Team:Valencia/Notebook/August

From 2010.igem.org

August 2nd

We visited the Astrobiology Center in Madrid and had some helpful discussions about our project with some of the researchers that nicely received us. We want to say a big thank you to Susanna Manrubia, Jesús Sobrado, Ángeles Aguilera and David Fernández-Remolar!

August 3rd

Unfortunatelly, the 2606 strain of Saccharomyces cerevisae did not grow for the third round of transformation. It did not grow in any of the three dishes so we there could be any problem with that strain. We move along. Don't want to get stuck in August!

August 6th

We decided to complete the transformation of the 5523 strain. It already has the plZ plasmid (GRE-LacZ -reporter-) and we want to get it together with the pG1 plasmid (the fusion of the prionic polypeptide plus the GR transcription factor). We hope it works!

We used the same protocol in order to transform the wild laboratory yeast strain W303 with the plasmid pG1-APC (with the prion from Aplysia), just to have an alternative to the original prion Sup35. It's better to be prudent, don't you think?

August 9th

The transformations were a pleasant success. We are sure you will understand our exclamation: "We have growth!". Now we are ready to start with the characterization of the prionic circuitery!

On the other hand, we tried to separate the p28a plasmid and the PM2 (LEA) insert. We had to digest the previously isolated plasmid with the BamH1 and XhoI restriction enzymes and let them disjoin in an agarose gel. Unfortunately, the isolation from an agarose did not work correctly, so we will have to repeat these steps tomorrow.

August 10th

We repeated the digestion and separation by electrophoresis that we conducted yesterday. The isolation has been made with a new kit that should work properly.

Finally, we obtained around 15 ng/uL of both plasmid and insert, which is not a great achievement, but allows us to continue with the procedure.

August 11th

The plasmid pUC19-ACT was digested in order to get the ACT constitutive promoter for our future PM2 construction. At the same time, we opened the shuttle centromeric and episomal plasmids YCP-XXXX and YEP-XXXX, where we plan to insert the promoter.

It did not turn out well, but we think we can get over that experimental drawback by repeating the procedure tomorrow.