09/16/2014

Hello!!! Today we used the spectrofluorometer for the first time!!! We used DH5α without genetic modifications as “blank” and DH5α with p26G as positive control, grown in liquid medium LB for 16h.

We used:

Equipment: Hidex Chameleon.

Excitation filter: 340nm

Emission filter: 500nm

For creation of a GFP insanity expression levels curve, we diluted the sample of DH5α with p26G in liquid medium LB for 10^-1, 10^-2, 10^-3. So we’ll be able to compare and calculate GFP expression intensity, our blank should also be diluted. The intensity levels of fluorescence showed by the Hidex Chameleon are:

In what DH5α with plasmid p26G in liquid medium LB grown for 16 hours non diluted shows fluorescence intensity of 283070 and diluted 10^-3 shows 1655. Espectrofluorimeter calibrated! Tomorrow we’re going to do our first test with GFP induced by mercury in DH5α!!!

We made pre inoculum in medium LB + chloramphenicol 34ug/ml of DH5α with constructions BB_Essential + E0840 in PSB1C3!! Now is the moment of truth! We also had a meeting with the professor Tereza Cristina from Chemistry department to discuss the possibilities and procedures for mercury quantification, and we could plan the methods to be applied for constructions characterization with the metal binding protein (MBP) and protein that volatilizes mercury (MerA). Was super good to know that we can work with minimum mercury quantities, and that there is a super sensitive equipment that is able to quantify that.