Difference between revisions of "Part:BBa K5136042"

(→‎Characterization)
(→‎Characterization)
Line 12: Line 12:
 
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.
−
   
+
  <center><html><https://static.igem.wiki/teams/5136/result/32.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 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>
 
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.
 
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.

Revision as of 07:42, 2 October 2024


LexRO

Biology

LexRO is a synthetic light-switchable repressor, based on a novel LOV light sensor domain, RsLOV. In the darkness, LexRO dimerizes and binds to its cognate operator sequence to repress promoter activity. Upon light exposure, the LexRO dimer dissociates, causing dissociation from the operator sequence, and initiates gene expression.

Usage and design

In the darkness, LexRO dimerizes and binds to the cognate operator sequence to repress the activity of pColE408. Upon blue light exposure, the LexRO dimer dissociates, causing dissociation from the operator sequence, and initiates the expression of ccdB， eventually leading to cell death. We used LexRO, pHybrid 2)-114 version, SD7, ccdA, and ccdB to construct the regulation system and obtained the composite part BBa_K5136231, which was assembled on the expression vector pSB4A5.

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).