Part:BBa_K5136043
pHybrid 2)-114 version
Biology
pHybrid 2)-114 version is an engineering promoter that is suppressed by the Aca2 repressor, which uses the -35 and -10 regions of J23114.
Usage and design
We use pHybrid 2)-114 version as the promoter of the ccdA.
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 Cytotoxicity verification of CcdB toxin. (A) The gene circuit to characterize the cytotoxicity of CcdB (BBa_K5136236) on pSB4A5 vector. (B) Agarose gel electrophoresis of the colony PCR products of BBa_K5136236_pSB4A5 and BBa_I0500_pSB4A5 in E. coli BL21(DE3) ΔaraBAD. (C) Cell viability was measured by CFU count and is displayed as a ratio of cells with L-arabinose to cells with D-glucose. p-value: 0.0007 (***). Various toxin-antitoxin (TA) systems have been widely utilized and engineered to construct kill switch for biocontainment (4,5). CcdB toxin of the CcdB-CcdA TA system, interferes with the activity of DNA gyrase and thus causes cell death (6), which will play the critical role of killing engineered bacteria. To verify the cytotoxicity of CcdB toxin used in the kill switch, we firstly constructed a gene circuit that the toxin encoding gene ccdB was placed downstream the L-arabinose inducible promoter (araC/pBAD) on the pSB4A5 vector. For convenience, the expression module of CcdA controlled by a weak constitutive engineering promoter p2)-114v was integrated into the circuit as well (Figure 1A), generating the composite part. While BBa_I0500 only on the pSB4A5 was set as control. Cytotoxicity tests were implemented in a BL21(DE3) strain in which the araBAD genes were knocked out (ΔaraBAD) in our lab before for minimizing the influence of L-arabinose metabolism. After transformation, positive transformants were selected and confirmed by colony PCR (Figure 1B) and sequencing. Spot assay (7) was performed for characterizing the killing effect (See more details in XMU-China experiment page), while cell viability was measured by colony forming unit (CFU) count and is displayed as a ratio of cells with L-arabinose to cells with D-glucose (survival ratio), in which the D-glucose could suppress the L-arabinose inducible promoter. Upon adding the inducer L-arabinose, the CcdB toxin (ccdBA) produced ~6 logs of killing for 6 hours′ culture (Figure 1C), which indicated the cytotoxicity of CcdB.
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. B. D. Tzschaschel, C. A. Guzmán, K. N. Timmis, V. D. Lorenzo, An Escherichia coli Hemolysin Transport System-based Vector for the Export of Polypeptides: Export of Shiga-like Toxin IIeb Subunit by Salmonella Typhimurium aroA. Nat. Biotechnol. 14, 765–769 (1996). 4. 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). 5. S.-I. Tan, I.-S. Ng, New Insight into Plasmid-driven T7 RNA Polymerase in Escherichia coli and Use as a Genetic Amplifier for a Biosensor. ACS Synth. Biol. 9, 613–622 (2020). 6. B. J. Feilmeier, G. Iseminger, D. Schroeder, H. Webber, G. J. Phillips, Green Fluorescent Protein Functions as a Reporter for Protein Localization in Escherichia coli. J. Bacteriol. 182, 4068–4076 (2000). 7. F. J. M. Mergulhão, D. K. Summers, G. A. Monteiro, Recombinant Protein Secretion in Escherichia coli. Biotechnol. Adv. 23, 177–202 (2005)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 30
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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