Coding

Part:BBa_K4247004:Design

Designed by: Akila Ravikumar   Group: iGEM22_UCopenhagen   (2022-09-23)
Revision as of 12:30, 23 September 2022 by Akila (Talk | contribs)


Minispidroin_NT-2rep-CT


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 469
    Illegal PstI site found at 475
    Illegal PstI site found at 490
    Illegal PstI site found at 544
    Illegal PstI site found at 562
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 469
    Illegal PstI site found at 475
    Illegal PstI site found at 490
    Illegal PstI site found at 544
    Illegal PstI site found at 562
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 469
    Illegal PstI site found at 475
    Illegal PstI site found at 490
    Illegal PstI site found at 544
    Illegal PstI site found at 562
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 469
    Illegal PstI site found at 475
    Illegal PstI site found at 490
    Illegal PstI site found at 544
    Illegal PstI site found at 562
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The DNA sequence coding for the N- and C-terminus would be separated by a spacer containing a BsaI site and the repetitive part of the protein would have 2 BsaI sites on each end such that when Golden Gate Cloning was performed, the repetitive part would be inserted in between the N- and C-terminus to give the whole minispidroin protein. The DNA sequence coding for the minispidroin protein will be contained in a pET24 expression vector containing a T7 promoter, terminator and a 6x His-tag following the C-terminus of the protein to facilitate protein purification.


However, since the type IIS assembly compatibility system forbids the presence of a BsaI recognition site within the sequence of a part, we have chose to split the N- and C-terminus into 2 basic parts.


Further, this sequence has been codon optimized as per E.coli's codon bias.


It is difficult to synthesise the DNA sequence coding for the central part of the minispidroin due to its repetitiveness. So, at the UCopenhagen team, we have decided to split the protein into the N-terminus and C-terminus in one and the repetitive part in another plasmid. Using Golden Gate Assembly, the repetitive part can be inserted in between the N and C terminus to get the coding sequence of the entire minispidroin protein. In this way, any sequence can be added in between the N and C terminus to get a whole protein.

Source

The sequence of this composite part is obtained from the following basic parts: BBa_K4247000 (Minispidroin_NT), BBa_K4247001 (Minispidroin_CT) and BBa_K4247002 (Minispidroin_2rep).

References

Andersson, M., Jia, Q., Abella, A. et al. Biomimetic spinning of artificial spider silk from a chimeric minispidroin. Nat Chem Biol 13, 262–264 (2017). https://doi.org/10.1038/nchembio.2269

Strickland, M., Tudorica, V., Řezáč, M. et al. Conservation of a pH-sensitive structure in the C-terminal region of spider silk extends across the entire silk gene family. Heredity 120, 574–580 (2018). https://doi.org/10.1038/s41437-018-0050-9