Team:Wroclaw/Notebook

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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

  1. After 48 h cultures were centrifuged, washed out with sterile distilled water twice and suspended in sterile water
  2. Optical Density was standardized (A600nm = 1,5)
  3. 1 uL of 1% methylene blue was added to 100 uL of suspended cells. After 5 minutes pictures were taken.
  4. 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


Figure 1. Yarrowia lipolityca A101 vitality - control culture. Cultivated in YPD for 48 h at room temperature (200 rpm). Cells stained with methylene blue.


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.


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.


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.

As shown on the chart the best responding primer appeared to be the one related to pYALI0C18481 gene expressing metallothionein IV protein. The best response was related to the exposure to Pb2+ ions. Full version HERE
  • Another step was to perform PCR reaction with primers for our best responding promoter sequence – from Yarrowia lipolityca A101 genome and for primers for GFP - taken from pHR plasmid.

From left: 1kb ladder maker, GFP PCR product, pYALI0C18481 PCR product
  • After cleaning-up our GFP PCR product, we performed reaction with restriction enzymes for both GFP coding sequence and our JMP62 series plasmid with TEF promoter.
  • Then, we performed ligation reaction and transformed plasmid into E. coli dh5 alpha cells.
  • We isolated our plasmid from correctly transformed cells and with the help of another restriction enzyme and ligation reaction we exchanged TEF promoter to our pYALI0C18481 promoter.
  • The last step we were able to do was to exchange the GFP coding sequence to four genes from the lycopene biosynthesis pathway. We inserted the expression cassettes into Yarrowia lipolityca A101 genome.
  • The transformants of Y. lipolityca A101 were cultured in YPG medium with and without (control) metal ions: a) Cd, b) Cu, c) Pb.
  • Each end every step of our project had to be carefully performed and repeated. As ambitious novices in the field of molecular biology and because of quite difficult task we chose for ourselves as our first contact with synthetic biology, dedicated time and the amount of repeats forced us to finish the project at this stage.