Part:BBa_K4286099

Classical oscillator composed of three genes encoding repressor protein

2022 SZU-China has designed a timed suicide switch mediated by a classical synthetic gene oscillator. In the negative feedback loop, the oscillator is composed of three genes encoding repressor protein -- TetR from the Tn10 transposon, cI from bacteriophage lambda, and LacI from the lactose operon -- each repressor protein is attached to an LVA degradation tag located at the carboxy terminus. The concentration of each kind of repressor protein changed cyclically: lacI inhibited the expression of tetR, tetR inhibited the expression of cI, and cI inhibited lacI expression.

Usage and Biology

Gene oscillation is a gene regulation mechanism, and the amplitude and period of oscillation reflect the gene expression. Its principle is that three gene modules whose encoded repressors inhibit each other are connected in series to form negative feedback, and the periodic change of the content of repressor is realized by the inhibition and deinhibition of gene modules. The oscillator contains three repressor proteins: TetR from the Tn10 transposon, λ cI from bacteriophage λ, and LacI from the lactose operon. Each repressor carries an LVA degradation tag at the C-terminus. The oscillator device is encoded on a low copy plasmid pSB3C5.

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

  Illegal BglII site found at 2188
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]