Difference between revisions of "Part:BBa K3771003"
 
(5 intermediate revisions by 2 users not shown)
Line 8: Line 8:
 
</b>
 
</b>
 
<br>
 
<br>
−
<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].  
+
<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].  
 
<br>
 
<br>
 
<br>
 
<br>
Line 42: Line 42:
 
" style="width:35%;">
 
" style="width:35%;">
 
</div>
 
</div>
−
   <p align="center">Fig. 3. Confirmation of  pSUI-Ptrc-CSAD by digestion.
+
   <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-Ptrc-CSAD (3674 bp)</p>
+
     M: Marker; Lane 1~3: Different colonies of pSUI-<i>P<sub>trc</sub>-csad</i> (3674 bp)</p>
  
 
    
 
    
Line 51: Line 51:
 
" style="width:35%;">
 
" style="width:35%;">
 
</div>
 
</div>
−
   <p align="center">Fig. 4. Transformation / CSAD in DH5α</p>
+
   <p align="center">Fig. 4. Transformation / CSAD in DH5α SDS-PAGE and western blot of CSAD enzyme to confirm protein expression.<br>
−
 
+
−
  <br>SDS-PAGE and western blot of CSAD enzyme to confirm protein expression.<br>
+
 
    
 
    
 
   <div style="width=100%; display:flex; align-items: center; justify-content: center;">
 
   <div style="width=100%; display:flex; align-items: center; justify-content: center;">
Line 59: Line 57:
 
" style="width:35%;">
 
" style="width:35%;">
 
</div>
 
</div>
−
   <p>Confirmation of protein expression of CSAD.
+
   <p>Fig. 5. Confirmation of protein expression of CSAD.
−
M: Marker; Lane1: whole cell of CSAD in DH5α; Lane2: soluble protein of CSAD in DH5α (~22 kDa)</p>
+
M: Marker; Lane 1: whole cell of CSAD in DH5α; Lane 2: soluble protein of CSAD in DH5α (~50 kDa)</p>
 
    
 
    
 
    
 
    
Line 68: Line 66:
 
    
 
    
 
    
 
    
−
  <html>
+
   
−
  <div style="width=100%; display:flex; align-items: center; justify-content: center;">
+
−
  <img src="https://2021.igem.org/wiki/images/a/aa/T--NCKU_Tainan--3.png" style="width:35%;">
+
−
</div>
+
−
</html>
+
−
<p align="center">Fig. 5.Taurine production of Ptrc-cdo1 +PlacI-csad +Ptrc-cs in different growth mediums.</p>
+
−
 
+
 
    
 
    
 
    
 
    

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