Difference between revisions of "Part:BBa K3771003"
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   <p align="center">Fig. 2. PCR of <i>csad</i> sequence (1500 bp).</p>
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   <p align="center">Fig. 2. Confirmation of <i>csad</p> fragment by PCR. M: Marker; Lane 1: csad (1368 bp)</p>
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  <p align="center">Fig. 3 Confirmation of  pSUI-Ptrc-CSAD by digestion.
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    M: Marker; Lane 1~3: Different colonies of pSUI-Ptrc-CSAD (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>Fig. 4 Transformation / CSAD in DH5α</p>
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  <p>Confirmation of protein expression of CSAD.
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M: Marker; Lane1: whole cell of CSAD in DH5α; Lane2: soluble protein of CSAD in DH5α (~22 kDa)</p>
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Revision as of 11:26, 20 October 2021


CSAD

Description

L-Cysteine sulfinic acid decarboxylase (CSAD) is an enzyme consisting of 493 amino acids and weighs 55.4 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

Confirmation of protein expression of CSAD. M: Marker; Lane1: whole cell of CSAD in DH5α; Lane2: soluble protein of CSAD in DH5α (~22 kDa)


SDS-PAGE and western blot of CSAD enzyme to confirm protein expression.

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


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 AgeI site found at 250
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 35