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

Revision as of 16:16, 26 September 2022

Minispidroin_NT

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 protein would have 2 BsaI sites on each end. In this way, the repetitive sequence was added in between the N and C terminus to get a whole protein.

The DNA sequence coding for the 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.

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