Coding

Part:BBa_K4339002

Designed by: Chloe Singleton   Group: iGEM22_Exeter   (2022-10-08)
Revision as of 09:43, 12 October 2022 by Matthewludwig (Talk | contribs) (→‎Usage and Biology)


MaSp1_K3264003mod_CT E. australis

A major ampullate spidroin 1 (MaSp1) based protein consisting of the Great Bay 2019 part BBa_K3264003 fused to a C-terminal domain from the African nursery web spider Euprosthenops australis. Due to the highly repetitive nature of spidroin proteins, the DNA coding sequences are also highly repetitive. Unfortunately we were unable to submit the original Great Bay sequence for synthesis, and therefore K3264003 had to be modified to allow for synthesis.


Usage and Biology

Sequence and Features K3264002mod original sequence originates from the common orb-weaving spider Araneus ventricosus [2]. This sequence is composed of 2 repeated poly-alanine and poly-glycine regions. CT domain sequence originates from the African nursery web spider Euprosthenops australis [1].


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

All MaSp parts (K4339002 - K4339010)

All MaSp parts were designed to be integrated into devices, fused with a a his tag and solubility tag TrxA (BBa_K3619001) with a thrombin cleavage site between them. TrxA is a small, highly soluble protein which, when fused with a protein of interest, can act as a molecular chaperon, guiding protein folding and preventing inclusion body formation [2], as well as increasing the solubility of the fusion partner. As per the methodology set out in [1], TrxA-fused MaSp proteins were designed to be soluble in the cytosol of E. coli when expressed, so could be extracted in the soluble portion cell lysate. MaSps would then be purified from the lysate using a Nickel-NTA column (which binds his-tagged proteins). Thrombin would then be added to the elution from the column to cleave off the TrxA tag, causing MaSp proteins to precipitate out of solution and spontaneously self-assemble into fibres.

References

[1] - Stark M et al. Macroscopic fibers self-assembled from recombinant miniature spider silk proteins. 2007;8(5): 1695–1701. doi: https://doi.org/10.1021/bm070049y.

[2] - LaVallie E et al. Thioredoxin as a fusion partner for production of soluble recombinant proteins in Escherichia coli. Methods in Enzymology. 2000;326: 322-340. doi: https://doi.org/10.1016/S0076-6879(00)26063-1

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