Difference between revisions of "Part:BBa K3771094"
 
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<br>This composite part consists of promoter <i>P<sub>soxS</sub></i> (BBa_K3771048) and the cs gene (BBa_K3771012), which encodes L-cysteine sulfonic acid synthase (CS). It is used to express CS to produce taurine under oxidative stress.
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<br>This composite part consists of promoter <i>P<sub>soxS</sub></i> (BBa_K3771048) and the <i>cs</i> gene (BBa_K3771012), which encodes L-cysteine sulfonic acid synthase (CS). It is used to express CS to produce taurine under oxidative stress.
 
The <i>soxS</i> gene is one component of the <i>soxRS</i> regulon, which is important for <i>E. coli</i> to sense and respond to the oxidants. The <i>soxS</i> promoter (<i>P<sub>soxS</sub></i>) is stimulated by oxidative stress, leading to the expression of the downstream gene. The L-cysteine sulfonic acid synthase (CS) is an enzyme that catalyzes the conversion of O-phospho-L-serine and sulfite into L-cysteine sulfonic acid (L-cysteate) and phosphate. In order to use anti-polyhistidine-tag antibodies to detect the production of CS by western blot, we add 6xHis-tag at the C-terminal of CS.
 
The <i>soxS</i> gene is one component of the <i>soxRS</i> regulon, which is important for <i>E. coli</i> to sense and respond to the oxidants. The <i>soxS</i> promoter (<i>P<sub>soxS</sub></i>) is stimulated by oxidative stress, leading to the expression of the downstream gene. The L-cysteine sulfonic acid synthase (CS) is an enzyme that catalyzes the conversion of O-phospho-L-serine and sulfite into L-cysteine sulfonic acid (L-cysteate) and phosphate. In order to use anti-polyhistidine-tag antibodies to detect the production of CS by western blot, we add 6xHis-tag at the C-terminal of CS.
  
 
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<br>This composite part was ligated with the pSAA vector and transformed into <i>E. coli</i>. We conducted colony PCR to verify whether <i>E. coli</i> uptake the correct plasmid. The size of the PCR product was as expected.The part has been confirmed by sequencing and has no mutations.
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<br>This composite part was ligated with the pSAA vector and transformed into <i>E. coli</i>. We conducted colony PCR to verify whether <i>E. coli</i> uptake the correct plasmid. The size of the PCR product was as expected. The part has been confirmed by sequencing and has no mutations.
 
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<img src="https://2021.igem.org/wiki/images/4/41/T--NCKU_Tainan--soxR-sfGFP_Colony_PCR.png"​ style="width:35%;">
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<img src="https://2021.igem.org/wiki/images/4/41/T--NCKU_Tainan--PsoxS-CS_Colony_PCR.png"​ style="width:25%;">
 
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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>-<i>sfgfp</i> (2691 bp);  Lane 2, 3: pSAA-<i>P<sub>soxS</sub>-cs-6xHis</i> (3240 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>-<i>sfgfp</i> (2691 bp);  Lane 2, 3: pSAA-<i>P<sub>soxS</sub>-cs-6xHis</i> (3240 bp).
 
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<br>The promoter strength under oxidative stress of <i>P<sub>soxS</sub></i> with its activator, SoxR, is determined by the expression level of sfGFP, which is their reporter. While using hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) as the inducer, the intensity of soxR-<i>P<sub>soxS</sub></i> shows no significant difference under different concentrations of hydrogen peroxide (Fig.2). However, soxR-<i>P<sub>soxS</sub></i> is well induced by using paraquat (PQ), which is a commonly used agent to induce oxidative stress for bacteria (Fig.3 & 4).
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<br>After the overnight incubation of <i>E. coli</i> with our plasmid, we diluted the bacteria culture and measured OD<sub>600</sub> once in a while. Until OD<sub>600</sub> reached 0.5, we added 0.5 mM paraquat, which served as an oxidative stress inducer, into the culture. We collected 1 ml culture each from the control group and the paraquat group at 2, 4, 6 hours after paraquat was added. Afterward, we conducted SDS-PAGE and western blot to confirm paraquat was added. Afterward, we conducted SDS-PAGE and western blot to confirm whether CS expressed successfully under the regulation of <i>P<sub>soxS</sub></i> by oxidative stress. The expression level of CS is too low to be seen on SDS-PAGE without its activator, SoxR (Fig. 2); nevertheless, the result of western blot shows more CS production in the paraquat group (Fig. 3).
 
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<img src="https://static.igem.org/mediawiki/parts/1/17/T--NCKU_Tainan--sfGFP_Expression_%28soxR-PsoxS%29.png"​ style="width:35%;">
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<img src="https://2021.igem.org/wiki/images/8/8f/T--NCKU_Tainan--PsoxS-CS_Page.png"​ style="width:25%;">
 
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<p align="center"> Fig.2. Relative fluorescence intensity of soxR-<i>P<sub>soxS</sub></i> after 4.5-hour incubation with hydrogen peroxide in various concentrations.
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<p align="center"> Fig. 2. The SDS-PAGE result. CS (~46 kDa); –: control; PQ: 0.5 mM paraquat.
 
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<img src="https://static.igem.org/mediawiki/parts/3/33/T--NCKU_Tainan--Del_Low_sfGFP_Expression_%28soxR-PsoxS%29.png"​ style="width:35%;">
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<img src="https://2021.igem.org/wiki/images/7/7b/T--NCKU_Tainan--PsoxS-CS_WB.png"​ style="width:40%;">
 
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<p align="center"> Fig.3. Relative fluorescence intensity of soxR-<i>P<sub>soxS</sub></i> after 4.5-hour incubation with hydrogen peroxide in low concentrations.
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<p align="center"> Fig. 3. The western blot result. CS (~46 kDa); –: control; PQ: 0.5 mM paraquat.
 
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<img src="https://static.igem.org/mediawiki/parts/0/0c/T--NCKU_Tainan--Del_High_sfGFP_Expression_%28soxR-PsoxS%29.png"​ style="width:35%;">
 
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<p align="center"> Fig.4. Relative fluorescence intensity of soxR-<i>P<sub>soxS</sub></i> after 4.5-hour incubation with hydrogen peroxide in high 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 J23100 shows no difference between the groups with and without paraquat. <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 J23100, <i>P<sub>katG</sub></i> (BBa_K37710XX), and <i>P<sub>soxS</sub></i> (BBa_K37710XX) solely (Fig.5). Moreover, we compared the efficiency of our design with (BBa_K37710XX), in which the concept came from the 2018 Imperial College London iGEM team. Different from our design, they used a mutant <i>P<sub>soxS</sub></i> promoter with RiboJ (BBa_K2862010) and expressed SoxR by J23101 promoter. However, the sfGFP expression by BBa_K2862010 was quite low  (Fig.6).
 
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<img src="https://static.igem.org/mediawiki/parts/c/c2/T--NCKU_Tainan--sfGFP_Expression_%28PQ_%404.5h%29.png"​ style="width:35%;">
 
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<p align="center"> Fig.5. Comparison of different promoter strengths by relative fluorescence intensity after 4.5-hour incubation with paraquat in high concentrations.
 
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<p align="center"> Fig.6. Comparison of different promoter strengths by relative fluorescence intensity after 4.5-hour incubation with paraquat in low concentrations.
 
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<br><b style="font-size:1.3rem">References</b>
 
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<br>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
 
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===Usage and Biology===
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3771094 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3771094 SequenceAndFeatures</partinfo>

Latest revision as of 03:06, 22 October 2021


PsoxS-CS-6xHis


Description


This composite part consists of promoter PsoxS (BBa_K3771048) and the cs gene (BBa_K3771012), which encodes L-cysteine sulfonic acid synthase (CS). It is used to express CS to produce taurine under oxidative stress. The soxS gene is one component of the soxRS regulon, which is important for E. coli to sense and respond to the oxidants. The soxS promoter (PsoxS) is stimulated by oxidative stress, leading to the expression of the downstream gene. The L-cysteine sulfonic acid synthase (CS) is an enzyme that catalyzes the conversion of O-phospho-L-serine and sulfite into L-cysteine sulfonic acid (L-cysteate) and phosphate. In order to use anti-polyhistidine-tag antibodies to detect the production of CS by western blot, we add 6xHis-tag at the C-terminal of CS.



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. The part has been confirmed by sequencing and has no mutations.


Fig. 1. The electrophoresis result of colony PCR. M: Marker; Lane 1: pSAA-PsoxS-sfgfp (2691 bp); Lane 2, 3: pSAA-PsoxS-cs-6xHis (3240 bp).


After the overnight incubation of E. coli with our plasmid, we diluted the bacteria culture and measured OD600 once in a while. Until OD600 reached 0.5, we added 0.5 mM paraquat, which served as an oxidative stress inducer, into the culture. We collected 1 ml culture each from the control group and the paraquat group at 2, 4, 6 hours after paraquat was added. Afterward, we conducted SDS-PAGE and western blot to confirm paraquat was added. Afterward, we conducted SDS-PAGE and western blot to confirm whether CS expressed successfully under the regulation of PsoxS by oxidative stress. The expression level of CS is too low to be seen on SDS-PAGE without its activator, SoxR (Fig. 2); nevertheless, the result of western blot shows more CS production in the paraquat group (Fig. 3).

Fig. 2. The SDS-PAGE result. CS (~46 kDa); –: control; PQ: 0.5 mM paraquat.

Fig. 3. The western blot result. CS (~46 kDa); –: control; PQ: 0.5 mM paraquat.


Sequence and Features


Assembly Compatibility:
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    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
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    COMPATIBLE WITH RFC[21]
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    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 604