Team:UNICAMP-Brazil/Notebooks/October 9

ColiGuard

Another PCR colony of BBa K112806 + BBa B0015, another failure...

• A lot of colonies have grown again, we selected 15 to do the Colony PCR, but none of them was positive.

• We are really worried with this, our time is finishing. We decided to chenge radically our strategy. We will change the terminator to BBa B0014 instead of BBa B0015 because the digestion of B0015 is not working. We let B0014 digesting ON with EcoRI and XbaI

Marcos

finO and finP with pGEM - confirmation

• We decided that we are going to confirm such ligations by performing:

-A Digestion: with XbaI and SpeI restriction enzymes, in order to release our parts from pGEM plasmid;

-A PCR: performed with the specific forward primer for our insert and with the reverse primer for pGEM plasmid (M13), in order to confirm that, once our inserts are indeed in there, they are also in the correct frame position.

• Seeing that, today we performed the digestion with EcoRI and SpeI for all of our minipreps samples that actually worked. Digestion lasted 3 hours.

• According to the picture, the digestion excised a fragment of expected size (600 bp for finO and 120 bp for finP) in almost all samples! That's an important clue on confirming our inserts's correctly ligation!

Gabriel and Marcelo

PY Promoter - Colony-PCR screening

• We selected 13 colonies of the transformation we did yesterday to perform a colony-PCR screening. We used 2 pairs of primers and analyzed the results of our PCR in an agarose gel:

- VF/VR, the verification primers of BBa_J23100 plasmid:

- Ppy-F-1 and Ppy-R, the pair of primers that amplify our inserted sequence (PY1):

- The expected size for PY1 + BBa_J23100 amplified with VF/VR primers is 1190 bp.

- The expected size for BBa_J23100 without our insert amplified with VF/VR primers is 1057 bp.

- The expected size for PY1 + BBa_J23100 amplified with PY primers is 133 bp.

• As we can observe in the gel photo, colonies 6 and 10 presented bands compatible with the size expected for both pairs of primers.

• So we inoculated these 2 colonies in liquid LB-AMP at 37ºC to perform a mini-prep for plasmid extraction tomorrow.

Fabi and Léo

Cre-Recombinase - Confirmation: Stage I

• After performing minipreps (on October 5th) for 20 inoculated cultures, possibly harbouring our Cre-Recombinase without ATG's Biobricks, it's time to start the confirmation procudures. Regarding "confirmation procudures", we mean a two stages procedure: (1) perform a specific PCR for Cre-Recombinase and (2) perform a digestion, aiming in excising a fragment of expectable size from the pSB1A3 vector.

• Today we did stage I, a PCR reaction using Cre's specific designed primers, running an agarose gel straight after reaction ended.

• Thereby, according to Stage I.... we did it! Several samples appears to contain our Cre's Biobrick!

• If we succeed in Stage II, we will finally be able to say we got a biobrick! =]

Víctor

YeastGuard

New Strategy: pGEM

• The plates containing E. coli transformed with pDLD didn’t grow. We repeated the ligation reaction and transformed competent E. coli again.

• We transformed the ligation between Lysozyme and biofusion, performed yesterday, in competent E. coli and plated in LB+Amp media.

• The pJEN1 plates showed colonies, we did the colony PCR to find a correct construction of the pJEN1 in Biofusion. We confirmed 12 of 20 colonies.

• We repeated the ligation reaction of JENorf with pGEM. We transformed and plated in LB+amp media.

YFP+Terminator

• We transformed the ligation between YFP+end and Adh1 promoter, performed yesterday, in competent E. coli and plated in LB+Amp media.

• We decided to check over the size of the terminator part at the registry, since we had to much work with it. We submited its sequence to the BLAST algorithm and discovered that this biobrick is the same as YFP one!!! =( We got so desapointed =/ It was the only terminator biobrick to be used in yeasts.

pADH1+YFP

• Today we started the experiments to obtain a new device in biobrick format. It’s the ADH1+YFP biobrick.

• First of all, we did these digestions: 1)YFP digestion using the enzymes XbaI and PstI; 2)pADH1 (biofusion) digestion using the enzymes SpeI and PstI.

Wesley

YEP358

We transformed E. coli with YEP358-β galactosidase plasmid (Protocol 3)