Part:BBa_K4202045

Sucrose regulated kill switch in Bacillus subtilis

Sucrose-induced promoters are linked to the toxin/antitoxin system. When the sucrose concentration is high enough, the promoter is efficient, and MazE continuously expresses the antagonistic toxin MazF; When the sucrose concentration is insufficient, the promoter efficiency is insufficient, MazE cannot be expressed continuously, and based on its instability, its cell concentration will decrease faster than MazF, causing MazF to exert its toxic effects and eventually lead to cell death. This part prevents engineering bacteria from escaping into the environment under certain stressful conditions.

Result

To characterized our kill switch, we transformed both the empty vector and the vector containing the kill switch into Bacillus subtilis WB600 and cultured them in the LB medium containing 1% sucrose. The OD600nm(L=1mm) of the cultures was measured every 40 minutes when the OD600nm(L=1mm) of cultures reached approximately 0.1.

Compared to the control group, the OD_600nm(L=1mm) of strains containing the kill switch began to decrease after the _600nm(L=1mm) reached 0.12. The early decrease of OD_600nm indicated that the bacteria are dying, which means our kill switch is working.

Fig 1The growth curve of two Bacillus subtilis WB600 strains.

Blue curve represents strains containing suicide switches and the yellow line represents strains containing the empty plasmid(CK)

Biology

The positive regulatory promoter PsacB is a weak endogenous promoter and can be induced by the sucrose [1]. It contains two parts, the core constitutive promoter and the leader RNA sequence. The leader RNA sequence contains a terminator overlaping with the binding site of sacY [2] [3]. When the sucrose existed, the sacY can bind to the bing site in the leader RNA and stablized the terminator, which enables the transcription. The absence of sucrose can lead to the formation of terminator structure, which will lead to the early termination of downstream genes. The antitoxin-toxin system is consisted by the coding sequence mazE and mazF. The mazF sequence used in our project is from E. coli and can degrade the mRNA nonspecificly. The antitoxin mazE can bind to the mazF and inhibit the activity of mazF.

Reference

[1] Aymerich, S., Gonzy-Tréboul, G., & Steinmetz, M. (1986). 5'-Noncoding region SACR is the target of all identified regulation affecting the LEVANSUCRASE gene in bacillus subtilis. Journal of Bacteriology, 166(3), 993–998. https://doi.org/10.1128/jb.166.3.993-998.1986

[2] Tortosa, P., & Le Coq, D. (1995). A ribonucleic antiterminator sequence (rat) and a distant palindrome are both involved in sucrose induction of the bacillus subtilis sacxy regulatory operon. Microbiology, 141(11), 2921–2927. https://doi.org/10.1099/13500872-141-11-2921

[3] Clerte, C., Declerck, N., & Margeat, E. (2013). Competitive folding of anti-terminator/terminator hairpins monitored by Single Molecule Fret. Nucleic Acids Research, 41(4), 2632–2643. https://doi.org/10.1093/nar/gks1315

Usage

Over the past decade, PsacB has been widely used as one of the few inducible promoters in Bacillus subtilis for protein expression, secondary metabolite production, and synthetic biology research. The mazE-mazF antitoxin-toxin system from E. coli has been widely used for density regulation of engineered bacterial populations and the construction of kill switches. In our project, the PsacB promoter was used together with the mazE-mazF antitoxin-toxin system to construct a kill switch for engineered Bacillus subtilis to commit suicide at a later stage of bacterial growth.

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 926
  Illegal AgeI site found at 654
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1139