Team:UNICAMP-Brazil/Notebooks/September 26

ColiGuard

New Strategy / finO and finP

• We are truly disappointed that we still couldn't make our finO and finP biobricks. We found a lot of issues that must be solved in order to achieve this objective, and we are totally running out of time =(

• We decided that we are going to leave finO and finP aside for a while, and we will focus on constructing our modified version of Paris 2007 Team's biobricks.

• We made a whole schedule for that, and we hope we would complete it in a week tops. The next step would be it's insertion into genomic DNA by homologous recombination.

Marcelo

Recovering More Biobricks

• We started our "new strategy" (related to modifying Paris 2007 team's biobricks) by recovering some biobricks that had now became useful for our project. We ressuspended biobricks BBa_E0840 (GFP device), BBa_I718017 (lox71), BBa_J61000 (chloramphenicol resistance cassette) and BBa_I718016 (lox66), and then transfomed them into electrocompetent E. coli cells, strain DH10B, according to Protocol 3.

• The transformed cells were plated in LB-AMP media, and were grown for an O/N period.

Marcelo and Victor

Cre-Recombinase and pSB1A3 - New digestion

• Today we repeated both Cre-Recombinase's and pSB1A3's digestion with XbaI and SpeI. Digestion lasted 3 hours.
• Then, we ran an 1% agarose gel in order to confirm that digestion actually worked.
• Sadly, it didn't. =/

Víctor

YeastGuard

Solving the recircularization problems: Dephosphorylation with CIAP

• The dephosphorylation of the vectors theoretically avoids its recircularization. We tried a diferent protocol from the one used by ColiGuard.

• We performed 5' dephosphorylation of a biobrick vector (previously digested with XbaI and SpeI) with CIAP (Protocol 10) in order to test the reaction efficiency and compare with SAP protocol (Protocol 9).

• We did two ligation reactions, one using the phosphorylated vector (control) and another using the dephosphorylated (Protocol 11).

• We transformed the electrocompetent E. coli (Protocol 3) with the ligations and plated in LB-AMP-KAN media.

Raíssa and Taís