Difference between revisions of "Part:BBa K4247011:Design"

Revision as of 15:21, 26 September 2022

Minispidroin_NT-4rep-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
Illegal PstI site found at 721
Illegal PstI site found at 727
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
Illegal PstI site found at 721
Illegal PstI site found at 727
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
Illegal PstI site found at 721
Illegal PstI site found at 727
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
Illegal PstI site found at 721
Illegal PstI site found at 727
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 two repetitive parts would be inserted in between the N- and C-terminus to give the whole minispidroin protein. We decided to produce a protein similar to Minispidroin_NT-2rep-CT (composite part BBa_K247007) because higher repeats in spider silks were shown to give better fibre performance. The DNA sequence coding for the minispidroin protein was then placed 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 chosen to split the N- and C-terminus into 2 basic parts.

Further, this sequence has been codon optimised 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), 2x BBa_K4247001 (Minispidroin_2rep) and BBa_K4247002 (Minispidroin_CT).

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

@@ Line 14: / Line 14: @@
 Further, this sequence has been codon optimised 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===