Team:UNICAMP-Brazil/Notebooks/October 6

ColiGuard

Another Miniprep, another digestion, another ligation and once again, another transformation

Transformation of the BBa K112806 + BBa B0015 ligation

• We did the miniprep of BBa B0015 and BBa K112806 according to Protocol 2

• With the protduct of the miniprep we made the digestion of BBa B0015 with EcoRI and XbaI and with EcoRI and SpeI to BBa K112806. After 3 hours of digestion we purified BBa K112806 from agarose gel and BBa B0015 directly from the digestion reaction.

• Using the purified digestions we did the ligation, Protocol 11, of BBa B0015 with BBa K112806 and to test the digestion of BBa B0015 we did a ligation only without BBa K112806, if colonies grow with this ligation it means BBa B0015 is recircularizing. We staterd to quantify our samples with the new lab's espectrophotometer.

• We transformed E. coli with the ligation, we hope this time it works!!

Ane and Marcos

PY Promoter - Mini-prep

• Today we performed mini-preps (Protocol 2) to extract the plasmids from the selected colonies inoculated yesterday.

Fabi and Léo

CeiB transforming in pGEM

• We got transforming cell. We selected the white colonies and we made PCR in order to prove the correct transformation. For CeaB, we performed the reaction with pGEM M13 primer forward and ColR primer reverse meanwhile for CeiB, we used pGEM M13 primer forward and anticol R primer reverse. We run the agarose gel and this time we confirmed one good transformation. The CeiB was correctly inserted in the pGEM vector. Unfortunately, the CeaB colicin was not confirmed. For CeiB, we expected and found ~360 bp band size.

Luige & Ane

YeastGuard

New strategy: pGEM

• We need more parts!! We did PCR from the DNA ressuspended from the kit plate corresponding to the "Terminator" biobrick and from the DNA extracted form Kluyveyromices lactis. We did PCR of Jen1orf as eall. We found the JENorf fragment, unfortunately we didn't find the terminator expected fragment.

• We inoculated JENorf-pGEM and Jen1promoter-pGEM transformed colonies in liquid media to perform the screening tomorrow.