Part:BBa_K5136236

I0500-B0034-ccdB-B0014-pHybrid 2)-114 version-B0034-ccdA

Biology

pHybrid 2)-114

pHybrid 2)-114 version is an engineering promoter that is suppressed by the Aca2 repressor, which uses the -35 and -10 regions of J23114.

ccdB

ccdB gene is located on the Escherichia coli F-factor plasmid and is part of the toxin-antitoxin system encoded by the ccd operon, responsible for plasmid maintenance during cell division. ccdB encodes a toxic protein that acts as a DNA gyrase poison. It can bind DNA gyrase to the broken double-stranded DNA, leading to cell death.

ccdA

ccdA is the gene found within the ccd operon, encoding the antidote protein (CcdA) that protects cells from the toxic effects of CcdB. CcdA protein is easily degraded by Lonprotease.The cell loses the ccdA gene due to the loss of the F plasmid, causing the cell to succumb to the toxicity of CcdB.

Characterization

Facing the threat that the unwanted survival and accumulation of engineered bacteria might happen once they escape to opening environment (1), we designed a light-triggered kill switch for biocontainment of the engineered bacteria. Rather than responding to some chemical inducers, the light-triggered kill switch will be turned to ON state after the engineered bacteria is exposed to the light illumination of specific wavelength. We chose a blue light-inducible optogenetic system, LexRO/pColE408 (2), to control the expression of CcdB toxin, in which an additional expression module of CcdA antitoxin was incorporated as well to neutralize the leaky toxin when the kill switch is in OFF state. Here, we firstly characterized the cytotoxicity of CcdB toxin and the blue light-inducible performance of LexRO/pColE408 system respectively, and then tested the killing effect of the blue light-induced kill switch. Further optimization for improving the killing effect of the switch was also tried primarily.

Figure 1 Characterization of blue light-induced kill switch. (A) The gene circuit of blue light-induced kill switch (BBa_K5136231) on pSB4A5 vector. (B) Agarose gel electrophoresis of the colony PCR products of BBa_K5136231_pSB4A5 (Kill Switch) and BBa_K5136234_pSB4A5 (Control) in E. coli BL21(DE3). (C) Cell viability was measured by CFU count and is displayed as a ratio of cells exposed to blue light to cells kept in dark condition. p-value: 0.0085 (**) for D/L, 0.0077 (**) for L.

After verifying the cytotoxicity of CcdB and blue light-inducible performance of LexRO/pColE408 system, we built the blue light-induced kill switch (BBa_K5136231), in which the toxin-antitoxin module is controlled by promoter pColE408 and LexRO is constitutively expressed as in BBa_K5136237 (Figure 1A). While the LexRO expression module only (BBa_K5136234) on the pSB4A5 was set as the control. Positive transformants were selected and confirmed by colony PCR (Figure 1B) and sequencing after transformed to BL21(DE3). Spot assay was also performed after cultured upon blue light illumination or kept in dark condition. A blue light illumination-dependent killing effect was observed, which indicates that this blue light-induced kill switch functioned to kill engineered bacteria when exposed to blue light (Figure 1C). Besides, when exposed to blue light for whole period (6 hours, “L”), the kill switch exhibited a slightly stronger killing effect than exposed to blue light for a shorter time (kept in dark for 2 hours 25 min first then switched on the blue light for 3 hours 35 min, “D/L”), which implied that the killing of engineered bacteria might be illuminating time-dependent.

Figure 2 Optimization of blue light-induced kill switch. (A) Optimized blue light-induced kill switch (BBa_K5136235) on pSB4A5 vector. The RBS of LexRO was changed to SD17. (B) Agarose gel electrophoresis of the colony PCR products of BBa_K5136235_pSB4A5 in E. coli BL21(DE3). (C) Cell viability was measured by CFU count and is displayed as a ratio of cells exposed to blue light to cells kept in dark condition.

Although we have verified the blue light-dependent killing effect of the kill switch, we still tried to optimize the gene circuit for further improving the killing effect. Since lower LexRO content were more sensitive to light illumination (3), we then changed the RBS of LexRO in the gene circuit to a weaker one (SD17, BBa_K5136049) to see whether this would improve the effect of killing or not, resulting in the generation of BBa_K5136235 composite part on pSB4A5 vector (Figure 2A). Colony PCR (Figure 2B) and sequencing were performed again to confirm the positive transformants of BL21(DE3). Similar test was done to the alternative kill switch. When lower the expression of LexRO, a slight decrease on survival ratio was obtained for the kill switch (Figure 2C), indicating that the strategy for optimizing the kill switch might be available and feasible.

Reference

1. R. Freudl, Signal Peptides for Recombinant Protein Secretion in Bacterial Expression Systems. Microb. Cell Fact. 17, 52 (2018).
2. H. Owji, N. Nezafat, M. Negahdaripour, A. Hajiebrahimi, Y. Ghasemi, A Comprehensive Review of Signal Peptides: Structure, Roles, and Applications. Eur. J. Cell Biol. 97, 422–441 (2018).
3. L. A. Fernández, I. Sola, L. Enjuanes, V. De Lorenzo, Specific Secretion of Active Single-chain Fv Antibodies into the Supernatants of Escherichia coli Cultures by Use of the Hemolysin System. Appl. Environ. Microbiol. 66, 5024–5029 (2000). Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  Illegal NheI site found at 1683
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1452
  Illegal SapI site found at 961