Difference between revisions of "Part:BBa K3771003"
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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 55.4 kDa. CSAD functions in the taurine biosynthesis pathway, converting L-Cysteine to taurine [1].  
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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:50%;">
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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><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). A 6xHis-tag is added to the C-terminal of the CSAD protein, allowing for confirmation of CSAD expression by western blot using the anti-6xHis antibody.
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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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  <p align="center">Fig. 2. PCR of <i>csad</i> sequence (1500 bp).</p>
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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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<p>Fig. 2 Confirmation of pSAA fragment by digestion. M: Marker; Lane 1: pSAA (2531 bp)</p>
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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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  <br>SDS-PAGE and western blot of CSAD enzyme to confirm protein expression.<br>
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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
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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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<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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<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. Journal of Agricultural and Food Chemistry. 2018;66(51):13454-13463. doi:10.1021/acs.jafc.8b05093
  
  
  
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Revision as of 19:02, 19 October 2021


CSAD-6xHis

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). A 6xHis-tag is added to the C-terminal of the CSAD protein, allowing for confirmation of CSAD expression by western blot using the anti-6xHis antibody.


Characterization

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

Fig. 2. PCR of csad sequence (1500 bp).


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