Difference between revisions of "Part:BBa K613016"

Latest revision as of 03:33, 22 September 2011

TetR P39K mutant

This is a TetR mutant that carries the P39K mutation.

In vitro characterization

Using the MITOMI technique we determined the DNA binding landscape of the TetR P39K mutant. To do so, first we designed and generated the library of double stranded DNA sequences that cover all possible single base substitution within the tetO operator sequence. Based on that library we measured the dissociation constants of the mutant to variable tetO-like sequences and determined the specificity of the mutant to the tet operator sequence (expressed as a PWM).

WebLogo we obtained for the P39K mutant:

The strong difference of the binding affinities between the P39K mutant and the wtTetR, might be due to the altered recognition of the P39K. This mutant was shown to have a new recognition specificity for the tetO-4C operator in [http://www.sciencedirect.com/science/article/pii/S0022283697915400 Helbl et al, 1998].

Workman CT, Yin Y, Corcoran DL, Ideker T, Stormo GD, Benos PV. enoLOGOS: a versatile web tool for energy normalized sequence logos. Nucleic Acids Res. 2005 Jul 1;33:W389-92.

Helbl, V., and Hillen, W. (1998). Stepwise selection of TetR variants recognizing tet operator 4C with high affinity and specificity. J Mol Biol 276, 313-318.

Position Weight Matrix

Each row represents the changes in binding energy, ΔΔG, compared to the reference sequence upon the substitution to the indicated nucleotide at certain position within the target DNA element. Values are indicated in kcal/mol.

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 25000 normalized RFUs, which is the far more than expression level of the wild-type TetR in the same conditions. This shows that the mutant can only poorly bind and inactivate pTet - in comparison to the wild-type and other mutants such as V36F or V36F W43S. With 2000 ng/mL of ATC in the cell culture, RFP expression is further increased, but this change is quite small. This indicates again that the P39K mutant is not repressing pTet. Interestingly, the P39K mutant resembles closely to the P39Q y42M mutant (K613019); both have a mutation on the 39 residue.

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.

The dose-response curve shows that ATC has almost no action on RFP expression. While we cannot know if this mutant still binds ATC in a normal way, it is clear that P39K has only little repressive action on the pTet promoter.

Sequence and Features