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><img src=" https://static.igem.wiki/teams/5136/result/33.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 kill switch</b>. (<b>A</b>) The gene circuit of blue light-induced kill switch (BBa_K5136231) on pSB4A5 vector. (<b>B</b>) Agarose gel electrophoresis of the colony PCR products of BBa_K5136231_pSB4A5 (Kill Switch) and BBa_K5136234_pSB4A5 (Control) in <i>E. coli</i> BL21(DE3). (<b>C</b>) 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. <i>p</i>-value: 0.0085 (**) for D/L, 0.0077 (**) for L.</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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After verifying the cytotoxicity of CcdB and blue light-inducible performance of LexRO/pColE408 system, we built the blue light-induced kill switch (<partinfo>BBa_K5136231</partinfo>), in which the toxin-antitoxin module is controlled by promoter pColE408 and LexRO is constitutively expressed as in <partinfo>BBa_K5136237</partinfo> (Figure 1A). While the LexRO expression module only (<partinfo>BBa_K5136234</partinfo>) 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).  
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Spot assay was also performed after cultured upon blue light illumination or kept in dark condition. A <b>blue light illumination-dependent killing effect</b> 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.
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===Reference===
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<br>1. R. Freudl, Signal Peptides for Recombinant Protein Secretion in Bacterial Expression Systems. Microb. Cell Fact. 17, 52 (2018).
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<center><html><img src=" https://static.igem.wiki/teams/5136/result/34.png" width="400px"></html></center>
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<br>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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<center><b>Figure 2 Optimization of blue light-induced kill switch.</b> (<b>A</b>) Optimized blue light-induced kill switch (BBa_K5136235) on pSB4A5 vector. The RBS of LexRO was changed to SD17. (<b>B</b>) Agarose gel electrophoresis of the colony PCR products of BBa_K5136235_pSB4A5 in <i>E. col</i>i BL21(DE3). (<b>C</b>) 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.</center>
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<br>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).
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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, <partinfo>BBa_K5136049</partinfo>) to see whether this would improve the effect of killing or not, resulting in the generation of <partinfo>BBa_K5136235</partinfo> 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 <b>lower the expression of LexRO</b>, 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.
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<br>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).
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<br>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).
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<br>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).
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<br>7. F. J. M. Mergulhão, D. K. Summers, G. A. Monteiro, Recombinant Protein Secretion in Escherichia coli. Biotechnol. Adv. 23, 177–202 (2005)
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==<b>Reference</b>==
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1. H. An et al., TEX264 Is an Endoplasmic Reticulum-Resident ATG8-Interacting Protein Critical for ER Remodeling during Nutrient Stress. <i>Molecular Cell</i> <b>74</b>, 891-908.e810 (2019).<br>
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2. Li X, Zhang C, Xu X, Miao J, Yao J, Liu R, Zhao Y, Chen X, Yang Y. A Single-component Light Sensor System Allows Highly Tunable and Direct Activation of Gene Expression in Bacterial Cells. Nucleic Acids Res. 2020 Apr 6;48(6):e33<br>
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3. Bernard P, Couturier M. Cell Killing by the F Plasmid CcdB Protein Involves Poisoning of DNA-topoisomerase II complexes. J Mol Biol. 1992 Aug 5;226(3):735-45.<br>
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4. Chan CT, Lee JW, Cameron DE, Bashor CJ, Collins JJ. 'Deadman' and 'Passcode' Microbial Kill Switches for Bacterial Containment. Nat. Chem. Biol. 2016 Feb;12(2):82-6. <br>
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5. Choi J, Ahn J, Bae J, Koh M. Recent Synthetic Biology Approaches for Temperature- and Light-Controlled Gene Expression in Bacterial Hosts. Molecules. 2022 Oct 11;27(20):6798.<br>
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6. Lalwani MA, Kawabe H, Mays RL, Hoffman SM, Avalos JL. Optogenetic Control of Microbial Consortia Populations for Chemical Production. ACS Synth Biol. 2021 Aug 20;10(8):2015-2029.<br>
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7. Zhang Y, Xue X, Fang M, Pang G, Xing Y, Zhang X, Li L, Chen Q, Wang Y, Chang J, Zhao P, Wang H. Upconversion Optogenetic Engineered Bacteria System for Time-Resolved Imaging Diagnosis and Light-Controlled Cancer Therapy. ACS Appl Mater Interfaces. 2022 Oct 19;14(41):46351-46361.<br>
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8.  Han C, Zhang X, Pang G, Zhang Y, Pan H, Li L, Cui M, Liu B, Kang R, Xue X, Sun T, Liu J, Chang J, Zhao P, Wang H. Hydrogel Microcapsules Containing Engineered Bacteria for Sustained Production and Release of Protein Drugs. Biomaterials. 2022 Aug;287:121619.
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 10:51, 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 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. H. An et al., TEX264 Is an Endoplasmic Reticulum-Resident ATG8-Interacting Protein Critical for ER Remodeling during Nutrient Stress. Molecular Cell 74, 891-908.e810 (2019).
2. Li X, Zhang C, Xu X, Miao J, Yao J, Liu R, Zhao Y, Chen X, Yang Y. A Single-component Light Sensor System Allows Highly Tunable and Direct Activation of Gene Expression in Bacterial Cells. Nucleic Acids Res. 2020 Apr 6;48(6):e33
3. Bernard P, Couturier M. Cell Killing by the F Plasmid CcdB Protein Involves Poisoning of DNA-topoisomerase II complexes. J Mol Biol. 1992 Aug 5;226(3):735-45.
4. Chan CT, Lee JW, Cameron DE, Bashor CJ, Collins JJ. 'Deadman' and 'Passcode' Microbial Kill Switches for Bacterial Containment. Nat. Chem. Biol. 2016 Feb;12(2):82-6. 
5. Choi J, Ahn J, Bae J, Koh M. Recent Synthetic Biology Approaches for Temperature- and Light-Controlled Gene Expression in Bacterial Hosts. Molecules. 2022 Oct 11;27(20):6798.
6. Lalwani MA, Kawabe H, Mays RL, Hoffman SM, Avalos JL. Optogenetic Control of Microbial Consortia Populations for Chemical Production. ACS Synth Biol. 2021 Aug 20;10(8):2015-2029.
7. Zhang Y, Xue X, Fang M, Pang G, Xing Y, Zhang X, Li L, Chen Q, Wang Y, Chang J, Zhao P, Wang H. Upconversion Optogenetic Engineered Bacteria System for Time-Resolved Imaging Diagnosis and Light-Controlled Cancer Therapy. ACS Appl Mater Interfaces. 2022 Oct 19;14(41):46351-46361.
8. Han C, Zhang X, Pang G, Zhang Y, Pan H, Li L, Cui M, Liu B, Kang R, Xue X, Sun T, Liu J, Chang J, Zhao P, Wang H. Hydrogel Microcapsules Containing Engineered Bacteria for Sustained Production and Release of Protein Drugs. Biomaterials. 2022 Aug;287:121619. Sequence and Features


Assembly Compatibility:
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    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]