Team:UNICAMP-Brazil/Notebooks/September 17

ColiGuard

Colicin Kill mechanisn

• We made two PCR reactions. The first reaction to CeaB gene amplification with VF2 and ColR primers and the second reaction to CeiB gene amplification with Anticol F and Anticol R. We got success with these PCR reactions. We obtained two different band size: 1800 bp for CeaB and 259 bp for CeiB.

Luige and Ane

PY Promoter - Selection of transformants

• Since the enzymes XbaI and SpeI produce compatible cohesive ends, the fragments PY1 and PY2 digested with this enzymes can be inserted in both orientations in the plasmid digested with the same enzymes. So, it will be necessary to do a screening of the transformants to find those ones that have the plasmid with the fragment inserted in the right orientation.

• We then selected 15 colonies of each transformation we did yesterday (PY1 + BBa_J23100 and PY2 + BBa_J23100) to perform a colony-PCR screening. In this PCR we used the forward primer of the inserted fragment and the reverse verification primer (VR) of the plasmid. Only in those colonies that have the plasmid with the fragment inserted in the right orientation this pair of primers would be able to amplify a fragment.

• The expected size for PY1 + BBa_J23100 amplified with these primers is 1060 bp.

• The expected size for PY2 + BBa_J23100 amplified with these primers is 1000 bp.

• We then chose 4 candidate colonies in the agarose gel of the PCR. 2 with PY1 and 2 with PY2.

• We inoculated these colonies in liquid LB-AMP at 37ºC to perform miniprep (Protocol 2) for plasmid extraction tomorrow.

Fabi and Léo