Team:Hong Kong HKUST/pTet
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 tetp is activated, 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.
Figure 1. tetp module
Promoter, Repressor and Inducer Specification
- 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
- (Chen & Wang, 2014)
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 (Lederer, Takahashi, & Hillen, 1995). 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 (Aleksandrov, Schuldt, Hinrichs, & Simonson, 2008).
- 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 (Gossen & Bujard, 1993). On top of that, it has also exhibited much lower antibiotic activity on Escherichia coli (Oliva, Gordon, McNicholas, Ellestad, & Chopra, 1992), making it a more suitable inducer for our Tristable Switch system.
Mechanism of tetp - TetR - aTc Interaction
Figure 2. Behaviour of TetR in the presence/absence of aTc
In the absence of aTc, TetR would bind to tetp, inhibiting transcription and thus bringing mTagBFP expression to a halt. In the presence of aTc, however, TetR would lose the ability to bind to tetp, allowing transcription to proceed, resulting mTagBFP expression.
References
1. Aleksandrov, A., Schuldt, L., Hinrichs, W., & Simonson, T. (2008). Tet Repressor Induction by Tetracycline: A Molecular Dynamics, Continuum Electrostatics, and Crystallographic Study. Journal Of Molecular Biology, 378(4), 898-912. http://dx.doi.org/10.1016/j.jmb.2008.03.022
2. Chen and Wang. (2014). Synthetic Gene Network: Modeling, Analysis and Robust Design Methods.
3. Gossen, M. & Bujard, H. (1993). Anhydrotetracycline, a novel effector for tetracycline controlled gene expression systems in eukaryotic cells. Nucleic Acids Research, 21(18), 4411-4412. http://dx.doi.org/10.1093/nar/21.18.4411
4. Lederer, T., Takahashi, M., & Hillen, W. (1995). Thermodynamic Analysis of Tetracycline-Mediated Induction of Tet Repressor by a Quantitative Methylation Protection Assay. Analytical Biochemistry, 232(2), 190-196. http://dx.doi.org/10.1006/abio.1995.0006
5. Oliva, B., Gordon, G., McNicholas, P., Ellestad, G., & Chopra, I. (1992). Evidence that tetracycline analogs whose primary target is not the bacterial ribosome cause lysis of Escherichia coli. Antimicrobial Agents And Chemotherapy, 36(5), 913-919. http://dx.doi.org/10.1128/aac.36.5.913
