://static.igem.wiki/teams/4890/wiki/images/engineering/05.png)

unpaired t-test: \*\*\*P<0.001,\*\*\*\*P<0.0001, ns: P>0.05

**Figure 5 mRNA levels of MTF-1 and Hid in Drosophila UAS-MTF-1/MRE-Hid/Ac-GAL4 cells treated with different concentrations of ZnCl2 or CdCl2**

![](https://static.igem.wiki/teams/4890/wiki/images/engineering/06.png)

Note: MTF-1 was detected by antibody of HA tag, which was co-expressed at the N-terminal of MTF-1, Hid was detected by antibody of Myc tag, which was co-expressed at the C-terminal of
Hid

**Figure 6 Protein levels of MTF-1 and Hid in Drosophila UAS-MTF-1/MRE-Hid/Ac-GAL4 cells treated with different concentrations of ZnCl2 or CdCl2**

**Mission 3: Generation of Drosophila lines with genotype of UAS-MTF-1;MRE-Hid/GMR-GAL4, UAS-MTF-1;MRE-Hid/Vg-GAL4, UAS-MTF-1;MRE-Hid/ptc-GAL4, and UAS-MTF-1;MRE-GFP/GMR-GAL4**

(1) Construction of Drosophila lines

pUAST-MTF-1, pMRE-Hid and pMRE-GFP were micro-injected into the embryos of Drosophila W1118 respectively to obtain Drosophila UAS-MTF-1, Drosophila MRE-Hid and Drosophila MRE-GFP
(Micro-injection was performed by Core Facility of Drosophila Resource and Technology, CEMCS, CAS).

Drosophila UAS-MTF-1 was crossed with Drosophila MRE-Hid to obtain the offspring with genotype of UAS-MTF-1;MRE-Hid. This progeny was crossed with Drosophila GMR-GAL4 or Drosophila
Vg-GAL4 or Drosophila ptc-GAL4 to obtain the progeny with genotype UAS-MTF-1;MRE-Hid/GMR-GAL4 or UAS-MTF-1;MRE-Hid/Vg-GAL4 or UAS-MTF-1;MRE-Hid/ptc-GAL4.

Drosophila UAS-MTF-1 was crossed with Drosophila MRE-GFP to obtain the offspring with genotype of UAS-MTF-1;MRE-GFP. This progeny was crossed with Drosophila GMR-GAL4 to obtain the
progeny with genotype UAS-MTF-1;MRE-GFP/GMR-GAL4

(2) Group assignment of Drosophila lines

Each cell line of Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 and Drosophila UAS-MTF-1;MRE-Hid/Vg-GAL4 was divided into 5 groups for larvae Acridine Orange (AO) staining and adult phenotype.
The control group received no treatment, and the other 4 groups were treated with 10μM ZnCl2, 100μM ZnCl2, 10μM CdCl2, and 100μM CdCl2, respectively.

Each cell line of Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 and Drosophila UAS-MTF-1;MRE-Hid/ptc-GAL4 were divided into 3 groups for larvae Death Caspase-1 (Dcp-1) staining. The control
group received no treatment, and the other 2 groups were treated with 10μM CdCl2, and 100μM CdCl2, respectively.

Drosophila UAS-MTF-1;MRE-GFP/GMR-GAL4 line was divided into 3 groups for detection of fluorescence intensity. The control group received no treatment, and the other 2 groups were treated
with 10μM CdCl2, and 100μM CdCl2, respectively.

For larvae AO staining and Dcp-1 staining, the 3rd instar larvae were collected in about 5 days and imaginal discs were dissected.

(3) Heavy metal response of Drosophila larvae: cell death

In Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4, AO staining detected increased cell death in the eye imaginal discs when cultured with ZnCl2 (100μM) or CdCl2 (10μM and 100μM) (P<0.01) (Figure
7). The level of cell death was correlated with the concentration of CdCl2 (P<0.05). More cell death was observed in Drosophila grown under 10μM and 100μM CdCl2 than in those under the
same concentration of ZnCl2 (P<0.05)

In Drosophila UAS-MTF-1;MRE-Hid/Vg-GAL4, AO staining detected increased cell death in the wing imaginal discs when cultured with 10μM and 100μM ZnCl2 or CdCl2 (P<0.05) (Figure 8). The
level of cell death was correlated with the concentration of metal ions (P<0.05). More cell death was observed in Drosophila grown under 10μM and 100μM CdCl2 than in those under the same
concentration of ZnCl2(P<0.05).

![](https://static.igem.wiki/teams/4890/wiki/images/engineering/07.png)

unpaired t-test (vs control): \*\*P<0.01, \*\*\*P<0.001, \*\*\*\*P<0.0001, ns: P>0.05

**Figure 7 Cell death in the eye imaginal discs of Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 cultured under ZnCl2 and CdCl2 for 5 days detected by AO staining**

![](https://static.igem.wiki/teams/4890/wiki/images/engineering/08.png)

unpaired t-test (vs control): \*P<0.05, \*\*P<0.01, \*\*\*P<0.001, \*\*\*\*P<0.0001

**Figure 8 Cell death in the wing imaginal discs of Drosophila UAS-MTF-1;MRE-Hid/Vg-GAL4 cultured under ZnCl2 and CdCl2 for 5 days detected by AO staining**

(4) Heavy metal response of Drosophila larvae: cell apoptosis

Dcp-1 staining detected enhanced cell apoptosis in the eye or wing imaginal discs of Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 and Drosophila UAS-MTF-1;MRE-Hid/ptc-GAL4 cultured under 100μM
CdCl2 (P<0.05)(Figure 9-10). Cultured under 10μM CdCl2, their discs also showed increased trend of cell apoptosis, but the difference was not statistically significant compared with the
control (P>0.05).

![](https://static.igem.wiki/teams/4890/wiki/images/engineering/09.png)

unpaired t-test (vs control): \*\*\*P<0.001, ns: P>0.05

**Figure 9 Cell apoptosis in the eye imaginal discs of Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 cultured under CdCl2 for 5 days detected by Dcp-1 staining**

![](https://static.igem.wiki/teams/4890/wiki/images/engineering/10.png)

unpaired t-test (vs control): \*\*\*\*P<0.0001, ns: P>0.05

**Figure 10 Cell apoptosis in the wing imaginal discs of Drosophila UAS-MTF-1;MRE-Hid/ptc-GAL4 cultured under CdCl2 for 5 days detected by Dcp-1 staining**

(5) Heavy metal response of Drosophila larvae: fluorescence intensity

The eye imaginal discs of Drosophila UAS-MTF-1;MRE-GFP/GMR-GAL4 cultured under 10μM and 100μM CdCl2 had higher fluorescence intensity than the control (P<0.05, Figure 11). The
fluorescence intensity was dependent on the concentration of CdCl2

![](https://static.igem.wiki/teams/4890/wiki/images/engineering/11.png)

unpaired t-test (vs control): \*\*\*\*P<0.0001

**Figure 11 Fluorescence intensity in the eye imaginal discs of Drosophila UAS-MTF-1;MRE-GFP/GMR-GAL4 cultured under CdCl2 for 5 days**

(6) Heavy metal response of Drosophila adults: eye/wing area

The F1 generation adults were collected in about 10 days.

Eye areas of Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 adults cultured under 10μM and 100μM ZnCl2 or CdCl2 were all smaller than the control (P<0.05) (Figure 12). With the increase of metal
ion concentration, the eye area decreased. Eye areas were more sensitive to CdCl2 than ZnCl2.

Wing areas of Drosophila UAS-MTF-1;MRE-Hid/Vg-GAL4 adults showed the similar results (Figure 13).

![](https://static.igem.wiki/teams/4890/wiki/images/engineering/12.png)

unpaired t-test (vs control): \*\*\*P<0.001, \*\*\*\*P<0.0001

**Figure 12 Eye areas of Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 adults cultured under ZnCl2 and CdCl2 for 10 days**

![](https://static.igem.wiki/teams/4890/wiki/images/engineering/13.png)

unpaired t-test (vs control): \*\*\*\*P<0.0001

**Figure 13 Wing areas of Drosophila UAS-MTF-1;MRE-Hid/Vg-GAL4 adults cultured under ZnCl2 and CdCl2 for 10 days**

## **3 Response modeling to heavy matals**

## **4 Conclusion and Future work**

This project involves the creation of a heavy metal pollution monitoring system that utilizes genetically modified Drosophila. The system is designed to visually determine whether a
sample contains high concentrations of heavy metals by observing changes in the sizes of the Drosophila's eyes and wings. The process involves rearing gene-edited adult Drosophila on
culture medium containing food, soil, or water samples in tubes for 5-10 days. If the samples contain high levels of heavy metals, the offspring Drosophila's eye and wing cells will
undergo apoptosis, leading to a reduction in eye and wing areas.

The primary advantage of this method is cost-effectiveness. Drosophila is relatively easy to cultivate and maintain, require minimal resources, and do not necessitate expensive
equipment. Furthermore, the method is user-friendly. Given their small size and short monitoring period, Drosophila can be easily transported to remote areas for long-term monitoring.
This method is also environmentally friendly and highly sensitive.

However, there are limitations to this monitoring system. It cannot accurately quantify heavy metal contamination, identify specific types of heavy metals, or detect multiple heavy
metals. Variations in sample concentrations within the Drosophila tubes may result in biased results.

In future research, we aim to develop a multi-metal monitoring technique for Drosophila and enhance their genetic makeup to exhibit distinguishable traits when exposed to multiple
metals. This will enable us to monitor different types of heavy metal contamination as well as contamination involving multiple heavy metals. We also plan to refine the monitoring
methods to ensure more uniform sample ingestion or exposure. Consideration is being given to incorporating sensor technology into the system for real-time monitoring of the Drosophila's
status.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

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).

## Inspirations

---

* [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)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## 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.

## Inspirations

---

* [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)

{% endblock %}