Difference between revisions of "Part:BBa K2856001"

Revision as of 07:05, 17 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.

Figure. 2 Validation of plasmid pNZ-gshF. M represented marker. 1, 2 and 3 represented three randomly picked colonies.

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.

Figure. 2 Validation of plasmid pNZ-gshF. M represented marker. 1, 2 and 3 represented three randomly picked colonies.

Validation of glutathione (GSH) by HPLC analysis

To confirm the synthetic glutathione in L. lactis/pNZ-gshF, HPLC was performed to analyze the extracts from the strain. Glutathione was identified on the basis of retention times related to standard sample. According to the retention time of standard glutathione sample, it can be confirmed that glutathione was synthesized in L. lactis/pNZ-gshF.

Figure. 4 Validation of glutathione (GSH) by HPLC.

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 751
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 495
Illegal BsaI.rc site found at 1032

@@ Line 17: / Line 17: @@
 [[File:T--H14Z1 Hangzhou--plasmid -gshF.jpeg|300px|thumb|centre| <p>'''Figure. 2 Validation of plasmid pNZ-gshF. M represented marker. 1, 2 and 3 represented three randomly picked colonies.'''</p>]]
-=== Protein Analysis ===
+=== Construction and validation of plasmid pNZ-gshF ===
-After transferring the plasmid pNZ-gshF to L. lactis NZ9000, SDS-PAGE was performed to detect the protein expression level of gshF gene. 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 supernatant of the samples were used to analysis the protein expression. As shown in Figure. 3, expected bands of the GshF protein were observed on the gel in the lane of recombinant L. lactis containing pNZ-gshF induced with different nisin concentration while no GshF protein existed in L. lactis NZ9000.
+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.
-[[File:T--H14Z1 Hangzhou--SDS-PAGE_gshF.jpeg|400px|thumb|centre| <p>'''Figure. 3 SDS PAGE validation of gene gshF expression in L. lactis. M represented marker. WT represented L. lactis NZ9000. 1-3 represented L. lactis/pNZ-gshF induced with 100, 50 and 20 ng/ml nisin.'''</p>]]
+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|300px|thumb|centre| <p>'''Figure. 2 Validation of plasmid pNZ-gshF. M represented marker. 1, 2 and 3 represented three randomly picked colonies.'''</p>]]
 === Validation of glutathione (GSH) by HPLC analysis ===