Difference between revisions of "Part:BBa K4339002"

Revision as of 09:23, 12 October 2022

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

The 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

@@ Line 13: / Line 13: @@
 The sequence originates from the African nursery web spider <i>Euprosthenops australis</i> [1].
 <partinfo>BBa_K4339002 SequenceAndFeatures</partinfo>
+All MaSp parts (K4339002 - K4339010)
-All MaSp parts (K4339002 - K4339010) were designed to be integrated into devices, fused with solubility tag TrxA (<partinfo>BBa_K3619001</partinfo>) with a thrombin cleavage site between them. TrxA is a small, highly soluble protein which can act as a molecular chaperon, guiding protein folding and preventing inclusion body formation
+All MaSp parts were designed to be integrated into devices, fused with a a his tag and solubility tag TrxA (<partinfo>BBa_K3619001</partinfo>) 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 <i>E. coli</i> 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===