Latest revision as of 02:59, 22 September 2011

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In-Vitro Characterization

In vitro Main | Why TetR? | Mutant TetRs | MITOMI Data | In-vivo & In-vitro outline

When we generate new libraries of transcription factors using site-directed mutagenesis, we need to have a precise and high-throughput characterization method. These two requirements are met by the microfluidics MITOMI device that we used.

Characterizing a lot of different mutants will allow us to get a better understanding of the mutations influencing the specificity and affinity of a transcription factor. With these parameters in hand, we will be able to fine-tune the characteristics of the newly produced transcription factors.

The new transcription factors mutants characterized in vitro then need to be tested in vivo. This last step is explained in the " in vivo characterization section".

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-{{:Team:EPF-Lausanne/Templates/TetRtemplate|title=Intro}}
+{{:Team:EPF-Lausanne/Templates/TetRtemplate|title=''In-Vitro'' Characterization}}
-The choice of the trial transcription factor for the pipeline developpement was made ....
+When we generate new libraries of transcription factors using site-directed mutagenesis, we need to have a precise and high-throughput characterization method. These two requirements are met by the microfluidics MITOMI device that we used.
-== Background Information about TetR ==
+Characterizing a lot of different mutants will allow us to get a better understanding of the mutations influencing the specificity and affinity of a transcription factor. With these parameters in hand, we will be able to fine-tune the characteristics of the newly produced transcription factors.
-Widespread among bacteria, TetR is a '''repressor''' that regulates enzymes essential for resistance against the antibiotic tetracycline. These enzymes are encoded on the tet operon (TetO), which is repressed by TetR in normal conditions. When tetracycline is present, the antibiotic molecule binds to TetR and inactivates it, thus allowing the expression of TetO.
+The new transcription factors mutants characterized in vitro then need to be tested ''in vivo''. This last step is explained in the " ''in vivo'' characterization section".
-TetR forms a dimer, each part of the dimer being involved in DNA binding. Consequently, the recognition sequence of TetR is symmetrical (the two boxes on the image below), with a base pair separating the two sub-sequences. [http://www.nature.com/doifinder/10.1038/73324 Orth et al, 2000]
-The whole promoter, comprising this recognition sequence, is called Ptet.
-[[File:EPFL_TetR_consensus.jpg]]
-The binding of each monomer to the recognition sequence has been studied thoroughly; we know which amino acid interacts to which nucleotide, as can be seen in the figure below. The amino acids directly involved in binding to the DNA are found in between position 26 and 48 of each monomer. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1197418/?tool=pubmed Ramos et al, 2005]
-[[File:EPFL_TetR_contact_map.jpg|500px]]
-The impact of one or several amino acids changes on the binding strength and specificity is less known. Amino acids in direct proximity to or the residues binding DNA are probably crucial - but more distant amino acids could also have an influence due to allosteric effects. Some TetR mutants that have an altered recognition sequence have been characterized in the literature.
-The effects of the mutations V36F, E37A, P39K and Y42F were described in [http://www.sciencedirect.com/science/article/pii/S0378111907004623 Krueger et al,2007]
+[[File:EPFL-Solange-MITOMI.jpg|700px]]
 {{:Team:EPF-Lausanne/Templates/Footer}}

Team:EPF-Lausanne/Our Project/TetR mutants

From 2011.igem.org

Latest revision as of 02:59, 22 September 2011

In-Vitro Characterization