Team:UNICAMP-Brazil/Notebooks/October 3

ColiGuard

Cre-Recombinase without ATG + pSB1A3 transformation

• Today we transformed the Cre-Recombinase without ATG + pSB1A3 ligation, performed yesterday, in E. coli DH10B eletrocompetent cells. We then plated on LB-AMP plate, according to information from registry page.

Victor

Ligation of finOP and Cre-Recombinase on pGEM vector

• In order to start our pGEM strategy on assembling biobricks, today we ligated PCR's products of finO, finP and Cre-Recombinase into pGEM Vector. There is no need for any digestion before this procedure, since pGEM has an T stick ends capable of pairing with the A stick ends left by Taq Polymerase on the PCR products.

• We followed Protocol 11, without modifications.

• Ligation lasted 1 hour.

Marcelo and Victor

Transformation of finOP's and Cre-Recombinase's ligations

• After performing the required ligations, we then transformed them into electrocompetent E. coli bacteria, strain DH10B, according to Protocol 3.

• We plated the trasformed cells in LB-AMP media, which already contained X-gal substrate.

• Plates were incubated at 37ºC for an O/N period.

Marcelo and Victor

Kamikaze’s parts digestion and ligation

• We made the digestion of BBa K112806 with EcoRI and SpeI, BBa B0015 with EcoRI and XbaI and BBa I746911 with SpeI and Pst.

• We purified the digestion and made the ligation of BBa K112806 in the BBa B0015 plasmid according to the Protocol 11

Marcos

PY Promoter - New strategy

• Our cloning strategy for inserting PY1 and PY2 fragments into the plasmid containing the RFP reporter didn’t work as we expected. We believe that one of our problems is the compatible cohesive ends produced by the enzymes XbaI and SpeI. As other members of our team are facing this same problem, our group decided to create another strategy to construct our biobricks. This new strategy is based on the pGEM vector system and consists basically in cloning our fragments in this vector and then excising them with EcoRI and SpeI. After the excision our fragment won’t have compatible cohesive ends anymore. (See pGEM cloning strategy for more information).

• So today we started to work in this new strategy. First of all we digested our fragments PY1 and PY2 (amplified by PCR from F plasmid) and pGEM vector with SpeI. The digestion lasted for 3 hours.

• After the digestion we performed 2 ligation reactions following Protocol 11: PY1 + pGEM and PY2 + pGEM.

Fabi and Léo

YeastGuard

New Strategy: pGEM

• The ligation reactions didn´t work. So today we decided not to digest the fragment and the pGEM vector with SpeI, but to ligate it directly, considering that we can confirm the correct insertion by PCR.

• We did new ligation reactions with our four parts (pJEN1, pDLD, Lysozyme and orfJEN1) in pGEM.

Taís