Composite

Part:BBa_K4274033

Designed by: Jiawen Chen   Group: iGEM22_KEYSTONE   (2022-09-30)
Revision as of 11:36, 12 October 2022 by Yolan (Talk | contribs) (→‎Usage and biology)

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/sup> 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

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]


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Categories
Parameters
None