Part:BBa_K4286100

Effector device in timed suicide switch

Usage and Biology

2022 SZU-China plans to design a timed suicide switch mediated by a classical synthetic gene oscillator. Effector is the device performing suicide. The MazEF system is an important component of the subtilisin-antitoxin system, which leads to programmed cell death. MazF is an mRNA endonuclease that specifically cleaves a five-base UACAU sequence on RNA. In the presence of MazE, MazF has a higher affinity for MazE than its RNA substrate. MazE combines with MazF at a ratio of 1:1 to occupy its active site and make it lose its toxicity.

Figure 1.0 The effector

The core of the effector is the MazEF toxin-antitoxin system, in which the antitoxin MazE is controlled by the Promoter tetR , and the antitoxin mazF is controlled by the constitutive promoter PJ23110. The effector is encoded in the high-copy plasmid colE1.

Assembly

The oscillator and the effector form a timed suicide switch.

The engineered bacteria with a timed suicide switch were placed in an IPTG-rich medium or in a dormant state before being applied in fields. The purpose of being placed in IPTG is to continuously activate the PlacI and make the oscillator unbalanced and stagnant， in which circumstance MazF does not express.

After being applied to the field, the oscillator is re-activated with the release of IPTG and the resuscitation of the engineering bacteria. The contents of three repressor proteins changed cyclically: lacI inhibited the expression of tetR, tetR inhibited the expression of λ cI, and λ cI inhibited lacI expression. That is, the three promoters PlacI, PtetR, and PλcI were alternately activated.

As for the effector, MazE was constitutively expressed and maintained at a certain concentration in the cytoplasm, while the expression of MazF was inhibited by tetR and showed a fluctuating increase. In a simplified model, MazE and MazF bind at the ratio of 1:1, resulting in toxin inactivation. When the concentration of toxin MazF is higher than that of antitoxin MazE, the extra toxin MazF plays the role of endonuclease to cut mRNA and kill the engineered microorganisms.

Sequencing

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 481
  Illegal NheI site found at 504
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 582
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]