Difference between revisions of "Part:BBa K2856001"
(→Construction and validation of plasmid pNZ-gshF) |
|||
| Line 14: | Line 14: | ||
<br> | <br> | ||
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. | 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. | + | 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. <br> |
[[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>]]<br><br> | [[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>]]<br><br> | ||
| Line 20: | Line 20: | ||
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. | 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. | + | 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. <br> |
| − | [[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>]] | + | [[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>]]<br> |
=== Validation of glutathione (GSH) by HPLC analysis === | === 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. | + | <br>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.<br> |
| − | [[File:T--H14Z1 Hangzhou--HPLC_gshF.jpeg|700px|thumb|centre| <p>'''Figure. 4 Validation of glutathione (GSH) by HPLC. '''</p>]] | + | [[File:T--H14Z1 Hangzhou--HPLC_gshF.jpeg|700px|thumb|centre| <p>'''Figure. 4 Validation of glutathione (GSH) by HPLC. '''</p>]]<br> |
<!-- --> | <!-- --> | ||
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
Revision as of 11:29, 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.[http://2018.igem.org/Team:H14Z1_Hangzhou Team H14Z1_Hangzhou 2018]
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.
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.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]