Part:BBa_K4656009

Plac-tetR-Ptet-lacI-MazF

TetR, the tetracycline repressor, can inhibit the promoter Ptet. That is to say, the promoter Ptet is constitutively ON and repressed by TetR. However, TetR repression is inhibited by the addition of tetracycline(TET) or its analog, aTc. LacI can bind to the PLac promotor and PLlac0-1 hybrid promotor and inhibit transcription. IPTG (isopropyl-β-d-thiogalactoside) binds to LacI and inhibits its operation, therefore promoting transcription.

Thus, tetR's ability to inhibit Ptet is weakened when TET is present, while lacI expression products inhibit the promoter Plac to produce less tetR to repress Ptet and enable MazF behind lacI on the gene route to express. MazF, as a toxin, is an endonuclease that cuts RNA at ACA sites and acts as a reporter gene to cause microbial death[1]. MazF can be characterized in a cell density-dependent manner in E. coli.

MazE, like MazF, encodes stable non-specific ribonuclease enzymes in Bacillus subtilis that are used in combination to deliver toxin-antitoxin suicide switches under a variety of stressful conditions. In the gene route, rhamnose activates the promoter PRha, which is expressed in the downstream MazE. Because MazE is an antitoxin, it can produce the opposite effect of MazF, that is, providing exogenous rhamnoose can eventually produce an antitoxin to fight the toxin and keep the E. coli alive.

Usage and Biology

This gene route can be used to control the production of both MazF toxin and MazE antitoxin in E. coli with exogenous TET or rhamnoose, to determine the survival or death of E. coli, and can be applied to the safety module to prevent E. coli from overbreeding when necessary or timely killing when there is a risk, and can also play a role in ensuring the survival of E.coli.

Reference

[1] Park, J. H., Yamaguchi, Y. & Inouye, M. Bacillus subtilis MazF-bs (EndoA) is a UACAU-specific mRNA interferase. FEBS Lett 585, 2526-2532 (2011).  https://doi.org:10.1016/j.febslet.2011.07.008 [2] Elowitz, M. B., & Leibler, S. (2000). A synthetic oscillatory network of transcriptional regulators. Nature, 403(6767), 335–338. https://doi.org/10.1038/35002125

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2237
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]