Difference between revisions of "Part:BBa K3771003"
 
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<br><b style="font-size:1.3rem">Description
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<b style="font-size:1.3rem">Description
 
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<br><i>PlacI-csad</i> is a composite part consisting of the lacI promoter and the csad sequences. This part was used in in vivo testing of taurine production. The sequence for csad enzyme and lacI promoter were ligated and transformed into E. coli to calculate taurine production using high-performance liquid chromatography (HPLC).<br>
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<br>L-Cysteine sulfinic acid decarboxylase (CSAD) is an enzyme consisting of 493 amino acids and weighs 50 kDa. CSAD functions in the taurine biosynthesis pathway, converting L-Cysteine to taurine [1].  
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<br><b style="font-size:1.3rem">Biology
 
<br><b style="font-size:1.3rem">Biology
 
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<br><i>lacI</i> promoter constitutively facilitates the expression of CSAD enzyme.<br>
 
  
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<img src="https://2021.igem.org/wiki/images/c/c9/T--NCKU_Tainan--taurine_pathway_1.png" style="width:35%;"></html>
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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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===Usage and Biology===
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<p align="center">Fig. 1. Taurine production pathway</p>
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<br>CSAD is part of the L-cysteine sulfinic acid pathway, one of two possible taurine synthesis pathways. CSAD catalyzes the decarboxylation of L-Cysteine sulfinic acid into hypotaurine, which is spontaneously oxidized to taurine [1].<br>
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<br><b style="font-size:1.3rem">Usage
 
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<br><i>cdo1</i> was used in in vivo testing of taurine production. The sequence for CDO1 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).
 
 
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  <br>CSAD was used in <i>in vivo</i> testing of taurine production. The sequence for CSAD 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).
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<img src="https://2021.igem.org/wiki/images/6/6b/T--NCKU_Tainan--invivo1.png" style="width:35%;"></html>
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<br><b style="font-size:1.3rem">Characterization
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<br><b style="font-size:1.3rem">Characterization  
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<br>The CDO1 fragment was synthesized by IDT and amplified by PCR. Agarose gel electrophoresis result is shown in Fig. 2.<br>
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<br>The CSAD fragment was synthesized by IDT and amplified by PCR. Agarose gel electrophoresis result is shown in Fig. 2.
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<img src="https://2021.igem.org/wiki/images/8/86/T--NCKU_Tainan--CSAD-PCR.png
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  <p align="center">Fig. 2. Confirmation of <i>csad</i> fragment by PCR. M: Marker; Lane 1: <i>csad</i> (1368 bp)</p>
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<img src="https://2021.igem.org/wiki/images/2/26/T--NCKU_Tainan--CSAD2-digestion.png
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  <p align="center">Fig. 3. Confirmation of  pSUI-<i>P<sub>trc</sub>-csad</i> by digestion.
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    M: Marker; Lane 1~3: Different colonies of pSUI-<i>P<sub>trc</sub>-csad</i> (3674 bp)</p>
  
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<img src="https://2021.igem.org/wiki/images/8/86/T--NCKU_Tainan--CSAD-PCR.png" style="width:35%;"></html>
 
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<p>Fig. 2 Confirmation of csad fragment by PCR. M: Marker; Lane 1: csad (1368 bp)</p>
 
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<img src="https://2021.igem.org/wiki/images/4/48/T--NCKU_Tainan--CSAD1-Vactor-digestion.png" style="width:35%;"></html>
 
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<p>Fig. 2 Confirmation of pSAA fragment by digestion. M: Marker; Lane 1: pSAA (2531 bp)</p>
 
 
    
 
    
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<img src="https://2021.igem.org/wiki/images/4/4e/T--NCKU_Tainan--CDO1-PAGE.png" style="width:35%;"></html>
 
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<p>Fig. 3 Confirmation of protein expression of CDO1.M: Marker; Lane1: CDO1 in DH5α without induction; Lane2: CDO1 in DH5α with induction (~22 kDa)</p>
 
 
    
 
    
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<img src="https://2021.igem.org/wiki/images/a/aa/T--NCKU_Tainan--3.png" style="width:35%;"></html>
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<img src="https://2021.igem.org/wiki/images/c/cd/T--NCKU_Tainan--CSAD2-plate%28DH5a%29.png
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<p>Fig. 4 Taurine production of Ptrc-cdo1 +PlacI-csad +Ptrc-cs in different growth mediums.</p>
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  <p align="center">Fig. 4. Transformation / CSAD in DH5α SDS-PAGE and western blot of CSAD enzyme to confirm protein expression.<br>
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<img src="https://2021.igem.org/wiki/images/9/92/T--NCKU_Tainan--CSAD-PAGE%28DH5a%29.png
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<br><b style="font-size:1.3rem">References
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<br>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<br>
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  <p>Fig. 5. Confirmation of protein expression of CSAD.
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M: Marker; Lane 1: whole cell of CSAD in DH5α; Lane 2: soluble protein of CSAD in DH5α (~50 kDa)</p>
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<br>Taurine production yield of CSAD with other production enzymes calculated by high-performance liquid chromatography (HPLC).<br>
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<br><b style="font-size:1.3rem">References</b>
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<br>1. 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. <i>Journal of Agricultural and Food Chemistry</i>. 2018;66(51):13454-13463. doi:10.1021/acs.jafc.8b05093
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===Usage and Biology===
 
  
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<span class='h3bb'>Sequence and Features</span>
 
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===Functional Parameters===
 
===Functional Parameters===
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Latest revision as of 03:56, 22 October 2021


CSAD

Description

L-Cysteine sulfinic acid decarboxylase (CSAD) is an enzyme consisting of 493 amino acids and weighs 50 kDa. CSAD functions in the taurine biosynthesis pathway, converting L-Cysteine to taurine [1].


Biology

Fig. 1. Taurine production pathway



CSAD is part of the L-cysteine sulfinic acid pathway, one of two possible taurine synthesis pathways. CSAD catalyzes the decarboxylation of L-Cysteine sulfinic acid into hypotaurine, which is spontaneously oxidized to taurine [1].


Usage

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


Characterization

The CSAD fragment was synthesized by IDT and amplified by PCR. Agarose gel electrophoresis result is shown in Fig. 2.

Fig. 2. Confirmation of csad fragment by PCR. M: Marker; Lane 1: csad (1368 bp)

Fig. 3. Confirmation of pSUI-Ptrc-csad by digestion. M: Marker; Lane 1~3: Different colonies of pSUI-Ptrc-csad (3674 bp)

Fig. 4. Transformation / CSAD in DH5α SDS-PAGE and western blot of CSAD enzyme to confirm protein expression.

Fig. 5. Confirmation of protein expression of CSAD. M: Marker; Lane 1: whole cell of CSAD in DH5α; Lane 2: soluble protein of CSAD in DH5α (~50 kDa)


Taurine production yield of CSAD with other production enzymes calculated by high-performance liquid chromatography (HPLC).

References

1. 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 BBa_K3771008 SequenceAndFeatures