Part:BBa_K4247027:Design

MAKI - marine minispidroin

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 469
  Illegal PstI site found at 811
  Illegal PstI site found at 871
  Illegal PstI site found at 895
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 469
  Illegal PstI site found at 811
  Illegal PstI site found at 871
  Illegal PstI site found at 895
  Illegal NotI site found at 609
  Illegal NotI site found at 885
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 516
  Illegal BamHI site found at 939
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 469
  Illegal PstI site found at 811
  Illegal PstI site found at 871
  Illegal PstI site found at 895
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 469
  Illegal PstI site found at 811
  Illegal PstI site found at 871
  Illegal PstI site found at 895
  Illegal NgoMIV site found at 630
  
• 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 MAKI-marine-minispidroin protein was obtained by finding in the Desis marina transcriptome (BioProject number: PRJNA510264) protein sequences similar to the original minispidroins. The protein was then aligned with the protein sequences of a number of terrestrial spiders, to decide which parts were to be included. The sequence aligned well and the differences were kept in case these were significant for resistance to marine water. The N and C terminus of minispidroin was then placed at the extremities of the repetitive region (poly A and glycine-rich motifs).

This protein was then synthesised with E.coli codon optimisation 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.

Source

The sequence of this composite part is obtained from the following basic parts: BBa_K4247000 (Minispidroin_NT), BBa_K4247026 (MAKI-marine-minispidroin_rep) and BBa_K4247002 (Minispidroin_CT).

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