1. Fluorescent Assay of a RING-type Ubiquitin Ligase Mississippi State University November 2007

2. Our Team

3. Our Team - Students Agricultural and Biological Engineering – Graduate Robert Morris – Undergraduate Lauren Beatty, Scott Tran, Joe Chen, Caleb Dulaney, Sam Pote, Karen Parks Biochemistry – Graduate Victor Ho – Undergraduate James Kastrantas

4. Our Team - Professors Agricultural and Biological Engineering – Dr. Filip To Biochemistry – Dr. Din-Pow Ma Electrical and Computer Engineering – Dr. Bob Reese Chemical Engineering – Dr. Todd French

5. Present Areas of Interest Departmental Goal – Discover lipid production pathways – Increase lipid synthesis in plants – Make biofuels more economically feasible iGEM 2007 Goal – Design a construction that simplifies and expedites the confirmation of ubiquitin ligase activity

6. Ubiquitin Proteasome Pathway The ubiquitination of target proteins for degradation requires the sequential activity of three enzymes: – E1, ubiquitin activating enzyme – E2, ubiquitin conjugating enzyme – E3, ubiquitin ligase

7. Ubiquitin Proteasome Pathway

8. Methodology A RING-type E3 gene, GhR1(1kb), was amplified by PCR and cloned into pGFPuv as an in-frame fusion at N-terminal with GFPuv. Chemically competent (CaCl 2 ) E. coli XL1- blue cells were transformed with recombinant plasmid pGFP/GhR1. Transformed XL1-blue cells were grown to OD 600 ~0.6 and lysed by sonnication.

9. Methodology Cell lysate was added to ubiquitin and wheat germ extract, which provides reagents (E1, E2, ATP) for ubiquitination. Reaction mixtures were analyzed by native PAGE. Excitation of the fusions revealed the presence of E3 and ubiquitination on the gel.

10. Project Justification Benefits: – Assays E3 ligase activity directly on native protein gel – Omits primary purification step, Western blot, and pull down assay – Saves time and resources

11. Desired Machine Function Rapidly report ubiquitin ligase activity via green fluorescence

12. Initial Idea for Plasmid Construction

15. Problems Encountered Upon inspection of our construction plans involving the insertion of ubiquitin next to the Registry part J01095, the S/X restriction site created with the ligation of these two parts would code for the stop codon UAG.

16. First Plasmid Construction

17. Confirmation of Constructions All constructions were sequenced using ABI 310 Prism Genetic Analyzer. All sequences were confirmed to be correct and in frame with the GFP gene.

18. Results of First Construction Lane ID 1 Protein Ladder 2 GhR1/GFP 3 GFP 4 GhR1/GFP/Ub 5 GFP/Ub 1 2 3 4 5 Ub – Ubiquitin

19. Analysis of First Construction The GhR1-GFP fusion protein in lanes 2 and 4 had fluorescence. No multiple protein bands were detected in lane 4 after Western blotting with anti-ubiquitin, suggesting that ubiquitination did not occur.

20. Second Plasmid Construction

21. Results of Second Construction Lane ID 1 Protein Ladder 2 Sh-GhR1/GFP 3 Fl- GhR1/GFP 4&5 GFP 1 2 3 4 5 Sh – shortFl – full

22. Analysis of Second Construction GFP without the fusion in lanes 4 and 5 showed fluorescence. There was no visible fluorescence in lanes 2 and 3, suggesting that the expression level of GhR1-GFP fusion was either too low or the fusion protein changed conformation with no fluorescence.

23. Conclusions The first construction was successful with the observation of fluorescence from the GhR1- GFP fusion protein. The first construction failed to detect poly- ubiquitin chains. GFP adjacent to the RING domain (C- terminal) of GhR1 might block ubiquitination.

24. Conclusions There was no expression of green fluorescence in the second construction. Expression of the GhR1-GFP fusion was not observed in either the short or full length form of GhR1. This could be the result of an alteration of protein structure from the fusion of GhR1 with GFP.

25. Future Work Fusion of RFP with GhR1 One single plasmid for both GFP-E3 and RFP-ubiquitin fusions Discovery of other protein-protein interactions – Interactions controlling lipid synthesis

26. Future Work Understanding of the regulation of lipid production will enable the design of new machines with predetermined lipid content – Higher lipid content in plant  Higher energy value of biofuel – Lower undesired lipid levels  Higher product value May lead to understanding of other regulatory pathways (polysaccharides)

27. Acknowledgements Advisors – Dr. Filip To, Dr. Din-Pow Ma MSU Bagley College of Engineering MSU College of Agriculture and Life Sciences USDA Strategic Research Initiative iGEM Staff

28. Questions?