Team:UNICAMP-Brazil/Notebooks/October 8

ColiGuard

Another try for Kamikaze System

• We purify from agarose gel the digestion that we let doing yesterday and prepare another digestion with XbaI, this digestion was purified and used in a new ligation of BBa B0015 BBa K112806.

• The ligation was used in a transformation, is the third time we do the same ligation, we hope this time we have luck!!!

Ane and Marcos

PY Promoter - Transformation

• Today we transformed the ligation PY1 + BBa_J23100 into electrocompetent E. coli bacteria, strain DH10B. We followed Protocol 3 (Electroporation).

• After the transformation we plated the transformed cells in LB-AMP plates and let them grow at 37ºC for an O/N period.

Fabi and Léo

YeastGuard

New Strategy: pGEM

• We did miniprep of pDLD-pGEM, pJEN1-pGEM and Lysozyme-pGEM. We digested these plasmids with EcoRI and SpeI to release the parts from the pGEM. All of the digestions worked so we ligated them with biofusion vector previously digested with EcoRI and SpeI. We transformed competent E. coli with the promoters+biofusion ligation reaction and plated in LB+Amp media. The lysozyme ligation reaction was performed late at night, so we left it O/N to transform tomorrow.

Lysozyme agarose gel.

YFP+Terminator

• We did mini-prep of the YFP+END negative colonies and then digested them with XbaI and PstI. We confirmed two digestions by electrophoresis and purified the correct fragment from the agarose gel (Protocol 7).

• We performed the ligation reaction of YFP+END in Adh1.

Raíssa

YEP

• Today we did the YEP358 digestion with PvuII to curt of a big fragment of β-galactosidase gene. We also did the electrophoresis gel and plasmid purification (Protocol 2)

• We did a ligation reaction to recircularize the YEP358 – β-galactosidase plasmid using T4 DNA ligase.