Part:BBa_K4286101
Oscillator device for improved version of timed suicide switch
Usage and Biology
2022 SZU-China has designed the second generation of timed suicide switch.
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.
The following improvements have been made to the second generation oscillator: the MazF expression device has been added; the LVA degradation tag has been removed. These changes create a concise and elegant oscillator with highly regular and longer period oscillations. What's more, the oscillator2.0 has a longer oscillation period and additionally has the function of expressing Toxin MazF. The oscillator2.0 is encoded on a low copy plasmid pSB3C5.
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.
Sequencing
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2662
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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