Difference between revisions of "Part:BBa K4247000:Design"

Revision as of 11:45, 24 September 2022

Minispidroin_NT

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]

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.

This sequence is similar to the sequence of part BBa_K3264001 but our sequence begins with the start codon (ATG) and further, part BBa_K3264001 includes some bases in the end that code for a linker that connects the N-terminus to the middle repetitive part whereas part BBa_K4247000 doesn't have those bases.

Source

The sequence of this part was taken from E.australis MaSP1 sequence (EMBL accession number: AM259067)

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 17: / Line 17: @@
 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.
 This sequence is similar to the sequence of part BBa_K3264001 but our sequence begins with the start codon (ATG) and further, part BBa_K3264001 includes some bases in the end that code for a linker that connects the N-terminus to the middle repetitive part whereas part BBa_K4247000 doesn't have those bases.
 ===Source===