Mississippi State/Project Description
- The search for renewable energy sources from alternative crops has led to the desire to understand the pathway by which lipids are synthesized. Higher lipid content has a direct correlation to higher energy value, so if the plant pathways involved in lipid metabolism can be modulated, biofuels could become more economically feasible. It has been reported that the ubiquitin proteasome pathway affects lipid metabolic processes. The ubiqination of target proteins for degradation requires the sequential activity of three enzymes: a ubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme (E2), and a ubiquitin ligase (E3). The objective of the 2007 MSU iGEM project is to develop a rapid and sensitive method for assaying the ubiquitin ligase (E3) activity.
- The E3 activity is generally determined by using an in vitro ubiquitination assay. The E3 is first incubated with ATP, ubiquitin, and wheat germ lysate containing E1 and E2 activities (or purified E1 and E2). The ligase reactions are then fractionated by electrophoresis on an SDS-PAGE gel and subjected to Western blotting for the detection of high molecular weight polyubiquitinated E3 proteins. This detection method is costly and time consuming.
- To develop a quicker assay of E3 activity, the E3 coding region is cloned into a plasmid and expressed as an in-frame C- (or N-) terminal fusion with a GFP protein. The E3 activity is then assayed by directly irradiating the protein gel with long wave ultraviolet light. The expression of green fluorescence in multiple, high molecular weight protein bands would indicate the ligase activity. To confirm that the multiple protein bands are ubiquinated, a plasmid containing RFP in frame with ubiquitin will also be constructed and transformed into E. coli cells harboring the E3-GFP fusion.
- Future work will involve in designing universal plasmids to provide for a quicker assay of any E3 ligases.