Difference between revisions of "Part:BBa K5136043"

Revision as of 07:52, 2 October 2024

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.

We turned to characterize the blue light-responding performance of LexRO/pColE408 optogenetic system used in the kill switch. The photosensor LexRO was controlled by a medium constitutive promoter J23106 and a medium RBS SD7 (3) (BBa_K5136045), while the mCherry fluorescent protein (BBa_J06504) was chosen as the reporter under the control of promoter pColE408 (BBa_K5136044) (Figure 1A), thus generating the composite part BBa_K5136237 on the pSB4A5 vector. BL21(DE3) was used to characterize this optogenetic system, and positive transformants were selected and confirmed by colony PCR (Figure 1B) and sequencing. Characterization was carried out in a self-made blue light (460 nm) illumination device. After cultured for about 17 hours upon blue light illumination (with a relative light intensity of 250) or kept in dark condition, red fluorescence intensity (λe_x = 585 nm, λ_em = 615 nm) and OD600 were measured. The normalized fluorescence intensity of “Light” group showed a significant higher value than that of “Dark” group (about 2 times), indicating that this optogenetic system could be induced by blue light (Figure 1C) indeed.

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).

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