Part:BBa_K4286103
Effector device for improved version of timed suicide switch
2022 SZU-China has designed the second generation of timed suicide switch. 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.
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. 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 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.
modeling
In order to establish the oscillator model, the following assumptions are necessary for the model: Assumption 1: The amount of DNA in the same cell is constant and the physiological behavior is same. The DNA in the same cell can be expressed at the corresponding gene fragment. Assumption 2: Transcription and translation processes are carried out under saturation conditions. In the process of transcription and translation, polymerases, ribosomes, amino acids and nucleotides are present in large amounts. Assumption 3: Degradation of protein and mRNA as well as reactive degradation. That is, proteins and mRNA can be degraded directly without intermediate products. Assumption 4: Transcription rate can be modelled by Hill equation. That is because the transcription rate of each gene is determined by the concentration of its protein product, and the repressor protein binds to the regulatory region of the gene faster than transcription and translation. Assumption 5: The Hill coefficient approximates the number of cooperative ligand binding sites on the receptor, and ligand molecules bind to a receptor simultaneously. Assumption 6: The translation rate of each gene is equal.
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]
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