Difference between revisions of "Part:BBa K5136043"

Revision as of 08:07, 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 No part name specified with partinfo tag.) 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 30
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 9: / Line 9: @@
 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.
-<center><html><img src=" https://static.igem.wiki/teams/5136/result/32.png" width="400px"></html></center>
+<center><html><img src=" https://static.igem.wiki/teams/5136/result/33.png" width="400px"></html></center>
-<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>
+<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.
-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.
+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).
-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.
+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.
+<center><html><img src=" https://static.igem.wiki/teams/5136/result/34.png" width="400px"></html></center>
+<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.
+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.
 ===Reference===
 . R. Freudl, Signal Peptides for Recombinant Protein Secretion in Bacterial Expression Systems. Microb. Cell Fact. 17, 52 (2018).
 . 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).
 . 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===
-<br>1. R. Freudl, Signal Peptides for Recombinant Protein Secretion in Bacterial Expression Systems. Microb. Cell Fact. 17, 52 (2018).
-<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).
-<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).
-<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).
-<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).
-<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).
-<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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