Difference between revisions of "Part:BBa K4247027"

Latest revision as of 16:35, 13 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.

Aim - To show the expression of the MAKI marine-minispidroin protein with a His-tag in the C-terminus or in the N-terminus.

Results - Cells expressing MAKI marine-minispidroin proteins with a His-tag in the N-terminus or C-terminus were grown to an OD600 of 0.1 and induced with 0.3mM IPTG to induce protein expression. Then, the cells were grown at 25°C post-induction ON. Further, different amounts of the cell lysate were loaded on the SDS-gel to differentiate the amount of proteins.

In the lanes of the protein with the His-tag in the C-terminus, we can clearly see a band only in the induced lane at the expected molecular weight. However, this band is absent in the lanes of the protein with the His-tag in the N-terminus.

Conclusion - Hence, it is evident that when the His-tag is in the C-terminus, the protein is expressed but when the His-tag was switched to the N-terminus, the protein was no longer expressed. To overcome this, we designed new User primers such that a PCR and User cloning would remove few additional base pairs we found between the RBS and the start codon of N-His marine. However, owing to the time constraints of the competition, we were unable to perform this cloning.

Simulating the assembly mechanism silk fiber networks

We simulated the formation of a spider silk fiber network during spinning and stretching of the silk fiber using dissipative particle dynamics. This was done on a different number of the characteristic hydrophobic alanine-repeat motif. The data from these simulations was then analysed by identifying clusters of the hydrophobic parts of the repeats and generalising them into nodes (red dots), and links between nodes was then indicated in bridges (black lines), where their thickness indicates the number of connections.

The simulation results indicate that spider silk proteins with 3 and 4 repeats seem to form the most stable fiber networks with large average beta sheet crystals and many connecting bridges, although our simulations were limited because we did not get a chance to simulate proteins with a larger number of repeats than 6.

Fig: Graph depicting the development of average beta-sheet crystals size and number of bridges for networks of spider silk proteins with different number of repeats. The average beta-sheet crystals size is measure in the number of hydrophibic beads that are included in beta-sheets, where each bead represent 3 hydrophobic amino acid residues.

Additionally we simulated spinning under the conditions of failed spinning attempts to try and troubleshoot and fix our mistakes for future attempts. We observed that the failed spinning case that we examined with a protein concentration of 15% had a lowered size of its average beta sheet crystals as well as a low connectivity. Through an additional simulation, with raised shear rate, which represents a higher shear force in spinning, accomplished by a narrower needle, it was observed that a raised shear rate could counteract the lowered average size of beta sheet crystals, but not the network connectivity.

Fig: Graph depicting the development of average beta-sheet crystals size and number of bridges for networks of spider silk proteins with different number of repeats, at different concentrations of spinning, and with different shear rates, which indicate how much shear force is applied to the proteins during spinning (lack of specific shear rate indicates a shear rate of 0.1). The average beta-sheet crystals size is measure in the number of hydrophibic beads that are included in beta-sheets, where each bead represent 3 hydrophobic amino acid residues.