Part:BBa_K4743030

Bacillus subtilis Hydrogen sulfide based kill switch

Kill switch’s mechanism

In Figure 1, we depict the initial exposure to normal conditions. In this scenario, when hydrogen sulfide levels are low, the translated SqrR protein (red) effectively represses the sqr promoter, resulting in the prevention of transcription for recombinases DRE or CRE ( gray) and the antitoxin (pink). Furthermore, the presence of terminator sequences labeled by rox or Lox sequences puts a halt to the transcription of the toxin. Consequently, under these conditions, the absence of both toxin and antitoxin prevents any cell death.

Figure 1: First exposure to the normal condition.

In the second condition, as illustrated in Figure 4 and 5, when the organism is exposed to the gut environment characterized by the presence of hydrogen sulfide (depicted as one yellow dot with two blue dots), the suppressive effect of the SqrR protein (red) decreases. This reduction in suppression leads to the transcription of recombinase DRE or CRE (gray) and the antitoxin. The recombinase, in turn, targets the terminator sequence labeled by rox/Lox and cleaves it, enabling the transcription of the toxin(blue). However, it's important to note that the transcribed antitoxin (pink) counteracts the toxin, neutralizing its toxic activity. In conclusion, under these conditions, the presence of both toxin and antitoxin allows the cell to remain viable.

Figure 2-3: exposure to the gut environment

In the third condition, depicted in Figure 4, we illustrate the organism's return to the normal environment, indicating its escape from the gut or excretion of feces. Under these circumstances, the SqrR suppressor (depicted in red) once again binds to the promoter, effectively inhibiting the transcription of the antitoxin. However, crucially, the terminator that normally halts toxin transcription is excised. This excision allows for the transcription of the toxin. In this scenario, the bacteria lose the protective antitoxin, leading to cell death as a consequence.

Figure 4: Second exposure to the normal environment

Then, We tailored our kill switch for Bacillus subtilis, selecting MazF as the toxin and MazE as the antitoxin, with DRE serving as our recombinase. Additionally, we carefully chose a promoter and terminator that are compatible with Bacillus subtilis, ensuring the effectiveness of our engineered system.

Figure 5: kill switch of Bacillus Subtilis

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 464
  Illegal AgeI site found at 1623
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 677
  

References

1.Shimizu, T., Shen, J., Fang, M., Zhang, Y., Hori, K., Trinidad, J. C., Bauer, C. E., Giedroc, D. P., & Masuda, S. (2017). Sulfide-responsive transcriptional repressor SqrR functions as a master regulator of sulfide-dependent photosynthesis. Proceedings of the National Academy of Sciences of the United States of America, 114(9), 2355–2360. https://doi.org/10.1073/pnas.1614133114