Difference between revisions of "Part:BBa K4247000"

Revision as of 16:10, 27 September 2022

Minispidroin_NT

This part codes for the N-terminal domain of minispidroin, a highly soluble spider silk protein. This part, together with BBa_K4247001 and BBa_K4247002 gives the full sequence of the minispidroin protein.

This part is one of a collection of compatible minispidroin parts: BBa_K4247000 (Minispidroin_NT), BBa_K4247001 (Minispidroin_2rep), BBa_K4247002 (Minispidroin_CT), BBa_K4247004 (Minispidroin_NT-2rep-CT), BBa_K247005 (Minispidroin_NT_N-6His), BBa_K247007 (Minispidroin_NT-2rep-CT_N-6His), BBa_K247010 (Minispidroin_NT-2rep-CT-SnoopTag_N-6His), BBa_K247011 (Minispidroin_NT-4rep-CT), BBa_K247012 (Minispidroin_NT-4rep-CT_N-6His), BBa_K247013 (Minispidroin_NT-4rep-CT-SnoopTag_N-6His).

Usage and Biology

Dragline silk produced by spiders is one of the strongest natural materials to exist and it is mainly made up of structural proteins called spidroins. These spidroins consist of non-repetitive N-terminal and C-terminal domains and a repetitive central part consisting of tandem repeats of a certain amino acid sequence. These sequences are rich in alanine and glycine to form the crystalline and amorphous parts of the fibre respectively.

There are many research articles whose authors could successfully produce recombinant spider silk proteins and spin them into fibres by mimicking the conditions of the spider’s silk gland where the fibers are formed naturally. But a major drawback in many of these recombinant spidroins was their low solubility. It has been found that the N-terminus of the spidroin is highly soluble at neutral pH which contributes to the solubility of the protein.

In the spider's silk gland, before spinning, the spidroins remain in a highly concentrated and soluble state. Then, this highly concentrated spidroin solution called spinning dope is subject to a gradual drop in pH from 7.6 to 5.7 along the gland which triggers the formation of the fiber. This drop in pH triggers the N-terminus to be more stable and form large network-like structures whereas the C-terminus becomes more unstable to form the beta-sheet fibrils which form the core of the fiber. The N-terminal domain restricts the formation of silk fibers to a precise point in the silk duct, preventing silk proteins stored in the silk gland from agglutinating.

This clearly shows us that the solubility and pH sensitivity have a huge effect on the N- and C-terminus of the spidroin which thus affects the formation of fibers. It has been found that the N-terminus of MaSp1 (Major ampullate spidroin 1) from Euprosthenops australis, shows extremely high solubility and pH sensitivity whereas the C-terminus has low solubility and is inert to pH changes and vice versa for the MiSp (Minor ampullate spidroin) of Araneus ventricosus.

So, minispidroin, a recombinant protein was produced by combining the N-terminal of E.australis Masp1, C-terminal of A.ventricosus Misp and the repetitive part of E.australis Masp. This chimeric protein has been designed for optimal spinning since it has high solubility and high sensitivity to pH changes which are 2 factors that are crucial for spinning.

Herein, part BBa_K4247000 contains the coding sequence for the N-terminus of the minispidroin protein.

Characterization

Optimization of inducer concentration

Aim - To determine the concentration of inducer required for optimal protein expression.

Results - Cell cultures were grown ON at 37°C. Then, the next day, the cultures were diluted to an OD600 of 0.1 and induced with 0.1, 0.3, 0.5 and 1mM IPTG and grew ON. We can clearly see that around 30kDa, there is a darker band in the induced lanes compared to the uninduced lane, showing that the protein is expressed upon induction with IPTG. Further, among the induced lanes, protein expression is maximum when the cultures were induced with 0.3mM IPTG.

Further, a western blot was done on the above SDS-gel to confirm that the proteins we see are indeed the minispidroin proteins. Since the proteins were expressed with a 6x His-tag, we used mouse anti-hexa his primary antibodies and goat anti-mouse HRP-conjugated secondary antibodies for the western blot. Once again, it is clear that 0.3mM IPTG is the optimal inducer concentration.

Conclusion - So, it is clear that 0.3mM IPTG is the optimal concentration for protein expression. This is further backed up by the results of the minispidroin literature since the authors found 0.3mM IPTG to the optimal concentration too.