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.