Difference between revisions of "Part:BBa K3771048"
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<p align="center">Fig.2. The mechanism of SoxR activation by superoxide<sup>[1]</sup>. The SoxR dimer has two forms, reduced and oxidized, both of them can bind with DNA. After SoxR is oxidized by oxidative stress, its conformation changes, modifies the local DNA topology at the promoter, and compensates for a dysfunctional spacing between promoter elements, and hence increasing the transcription of <i>soxS</i><sup>[1]</sup>.</p>
 
<p align="center">Fig.2. The mechanism of SoxR activation by superoxide<sup>[1]</sup>. The SoxR dimer has two forms, reduced and oxidized, both of them can bind with DNA. After SoxR is oxidized by oxidative stress, its conformation changes, modifies the local DNA topology at the promoter, and compensates for a dysfunctional spacing between promoter elements, and hence increasing the transcription of <i>soxS</i><sup>[1]</sup>.</p>
 
    
 
    
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<br>After conducting colony PCR from the E.coli MG1655, the PsoxS fragment can be amplified from the chromosome and the experiment result can be checked by agarose gel electrophoresis. The result is shown in Figure 3.<br>
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<br>After conducting colony PCR from the <i>E.coli</i> MG1655, the <i>P<sub>soxS</sub></i> fragment can be amplified from the chromosome and the experiment result can be checked by agarose gel electrophoresis. The result is shown in Fig.3.The part has been confirmed by sequencing and has no mutations.<br>
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<p align="center">Figure 3. The electrophoresis result of PsoxS fragment from the chromosome PCR. M: Marker; Lane 1:  soxS promoter (PsoxS) (220 bp).</p>
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<img src="https://2021.igem.org/wiki/images/e/ee/T--NCKU_Tainan--PsoxS_PCR.png" style="width:50%;"></html>
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<p align="center">Figure 3. The electrophoresis result of <i>P<sub>soxS</sub></i> fragment from the chromosome PCR. M: Marker; Lane 1:  <i>soxS</i> promoter (<i>P<sub>soxS</sub></i>) (220 bp).</p>
 
      
 
      
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<br>The part has been confirmed by sequencing and has no mutations.<br>
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<br> The promoter strength of PsoxS is determined by the expression level of the reporter, sfGFP, under oxidative stress. While using hydrogen peroxide (H2O2) as the inducer, the strength of PsoxS shows no significant difference under different concentrations of hydrogen peroxide (Figure 4). However, PsoxS is well induced by using paraquat (PQ), which is a commonly used agent to induce oxidative stress for bacteria (Figure 5 & 6).<br>
 
<br> The promoter strength of PsoxS is determined by the expression level of the reporter, sfGFP, under oxidative stress. While using hydrogen peroxide (H2O2) as the inducer, the strength of PsoxS shows no significant difference under different concentrations of hydrogen peroxide (Figure 4). However, PsoxS is well induced by using paraquat (PQ), which is a commonly used agent to induce oxidative stress for bacteria (Figure 5 & 6).<br>
 
      
 
      

Revision as of 14:18, 17 October 2021


PsoxS


Description

The soxS gene is one component of the soxRS regulon (Fig.1), which is important for E. coli<i> to sense and respond to the oxidants. The <i>soxS promoter (PsoxS) is regulated by SoxR and SoxS protein. SoxR protein binds to the site between the -10 and -35 elements of the soxS promoter. The distance between the ends of the -10 and -35 hexamers of the soxS promoter is 19 bp, which is different from 17±1 bp spacers for most E. coli promoters. Therefore, transcription of soxS is low without oxidized SoxR[1]. Oxidized SoxR induces target gene expression through distorting soxS promoter (Fig.2)[2]. In addition, SoxS protein serves as a negative autoregulator and diminishes soxS transcription[3,4].


Fig.1. Transcription unit of the soxRS regulon in E. coli K-12[5].

Fig.2. The mechanism of SoxR activation by superoxide[1]. The SoxR dimer has two forms, reduced and oxidized, both of them can bind with DNA. After SoxR is oxidized by oxidative stress, its conformation changes, modifies the local DNA topology at the promoter, and compensates for a dysfunctional spacing between promoter elements, and hence increasing the transcription of soxS[1].


Usage and Biology </br>


After conducting colony PCR from the E.coli MG1655, the PsoxS fragment can be amplified from the chromosome and the experiment result can be checked by agarose gel electrophoresis. The result is shown in Fig.3.The part has been confirmed by sequencing and has no mutations.

Figure 3. The electrophoresis result of PsoxS fragment from the chromosome PCR. M: Marker; Lane 1: soxS promoter (PsoxS) (220 bp).



The promoter strength of PsoxS is determined by the expression level of the reporter, sfGFP, under oxidative stress. While using hydrogen peroxide (H2O2) as the inducer, the strength of PsoxS shows no significant difference under different concentrations of hydrogen peroxide (Figure 4). However, PsoxS is well induced by using paraquat (PQ), which is a commonly used agent to induce oxidative stress for bacteria (Figure 5 & 6).

Figure 4. Relative fluorescence intensity of PsoxS after 4.5-hour incubation with hydrogen peroxide in various concentrations.

Figure 5. Relative fluorescence intensity of PsoxS after 4.5-hour incubation with paraquat in low concentrations.

Figure 6. Relative fluorescence intensity of PsoxS after 4.5-hour incubation with paraquat in high 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

2. Koo MS, Lee JH, Rah SY, et al. A reducing system of the superoxide sensor SoxR in Escherichia coli. EMBO J. 2003;22(11):2614-2622. doi:10.1093/emboj/cdg252

3. Nunoshiba T, Hidalgo E, Li Z, Demple B. Negative autoregulation by the Escherichia coli SoxS protein: a dampening mechanism for the soxRS redox stress response. J Bacteriol. 1993;175(22):7492-7494. doi:10.1128/jb.175.22.7492-7494.1993

4. Seo SW, Kim D, Szubin R, Palsson BO. Genome-wide Reconstruction of OxyR and SoxRS Transcriptional Regulatory Networks under Oxidative Stress in Escherichia coli K-12 MG1655. Cell Rep. 2015;12(8):1289-1299. doi:10.1016/j.celrep.2015.07.043

5. Keseler IM, Mackie A, Santos-Zavaleta A, et al. The EcoCyc database: reflecting new knowledge about Escherichia coli K-12. Nucleic Acids Res. 2017;45(D1):D543-D550. doi:10.1093/nar/gkw1003


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]