Composite

Part:BBa_K4274031

Designed by: Jiawen Chen   Group: iGEM22_KEYSTONE   (2022-09-30)
Revision as of 10:47, 12 October 2022 by EmmaChen (Talk | contribs)

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

ptac-RiboJ-B0034-ClSS_S533A-B0034-ERG20_F96W-B0015 was used to complete the pathway of IPP ⇄DMAPP → GPP → FPP → alpha-santalene. This part expresses the necessary enzymes (C. lansium santalene synthase and ERG20) participating in the formation of alpha-santalene. This composite part utilizes the promoter ptac-RiboJ (BBa_K3552015), which has an optimized productivity and is IPTG-inducible to initiate the later sequence. ptac-RiboJ is followed by the ribosome binding site (BBa_B0034), derived from the Elowitz repressilator. Then, we coded in the ClSS_S533A(BBa_K4274000), which mutated its 533th amino acid from serine to alanine, reaching the optimization of its effectiveness. C. lansium santalene synthase plays a crucial role to assist the transformation from FPP (farnesyl diphosphate) to alpha-santalene (the precursor of santalol). Then, after an identical ribosome binding site as before (BBa_B0034), we inserted the sequence for ERG20_F96W, which can catalyze the formation of FPP (farnesyl diphosphate). Compared with ERG20（BBa_K849001），ERG20_F96W’s 96th amino acid being mutated from phenylalanine to tryptophan turns out to significantly improve the yield of a-santalene. This part ends with the double terminator (BBa_B0015).

We have created a parts collection for all parts optimizing the production of alpha-santalene. This collection includes the series to optimize the activity of farnesyl pyrophosphate synthase, ERG20_F96W (Part: BBa_K4274002) and ptac-RiboJ-B0034-ClSS_S533A-B0034-ERG20-B0015 (Part: BBa_K4274030). A series conducting the secretion of ClSS and ERG20, consisting of CmR29M2-ClSS_S533A (Part: BBa_K4274001) and ptac-RiboJ-B0034-CmR29M2-ClSS_S533A -B0034-ERG20_F96W-B0015 (Part: BBa_K4274032). However, the comparative results revealed that this modification does not optimize the production of alpha-santalene. Finally, the series of parts expressing fusion protein ClSS_S533A-FL-ERG20_F96W (Part: BBa_K4274033), and BBa_K4274003.

We aim to assist future teams in their journey, providing a collection that optimizes the production of santalene.


Usage and Biology

ClSS_S533A refers to the mutated and optimized C. lansium santalene synthase, an enzyme that assists the transformation from FPP (farnesyl diphosphate) to alpha-santalene. ERG20_F96W is a mutated synthase that catalyzes the formation of FPP in the pathway that produces santalene. This composite part can express both optimized enzymes for the higher production of a-santalene.

Source

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

Sequence and features

Sequence and Features


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


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Categories
Parameters
None