iGEM 2019 — Team Wroclaw

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Wroclaw/Notebook

[![](https://static.igem.org/mediawiki/2019/f/f3/T--Wroclaw--home.png)](https://2019.igem.org/Team%3AWroclaw)

project

[description](https://2019.igem.org/Team%3AWroclaw/Description)
[health threat](https://2019.igem.org/Team%3AWroclaw/Metal)
[filter](https://2019.igem.org/Team%3AWroclaw/Filter)
[lab diary](https://2019.igem.org/Team%3AWroclaw/Notebook)

human practices

[human practices](https://2019.igem.org/Team%3AWroclaw/Human_Practices)
[public engagement](https://2019.igem.org/Team%3AWroclaw/Public_Engagement)
[collaborations](https://2019.igem.org/Team%3AWroclaw/Collaborations)
[comic](https://2019.igem.org/Team%3AWroclaw/Comic)
[meetings](https://2019.igem.org/Team%3AWroclaw/Meetings)

[parts](https://2019.igem.org/Team%3AWroclaw/Parts)
[entrepreneurship](https://2019.igem.org/Team%3AWroclaw/Entrepreneurship)
[safety](https://2019.igem.org/Team%3AWroclaw/Safety)

people

[team](https://2019.igem.org/Team%3AWroclaw/Team)
[attributions](https://2019.igem.org/Team%3AWroclaw/Attributions)

judging

[judging form](https://igem.org/2019_Judging_Form?team=Wroclaw)
[medals](https://2019.igem.org/Team%3AWroclaw/Medals)

![](https://static.igem.org/mediawiki/2019/a/a7/T--Wroclaw--logo2.png)

# Lab Diary

## Cell vitality

First thing we had to do was to investigate Yarrowia lipolytica A101 vitality in different metal suspensions. Chosen concentrations of heavy metals were based on literature (Hosiner et al., 2014; Garcia, Prado, Dégano & Dominguez,2002)

* **Protocol:**

1. Erlenmeyer flask containing 50 ml of YPG medium was inoculated with Yarrowia lipolityca A101 yeast. Pre-cultivation was carried out (24 h, room temperature, 200 rpm).
2. After that time, 1 ml of inoculated medium culture was carried to 4 different Erlenmeyer flasks containing 50 ml YPG medium. Three flasks additionally contained respectively: 50 mM Pb2+ , 10 mm Cd2+ , 28mM Cu2+. Cultures were cultivated for 48 h at room temperature (200 rpm).

| Erlenmeyer flask no. | control | 2 | 3 | 4 |
| --- | --- | --- | --- | --- |
| Medium | YPD | YPD | YPD | YPD |
| Inoculum volume | 1 ml | 1 ml | 1 ml | 1 ml |
| Salt concentration | - | 50 mM Pb(NO3)2 | 10 mM CdCl2\*2,5H2O | 28 mM CuCl2\*2,5H2O ||

3. After 48 h cultures were centrifuged, washed out with sterile distilled water twice and suspended in sterile water
4. Optical Density was standardized (A600nm = 1,5)
5. 1 uL of 1% methylene blue was added to 100 uL of suspended cells. After 5 minutes pictures were taken.
6. Cells with no blue color are vital.

* **Results**

The vitality of cells cultured in medium containing cadmium was the lowest. Lead and copper
in YPG medium had no significant effect on cell survival comparing to the control sample.

* **References**

* García, S., Prado, M., Dégano, R., & Domínguez, A. (2002). A copper-responsive transcription factor, CRF1, mediates copper and cadmium resistance in Yarrowia lipolytica. Journal of Biological Chemistry, 277(40), 37359–37368. https://doi.org/10.1074/jbc.M201091200
* Hosiner, D., Gerber, S., Lichtenberg-Fraté, H., Glaser, W., Schüller, C., & Klipp, E. (2014). Impact of acute metal stress in Saccharomyces cerevisiae. PLoS ONE, 9(1), 1–14. https://doi.org/10.1371/journal.pone.0083330

![](https://static.igem.org/mediawiki/2019/e/e5/T--Wroclaw--fig1.png)
Figure 1. Yarrowia lipolityca A101 vitality - control culture. Cultivated in YPD for 48 h at room temperature (200 rpm). Cells stained with methylene blue.

![](https://static.igem.org/mediawiki/2019/0/0c/T--Wroclaw--fig2.png)
Figure 2. Yarrowia lipolityca A101 vitality – Cells cultivated in YPD with Pb2+ for 48 h at room temperature (200 rpm). Cells stained with methylene blue.

![](https://static.igem.org/mediawiki/2019/4/4a/T--Wroclaw--fig3.png)
Figure 3. Yarrowia lipolityca A101 vitality – Cells cultivated in YPD with Cd2+ for 48 h at room temperature (200 rpm). Cells stained with methylene blue.

![](https://static.igem.org/mediawiki/2019/4/41/T--Wroclaw--fig4.png)
Figure 4. Yarrowia lipolityca A101 vitality – Cells cultivated in YPD with Cu2+ for 48 h at room temperature (200 rpm). Cells stained with methylene blue.

## Promoter identification

* We have prepared flasks with YPD medium. After the sterilization process, we inoculated the medium with the strain Yarrowia lipolytica A101. The flasks were placed on a shaker at 28 ° C, 200 rpm.

* We prepared 21 flasks with YPD substrate. The flasks were then subjected to a sterilization process. They were inoculated with 1 ml of inoculation culture. We also prepared heavy metal salt solutions, which were then added to the flasks in the right amount. The tests were divided into those where heavy metals were added to the substrate immediately and those to which solutions were added after 18 hours:

1. 3× control
2. as in table:

| Salt solutions added right away | Salt solutions added after 18 hours |
| --- | --- |
| 3× ions Pb2+ | 3× ions Pb2+ |
| 3× ions Cd2+ | 3× ions Cd2+ |
| 3× ions Cu2+ | 3× ions Cu2+ |

Salt solutions:

|  |  |
| --- | --- |
| For lead 50mM | Pb(NO3)2 |
| For cadmium 10mM | CdCl2 × 2.5H2O |
| For copper 28mM | CuCl2 × 2H2O |

* We took 2 ml of the solution from twenty-four hour cultures and subjected it to the purification process. After cleaning, the samples were frozen in liquid nitrogen.

* In full concentration, two people from our team sat at the laminar chamber. Our hearts killed us all a bit harder than usual. We only had one question in our minds: "Can we get what we want the most?" Using a ready-made kit, we carried out the RNA isolation process. We performed all the operations with particular precision, like agent 007 and with sterile working conditions. Obtained results were extremely satisfying. This is our first success! We are all very excited! :)

* On this day, we focused on carefully cleaning DNA from RNA samples. We used DNAse enzyme for this. Thanks to it, following the instructions, we removed the DNA leaving pure RNA. After doing this, we prepared the reaction mixture to carry out the reverse transcription processes - all previously isolated RNA was prescribed for cDNA. It was a very long-lasting job but as satisfying!

* After long fight with prescribing our cDNA, we performed numerous real-time PCRs to search for the best responding metallothionein primer.

![](https://static.igem.org/mediawiki/2019/a/ab/T--Wroclaw--yali_qPCR.png)

![](https://static.igem.org/mediawiki/2019/6/64/T--Wroclaw--sknbif.png)
![](https://static.igem.org/mediawiki/2019/0/0e/T--Wroclaw--orgchem.png)
![](https://static.igem.org/mediawiki/2019/e/ea/T--Wroclaw--sknbtch.png)
![](https://static.igem.org/mediawiki/2019/d/df/T--Wroclaw--uni.png)


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Wroclaw/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.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Wroclaw/Results

# 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 also be on the part's Main Page on the Registry.** 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.

* [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)