Difference between revisions of "Part:BBa K4247026:Design"

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===References===
 
===References===
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Andersson, Marlene, et al. “Biomimetic Spinning of Artificial Spider Silk from a Chimeric Minispidroin.” /Nature Chemical Biology/, vol. 13, no. 3, Sept. 2017, pp. 262–264., doi:10.1038/nchembio.2269.
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Cecilia Boutry, Todd Alan Blackledge; Evolution of supercontraction in spider silk: structure–function relationship from tarantulas to orb-weavers. J Exp Biol 15 October 2010; 213 (20): 3505–3514. doi: https://doi.org/10.1242/jeb.046110
 +
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Correa-Garhwal, S.M., Clarke, T.H., Janssen, M. et al. Spidroins and Silk Fibers of Aquatic Spiders. Sci Rep 9, 13656 (2019). https://doi.org/10.1038/s41598-019-49587-y

Revision as of 18:23, 2 October 2022


Marine-minspidroin_rep


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 71
    Illegal PstI site found at 413
    Illegal PstI site found at 473
    Illegal PstI site found at 497
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 71
    Illegal PstI site found at 413
    Illegal PstI site found at 473
    Illegal PstI site found at 497
    Illegal NotI site found at 211
    Illegal NotI site found at 487
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 118
    Illegal BamHI site found at 541
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 71
    Illegal PstI site found at 413
    Illegal PstI site found at 473
    Illegal PstI site found at 497
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 71
    Illegal PstI site found at 413
    Illegal PstI site found at 473
    Illegal PstI site found at 497
    Illegal NgoMIV site found at 232
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

It is difficult to synthesise the entire DNA sequence of minispidroins due to the repetitiveness of the central motifs. So, at the UCopenhagen team, we have decided to split the protein into the N-terminus and C-terminus in an expression plasmid and the repetitive part in another cloning plasmid which is easier to produce. The DNA sequence coding for the N- and C-terminus was designed to be separated by a spacer containing two BsaI sites while the repetitive (central) part of the final Marine-minispidroin protein would have 2 BsaI sites on each end.

The DNA sequence coding for the Marine-minispidroin protein was also contained in a pET24 expression vector having a T7 promoter, terminator and KAN resistance gene. Some proteins are expressed better if they have the His-tag in the N-terminus or vice versa. Our expression vector - pET24 (+) - has a 6x His-tag in the C-terminus.

Since the type IIS assembly compatibility system forbids the presence of a BsaI recognition site within the sequence of a part, we have chosen to split the N- and C-terminus into 2 basic parts here in the Registry.

Further, this sequence was codon optimised as per E.coli's codon bias.

Source

The sequence of this part was taken from the transcriptome shotgun assembly of Desis Marina (BioProject number: PRJNA510264)

References

Andersson, Marlene, et al. “Biomimetic Spinning of Artificial Spider Silk from a Chimeric Minispidroin.” /Nature Chemical Biology/, vol. 13, no. 3, Sept. 2017, pp. 262–264., doi:10.1038/nchembio.2269.

Cecilia Boutry, Todd Alan Blackledge; Evolution of supercontraction in spider silk: structure–function relationship from tarantulas to orb-weavers. J Exp Biol 15 October 2010; 213 (20): 3505–3514. doi: https://doi.org/10.1242/jeb.046110

Correa-Garhwal, S.M., Clarke, T.H., Janssen, M. et al. Spidroins and Silk Fibers of Aquatic Spiders. Sci Rep 9, 13656 (2019). https://doi.org/10.1038/s41598-019-49587-y