Difference between revisions of "Part:BBa K5136043"

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===Characterization===
 
===Characterization===
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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.
 
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.
 
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.
 
   
 
   
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<b>Figure 1 Cytotoxicity verification of CcdB toxin.</b> (<b>A</b>) The gene circuit to characterize the cytotoxicity of CcdB (BBa_K5136236) on pSB4A5 vector. (<b>B</b>) Agarose gel electrophoresis of the colony PCR products of BBa_K5136236_pSB4A5 and BBa_I0500_pSB4A5 in <i>E. coli</i> BL21(DE3) Δ<i>araBAD</i>. (<b>C</b>) Cell viability was measured by CFU count and is displayed as a ratio of cells with <i>L</i>-arabinose to cells with <i>D</i>-glucose. <i>p</i>-value: 0.0007 (***).
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<center><html><https://static.igem.wiki/teams/5136/result/32.png" width="400px"></html></center>
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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 <i>ccdB</i> was placed downstream the <i>L</i>-arabinose inducible promoter (<i>araC</i>/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.
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<center><b>Figure 1 Characterization of blue light-induced LexRO/pColE408</b>. (<b>A</b>) The gene circuit to characterize blue light-responding performance of LexRO/pColE408 system (BBa_K5136237) on pSB4A5 vector. (<b>B</b>) Agarose gel electrophoresis of the colony PCR products of BBa_K5136237_pSB4A5 in <i>E. coli</i> BL21(DE3). (<b>C</b>) The relative fluorescence units (RFU) of bacterial culture subtracted the background fluorescence of growth media, resulting in the RFU<sub>mCherry</sub>. p-value: 0.0029 (**).</center>
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Cytotoxicity tests were implemented in a BL21(DE3) strain in which the <i>araBAD</i> genes were knocked out (Δ<i>araBAD</i>) in our lab before for minimizing the influence of <i>L</i>-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 <b>a ratio of cells with L-arabinose to cells with D-glucose (survival ratio)</b>, in which the <i>D</i>-glucose could suppress the <i>L</i>-arabinose inducible promoter. Upon adding the inducer <i>L</i>-arabinose, the CcdB toxin (<i>ccdBA</i>) produced ~6 logs of killing for 6 hours′ culture (Figure 1C), which indicated the cytotoxicity of CcdB.
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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) (<partinfo>BBa_K5136045</partinfo>), while the mCherry fluorescent protein (<partinfo>BBa_J06504</partinfo>) was chosen as the reporter under the control of promoter pColE408 (<partinfo>BBa_K5136044</partinfo>) (Figure 1A), thus generating the composite part <partinfo>BBa_K5136237</partinfo> 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.
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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 (<i>λe</i><sub>x</sub> = 585 nm, <i>λ</i><sub>em</sub> = 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 <b>optogenetic system could be induced by blue light</b> (Figure 1C) indeed.
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===Reference===
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1. R. Freudl, Signal Peptides for Recombinant Protein Secretion in Bacterial Expression Systems. Microb. Cell Fact. 17, 52 (2018).
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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).
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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===
 
===Reference===

Revision as of 07:48, 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.

Figure 1 Characterization of blue light-induced LexRO/pColE408. (A) The gene circuit to characterize blue light-responding performance of LexRO/pColE408 system (BBa_K5136237) on pSB4A5 vector. (B) Agarose gel electrophoresis of the colony PCR products of BBa_K5136237_pSB4A5 in E. coli BL21(DE3). (C) The relative fluorescence units (RFU) of bacterial culture subtracted the background fluorescence of growth media, resulting in the RFUmCherry. p-value: 0.0029 (**).

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 (λex = 585 nm, λem = 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]