Difference between revisions of "Part:BBa K1653003"
| Line 8: | Line 8: | ||
Farnesyl diphosphate synthase can utilize both dimethylallyl and geranyl diphosphates as substrates, generating geranyl and farnesyl diphosphate, respectively. Therefore the enzyme can catalyze two sequential reactions in the polyisoprenoid biosynthetic pathway. | Farnesyl diphosphate synthase can utilize both dimethylallyl and geranyl diphosphates as substrates, generating geranyl and farnesyl diphosphate, respectively. Therefore the enzyme can catalyze two sequential reactions in the polyisoprenoid biosynthetic pathway. | ||
| − | |||
===Usage and Biology=== | ===Usage and Biology=== | ||
| + | <p>Compare strain H1 and H2</p> | ||
| + | https://2019.igem.org/wiki/images/1/1c/T--SEFLS_Shanghai--BBa_K1653002-fig1.jpg | ||
| + | <p>Genes idi and ispA were added to the plasmid pET-YSS, yielding pET-IAY. Co-transformation of the plasmid pET-IAY with p35151 resulted in squalene production of 69.3 mg/L (strain H2), an approximately 3.7-fold increase compared to BL21(DE3) harboring pET-YSS and p35151(Figure. 2). This result indicated that the overexpression of idi and ispA increased the supply of precursor, and thus increasing the yield of squalene. </p> | ||
| + | https://2019.igem.org/wiki/images/0/07/T--SEFLS_Shanghai--BBa_K1653002-fig2.jpg | ||
| + | <p>Figure.2 construction of strains H1, H2 & H3</p> | ||
| + | <p> Compare plasmids pETDuet-1T7-YSS-2T7-CrtN <strong>BBa_K3166998 </strong>with<strong> </strong>pETDuet-rbs-idi-rbs-ispA-rbs-YSS-2T7-CrtN <strong>BBa_K3166808</strong></p> | ||
| + | <p>By testing the pigmentation level，We discovered that the pigmentation level in the expression of YSS is higher than other strains expressing NSS KSS or thSQS, showing that YSS is relatively active, while the pigmentation level in strains containing pET-IAY-CN is 2.2 times that of strains expressing pET-YN, demonstrating that we can significantly increase the metabolic flux towards FPP by expressing idi and ispA.</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 | ||
| − | + | --> | |
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K1653003 SequenceAndFeatures</partinfo> | <partinfo>BBa_K1653003 SequenceAndFeatures</partinfo> | ||
Revision as of 22:58, 21 October 2019
ispA
ispA(from Escherichia coli JM109)
ispA encodes not only geranyl diphosphate synthase but also farnesyl diphosphate synthase.
Farnesyl diphosphate synthase can utilize both dimethylallyl and geranyl diphosphates as substrates, generating geranyl and farnesyl diphosphate, respectively. Therefore the enzyme can catalyze two sequential reactions in the polyisoprenoid biosynthetic pathway.
Usage and Biology
Compare strain H1 and H2
Genes idi and ispA were added to the plasmid pET-YSS, yielding pET-IAY. Co-transformation of the plasmid pET-IAY with p35151 resulted in squalene production of 69.3 mg/L (strain H2), an approximately 3.7-fold increase compared to BL21(DE3) harboring pET-YSS and p35151(Figure. 2). This result indicated that the overexpression of idi and ispA increased the supply of precursor, and thus increasing the yield of squalene.
Figure.2 construction of strains H1, H2 & H3
Compare plasmids pETDuet-1T7-YSS-2T7-CrtN BBa_K3166998 with pETDuet-rbs-idi-rbs-ispA-rbs-YSS-2T7-CrtN BBa_K3166808
By testing the pigmentation level，We discovered that the pigmentation level in the expression of YSS is higher than other strains expressing NSS KSS or thSQS, showing that YSS is relatively active, while the pigmentation level in strains containing pET-IAY-CN is 2.2 times that of strains expressing pET-YN, demonstrating that we can significantly increase the metabolic flux towards FPP by expressing idi and ispA.
--> Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 867
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 163
- 1000COMPATIBLE WITH RFC[1000]