Team:UNICAMP-Brazil/Notebooks/September 4

MicroGuards

Electrocompetent E. coli

• Today we prepared electrocompetent E. coli to use in our transformations according to Protocol 4.

Hemolysin Operon Amplifications

• We tried again to amplify the hemolysin operon, but this time we reduced the annealing temperature to 50, 48, 46, 44, 42, 40 and 38 Celsius degrees. Again we do not had positive results.

The DNA used was extracted from wild strains alpha hemolysin positive, we decided to use a already known strain, O26H-, which probably has the plasmidial hemolysin.

ColiGuards

Cre-Recombinase without ATG

• Today we started the construction of Cre-Recombinase without ATG, in order to made a new biobrick involving this construction. We ressuspended the biobrick of Cre-Recombinase: BBa_J61047 and transformed in E. coli DH10B eletrocompetent cells. We then plated on LB Amp plate, according to information from registry page.

Víctor

PY Promoter - PCR amplification

• Today we performed the PCR amplification of the PY promoter from the F plasmid previously extracted from the conjugative strain (July 16). We did two different reactions, using two types of forward primers (Ppy-F-1 and Ppy-F-2) for the same reverse primer (Ppy-R). The product amplified with the Ppy-F-1/Ppy-R primers has 133 bp and the one amplified with the Ppy-F-2/Ppy-R has 75 bp. We called these products PY1 and PY2 respectively. The sizes of the products were confirmed by an agarose gel:

Fabi and Léo