Purification of the CusS protein into liquid nanodisks
Dr. Misty Thomas
Background: Silver is an antimicrobial agent that helps reduce the rate of bacteria growth. Silver kills bacteria by interacting with the cell wall and membrane, as well as disrupting DNA replication, transcription, and translation. Previous work using experimental evolution found four mutations leading to silver resistance in the cusS gene.The purpose of this is to determine if any of the mutations in the cusS gene will change the function of the CusS protein and to determine if these changes will indeed make the bacteria resistant to silver.
Question: How do mutations in the CusS gene change the function of the CusS protein? How do these changes make the bacteria resistant to silver?
Methods: The wild-type and mutant cusS genes were previous cloned into a protein expression vector. These samples were used to grow overnight cultures and then subsequently used to inoculate 1L cultures and grown to an optical density of 0.5 at 600nm. Once this was reached, cultures were induced with 1mM IPTG overnight at room temperature. Samples were then run on an SDS-PAGE gels to test for expression of the wild type protein.
Conclusions: The wild type protein was successfully purified into the insoluble fraction of the cell lysate.
Future Directions: After purifying the wild type CusS protein, the CusS protein would be isolated from the membrane to study it biochemically by inserting the CusS protein into a liquid nanodisk. Lipid nano disk keeps the membrane proteins in their natural form to be studied. This will be completed again using the mutant type CusS protein to compare the function from the wild type CusS protein.
Broader Impact: Two-component response systems have been proposed to be a potential target for drug development and therefore this work will help us to better preventively understand the mechanisms of silver resistance before it is widespread in nature and possibly have the opportunity to keep it under control.
Dickens, Jourdan, "Purification of the CusS protein into liquid nanodisks" (2018). Undergraduate Research and Creative Inquiry Symposia. 2.