Part:BBa_K1075048

AraC-pBAD-RBS32-mazF-TT-pLac-RBS32-mazE-(Ec)ssrA(DAS+4)-TT

Structure

This part consists of AraC-pBAD-RBS-mazF-TT-pLac2-RBS-mazE-(ec)ssrA(DAS+4)-TT

Biology

MazF is part of the biological toxin-antitoxin system mazEF. This system is derived from the bacterium Escherichia coli, where it serves as a stress inducable killswitch. mazF, a RNA-degrading enzyme, ist he toxin and is normally inhibited by the presence of mazE. Absence of mazE leads to cell death. MazF is part of the biological toxin-antitoxin system mazEF. mazE is the antitoxin of this part and normally inhibits the activity of mazF. Absence of mazE leads to cell death, lest it be expressed in high levels during a certain timeframe [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC532418/]. The Escherischia Coli ssrA tag is part of the ClpXP system of E. Coli. In this system, it serves as a degradation tag that is recognized either by sspB or the ClpXP protease, both resulting in the degradation of the tagged protein. The DAS+4 mutation decreases the sensitivity of the ClpXP protease for the ssrA tag, thus increasing the dependency of mediation via sspB for degradation [http://www.ncbi.nlm.nih.gov/pubmed/16762842].

Application

This construct can be used to gain IPTG and Lactose inducible expression of mazE(ec)ssrA(DAS+4) and arabinose inducible expression of MazF. Thus, both parts of the toxin-antitoxin system mazEF are inducible and this construct can be used to further examine this system. We used this construct as part of our proof of concept. Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 2244
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  Illegal BamHI site found at 1994
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961