Promoter, Repressor and Inducer Specification

• Promoter name: lacp (Lac repressible promoter)
• Length: 200 base pairs
• Strength with:
  • Weak RBS (BBa_B0032): 5.6 - 517.5 (+17) RPU
  • Medium RBS (BBa_B0030): 25.5 - 2323.1 (+259.5) RPU
  • Strong RBS (BBa_B0034): 39.6 - 4041.5 (+247.8) RPU

In gram negative bacteria, LacI represses lacp which regulates tetracycline resistance by driving the expression of TetR and TetA, the latter of which codes for a tetracycline-magnesium complex that pumps tetracycline out of the cell.

• Repressor name: LacI (LacI repressor)
• Part Length: 1153 base pairs
• Structure: Homo-tetramer
• Binding affinity: To lacp, very light, 0-1 µl/ml has been shown to cause a 5 order of magnitude change in fluorescent protein production

The TetR repressor exists in 2 states: silent and active. In the absence of inducers, the active state of TetR binds to the TetO1 and TetO2 operator sites of the tetp promoter, thus inhibiting transcription. In the presence of inducers, however, TetR undergoes allosteric rearrangement and switches to its silent state, resulting in its release from the promoter, thus allowing transcription to take place.

• Inducer name: ATc (Anhydrotetracycline)
• Binding Affinity: (to TetR) Ka ~ 109 M-1
• Effective range: 250 - 25000 ng/ml

In our project, anhydrotetracycline (aTc) was used to induce tetp driven expressions. aTc is a derivative of tetracycline, tetp’s native inducer, which has shown stronger binding affinity to TetR. On top of that, it has also exhibited much lower antibiotic activity on Escherichia coli, making it a more suitable inducer for our tristable switch system.