Making a 4 monomer silk construct
This page describe how we intended to proceed with our project if we had been able to produce a longer silk construct.
Insertion of ICA product into E. coli.
The assembled genes from the ICA method contain the two restriction sites, EcoRI and PstI. The gene can therefore be digested and ligated into a vector, pSB1C3, with corresponding restriction sites. The ligated product can hereafter be inserted into E. coli. The cloning should be checked by restriction digest and agarose gel electrophoresis and confirmed by DNA sequencing. To prove that the protein can be correctly expressed, the size of the proteins should be checked by SDS-PAGE electrophoresis followed by Coomassie blue staining. The proteins should also be detected on Western blots using a monoclonal anti-histidine tag antibodyAlbertson, A. E., et al. (2014). "Effects of different post-spin stretching conditions on the mechanical properties of synthetic spider silk fibers" Journal of the Mechanical Behavior of Biomedical Materials 29: 225-234..
Purification of silk from E. coli.
For the extracting of the silk from the bacteria, it has been decided to use a purification method without the use of His-tag. The method is choosen, because earlier literature have demonstrated that recombinant silk produced without a His-tag have better mechanical properties compared to the fibres made from silk proteins with a His-tag Tokareva, O., et al. (2013). "Recombinant DNA production of spider silk proteins." Microbial Biotechnology 6(6): 651-663..
The first step in the purification method is to cultivate the bacteria containing the silk construct in LB broth at 37 celsius degrees until it reaches an OD600 at 0.6-0.8. Here the gene expression will be induced with IPTG for 2-4 hours Teulé, F., et al. (2013). "A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning." Nature Protocols 4(3): 341-355.. For large scale expression a fermentor can be used. The cells can be grown in minimal medium with 1% yeast extract and 100 μg/ml chloramphenicol. The pH should be kept at 6.8 by addition of ammonia water except for periods when the pH increases above 6.88 due to glucose depletion, whereby the feed solution (50 % glucose, 10 % yeast extract, 2 % MgSO4, 100 μg/ml chloramphenicol) was added. The dissolved oxygen level was sustained at 40 % by automatically increasing the agitation speed to 850 rpm and by supplying compressed air. The cells will be grown until it reaches an OD600 of 9-11, where it will be induced with 1mM IPTG. After 4 hours of incubation, the cells can be harvested by centrifugation at 3500g Dams-Kozlowska, H., et al. (2013). "Purification and cytotoxicity of tag-free bioengineered spider silk proteins." Journal of Biomedical Materials Research. Part A 101(2): 456-464..
The purification method is based on the organic spider silk solubility. First 1 g of wet pellet can be combined with 1 ml 13.3N propionic acid, and thereafter diluted to 2.3N acid with ultrapure water. The solution is then set for stirring for 1 hour at room temperature. The precipitated proteins can be clarified by centrifugation at 50,000g for 30 min. The clarified supernatant will thereafter be dialyzed extensively into 10 mM Tris pH 7.5. The aggregates formed during this dialysis can be removed by sedimentation at 125,000g for 30 min. Now the silk proteins are solubilized and they can then be applied to a strong anion exchange resin, which will be equilibrated with 10 mM Tris pH 7.5. After 1 hour agitation the resin will be transferred to the column, where after the silk proteins can be rinsed. The column should be washed with 10 mM Tris pH 7.5 to recover any remaining silk protein. The silk proteins are then ready for spinning Dams-Kozlowska, H., et al. (2013). "Purification and cytotoxicity of tag-free bioengineered spider silk proteins." Journal of Biomedical Materials Research. Part A 101(2): 456-464..
Spinning of the silk
For the creation of functionalized silk fibers, several biotechnological methods have been analyzed during the last decades. The silk fibers can be solubilized with a variety of solvents like water, organic solvents and ionic liquids. Once they are solubilized they can be processed into a range of different structures such as films, sponges, hydrogels, electrospun mats and tubes Kluge, J. A., et al. (2008). "Spider silks and their applications" Trends in Biotechnology 26(5): 244-251.. The type of structure applied, depends on the purpose of the silk. For example, is a porous feature preferred for wound healing biomaterials, since it allows the promotion of oxygen exchange, which is vital for fibroblast proliferation, collagen synthesis, and polymorphonuclear cell functions Gil, E. S., et al. (2013). "Functionalized silk biomaterials for wound healing." Advanced Healthcare Materials 2(1): 206-217.. This type of scaffold does also support cells, allow transport of nutrients and metabolic wastes, and promote tissue development Kluge, J. A., et al. (2008). "Spider silks and their applications" Trends in Biotechnology 26(5): 244-251..
In nature spiders draw the the thread with the hind legs. By copying this method in the laboratory it has been found that the spinning speed and temperature can cause differences in resilience, ductility and the diameter of the thread Römer, L., et al. (2008). "The elaborate structure of spider silk" Prion 2(4): 154-161.. The spinning conditions can therefore be varied in order to optimize the mechanical properties of the silk.
For this project the goal had been to form a thread of silk by wet-spinning, which is a very simple and inexpensive method. First the recombinant silk proteins must be completely dissolved in 100 % hexafluoro-2-propanol (HFIP) to make a 25-30 % (wt/vol) silk spinning dope. It should be noted that HFIP is a very toxic and volatile component and proper precautions should therefore be taken. The silk protein concentration should be about 30-40 mg/ml to make sure that the concentration is high enough for the polymer to form. A syringe pump will be used to extrude the silk through a needle into a 90 % isopropanol bath at a rate of 0.6-1 mm/min. The spinning apparatus to be used is a red PEEK (polyetheretherketone) tube and it has an internal diameter of 0.127 mm. After the fiber has formed it can be drawn out of the bath with either a pair of tweezers or a motorized godet Teulé, F., et al. (2013). "A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning." Nature Protocols 4(3): 341-355..
In relation to using the recombinant silk for wound healing, it could also be interesting to prepare a three dimensional scaffold of the silk. Here a number of methods, such as salt leaching, gas forming or freeze-drying, have been reported to generate porous three-dimensional matrices. Aqueous-derived silk ﬁbroin scaffolds can be prepared by adding 4 g of granular NaCl (particle size; 300–1180 mm) into 2 ml of an aqueous solution of silk (4–10 wt%) in disk-shaped containers. The container will be covered and left at room temperature for 24 hours. Hereafter the container will be immersed in cold water and then the NaCl will be extracted for 2 days. At last the porous silk scaffolds will be air-dried and then placed in a vacuum-dryer for 24 hours. The morphological and functional properties of the scaffold can be controlled by adjusting the concentration of silk ﬁbroin in water, and the particle size of the granular NaCl Nazarov, R., et al. (2004). "Porous 3-D Scaffolds from Regenerated Silk Fibroin" Biomacromolecules 5: 718-727. Ung-Jin, K., et al. (2005). "Three-dimensional aqueous-derived biomaterial scaffolds from silk fibroin" Biomaterials 26(15): 2775-2785..