Team:UNICAMP-Brazil/Notebooks/October 5

ColiGuard

CRE-Recombinase without ATG - Miniprep

• Today we performed the miniprep of the 20 grown cultures from yesterday with our new biobrick: Cre-Recombinase without ATG. We confirmed it by running an electrophoresis gel.

Victor

finOP and Cre-Recombinase with pGEM strategy

• Today we performed minipreps from yesterday's inoculated cultures, according to Protocol 2.
• We expect to recover pGEM plasmids finally containing singly our inserts for finO, finP and Cre-Recombinase w/o ATG.
• As both sides of the inserts contains the A stick end, it's possible that we will find in some samples unwanted ligations (it could be ligate in an upside down position). Therefore, we will also need to perform PCRs to confirm that our inserts indeed are in the correct frame position.

Marcelo and Victor

Colony PCR to confirm the BBa K112806 + BBa B0015 ligation

• A lot of colonies have grown, it looks like our ligation were ok.

• We selected 10 colonies to a PCR colonies,we used BBa B0015 as our positive control.

• All the colonies didn't have the insertion of BBa K112806, but the size of the fragment was ok to BBa B0015.

• We don't have material enough of BBa B0015 for other digestion, so we did another inoculum to do a miniprep tomorrow.

Marcos, Luige and Ane

PY Promoter - Colony-PCR screening

• We selected 5 white colonies of each transformation we did yesterday to perform a colony-PCR screening. In this PCR we used the M13 primers of pGEM vector. We analyzed the results of our PCR in an agarose gel:

• The expected size for PY1 + pGEM amplified with M13 primers is 390 bp.

• The expected size for PY2 + pGEM amplified with M13 primers is 332 bp.

• Unfortunately, just the colonies with PY1 displayed bands with the expected size in the gel. Since the sequence of PY1 is more complete than the sequence of PY2, we decided to not try again with PY2 and use just the colonies that have pGEM vector with PY1.

• So we selected 2 colonies with PY1 + pGEM (2 and 3) and inoculated them in liquid LB-AMP at 37ºC to perform a mini-prep for plasmid extraction tomorrow.

Fabi and Léo

CeaB and CeiB: pGEM cloning strategy

• Due to the unsuccessful transformations in the backbone plasmid, we decided to change the strategy. Then, today we started the pGem cloning strategy (Protocol 15). We are going to insert our DNA colicins in the pGEM plasmid. We cut CeaB, CeiB and pGEM plasmid with SpeI restriction enzyme separately. After purification, we started the ligation reaction. We joined pGEM plasmid with CeaB and CeiB respectively. We made dyalisis and finally transformed into competent E.coli. We expect a good result this time.

Ane & Luige

YeastGuard

New Strategy: pGEM

• <p style=”text-align:justify;”>We digested the biofusion vector with two combinations of enzymes: EcoRI and SpeI; XbaI and PstI to use in the new strategy (Protocol 14).

• We repeated the part's PCR to be sure that we will have enough material to perform the following ligation reactions to achieve the biobrick format. We obtained Lysozyme, pJEN1 and JENorf amplicons.

• We confirmed the correct insertion of pDLD in pGEM by colony PCR using the part's internal forward primer and the vector's reverse primer (M13). The Lysozyme insertion wasn’t confirmed yet.

Raíssa and Taís