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.
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