Difference between revisions of "Part:BBa K3771003"
 
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<b style="font-size:1.3rem">Description
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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
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<br><b style="font-size:1.3rem">Achievements</b>
 
 
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<br>Confirmation of CSAD enzyme expression under oxidative stress by western blot.<br>
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<br><b style="font-size:1.3rem">Process</b>
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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">Fig. 1. Taurine production pathway</p>
 
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<br>To confirm that the CSAD enzyme is successfully expressed when induced by paraquat, the western blot was performed. When the OD600 of the bacterial culture reached 0.5, we added 0.1 mM paraquat into the culture and collected samples at 2, 4, 6 hours after paraquat was added. The result confirmed that CSAD was expressed under oxidative stress. The sample collected at 6 hours after paraquat was added had the greatest expression, and CSAD expression increases as the time allowed for paraquat to induce the SoxS promoter increases.  (Figure #).<br>
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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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<img src="https://2021.igem.org/wiki/images/5/58/T--NCKU_Tainan--soxR-CSAD_WB.png" style="width:35%;"></html>
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<br><b style="font-size:1.3rem">Usage
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<p align="center">Figure #. The western blot result. CSAD (~55 kDa); –: control; PQ: 0.1 mM paraquat.</p>
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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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<br><b style="font-size:1.3rem">Characterization
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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/c/cd/T--NCKU_Tainan--CSAD2-plate%28DH5a%29.png
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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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  <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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<partinfo>BBa_K3771008 parameters</partinfo>
 
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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