Difference between revisions of "Part:BBa K4286100"

Revision as of 14:50, 9 October 2022

Effector device in timed suicide switch

2022 SZU-China plans to design a timed suicide switch mediated by a classical synthetic gene oscillator. 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 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 binds to MazF to occupy its active site, rendering it unable to bind or cleave the mRNA.

Usage and Biology

The core of the effector is the MazEF toxin-antitoxin system from Bacillus subtilis, which can lead to programmed cell death. The antitoxin MazE is controlled by the Promoter tetR, and the toxin MazF is controlled by the constitutive Promoter PJ23110. MazF is an endonuclease that specifically cleaves UACAU sites on mRNA. MazE combines with MazF at a ratio of 1:1 to occupy its active site and make it lose its toxicity. 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.

modeling

Sequencing

Sequence and Features