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Effector device for improved version of timed suicide switch

2022 SZU-China has designed the second generation of timed suicide switch.

See more about Oscillator1.0 in BBa_K4286099 and Effector1.0 in BBa_K4286100.

Usage and Biology

Effector is the device performing suicide. 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 following improvements have been made to the second-generation effector: the MazF expression device has been deleted; high efficiency tetR binding sites have been added. what's more, the effector2.0 removes the function of periodically expressing MazF and adds the function of binding tetR, acting as a tetR sponge. The effector2.0 is encoded in the high-copy plasmid colE1.

Sequencing

Sequence and Features

Assembly

The oscillator2.0 and the effector2.0 form a timed suicide switch2.0.

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.

Prediction

In theory, the oscillating rise of MazF concentration and the stable concentration of MazE can be predicted by the mathematical model. That is, we can predict the exact time point of suicide as long as we have accurate parameters. We performed a modeling analysis of the timed suicide switch1.0, mathematically demonstrating that the engineered bacteria will begin to suicide after approximately 770.1 minutes, when the concentration of toxin mazF is higher than that of antitoxin MazE reversely.

Furthermore, we may also be able to achieve different suicide time by changing the parts of the gene device. For example, by replacing the constitutive promoter with different transcriptional activity, we can change the stable concentration of antitoxin MazE, so as to change the time of concentration reversal of MazF and MazE. Thereby changing the suicide time of the engineering microorganisms.

See more about the model of timed suicide switch1.0 in BBa_K4286099.

References

[1]Elowitz MB, Leibler S. A synthetic oscillatory network of transcriptional regulators. Nature. 2000 Jan 20;403(6767):335-8. doi: 10.1038/35002125. PMID: 10659856.

[2]Purcell O, di Bernardo M, Grierson CS, Savery NJ. A multi-functional synthetic gene network: a frequency multiplier, oscillator and switch. PLoS One. 2011 Feb 17;6(2):e16140. doi: 10.1371/journal.pone.0016140. PMID: 21359152; PMCID: PMC3040778.

[3]Potvin-Trottier L, Lord ND, Vinnicombe G, Paulsson J. Synchronous long-term oscillations in a synthetic gene circuit. Nature. 2016 Oct 27;538(7626):514-517. doi: 10.1038/nature19841. Epub 2016 Oct 12. PMID: 27732583; PMCID: PMC5637407.

[4]Hoseini S, Kalani BS, Ghafourian S, Maleki A, Asadollahi P, Badakhsh B, Pakzad I. In Vitro and In Silico Investigation of some Type II TA Genes in H. Pylori. Clin Lab. 2022 Aug 1;68(8). doi: 10.7754/Clin.Lab.2021.211002. PMID: 35975492.

[5]Nigam A, Ziv T, Oron-Gottesman A, Engelberg-Kulka H. Stress-Induced MazF-Mediated Proteins in Escherichia coli. mBio. 2019 Mar 26;10(2):e00340-19. doi: 10.1128/mBio.00340-19. PMID: 30914510; PMCID: PMC6437054.