Part:BBa_K4286103:Design
Effector device for improved version of timed suicide switch
- 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]
Design Notes
2022 SZU-China has modeled the timed suicide switch. Although the mathematical model shows a continuous and stable oscillation, the experimental evidence shows that the oscillation of the repressor in the engineering bacteria is still irregular in phase and amplitude. We investigated and identified three factors that contributed to the noise in the first generation of the timed suicide switch and come out with improvements. Based on that, we made some improvements on the tentative timed suicide switch：
1.The number of effector plasmids fluctuates at a high level；
Therefore, we transferred the MazF expression device from the effector plasmid to the low-copy oscillator plasmid, which can 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；
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
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.
To sum up, the upwards improvements have been made to the tentative timed suicide switch, creating the stable and elegant timed suicide switch2.0.
Source
The improved version of the effector is independently designed by SZU-China.
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.