Difference between revisions of "Part:BBa K3771080"
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<br><i>T7</i> promoter constitutively facilitates the expression of CoaBC enzyme.
 
<br><i>T7</i> promoter constitutively facilitates the expression of CoaBC enzyme.
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<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:50%;">
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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 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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<img src="https://2021.igem.org/wiki/images/7/7f/T--NCKU_Tainan--CoaBC-PCR.png" style="width:35%;">
 
<img src="https://2021.igem.org/wiki/images/7/7f/T--NCKU_Tainan--CoaBC-PCR.png" style="width:35%;">
 
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<p align="center">Fig. 1 Confirmation of coaBC fragment by PCR. M: Marker; Lane 1: coaBC (1221 bp)</p>
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  <p align="center">Fig. 2. Confirmation of <i>coaBC</i> fragment by PCR. M: Marker; Lane 1: <i>coaBC</i> (1221 bp)</p>
  
 
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<div style="width=100%; display:flex; align-items: center; justify-content: center;">
 
<img src="https://2021.igem.org/wiki/images/5/53/T--NCKU_Tainan--CoaBC-plate%28DH5a%29.png" style="width:35%;">
 
<img src="https://2021.igem.org/wiki/images/5/53/T--NCKU_Tainan--CoaBC-plate%28DH5a%29.png" style="width:35%;">
 
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<p align="center">Fig. 2 Transformation/CoaBC in DH5α</p>
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<p align="center">Fig. 3. Transformation/CoaBC in DH5α</p>
  
 
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<div style="width=100%; display:flex; align-items: center; justify-content: center;">
 
<img src="https://2021.igem.org/wiki/images/5/59/T--NCKU_Tainan--CoaBC-digestion.png" style="width:35%;">
 
<img src="https://2021.igem.org/wiki/images/5/59/T--NCKU_Tainan--CoaBC-digestion.png" style="width:35%;">
 
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<p align="center">Fig. 3 Confirmation of  pET28a-T7-CoaBC by digestion.
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  <p align="center">Fig. 4. Confirmation of  pET28a-<i>P<sub>T7</sub>-coaBC</i> by digestion.
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M: Marker; Lane 1~3: Different colonies of pET28a-T7-CoaBC (5398 bp)
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M: Marker; Lane 1~3: Different colonies of pET28a-<i>P<sub>T7</sub>-coaBC</i> (5398 bp)
 
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<img src="https://2021.igem.org/wiki/images/d/d1/T--NCKU_Tainan--CoaBC-plate%28BL21%28DE3%29%29.png" style="width:35%;">
 
<img src="https://2021.igem.org/wiki/images/d/d1/T--NCKU_Tainan--CoaBC-plate%28BL21%28DE3%29%29.png" style="width:35%;">
 
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<p align="center">Fig. 4 Transform into BL21(DE3)</p>
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<p align="center">Fig. 5. Transform into BL21(DE3)</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/3/39/T--NCKU_Tainan--CoaBC-PAGE.png" style="width:35%;">
 
<img src="https://2021.igem.org/wiki/images/3/39/T--NCKU_Tainan--CoaBC-PAGE.png" style="width:35%;">
 
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<p align="center">Fig. 5 Confirmation of protein expression of CoaBC.
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<p align="center">Fig. 6. Confirmation of protein expression of CoaBC.
 
M: Marker; Lane1: whole cell of CoaBC in BL21(DE3); Lane2: soluble protein of CoaBC in BL21(DE3) (~45 kDa)
 
M: Marker; Lane1: whole cell of CoaBC in BL21(DE3); Lane2: soluble protein of CoaBC in BL21(DE3) (~45 kDa)
 
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</p>

Revision as of 01:53, 22 October 2021


T7-CoaBC


Description

PT7-coaBC is a composite part consisting of the T7 promoter and the coaBC sequences. This part was used in in vivo testing of taurine production. The sequence for jju and coaBC 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 CoaBC 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

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

Taurine production of CoaBC in BL21(DE3) and JJU in BL21(DE3) in supernatant and whole cell samples.

Characterization

Fig. 2. Confirmation of coaBC fragment by PCR. M: Marker; Lane 1: coaBC (1221 bp)

Fig. 3. Transformation/CoaBC in DH5α

Fig. 4. Confirmation of pET28a-PT7-coaBC by digestion. M: Marker; Lane 1~3: Different colonies of pET28a-PT7-coaBC (5398 bp)

Fig. 5. Transform into BL21(DE3)

Fig. 6. Confirmation of protein expression of CoaBC. M: Marker; Lane1: whole cell of CoaBC in BL21(DE3); Lane2: soluble protein of CoaBC in BL21(DE3) (~45 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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 632
  • 1000
    COMPATIBLE WITH RFC[1000]