Difference between revisions of "Part:BBa K3771094"
 
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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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<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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<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://2021.igem.org/wiki/images/8/8f/T--NCKU_Tainan--PsoxS-CS_Page.png"​ style="width:25%;">
 
<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. The SDS-Page result. CS (~46 kDa); –: control; PQ: 0.5 mM paraquat.
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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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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:
  • 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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 604