Part:BBa_K2066550

85x tetO Binding Array

Part containing 85 tetO (tetR binding) sites. This part is used to induce a rightward shift in a transfer function by way of molecular titration.

Usage and Biology

Molecular titration as the name implies is the process of titrating out molecules of transcription factor. That means, for some amount of transcription factor, a constant amount is taken away, such that for any given amount of transcription factor concentration, we are actually working with functionally less of said transcription factor (Figure 1).

Figure 1: Diagram showing the interactions between an activator transcription factor and decoy binding array, which as a molecular titrator. Note that the number of decoy binding sites impacts the equilibrium of free transcription factor, which in turn impacts the equilibrium of the amount bound to the promoter. Diagram adapted from Lee et al. 2012 (“A regulatory role for repeated decoy transcription factor binding sites in target gene expression”)

To accomplish this shift in E. coli we used decoy binding arrays, which are plasmids containing many repeated sequences of transcription factor binding sites. These repeated binding sites cause a large number of transcription factors to be bound to sites which produce no product, thus titrating them out - see Brewster et al. 2014 (“The Transcription Factor Titration Effect Dictates Levels of Gene Expression”). This causes a rightward shift in the graph of transcription factor vs. gene product. If we then graph that same gene product versus a small molecule inducer for said transcription factor, then depending on the type of transcription factor we will either get a shift to the right (activator) or the the left (repressor). As you can see in the figure below, we used a repressor as the graph was shifted to the left.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 2434
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 2434
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1593
  Illegal BamHI site found at 825
  Illegal BamHI site found at 959
  Illegal BamHI site found at 1109
  Illegal BamHI site found at 1726
  Illegal BamHI site found at 1934
  Illegal BamHI site found at 2515
  Illegal BamHI site found at 3081
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 2434
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 2434
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2893
  Illegal SapI.rc site found at 160
  Illegal SapI.rc site found at 2506