Difference between revisions of "Team:Hong Kong HKUST/pTet"

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

Revision as of 16:09, 18 October 2016

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tetp Module

The tetp module consists of a Tet repressible promoter upstream of the CDSs of PhlF and LacI, which are then followed by the reporter gene, mTagBFP. When the tetp module is active, the two repressor proteins, PhlF and LacI will be expressed along with the reporter gene, mTagBFP. This results in the repression of the two other modules in our system, phlFp and lacp.

Promoter, Repressor and Inducer Specification


tetp promoter
  • Promoter name: tetp (Tet repressible promoter)
  • Length: 54 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, TetR represses tetp 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: TetR (Tet repressor)
  • Part Length: 685 base pairs
  • Structure: Homodimer of alpha-helices
  • Binding affinity: To tetp, 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.

Mechanism of tetp - TetR - aTc Interaction


Figure 5. Behaviour of TetR in the presence/absence of aTc