Difference between revisions of "Part:BBa K1088004"
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"IspG catalyzes the conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate. IspG activity depends on as-yet unidentified additional proteins, most likely involved in the oxidation portion of the reaction" [http://www.ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG10370 (Link)] | "IspG catalyzes the conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate. IspG activity depends on as-yet unidentified additional proteins, most likely involved in the oxidation portion of the reaction" [http://www.ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG10370 (Link)] | ||
| − | + | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
| + | <p>The genes dxs and ispG were placed on the backbone of low-copy plasmid pBBR1MCS-2, and the promoter was medium strength lac promoters, to construct plasmid pBBR1MCS-2-dxs-ispG (pMEP-DG).</p> | ||
| + | <p>Genes ispH, idi, and ispA were placed on the backbone of high-copy plasmid pETDuet-1, and the promoter was strong promoter T7 to construct plasmid pETDuet-1-1T7-IspH-Idi-IspA-Yss (pET-HIAY).</p> | ||
| + | <p>When Dxs and IspG were overexpressed, the squalene yield increased by 4.3 times. We speculated that the overexpressed gene ispG was constructed on low-copy plasmids and was controlled by promoters of medium strength, so it didn’t produce large amounts of harmful intermediate HMBPP. And overexpressing Dxs promoted the metabolic flow, increasing the squalene yield.</p> | ||
| + | https://2019.igem.org/wiki/images/9/91/T--SEFLS_Shanghai--BBa_K1088004-fig1.png | ||
| + | <p>H3: p35151/pET-HIAY</p> | ||
| + | <p>H5: p35151/pMEP-DG/pET-HIAY</p> | ||
| + | https://2019.igem.org/wiki/images/thumb/8/8a/T--SEFLS_Shanghai--BBa_all-fig1.png/781px-T--SEFLS_Shanghai--BBa_all-fig1.png.jpeg | ||
| + | https://2019.igem.org/wiki/images/thumb/d/d5/T--SEFLS_Shanghai--BBa_all-fig2.png/800px-T--SEFLS_Shanghai--BBa_all-fig2.png.jpeg | ||
| + | https://2019.igem.org/wiki/images/thumb/b/b3/T--SEFLS_Shanghai--BBa_all-fig3.png/799px-T--SEFLS_Shanghai--BBa_all-fig3.png.jpeg | ||
| + | https://2019.igem.org/wiki/images/7/78/T--SEFLS_Shanghai--BBa_all-fig4.jpg | ||
| + | https://2019.igem.org/wiki/images/1/17/T--SEFLS_Shanghai--BBa_all-fig5.jpg | ||
| + | https://2019.igem.org/wiki/images/0/03/T--SEFLS_Shanghai--BBa_all-fig6.jpg | ||
| + | https://2019.igem.org/wiki/images/f/f5/T--SEFLS_Shanghai--BBa_all-fig7.jpg | ||
| + | https://2019.igem.org/wiki/images/0/0a/T--SEFLS_Shanghai--BBa_all-fig8.jpg | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K1088004 SequenceAndFeatures</partinfo> | <partinfo>BBa_K1088004 SequenceAndFeatures</partinfo> | ||
Revision as of 23:01, 21 October 2019
IspG (1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase)
1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG) is part of the bacterial MEP (methylerythritol phosphate) pathway used for production of isoprenoids.
From ecocyc: "IspG catalyzes the conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate. IspG activity depends on as-yet unidentified additional proteins, most likely involved in the oxidation portion of the reaction" [http://www.ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG10370 (Link)]
Usage and Biology
The genes dxs and ispG were placed on the backbone of low-copy plasmid pBBR1MCS-2, and the promoter was medium strength lac promoters, to construct plasmid pBBR1MCS-2-dxs-ispG (pMEP-DG).
Genes ispH, idi, and ispA were placed on the backbone of high-copy plasmid pETDuet-1, and the promoter was strong promoter T7 to construct plasmid pETDuet-1-1T7-IspH-Idi-IspA-Yss (pET-HIAY).
When Dxs and IspG were overexpressed, the squalene yield increased by 4.3 times. We speculated that the overexpressed gene ispG was constructed on low-copy plasmids and was controlled by promoters of medium strength, so it didn’t produce large amounts of harmful intermediate HMBPP. And overexpressing Dxs promoted the metabolic flow, increasing the squalene yield.
H3: p35151/pET-HIAY
H5: p35151/pMEP-DG/pET-HIAY
