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): 651-63..

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 OD_600 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): 341-55.. 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 OD_600 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): 456-64..

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): 456-64..