Difference between revisions of "Part:BBa K2856001"

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<partinfo>BBa_K2856001 short</partinfo>
 
<partinfo>BBa_K2856001 short</partinfo>
  
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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.
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The BBa_K2856001 harbors a coding sequence of bi-functional 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===
 
===Usage and Biology===
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Bifunctional glutamate--cysteine ligase/glutathione synthase (gshF) is an enzyme involved and responded to synthetic reaction of GSH.In this
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Bifunctional glutamate--cysteine ligase/glutathione synthase (gshF) is an enzyme involved and responded to synthetic reaction of GSH. In this reaction, one Cysteine and one Glutamate are converted to one γ-GC, then one γ-GC and one Glycine 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 produce GSH in Lactococcus Lactis NZ9000.
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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.
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[[File:T--H14Z1 Hangzhou--Reaction_gshF.jpeg|500px|thumb|centre| <p>'''Figure. 1  Enzymatic reaction catalyzed by gshF'''</p>]]
 
[[File:T--H14Z1 Hangzhou--Reaction_gshF.jpeg|500px|thumb|centre| <p>'''Figure. 1  Enzymatic reaction catalyzed by gshF'''</p>]]
  
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===Protein Analysis ===
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=== Construction and validation of plasmid pNZ-gshF ===
  
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SDS-PAGE was performed to detect the protein expression level of gshF. The cells were washed  twice with 0.1 M PBS after centrifugation.Crude protein was extracted through cell breaking using ultrasonication and centrifugation. Then the samples were added into gel to analysis the protein.
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Gene gshF was amplified from genomic DNA of S. agalactiae and cut with restriction enzyme Hind III and NcoI, and ligased with plasmid pNZ8148 cut with the same enzyme. Then the ligation product was transferred to E.coli and spread on plates containing 10 mg/L chloramphenicol.
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Colonies on the plates were randomly picked and inoculated in 1ml LB medium for 3 hours at 37℃, 200 rpm. 1 μl culture were added to the PCR system as template. As shown in Figure. 2, all the picked colonies had gene gshF, illustrating that the plasmid pNZ-gshF was successfully constructed.  
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[[File:T--H14Z1 Hangzhou--SDS-PAGE-gshF.jpeg|500px|thumb|centre| <p>'''Figure. 2 gshF protein analysis'''</p>]]
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[[File:T--H14Z1 Hangzhou--plasmid -gshF.jpeg|500px|thumb|centre| <p>'''Figure. 2 Validation of plasmid pNZ-gshF. M repres
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===Validation of GSH by HPLC analysis===
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　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.Compared with engineered strain,we didn't detect GSH in wild type  Lactococcus lactis NZ9000.
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[[File:T--H14Z1 Hangzhou--HPLC.jpg|500px|thumb|centre|<p>'''Figure. 4　　Validation of GSH 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>]]
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2856001 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K2856001 parameters</partinfo>
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Revision as of 11:56, 14 October 2018


Bifunctional glutamate-cysteine ligase/glutathione synthase (gshF)

The BBa_K2856001 harbors a coding sequence of bi-functional 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 Cysteine and one Glutamate are converted to one γ-GC, then one γ-GC and one Glycine 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 produce GSH in Lactococcus Lactis NZ9000.

Figure. 1 Enzymatic reaction catalyzed by gshF

Construction and validation of plasmid pNZ-gshF

Gene gshF was amplified from genomic DNA of S. agalactiae and cut with restriction enzyme Hind III and NcoI, and ligased with plasmid pNZ8148 cut with the same enzyme. Then the ligation product was transferred to E.coli and spread on plates containing 10 mg/L chloramphenicol. Colonies on the plates were randomly picked and inoculated in 1ml LB medium for 3 hours at 37℃, 200 rpm. 1 μl culture were added to the PCR system as template. As shown in Figure. 2, all the picked colonies had gene gshF, illustrating that the plasmid pNZ-gshF was successfully constructed.

[[File:T--H14Z1 Hangzhou--plasmid -gshF.jpeg|500px|thumb|centre|

Figure. 2 Validation of plasmid pNZ-gshF. M repres