Title:

Purification of EcoRI-RTX Through Calcium Precipitation

Double-stranded DNA (dsDNA) is a basic polymer that is often overlooked in biomaterial synthesis. It comes in a wide variety of sizes while retaining water solubility, making it an attractive material for water-based biomaterials such as hydrogels. dsDNA-based hydrogels are one of the main product goals of the Oldenhuis lab. Another feature that makes dsDNA an attractive polymer is its ability to be altered enzymatically. In order to do that in our lab, we will need a library of enzymes that will allow us to reliably, predictably, and selectively manipulate the topological state of DNA. For this project, we will be working to purify multiple enzymes, starting with the restriction enzyme EcoRI-RTX. The RTX tag appended to the enzyme allows the protein to be purified via calcium precipitation, making it a good starting point for this work. Sonication was used as a method of mechanical lysis but has not yet yielded soluble protein, preventing purification of any EcoRI-RTX. Cell lysis and purification methods will need to be optimized to produce functional enzyme. Following the development of an optimized EcoRI-RTX purification protocol, we will work to purify enzymes with more rigorous purification protocols such as topoisomerase and condensin.

Name

Comment*

Comments are viewable only by submitter

Captcha*