Part:BBa_K4286103
Effector device for improved version of timed suicide switch
2022 SZU-China has designed the second generation of timed suicide switch. Improvements have been made to the tentative timed suicide switch, creating the stable and elegant timed suicide switch2.0. See more about effector2.0 in BBa_K4286103.
See more about Oscillator1.0 in BBa_K4286099 and Effector1.0 in BBa_K4286100.
Sequencing
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 108
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
Effector is the device performing suicide. The core of the effector1.0 is the MazEF toxin-antitoxin system from Bacillus subtilis, which can lead to programmed cell death. The antitoxin MazE is controlled by the Promoter PJ23110, and the toxin MazF is controlled by the constitutive Promoter tetR. 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.
Assembly
The improved timed suicide switch consists of oscillator2.0 and effector2.0.
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.
Reasons for improvement
1. The number of effector plasmids fluctuates at a high level
First, in the timed suicide switch1.0, the oscillator is placed on a rigor type plasmid and the effector is placed on a relaxation type plasmid. The two devices are in a separated state and communicate with each other through tetR (tet repressor). Due to the poor replication control of effector plasmids used, the number of effector plasmids fluctuated at a high level, and the number of effector plasmids in each cell of the engineered bacteria population was not strictly the same. This difference sets the activation and inhibition of Promoter tetR on the oscillator and effector at variance, making it difficult to achieve synchronous oscillation of toxin mazF's concentration in the population.
Therefore, we transferred the MazF expression device from the effector plasmid to the low-copy oscillator plasmid, which could greatly reduce the amplitude standard deviation of MazF oscillation and help to achieve the synchronization of engineering bacteria.
2.Overloading of Proteolytic System in engineering bacteria
Secondly, for the purpose of realizing the rapid oscillation, the C-terminal of the three repressor proteins of the oscillator in the timed suicide switch1.0 is added with a rapid degradation tag. However, because these repressor proteins with the tag share the same Proteolytic System with other proteins, this leads to an overload. Theory shows that the supersaturation of the Proteolytic System will lead to large random fluctuations in the oscillation of individual cell, and the fluctuation caused by the delayed degradation of repressor will be fed back to the repressor protein through the Proteolytic System, thus interfering with the oscillation.
Therefore, we deleted the LVA degradation tag at the C-terminus of repressor proteins, eliminating the degradation competition between repressor proteins, which will reduce the noise of single cell oscillation and prolong the oscillation period. This will also extend the suicide time of engineered bacteria, so that the engineered bacteria can survive longer, which is good for our project.
3. Low disinhibition prefabricated of Promoter tetR
Third, previous studies have shown that the noisiest phase of oscillations is when the level of TetR is the lowest. At this time, the disinhibition of Promoter tetR occurs at a very low threshold of tetR. Theory suggests that if this threshold is raised, the regularity of oscillations may be greatly improved.
We added five highly efficient tetR binding sites to the effector plasmid. The modified effector plasmid can be used as a sponge of tetR to adsorb tetR. The tetR binding site was derived from Promoter tetR and artificially engineered to increase binding efficiency. In theory, the introduction of tetR molecular sponge will reduce the effective concentration of tetR in the cytoplasm, thus increasing the apparent threshold of Promoter tetR disinhibition and greatly improving the regularity of the oscillator.
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.
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