Difference between revisions of "Part:BBa K613018"

Revision as of 21:53, 21 September 2011

TetR Y42F K108E mutant

This is a TetR mutant, who carries the YF42KE108 mutation.

In vivo characterization

This TetR mutant was characterized in vivo by putting it into pSB3K1 under a constitutive promoter (J23116). This plasmid was cotransformed with J61002 harbouring RFP under pTet promoter (B0040) in DH5alpha cells. Cells were grown in a medium containing Kanamycin & Amplicillin plus different concentrations of ATC, ranging from 0 to 2000 ng/mL. OD600 absorbence and RFP fluorescence were measured every 10 minutes during 12 hours on a platereader machine.

Induction curves

Fluorescence measurements (RFUs) were normalized by OD600 values.

In the absence of ATC, RFP expression in presence of the mutant goes up to 5000 normalized RFUs, which is slightly more than what is observed with the wild-type or the V36F mutant(K613013) for example. This indicates that the mutant is able to strongly bind and inactivate pTet, although less than the wild-type. With 2000 ng/mL of ATC in the cell culture, RFP expression rises up to 25000 normalized RFUs - a value observed for the wild-type and the P39K mutant(K613016) - showing that the P39K mutant is repressed by ATC in a normal way.

Dose-response curve

Fluorescence measurements (RFUs) were normalized by OD600 values. For each ATC concentration, we estimated the steady-state fluorescence expression by averaging the measurements over the last hour.

Sequence and Features