Part:BBa_K4644016
pZE-glnAp2-ttgR-asrG-Pttg-bsrG
kill switch
‘’‘Ammonia Suicide’‘’
The engineered bacterial strains designed by BUCT operate within the human intestinal tract. Human gut bacteria naturally enter the urban sewage system with excreta, eventually reaching centralized wastewater treatment facilities or being exposed directly to the natural environment.This kill switch function in septic tanks before the engineered bacteria reach wastewater treatment facilities， ensuring the termination of the bacterial strains.
design
To meet our biosafety requirements, we identified the bsrG-asrG toxin-antitoxin system, specifically the Type I toxin-antitoxin system bsrG/asrG(SR4), in Bacillus subtilis strain A-5. The antitoxin, asrG, is a cis-encoded regulatory RNA that neutralizes the action of the BsrG toxin. It prevents toxin expression by promoting the degradation of toxin mRNA and inhibiting its translation.
The transcriptional regulator TtgR from Pseudomonas putida NBRC 14164 belongs to the TetR family of transcriptional repressors. It inhibits the transcription of the TtgABC operon and its own transcription, thereby regulating the efflux of harmful chemicals from bacterial cells through efflux pumps. Due to the flexibility of TtgR's ligand-binding domain, it can bind to a wide variety of structurally diverse ligands.
Therefore, we designed a gene circuit for the suicide system (as shown in the figure below). In the human intestinal tract, which is a relatively low-nitrogen environment, the expression of glnAP2 is relatively high. At this time, PttgA is inhibited by TtgR. We also considered the possibility of leakage expression, so the antitoxin of bsrG will be co-expressed, and its expression level will be much higher than that of the toxin. In this state, the bacteria can survive normally.
In the septic tank, the bacteria will be in a high-nitrogen environment, and the expression of glnAP2 will be inhibited, which will release the inhibition on PttgA. As a result, the toxin will be expressed, and the bacteria will die.
- Fig 1. Gene circuit of "ammonia suicide" system.
Experiment
We obtained the corresponding gene sequences from the genomes of Pseudomonas putida and Bacillus subtilis (primer sequences provided in the supporting information). Using the pZe plasmid as a vector, we successfully constructed the pathway.
To validate the effectiveness of the suicide system, we transformed this plasmid into cells and cultured them on a nitrogen-free solid medium supplemented with gradient concentrations of ammonium chloride (0, 50, 100, 150, 200, 250, 300 ,350, 400, 450 mg/L) at 37°C, 12h.
The results are as follows:
- Fig 2. Verification results of suicide system.
It can be found that with the increase of the concentration of ammonium chloride, the OD600 of the culture medium reached the highest at 150mg/l, and then reached the lowest at 450mg/l, which is almost completely consistent with our expectation, which also proves that the construction of our "ammonia suicide" system is successful.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1936
Illegal SpeI site found at 1879
Illegal PstI site found at 875
Illegal PstI site found at 1354 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1936
Illegal SpeI site found at 1879
Illegal PstI site found at 875
Illegal PstI site found at 1354 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1936
Illegal BamHI site found at 2080
Illegal XhoI site found at 150 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1936
Illegal SpeI site found at 1879
Illegal PstI site found at 875
Illegal PstI site found at 1354 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1936
Illegal SpeI site found at 1879
Illegal PstI site found at 875
Illegal PstI site found at 1354
Illegal AgeI site found at 1663 - 1000COMPATIBLE WITH RFC[1000]
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