Difference between revisions of "Part:BBa K3771078"
 
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  <img src="https://2021.igem.org/wiki/images/c/c9/T--NCKU_Tainan--taurine_pathway_1.png" style="width:35%;">
 
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   <p>Fig. 1 L-cysteine sulfonic acid synthase catalyzes the reaction that turns O-phospho-L-serine into L-cysteine sulfonic acid. CDO1, L-cysteine dioxygenase; CSAD, L-cysteine sulfinic acid decarboxylase.
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   <p align="center">Fig. 1. L-cysteine sulfonic acid synthase catalyzes the reaction that turns O-phospho-L-serine into L-cysteine sulfonic acid. CDO1, L-cysteine dioxygenase; CSAD, L-cysteine sulfinic acid decarboxylase.
 
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   <br>CS was used in in vivo testing of taurine production. The sequence for CS enzyme and <i>trc</i> promoter were ligated and transformed into <i>E. coli</i> to calculate taurine production using high-performance liquid chromatography (HPLC). <br>
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   <br>CS was used in <i>in vivo</i> testing of taurine production. The sequence for CS enzyme and <i>trc</i> promoter were ligated and transformed into <i>E. coli</i> to calculate taurine production using high-performance liquid chromatography (HPLC). <br>
 
   
 
   
 
  <div style="width=100%; display:flex; align-items: center; justify-content: center;">
 
  <div style="width=100%; display:flex; align-items: center; justify-content: center;">
 
  <img src="https://2021.igem.org/wiki/images/a/aa/T--NCKU_Tainan--3.png" style="width:35%;">
 
  <img src="https://2021.igem.org/wiki/images/a/aa/T--NCKU_Tainan--3.png" style="width:35%;">
 
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   <p>Fig. 2 Taurine production of <i>P<sub>trc</sub>-cdo1</i>+<i>P<sub>lacI</sub>-csad</i> +<i>P<sub>trc</sub>-cs</i> in different growth mediums.</p>
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   <p align="center">Fig. 2. Taurine production of <i>P<sub>trc</sub>-cdo1</i>+<i>P<sub>lacI</sub>-csad</i> +<i>P<sub>trc</sub>-cs</i> in different growth mediums.</p>
 
    
 
    
 
<br><b style="font-size:1.3rem">Characterization</b>
 
<br><b style="font-size:1.3rem">Characterization</b>
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<img src="https://2021.igem.org/wiki/images/3/39/T--NCKU_Tainan--CS-PCR.png" style="width:35%;">
 
<img src="https://2021.igem.org/wiki/images/3/39/T--NCKU_Tainan--CS-PCR.png" style="width:35%;">
 
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<p align="center">Fig. 2 Confirmation of <i>cs</i> fragment by PCR. M: Marker; Lane 1: <i>cs</i> (1272 bp)</p>   
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<p align="center">Fig. 3. Confirmation of <i>cs</i> fragment by PCR. M: Marker; Lane 1: <i>cs</i> (1272 bp)</p>   
 
    
 
    
 
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<img src="https://2021.igem.org/wiki/images/e/e3/T--NCKU_Tainan--CS-Vactor-digestion.png" style="width:35%;">
 
<img src="https://2021.igem.org/wiki/images/e/e3/T--NCKU_Tainan--CS-Vactor-digestion.png" style="width:35%;">
 
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<p align="center">Fig. 3 Confirmation of pSUI fragment by digestion. M: Marker; Lane 1: pSUI (3664 bp)</p>
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<p align="center">Fig. 4. Confirmation of pSUI fragment by digestion. M: Marker; Lane 1: pSUI (3664 bp)</p>
  
 
<div style="width=100%; display:flex; align-items: center; justify-content: center;">
 
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<img src="https://2021.igem.org/wiki/images/3/35/T--NCKU_Tainan--CS-plate%28DH5a%29.png" style="width:35%;">
 
<img src="https://2021.igem.org/wiki/images/3/35/T--NCKU_Tainan--CS-plate%28DH5a%29.png" style="width:35%;">
 
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<p align="center">Fig. 4 Transformation/CS in DH5α</p>
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<p align="center">Fig. 5. Transformation/CS in DH5α</p>
  
 
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<img src="https://2021.igem.org/wiki/images/9/9b/T--NCKU_Tainan--CS-colony-PCR.png" style="width:35%;">
 
<img src="https://2021.igem.org/wiki/images/9/9b/T--NCKU_Tainan--CS-colony-PCR.png" style="width:35%;">
 
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<p align="center">Fig. 5 Confirmation of <i>cs</i> fragment by colony PCR.
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<p align="center">Fig. 6. Confirmation of <i>cs</i> fragment by colony PCR.
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M: Marker; Lane 1~6: Different colonies of <i>pSUI-P<sub>trc</sub>-CS</i> (1272 bp)
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M: Marker; Lane 1~6: Different colonies of <i>pSUI-P<sub>trc</sub>-cs</i> (1272 bp)
 
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<img src="https://2021.igem.org/wiki/images/1/1d/T--NCKU_Tainan--CS-digestion.png" style="width:35%;">
 
<img src="https://2021.igem.org/wiki/images/1/1d/T--NCKU_Tainan--CS-digestion.png" style="width:35%;">
 
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<p align="center">Fig. 6 Confirmation of <i>pSUI-P<sub>trc</sub>-CS</i> by digestion.
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<p align="center">Fig. 7. Confirmation of <i>pSUI-P<sub>trc</sub>-CS</i> by digestion.
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M: Marker; Lane 1: Colony of <i>pSUI-P<sub>trc</sub>-CS</i>
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M: Marker; Lane 1: Colony of <i>pSUI-P<sub>trc</sub>-cs</i>
 
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<img src="https://2021.igem.org/wiki/images/6/64/T--NCKU_Tainan--CS-PAGE%28DH5a%29.png" style="width:35%;">
 
<img src="https://2021.igem.org/wiki/images/6/64/T--NCKU_Tainan--CS-PAGE%28DH5a%29.png" style="width:35%;">
 
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<p align="center">Fig. 7 Confirmation of the protein expression of CS
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<p align="center">Fig. 8. Confirmation of the protein expression of CS
 
M: Marker; Lane 1: CS in DH5α with induction (~46 kDa)
 
M: Marker; Lane 1: CS in DH5α with induction (~46 kDa)
 
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</p>

Latest revision as of 03:11, 22 October 2021


Ptrc-CS


Description

Ptrc-cs is a composite part consisting of the trc promoter and the cs sequences. This part was used in in vivo testing of taurine production. The sequence for cs and trc promoter were ligated and transformed into E. coli to calculate taurine production using high-performance liquid chromatography (HPLC).

Biology

trc promoter constitutively facilitates the expression of CS enzyme.

Fig. 1. L-cysteine sulfonic acid synthase catalyzes the reaction that turns O-phospho-L-serine into L-cysteine sulfonic acid. CDO1, L-cysteine dioxygenase; CSAD, L-cysteine sulfinic acid decarboxylase.


Usage

CS was used in in vivo testing of taurine production. The sequence for CS enzyme and trc promoter were ligated and transformed into E. coli to calculate taurine production using high-performance liquid chromatography (HPLC).

Fig. 2. Taurine production of Ptrc-cdo1+PlacI-csad +Ptrc-cs in different growth mediums.


Characterization

Fig. 3. Confirmation of cs fragment by PCR. M: Marker; Lane 1: cs (1272 bp)

Fig. 4. Confirmation of pSUI fragment by digestion. M: Marker; Lane 1: pSUI (3664 bp)

Fig. 5. Transformation/CS in DH5α

Fig. 6. Confirmation of cs fragment by colony PCR. M: Marker; Lane 1~6: Different colonies of pSUI-Ptrc-cs (1272 bp)

Fig. 7. Confirmation of pSUI-Ptrc-CS by digestion. M: Marker; Lane 1: Colony of pSUI-Ptrc-cs

Fig. 8. Confirmation of the protein expression of CS M: Marker; Lane 1: CS in DH5α with induction (~46 kDa)


References

Joo Y-C, Ko YJ, You SK, et al. Creating a New Pathway in Corynebacterium glutamicum for the Production of Taurine as a Food Additive. Journal of Agricultural and Food Chemistry. 2018;66(51):13454-13463. doi:10.1021/acs.jafc.8b05093
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 580