Difference between revisions of "Part:BBa K4247004"

Revision as of 19:38, 23 September 2024

Minispidroin_NT-2rep-CT

This composite part codes for the full minispidroin protein, a highly soluble spider silk protein. This is a composite part consisting of the following basic parts: BBa_K4247000, BBa_K4247001 and BBa_K4247002. Part BBa_K4247004 contains the coding sequence for the full minispidroin protein with 2 repeats of the central repetitive domain.

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_K4247005 (Minispidroin_NT_N-6His), BBa_K4247007 (Minispidroin_NT-2rep-CT_N-6His), BBa_K4247010 (Minispidroin_NT-2rep-CT-SnoopTag_N-6His), BBa_K4247011 (Minispidroin_NT-4rep-CT), BBa_K4247012 (Minispidroin_NT-4rep-CT_N-6His), BBa_K4247013 (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 drive spontaneous fibre formation by forming 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.

Andersson et al., 2017 show how minispidroin can be spun into long fibers

Herein, part BBa_K4247004 is a composite part formed from the following basic parts: BBa_K4247000 (Minispidroin_NT), BBa_K4247001 (Minispidroin_2rep) and BBa_K4247002 (Minispidroin_CT). BBa_K4247004 contains the coding sequence for the full minispidroin protein with 2 repeats of the central repetitive domain.

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.

Optimization of temperature after induction

Aim - To determine the optimal temperature for growing the cells post-induction.

Results - The cultures were grown ON at 37°C. Then, the next day, the cultures were diluted to an OD600 of 0.1 and induced with 0.3mM IPTG and grew ON at 3 different temperatures - 20, 28 and 37°C. Similarly, uninduced controls were grown in identical conditions. The cells either have the 6x His-tag in the N-terminus or the C-terminus.

Conclusion - It can be seen that at all temperatures, the induced cells produce the proteins whereas the uninduced controls don’t and among the induced cultures, protein expression is highest when the cells are incubated at 28°C post-induction. Further, it is clear that more protein is expressed when the 6x His-tag is in N-terminus rather than the C-terminus of the protein.

Optimization of lysis buffer

Aim - To determine the best buffer for cell lysis that provides most of the protein in a soluble state.

Results - In order to lyse our cells by sonication, we used 2 different lysis buffers to decide which lysis buffer gave the most proteins in the soluble fraction. The recipes of the buffers are as follows, Buffer 1: 50 mM NaH2PO4 + 500 mM sodium chloride + 10 mM imidazole + 0.5% Triton X-100 + 10% glycerol + 2 mM DTT (added fresh, right before use), pH 8.0 Buffer 2: 20mM Tris-Cl, pH 8.0

The cell cultures were centrifuged to obtain the cell pellets which were resuspended in the cell lysis buffer and then sonicated until a clear lysate was obtained. The lysate was centrifuged to obtain the insoluble and soluble fractions in the pellet and supernatant respectively. In the SDS-gel, we can clearly see that most of the protein is in the soluble fraction for both the buffers. Further, it is also clear that we obtain more of the protein in the soluble fraction with buffer 2 which is also the buffer used in the minispidroin literature.

Further, a western blot was done on the above SDS-gel to confirm that the proteins we see are indeed the minispidroin proteins using the above mentioned antibodies. Once again, it is clear that most of the protein is obtained in the soluble fraction with buffer 2.

Conclusion - From the SDS-gel and western blot, we can conclude that buffer 2 is better than buffer 1 since it provides most of the protein in the soluble fraction. This is further backed up by the results of the minispidroin literature since the authors used buffer 2 as well.

Heat purification

Aim - To determine if there is a temperature that would precipitate only the minispidroin proteins without the other E.coli proteins, to facilitate an easy purification method using heat treatment.

Results - The soluble fraction of the lysate was subject to different temperatures (37, 40, 45, 50, 60°C). Then, after heat treatment, the samples were centrifuged and the supernatants and pellets obtained after different temperatures were run on an SDS-gel. It is clear that minispidroin-NT_2rep_CT remains soluble upto 50°C since most of it is found in the supernatant and not the pellet. At 50 and 60°C, most of the protein is found in the pellet showing that it precipitates at temperatures above 50°C. However, a lot of other proteins also precipitate at those temperatures, so it is not possible to obtain the pure protein.

To test if higher temperatures would yield puree proteins, the lysate was subject to 70 and 80°C. It is clear that most of the protein precipitates at 70 and 80°C but like before, it is not very pure since a lot of other proteins are also precipitating.

Further, a western blot was done on the above SDS-gel to confirm that the proteins we see are indeed the minispidroin proteins using the above mentioned antibodies. Once again, it is clear that most of the protein precipitates at 70 and 80°C.

Conclusion - So, although most of the protein precipitates at temperatures above 50°C, heat treatment is not a suitable method for protein purification since there is no temperature that precipitates minispidroin without also precipitating other proteins.

pH purification

Aim - To determine if there is a certain pH at which minispidroin_NT-2rep-CT precipitates without the other E.coli proteins, to facilitate an easy purification method using changes in pH.

Results - The soluble fraction of the lysate was adjusted to different pH (5.5, 6, 6.5, 7, 7.5) and incubated for 1h at room temperature. Then, the samples were centrifuged and the supernatants and pellets obtained at different pH values were run on an SDS-gel. It is clear that minispidroin_NT-2rep-CT does not precipitate at any pH since the lanes with pellets do not have any bands around 30kDa.

Conclusion - Since there is no pH value at which the protein precipitates, the method of adjusting the pH cannot be used for purification.

Optimization of media

Aim - To determine which media, LB (Luria broth) or TB (Terrific broth) is better for protein expression.

Results - The cells were inoculated in either LB or TB media and grown ON at 37°C in identical conditions. The cultures were induced with 0.3mM IPTG and allowed to express the protein ON.

Conclusion - It is clear that more of the protein is expressed when the cells are grown in LB rather than TB.

Protein purification by IMAC

Aim - To purify the protein by IMAC (immobilized metal ion chromatography) using Ni-NTA resin.

Results - Mini columns were loaded with Ni-NTA resin and the soluble fraction of the lysate was added to the columns. Then, the columns were washed twice and eluted to obtain the purified protein. An SDS-gel was run on the different purification fractions and it is clear that most of the protein was lost in the flowthrough and washes and almost nothing was eluted. This shows that the proteins did not bind to the Ni-NTA column at all.

Lysis buffer - 20mM Tris-Cl, pH 8.0

Wash buffer - 20mM Tris-Cl, 5mM Imidazole, pH 8.0

Elution buffer - 20mM Tris-Cl, 200mM Imidazole, pH 8.0

Dialysis buffer - 20mM Tris-Cl, pH 8.0

Since most of the protein was lost in the flowthrough and washes in the previous attempt, the soluble fraction containing the proteins was allowed to incubate with the Ni-NTA resin under shaking conditions for 30-60 mins. This would provide sufficient time for the resin to bind the protein. Then, the columns were washed twice and eluted to obtain the purified protein. An SDS-gel was run on the different purification fractions and it is clear that most of the protein is obtained in the eluate and none of it is lost in the flowthrough or washes. Further, the purity of the obtained protein is high since there are no other bands.

Conclusion - So, with a sufficient incubation time (30-60mins) that allows the Ni-NTA resin to bind all the proteins, it is possible to elute the proteins with very high purity.

Protein yields

Aim - To do a BCA assay after dialysis of the protein to quantify the final yield.

Results -

Minispidroin_NT-2rep-CT was inoculated in LB, grown at 37°C, induced with 0.3mM IPTG and allowed to express the protein ON in a flask. The cells were lysed, the proteins purified by IMAC and dialysed ON. Then, a BCA assay was done to estimate the protein yields.

Conclusion - The yield of Minispidroin_NT-2rep-CT was found to be 5.85 mg/L of culture.

Comparison of protein production with respect to His-tag location

Aim - To compare the protein production of the Minispidroin_NT-2rep_CT when the 6x His-tag was at the N-terminus or the C-terminus.

Results - Cells expressing 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 28°C post-induction ON. Then, the cells were lysed and the proteins were purified by IMAC. Further, the different purification fractions were run on an SDS-gel to compare the amount of proteins eluted.

Further, a BCA assay was done on the purified proteins to quantify the yields.

Conclusion - It is very evident that switching the His-tag from the C-terminus to N-terminus increased the protein production of the Minispidroin protein substantially, by nearly 8 times.

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.

Team iGEM CNPEM-Brazil 2024 literature review

• Coacervate Microdroplets: A study published in June 2023 focuses on the construction of coacervate microdroplets using recombinant spidroin NT2RepCT. This research emphasizes the ability of these microdroplets to respond to environmental stimuli, making them suitable for simulating advanced life behaviors. The morphology of these droplets can be manipulated by altering conditions such as protein concentration, pH, and temperature, which is crucial for understanding material properties and phase behavior in artificial cell systems. [1]
• Nt2RepCt hydrogel: This study from May, 2023 discusses the innovative development of hydrogels derived from recombinant spider silk proteins, which can be tailored for specific biomedical applications. The researchers demonstrate how these hydrogels can be engineered to possess adjustable mechanical and physical properties, making them suitable for controlled drug release and 3D cell culture environments. The study emphasizes the potential of these spider silk-based hydrogels to mimic the extracellular matrix, thereby enhancing cell growth and interaction. This work highlights the versatility and biocompatibility of spider silk proteins, positioning them as promising materials for future applications in tissue engineering and regenerative medicine. [2]
• Synthetic spider silk: a study published in July, 2024 addresses the challenge of replicating the exceptional mechanical properties of natural spider silk through synthetic methods. It introduces a novel approach using multiarm polyethylene glycol (PEG) to create synthetic spider silk that retains biomimetic qualities while enhancing tensile strength and extensibility. The study highlights the versatility of bioconjugation techniques in modifying protein structures, facilitating spidroin crosslinking, and chemical functionalization. By employing this method, the researchers aim to improve the toughness of the fibers without needing to develop new mini-spidroin constructs, thus potentially mitigating the adverse effects of environmental conditions on synthetic spider silk applications. [3]
• Structural variants: the study from september, 2023, investigates the role of a spider silk protein, NT2RepCT, in promoting liquid-liquid phase separation, a critical process in the formation of intracellular condensates within yeast cells (Saccharomyces cerevisiae). The study explores how different structural variants of NT2RepCT behave under varying intracellular conditions, revealing that factors such as molecular crowding and pH significantly influence the condensation properties and growth of these protein assemblies. By conducting parallel in vivo and in vitro experiments, the researchers demonstrate that yeast cells can provide a unique environment for studying protein condensation mechanisms, which could inform future designs of biomolecular materials for synthetic biology applications.