Composite

Part:BBa_K4274033

Designed by: Jiawen Chen   Group: iGEM22_KEYSTONE   (2022-09-30)
Revision as of 11:38, 12 October 2022 by Yolan (Talk | contribs) (→‎Source)

ptac-RiboJ-B0034-ClSS_S533A-FL-ERG20_F96W-B0015

BBa_K3552015, BBa_B0034, BBa_K4274003,BBa_B0015

Usage and Biology

This composite part is designed for the pathway IPP ⇄DMAPP → GPP → FPP → alpha-santalene. It is used as a trial to express enzymes ClSS and ERG20 to its maximized extent, hence reaching the improvement of alpha-santalene production. It starts with the promoter ptac-RiboJ (Part: BBa_K3552015), following the ribosome binding site B0034 (Part: B0034). Then, we added the coding sequence for ClSS_S533A, followed by a flexible linker, and then ERG20_F96W. The part ends with the terminator B0015 (Part: BBa_B0015).
This part falls under our collection that produces alpha-santalene. We also have two parts that optimizes the activity of FPP (farnesyl diphosphate), consisting of ERG20_F96W (Part: BBa_K4274002), and ptac-RiboJ-B0034-ClSS_S533A-B0034-ERG20-B0015 (Part: BBa_K4274030). Parts secreting ClSS and ERG20, CmR29M2_ClSS_S533A (PART: BBa_K4274001) and ptac-RiboJ-B0034-CmR29M2_ClSS_S533A-B0034-ERG20_F96W-B0015 (Part: BBa_K4274032). Finally, this part, exploring the effects of fusion protein on secretion and santalene production (Part: BBa_K4274033).
This part collection aims to optimize the production of alpha-santalene, the precursor of santalol.

Usage and biology

ClSS_S533A (C. lansium santalene synthase) is the enzyme that catalyzes the transformation from FPP (farnesyl diphosphate) to alpha-santalene. Its 533rd amino acid has been artificially mutated from serine to alanine for the sake of optimization. ERG20_F96W is the enzyme that catalyzes the formation of FPP (farnesyl diphosphate). The 86th amino acid was mutated from phenylalanine to tryptophan. FL refers to the flexible linker that we have added in between the two proteins. It was hoped that adding a flexible linker can maximize alpha-santalene production. However, our results demonstrate the opposite, that alpha-santalene production decreased after adding a flexible linker.

Source

ClSS_S533A (Part: BBa_K4274000) is from Clausena lansium, ERG20_F96W (Part: BBa_K849001) is from S. cerevisiae, and CmR29 is from Synechocystis.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3943
    Illegal BglII site found at 4004
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1541
    Illegal AgeI site found at 3659
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None