Part:BBa_K1877011

Tetr Promoter+RBS+GFP+Terminator

The construct is designed to test along with BBa_K1877001. This is the testing construct for the repressible mechanism of 2016 Gaston Day School iGEM team's project, killswitch, which serves as a suicide program to prevent bacteria from escaping the laboratory environment and contaminating food and water system. If this construct is placed by itself in medium containing arabinose, it should be producing GFP nonstop, and we tested it with fluorescences per OD 600. After testing its individual function, we, then, ligate BBa_K1877031 along with this composite part. Then, we put them into different concentrations of arabinose. With the Tetr repressor repressing the Tetr repressible promoter, the GFP concentration should decrease in response to an increase of arabinose concentration.

Usage and Biology (added by CCA_San_Diego 2020)

Author: Ayush Agrawal, Andrew Sun

Summary: Provides specific background about the functionality and binding of the TetR repressible promoter.

Documentation:

The TetR repressible promoter is part of the TetR transcription regulation system. The tetracycline repressor TetR binds to the TetO operating sequence in its natural state. However, in the presence of tetracycline (Tet), TetR will detach from TetO to leave TetO able to induce expression. The TetR system is also incredibly tight in terms of repression, and yet sensitive in terms of induction, and therefore is an optimal choice as a promoter system for many projects and designs (Bertram & Hillen, 2007).

Tetracyclines generally have four hydrocarbon rings. They are a commonly used antibiotic, and often used to treat human infections as an antibiotic. TetA is a tetracycline resistance efflux pump that resides on the cell membrane. TetA is an effective solution to provide resistance against Tet in bacteria. The genes encoding for TetA and TetR are placed in the opposite direction, and the regulatory region for these two genes are shared (Das, Tenenbaum, Berkhout, 2016).

The TetR system arises in gram-negative bacteria. It is an alpha helical protein and is active when bound to TetO or when induced. Because of the nature of its development in bacteria, the TetR system is extremely efficient at its repressor function. Even a low level expression of TetA that is unnecessary will be detrimental to the cell. Also, due to the actual mechanism behind the toxicity of Tet to bacteria, being that it blocks protein synthesis, the induction for the system must be extremely sensitive. Due to its reproducibility in changing the expression levels of other repressors and activators, TetR is commonly used in synthetic biology constructs (Bertram & Hillen, 2007).

References

[1] Bertram, R., & Hillen, W. (2008, January). The application of Tet repressor in prokaryotic gene regulation and expression. Retrieved October 22, 2020, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3864427/ [2] Das, A. T., Tenenbaum, L., & Berkhout, B. (2016). Tet-On Systems For Doxycycline-inducible Gene Expression. Current gene therapy, 16(3), 156–167. https://doi.org/10.2174/1566523216666160524144041

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 724