Difference between revisions of "Part:BBa K3771099"

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<br>This composite part consists of promoter <i>P<sub>soxS</sub></i> Mutant 3 (BBa_K2862010),  genetic insulator RiboJ (BBa_K1679038), sfGFP (BBa_K1321337), constitutive promoter J23101 (BBa_J23101), and the soxR gene (BBa_K223000). The concept of this composite part comes from the 2018 Imperial College London iGEM team.
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<br>This composite part consists of promoter <i>P<sub>soxS</sub></i> Mutant 3 (BBa_K2862010),  genetic insulator RiboJ (BBa_K1679038), sfGFP (BBa_K1321337), constitutive promoter J23101 (BBa_J23101), and the <i>soxR</i> gene (BBa_K223000). The concept of this composite part comes from the 2018 Imperial College London iGEM team.
 
The <i>soxR</i> and the <i>soxS</i> gene are components of the <i>soxRS</i> regulon, which is important for <i>E. coli</i> to sense and respond to the oxidants. When SoxR protein is fully oxidized, it becomes a powerful transcription activator of the <i>soxS</i> promoter (<i>P<sub>soxS</sub></i>) (up to 100-fold), leading to the expression of the downstream gene<sup>[1]</sup>. RiboJ is a genetic insulator that can increase the expression of insulated genes. sfGFP serves as a reporter to evaluate the strength of <i>P<sub>soxS</sub></i>-Riboj.  
 
The <i>soxR</i> and the <i>soxS</i> gene are components of the <i>soxRS</i> regulon, which is important for <i>E. coli</i> to sense and respond to the oxidants. When SoxR protein is fully oxidized, it becomes a powerful transcription activator of the <i>soxS</i> promoter (<i>P<sub>soxS</sub></i>) (up to 100-fold), leading to the expression of the downstream gene<sup>[1]</sup>. RiboJ is a genetic insulator that can increase the expression of insulated genes. sfGFP serves as a reporter to evaluate the strength of <i>P<sub>soxS</sub></i>-Riboj.  
 
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<img src="https://2021.igem.org/wiki/images/2/29/T--NCKU_Tainan--RCP_Colony_PCR.png"​ style="width:35%;">
 
<img src="https://2021.igem.org/wiki/images/2/29/T--NCKU_Tainan--RCP_Colony_PCR.png"​ style="width:35%;">
 
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<p align="center"> Fig.1. The electrophoresis result of colony PCR. M: Marker; Lane 1: pSAA-pSAA-<i>P<sub>soxS</sub>-RiboJ-rcp<i>-J23101-<i>soxR</i>  (1103 bp); Lane 2, 3: pSAA-<i>P<sub>soxS</sub>-RiboJ-sfgfp</i>-J23101-<i>soxR</i>(998 bp).
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<p align="center"> Fig. 1. The electrophoresis result of colony PCR. M: Marker; Lane 1: pSAA-<i>P<sub>soxS</sub></i>-RiboJ-<i>rcp</i>-J23101-<i>soxR</i>  (1103 bp); Lane 2, 3: pSAA-<i>P<sub>soxS</sub>-RiboJ-sfgfp</i>-J23101-<i>soxR</i>(998 bp).
 
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<br>With its activator, SoxR, the promoter strength of <i>P<sub>soxS</sub></i>-Riboj under oxidative stress, is determined by the expression level of sfGFP, which is their reporter. While using paraquat (PQ), which is a commonly used agent to induce oxidative stress for bacteria, as the inducer, the intensity of <i>P<sub>soxS</sub></i>-Riboj shows no significant difference under different concentrations of paraquat (Fig.2).
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<br>With its activator, SoxR, the promoter strength of <i>P<sub>soxS</sub></i>-Riboj under oxidative stress, is determined by the expression level of sfGFP, which is their reporter. While using paraquat (PQ), which is a commonly used agent to induce oxidative stress for bacteria, as the inducer, the intensity of <i>P<sub>soxS</sub></i>-Riboj shows no significant difference under different concentrations of paraquat (Fig. 2).
 
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<img src="https://static.igem.org/mediawiki/parts/0/0b/T--NCKU_Tainan--Del_Low_sfGFP_Expression_%28PsoxS-RiboJ_%2B_J23101-soxR%29.png"​ style="width:35%;">
 
<img src="https://static.igem.org/mediawiki/parts/0/0b/T--NCKU_Tainan--Del_Low_sfGFP_Expression_%28PsoxS-RiboJ_%2B_J23101-soxR%29.png"​ style="width:35%;">
 
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<p align="center"> Fig.2. Relative fluorescence intensity of <i>P<sub>soxS</sub></i>-Riboj after 4.5-hour incubation with paraquat in various concentrations.
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<p align="center"> Fig. 2. Relative fluorescence intensity of <i>P<sub>soxS</sub></i>-Riboj after 4.5-hour incubation with paraquat in various concentrations.
 
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<br>We compared the relative fluorescence intensity of sfGFP expressed by different promoters under the stimulation by different concentrations of paraquat. As expected, sfGFP expression level of <i>P<sub>soxS</sub></i> with its activator, SoxR (BBa_K37710XX), is highly activated under oxidative stress and has the highest gene expression level compared to  <i>P<sub>skatG</sub></i> (BBa_K37710XX), <i>P<sub>soxS</sub></i> (BBa_K37710XX) solely, and this composite part. The sfGFP expression by this composite part was quite low (Fig.3).
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<br>We compared the relative fluorescence intensity of sfGFP expressed by different promoters under the stimulation by different concentrations of paraquat. As expected, sfGFP expression level of <i>P<sub>soxS</sub></i> with its activator, SoxR (BBa_K37710XX), is highly activated under oxidative stress and has the highest gene expression level compared to  <i>P<sub>skatG</sub></i> (BBa_K37710XX), <i>P<sub>soxS</sub></i> (BBa_K37710XX) solely, and this composite part. The sfGFP expression by this composite part was quite low (Fig. 3).
 
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<img src="https://static.igem.org/mediawiki/parts/0/0d/T--NCKU_Tainan--Low_sfGFP_Expression_%28PQ_%404.5h%29.png"​ style="width:35%;">
 
<img src="https://static.igem.org/mediawiki/parts/0/0d/T--NCKU_Tainan--Low_sfGFP_Expression_%28PQ_%404.5h%29.png"​ style="width:35%;">
 
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<p align="center"> Fig.3.Comparison of different promoter strengths by relative fluorescence intensity after 4.5-hour incubation with paraquat in various concentrations.
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<p align="center"> Fig. 3.Comparison of different promoter strengths by relative fluorescence intensity after 4.5-hour incubation with paraquat in various concentrations.
  
 
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Revision as of 12:46, 18 October 2021


PsoxS-Riboj-sfGFP-J23101-SoxR


Description


This composite part consists of promoter PsoxS Mutant 3 (BBa_K2862010), genetic insulator RiboJ (BBa_K1679038), sfGFP (BBa_K1321337), constitutive promoter J23101 (BBa_J23101), and the soxR gene (BBa_K223000). The concept of this composite part comes from the 2018 Imperial College London iGEM team. The soxR and the soxS gene are components of the soxRS regulon, which is important for E. coli to sense and respond to the oxidants. When SoxR protein is fully oxidized, it becomes a powerful transcription activator of the soxS promoter (PsoxS) (up to 100-fold), leading to the expression of the downstream gene[1]. RiboJ is a genetic insulator that can increase the expression of insulated genes. sfGFP serves as a reporter to evaluate the strength of PsoxS-Riboj.



Usage and Biology


This composite part was ligated with the pSAA vector and transformed into E. coli. We conducted colony PCR to verify whether E. coli uptake the correct plasmid. The size of the PCR product was as expected.

Fig. 1. The electrophoresis result of colony PCR. M: Marker; Lane 1: pSAA-PsoxS-RiboJ-rcp-J23101-soxR (1103 bp); Lane 2, 3: pSAA-PsoxS-RiboJ-sfgfp-J23101-soxR(998 bp).


With its activator, SoxR, the promoter strength of PsoxS-Riboj under oxidative stress, is determined by the expression level of sfGFP, which is their reporter. While using paraquat (PQ), which is a commonly used agent to induce oxidative stress for bacteria, as the inducer, the intensity of PsoxS-Riboj shows no significant difference under different concentrations of paraquat (Fig. 2).

Fig. 2. Relative fluorescence intensity of PsoxS-Riboj after 4.5-hour incubation with paraquat in various concentrations.


We compared the relative fluorescence intensity of sfGFP expressed by different promoters under the stimulation by different concentrations of paraquat. As expected, sfGFP expression level of PsoxS with its activator, SoxR (BBa_K37710XX), is highly activated under oxidative stress and has the highest gene expression level compared to PskatG (BBa_K37710XX), PsoxS (BBa_K37710XX) solely, and this composite part. The sfGFP expression by this composite part was quite low (Fig. 3).

Fig. 3.Comparison of different promoter strengths by relative fluorescence intensity after 4.5-hour incubation with paraquat in various concentrations.


References


1. Pomposiello PJ, Demple B. Redox-operated genetic switches: the SoxR and OxyR transcription factors. Trends Biotechnol. 2001;19(3):109-114. doi:10.1016/s0167-7799(00)01542-0


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 922
    Illegal NheI site found at 945
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 206