Difference between revisions of "Part:BBa K3771003"
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<partinfo>BBa_K3771003 short</partinfo> | <partinfo>BBa_K3771003 short</partinfo> | ||
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| − | <br><b style="font-size:1.3rem">Description | + | <br> |
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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 | <br><b style="font-size:1.3rem">Biology | ||
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| − | + | <div style="width=100%; display:flex; align-items: center; justify-content: center;"> | |
| − | + | <img src="https://2021.igem.org/wiki/images/c/c9/T--NCKU_Tainan--taurine_pathway_1.png" style="width:60%;"> | |
| − | + | </div> | |
| − | + | <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 | <br><b style="font-size:1.3rem">Usage | ||
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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><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. | ||
| + | <div style="width=100%; display:flex; align-items: center; justify-content: center;"> | ||
| + | <img src="https://2021.igem.org/wiki/images/8/86/T--NCKU_Tainan--CSAD-PCR.png | ||
| + | " style="width:35%;"> | ||
| + | </div> | ||
| + | <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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| + | <div style="width=100%; display:flex; align-items: center; justify-content: center;"> | ||
| + | <img src="https://2021.igem.org/wiki/images/2/26/T--NCKU_Tainan--CSAD2-digestion.png | ||
| + | " style="width:35%;"> | ||
| + | </div> | ||
| + | <p align="center">Fig. 3. Confirmation of pSUI-<i>P<sub>trc</sub>-csad</i> by digestion. | ||
| + | M: Marker; Lane 1~3: Different colonies of pSUI-<i>P<sub>trc</sub>-csad</i> (3674 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/c/cd/T--NCKU_Tainan--CSAD2-plate%28DH5a%29.png | |
| − | + | " style="width:35%;"> | |
| − | + | </div> | |
| − | + | <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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| − | + | <div style="width=100%; display:flex; align-items: center; justify-content: center;"> | |
| − | + | <img src="https://2021.igem.org/wiki/images/9/92/T--NCKU_Tainan--CSAD-PAGE%28DH5a%29.png | |
| − | <br><b style="font-size:1.3rem">References | + | " style="width:35%;"> |
| − | </b> | + | </div> |
| − | <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> | + | <p>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)</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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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
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===Functional Parameters=== | ===Functional Parameters=== | ||
| − | <partinfo> | + | <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