Difference between revisions of "Part:BBa K4247029:Design"

Revision as of 17:45, 9 October 2022

MAKI_marine_minispidroin_N-6His

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 Marine-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. For this part, the N terminus 6His-tag was much better for production.

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.

Since for some proteins, the expression is better with the His-tag on the N-terminus instead of the C-terminus we used primers to switch the 6x His-tag. This part codes for a 6x His-tag followed by the N-terminal domain of minispidroin and when combined with BBa_K4247026 and BBa_K4247002, we get the full sequence of the MAKI marine minispidroin protein with the 6x His-tag in the N-terminus.

Source

The sequence of this composite part is obtained from the following basic parts: BBa_K4247005 (Minispidroin_NT_N-6His), BBa_K4247026 (Marine-minispidroin_rep) and BBa_K4247002 (Minispidroin_CT).

References