Difference between revisions of "Part:BBa K4247027"

Revision as of 21:39, 9 October 2022

MAKI_marine_minispidroin

This part codes for the full MAKI marine-minispidroin, a chimeric protein formed by combining the sequences of Desis marina, a marine spider's spider silk proteins and minispidroin, a highly soluble spider silk protein. This is a composite part consisting of the following basic parts: BBa_K4247000 (Minispidroin_NT), BBa_K4247026 (MAKI_marine_minispidroin_rep) and BBa_K4247002 (Minispidroin_CT).

This part is one of a collection of compatible Marine-minispidroin parts: BBa_K4247000 (Minispidroin_NT), BBa_K4247002 (Minispidroin_CT), BBa_K4247005 (Minispidroin_NT_N-6His), BBa_K4247026 (MAKI_marine_minispidroin_Rep), BBa_K4247027 (MAKI_marine_minispidroin) and BBa_K4247029 (MAKI_marine_minispidroin_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.

It has been shown that spider webs from terrestrial spiders undergo structural changes with humidity wherein high humidity causes supercontraction. Supercontraction is a phenomenon where when spider silk is exposed to water, water infiltrates the fibre and causes it to reduce in length to nearly half of it’s length when dry. Major ampullate silk proteins contain a lot of GPGXX motifs wherein G is glycine, P is proline and X can be any amino acid from a small set of amino acids. These motifs form the non-crystalline fractions of the spider silk and when the silk is in a dry state, hydrogen bonds keep these non-crystalline fractions parallel to the fiber axis whereas when the silk is wet, these hydrogen bonds are disrupted which causes a loss of orientation and drives the shrinking and thickening of the fiber.

Hence, supercontraction hinders the use of spiders for underwater applications. However, there are certain spiders in nature that can produce silk in water such as Argyroneta aquatica (freshwater) and Desis marina (marine). Desis marina spiders construct retreats with their silk for protection from tides and pressure. Further, they can trap air in their retreat and remain submerged for upto 19 days.

Since D.marina’s silk is produced under water, it would be expected that these silks would not supercontract since that is not beneficial for the spiders. Just as expected, a transcriptomics study on D.marina revealed that the silk sequences of D.marina lack the amino acid motifs associated with supercontraction.

It is well known that solubility and pH sensitivity affect the N- and C-terminus which in turn plays a huge role in spinning. So, minispidroin was designed in such a way that it combined the N-terminus and C-terminus from 2 different spiders (E.australis and A.ventricosus) to have high solubility and pH sensitivity to ensure optimal spinning. Considering that the N- and C-terminus have been optimised for spinning, we decided to design a chimeric protein by combining the sequence of D.marina’s MaSp and the minispidroin’s repetitive region (E.australis Masp). This chimeric protein would not only have good solubility and pH sensitivity for optimal spinning but also the ability to persist underwater without undergoing supercontraction.

Herein, part BBa_K247027 codes for the full chimeric protein, MAKI marine-minispidroin.

Characterization

Optimization of inducer concentration and temperature post-induction

Aim - To determine the concentration of inducer and temperature after induction 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 0.3, 0.5 and 1mM IPTG and grew ON at 20, 25, 30 and 37°C. We can clearly see that around 43kDa, 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 incubated at 25°C after induction.

Conclusion - So, it is clear that induction with 0.3mM IPTG and incubation at 25°C post-induction are the optimal conditions for protein expression.