Difference between revisions of "Part:BBa K2856001"
Line 3: Line 3:
 
<partinfo>BBa_K2856001 short</partinfo>
 
<partinfo>BBa_K2856001 short</partinfo>
  
−
The BBa_K2856001 harbors a coding sequence of bifunctional glutamate--cysteine ligase/glutathione synthase (gshF) derived from Streptococcus. Codon-optimization has been made for Lactococcus Lactis. gshFp catalyzes the conversion of  Cys,Glu and Gly to GSH.
+
The BBa_K2856001 harbors a coding sequence of bifunctional glutamate--cysteine ligase/glutathione synthase (gshF) derived from S.agalactiae. Codon-optimization has been made for Lactococcus Lactis. gshFp catalyzes the conversion of  Cys,Glu and Gly to GSH.
  
  
Line 13: Line 13:
  
 
===Protein Analysis ===
 
===Protein Analysis ===
 +
  
 
[[File:T--H14Z1 Hangzhou--SDS-PAGE-gshF.jpeg|500px|thumb|centre| <p>'''Figure. 2  gshF protein analysis'''</p>]]
 
[[File:T--H14Z1 Hangzhou--SDS-PAGE-gshF.jpeg|500px|thumb|centre| <p>'''Figure. 2  gshF protein analysis'''</p>]]
 +
 +
===Validation of GSH by HPLC analysis===
 +
　To further confirm the synthetic GSH in Lactococcus lactis, HPLC was performed to analyze the product. GSH was identified on the basis of retention times related to standard sample. According to the retention time of standard GSH sample, GSH compound of extracts from engineered Lactococcus lactis can be confirmed.
 +
 +
[[File:T--H14Z1 Hangzhou--HPLC.jpg|500px|thumb|centre|<p>'''Figure. 4　　Validation of astaxanthin by HPLC.'''<br>HPLC chromatogram of methanol extracts from theengineered  Lactococcus lactis  and wild-type Lactococcus lactis. HPLC analysis recorded at 480 nm of extracts. </p>]]
 +
  
 
<!-- -->
 
<!-- -->

Revision as of 05:27, 6 October 2018


Bifunctional glutamate-cysteine ligase/glutathione synthase (gshF)

The BBa_K2856001 harbors a coding sequence of bifunctional glutamate--cysteine ligase/glutathione synthase (gshF) derived from S.agalactiae. Codon-optimization has been made for Lactococcus Lactis. gshFp catalyzes the conversion of Cys,Glu and Gly to GSH.


Usage and Biology

Bifunctional glutamate--cysteine ligase/glutathione synthase (gshF) is an enzyme involved and responded to synthetic reaction of GSH.In this reaction,one Cys and one Glu are converted to one γ-GC,then one γ-GC and one Gly are converted to one GSH.(Figure 1) The Lactococcus Lactis NZ9000 has inability to synthesis GSH.In our project,we construct a plasmid harboring gshF in order to produnce GSH in Lactococcus Lactis NZ9000.

Figure. 1 Enzymatic reaction catalyzed by gshF

Protein Analysis

Figure. 2 gshF protein analysis

Validation of GSH by HPLC analysis

　To further confirm the synthetic GSH in Lactococcus lactis, HPLC was performed to analyze the product. GSH was identified on the basis of retention times related to standard sample. According to the retention time of standard GSH sample, GSH compound of extracts from engineered Lactococcus lactis can be confirmed.

Figure. 4　　Validation of astaxanthin by HPLC.
HPLC chromatogram of methanol extracts from theengineered Lactococcus lactis and wild-type Lactococcus lactis. HPLC analysis recorded at 480 nm of extracts.


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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]