Difference between revisions of "Part:BBa K3771079"
 
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<br><i>T7</i> promoter constitutively facilitates the expression of JJU enzyme.
 
<br><i>T7</i> promoter constitutively facilitates the expression of JJU enzyme.
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<div style="width=100%; display:flex; align-items: center; justify-content: center;">
 
<div style="width=100%; display:flex; align-items: center; justify-content: center;">
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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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<img src="https://2021.igem.org/wiki/images/c/c9/T--NCKU_Tainan--taurine_pathway_1.png" style="width:60%;">
 
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<p align="center">圖片描述</p>
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<p align="center">Fig. 1. Taurine pathways in <i>E. coli</i>
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<br><b style="font-size:1.3rem">Usage</b>
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  <br>JJU enzyme was used in <i>in vitro</i> testing of taurine production. The sequence for JJU enzyme and trc promoter (BBa_K864400) were ligated and transformed into <i>E. coli</i>. By adding L-cysteine substrate into a reaction solution, JJU can help catalyze the conversion of L-cysteine to taurine. Taurine concentrations were determined using high-performance liquid chromatography (HPLC).<br>
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<br><b style="font-size:1.3rem">Characterization</b>
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<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/0/09/T--NCKU_Tainan--jju-fragment-pcr.png" style="width:35%;">
 
<img src="https://2021.igem.org/wiki/images/0/09/T--NCKU_Tainan--jju-fragment-pcr.png" style="width:35%;">
 
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<p align="center">Fig 1. Confirmation of <i>jju</i> fragment by PCR. M: Marker; Lane 1: <i>jju</i> (1180bp)</p>
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<p align="center">Fig. 2. Confirmation of <i>jju</i> fragment by PCR. M: Marker; Lane 1: <i>jju</i> (1180bp)</p>
  
 
<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/e/ec/T--NCKU_Tainan--jju-vactor-digestion.png" style="width:35%;">
 
<img src="https://2021.igem.org/wiki/images/e/ec/T--NCKU_Tainan--jju-vactor-digestion.png" style="width:35%;">
 
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<p align="center">Fig 2. Confirmation of pET28a fragment by digestion. M: Marker; Lane 1: pET28a (~5280 bp)</p>
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<p align="center">Fig. 3. Confirmation of pET28a fragment by digestion. M: Marker; Lane 1: pET28a (~5280 bp)</p>
  
 
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<img src="https://2021.igem.org/wiki/images/4/44/T--NCKU_Tainan--jju-plate%28DH5a%29.png" style="width:35%;">
 
<img src="https://2021.igem.org/wiki/images/4/44/T--NCKU_Tainan--jju-plate%28DH5a%29.png" style="width:35%;">
 
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<p align="center">Fig. 3 Transformation/JJU in DH5α</p>
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<p align="center">Fig. 4. Transformation/JJU in DH5α</p>
  
 
<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/c/c2/T--NCKU_Tainan--jjudigestion.png" style="width:35%;">
 
<img src="https://2021.igem.org/wiki/images/c/c2/T--NCKU_Tainan--jjudigestion.png" style="width:35%;">
 
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<p align="center">Fig. 4 Confirmation of  pET28a-T7-JJU by digestion.
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  <p align="center">Fig. 5. Confirmation of  pET28a-<i>P<sub>T7</sub>-jju</i> by digestion.
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M: Marker; Lane 1: Colony of pET28a-T7-JJU (~6466 bp)
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  M: Marker; Lane 1: Colony of pET28a-<i>P<sub>T7</sub>-jju</i> (~6466 bp)
 
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<img src="https://2021.igem.org/wiki/images/e/e6/T--NCKU_Tainan--jju-plate%28BL21%28DE3%29%29.png" style="width:35%;">
 
<img src="https://2021.igem.org/wiki/images/e/e6/T--NCKU_Tainan--jju-plate%28BL21%28DE3%29%29.png" style="width:35%;">
 
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<p align="center">Fig. 5 Transform into BL21(DE3)</p>
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<p align="center">Fig. 6. Transform into BL21(DE3)</p>
  
 
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<img src="圖片網址" style="width:35%;">
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<img src="https://2021.igem.org/wiki/images/1/19/T--NCKU_Tainan--jju-PAGE%28BL2%28DE3%29.png" style="width:35%;">
 
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<p align="center">Fig. 6 Confirmation of protein expression of JJU.
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<p align="center">Fig. 7. Confirmation of protein expression of JJU.
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M: Marker; Lane1: whole cell of JJU in BL21(DE3); Lane2: soluble protein of JJUBL21(DE3) (~43 kDa)
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M: Marker; Lane1: whole cell of JJU in BL21(DE3); Lane2: soluble protein of JJU in BL21(DE3) (~43 kDa)
 
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<img src="https://2021.igem.org/wiki/images/1/1b/T--NCKU_Tainan--jju-plate%28BD7G%29.png" style="width:35%;">
 
<img src="https://2021.igem.org/wiki/images/1/1b/T--NCKU_Tainan--jju-plate%28BD7G%29.png" style="width:35%;">
 
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<p align="center">Fig. 7 Transform into BD7G</p>
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<p align="center">Fig. 8. Transform into BD7G</p>
  
 
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<img src="圖片網址" style="width:35%;">
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<img src="https://2021.igem.org/wiki/images/3/32/T--NCKU_Tainan--jju-PAGE%28BD7G%29.png" style="width:35%;">
 
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<p align="center">Fig. 8 Confirmation of protein expression of JJU.
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<p align="center">Fig. 9. Confirmation of protein expression of JJU.
 
M: Marker; Lane1: whole cell of JJU in BD7G; Lane2: soluble protein of JJU in BD7G(~43 kDa)
 
M: Marker; Lane1: whole cell of JJU in BD7G; Lane2: soluble protein of JJU in BD7G(~43 kDa)
 
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<br><b style="font-size:1.3rem">Usage</b>
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<br><b style="font-size:1.3rem">References</b>
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<br>BRENDA - Information on EC 4.4.1.10 - cysteine lyase. Brenda-enzymes.org. Published 2021. Accessed October 6, 2021. https://www.brenda-enzymes.org/enzyme.php?ecno=4.4.1.10<br>
  
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<br><b style="font-size:1.3rem">Characterization</b>
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<br><b style="font-size:1.3rem">References</b>
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<!-- Add more about the biology of this part here
 
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Latest revision as of 03:01, 22 October 2021


T7-JJU


Description

PT7-jju is a composite part consisting of the T7 promoter and the cs sequences. This part was used in in vivo testing of taurine production. The sequence for jju and T7 promoter were ligated and transformed into E. coli to calculate taurine production using high-performance liquid chromatography (HPLC).

Biology

T7 promoter constitutively facilitates the expression of JJU enzyme.

Fig. 1. Taurine pathways in E. coli


Usage

JJU enzyme was used in in vitro testing of taurine production. The sequence for JJU enzyme and trc promoter (BBa_K864400) were ligated and transformed into E. coli. By adding L-cysteine substrate into a reaction solution, JJU can help catalyze the conversion of L-cysteine to taurine. Taurine concentrations were determined using high-performance liquid chromatography (HPLC).

Characterization

Fig. 2. Confirmation of jju fragment by PCR. M: Marker; Lane 1: jju (1180bp)

Fig. 3. Confirmation of pET28a fragment by digestion. M: Marker; Lane 1: pET28a (~5280 bp)

Fig. 4. Transformation/JJU in DH5α

Fig. 5. Confirmation of pET28a-PT7-jju by digestion. M: Marker; Lane 1: Colony of pET28a-PT7-jju (~6466 bp)

Fig. 6. Transform into BL21(DE3)

Fig. 7. Confirmation of protein expression of JJU. M: Marker; Lane1: whole cell of JJU in BL21(DE3); Lane2: soluble protein of JJU in BL21(DE3) (~43 kDa)

Fig. 8. Transform into BD7G

Fig. 9. Confirmation of protein expression of JJU. M: Marker; Lane1: whole cell of JJU in BD7G; Lane2: soluble protein of JJU in BD7G(~43 kDa)


References
BRENDA - Information on EC 4.4.1.10 - cysteine lyase. Brenda-enzymes.org. Published 2021. Accessed October 6, 2021. https://www.brenda-enzymes.org/enzyme.php?ecno=4.4.1.10
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]