This research was conducted within the Miami University Department of Chemistry and Biochemistry, and specifically looked at using common biochemical techniques to modify a protein for ease of study. Proteins are the workhorses of all cells. In order to do their job, proteins need to reach a specific site of activity within the cell, and other proteins are required to help them get there. One such protein transporter is the Tat system of plant thylakoids. Tat is made up of three proteins – Tha4, Hcf106, and cpTatC. The exact mechanism by which Tat transports other proteins remains a subject of considerable study. The Dabney-Smith group within the Department of Chemistry has a DNA construct that codes for a fused version of cpTatC-Hcf106; this could be very useful in future Tat research. However, this constuct contains problematic DNA which makes working with the protein difficult. Polymerase chain reaction (PCR) is used here to mutate and amplify the DNA to eliminate those problematic points. After PCR is performed, the sample is digested with an enzyme in order to cut up interfering DNA and then the sample is analyzed with agarose gel electrophoresis. This allows for the presence and size of DNA in the sample to be determined. Once PCR is confirmed to have worked, the DNA is taken up by an E. coli colony, which further amplifies the DNA. The DNA is then isolated from the bacteria and the process is repeated for the next mutation. Currently, two out of the four desired mutations are completed and the third is in progress. Once all four are completed and verified by an independent laboratory, work can begin on overexpressing the protein (mass-producing it in bacteria) and verifying that it functions like the natural versions before using it in mechanistic studies.
Author: Andrew Abata
Advisor: Carole Dabney-Smith, Chemistry and Biochemistry
Graduate Advisor: Jorge Escobar, Biology
You must be logged in to post a comment.