• Introduction
  • Home
  • The Team
  • Photo Gallery
• Our Project
  • Results Summary
  • Selection System
  • In-Vitro Characterization
  • In-Vivo Characterization
  • Microfluidics
  • Data
  • Attributions
• Tools
  • Gibson Assembly
  • MITOMI
• Notebook
  • Protocols
  • May
  • June
  • July
  • August
  • September
  • October
• Considerations
  • Human Practices
  • Safety
• Acknowledgements
  • Sponsors
  • Partners

Intro

to write here: how we got TetR mutants, with which method (we can also explain the method in the "tools" section)
The MITOMI characterization will be explained in the "tool" section?

Background Information about TetR

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.

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. Orth et al, 2000 The whole promoter, comprising this recognition sequence, is called Ptet.

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. Ramos et al, 2005

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.

Lilia, please add a list... I have 4C, 6C (Helbl) and double from Krueger