Team:UCLA/Project/Protein Expression and Processing
Protein Expression and Processing
Background
Abstract
Following genetic design of our constructs, we must express and process them into functional materials. Here, we highlight the methods that we used to take our silks from DNA to proteins and ultimately to fibers and films.
Introduction
To create functionalized fibers, we co-spun the NCSilkGFP with native Bombyx mori silk. The NCSilkGFP was designed such that the N and C termini have affinity to b.mori silk, allowing it to bind to the native silk upon spinning.
Methodology
In brief, we followed the standard process as highlighted in the literature [1] to produce a concentrated aqueous solution, or dope, of commercially purchased b.mori silk. We then added aqueous NCSilkGFP to the concentrated b.mori dope so that the final solution had 750 grams of b.mori silk to 1 gram of NCSilkGFP. We then extruded this dope into a coagulation bath of 90% v/v isopropanol and water and collected the resulting fiber on a godet. Detailed steps are as follows:
Degumming
Silk is comprised of two main proteins, fibroin and sericin. Fibroin is the structural protein of the silk and our protein of interest. Sericin serves as the ‘glue’ of the silk. Degumming separates and removes sericin and is an essential preparation step before dissolving the silk. We found that commercially degummed silk was not properly degummed for our purposes and this step had to be carried out in-lab.
To degum, we boiled 2.5 grams of B.mori silk in 0.02M sodium carbonate solution for 30 minutes.