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Barrett presentation valerie metea

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Barrett presentation valerie metea

  1. 1. Bioengineering fluorescent protein tags from cyanobacteriochromes from T. elongatus: Protein Purification Valerie Metea Summer 2011 CBST Summer Research Project PI: Dr. Susan Spiller
  2. 2. Background <ul><li>Expression system: E. coli </li></ul><ul><li>Tag from: a cyanobacteriochrome found in Thermosynechococcus elongatus </li></ul>
  3. 3. Five Distinct Genes of Interest Rockwell, Nathan. Biochemistry  (2008)
  4. 4. Overview: Start to Finish RE & Ligation PCR DNA SDS-PAGE Gel Protein Purification Protein Expression Transformation Site-Directed Mutagenesis Transfection Credit: Monica Bower
  5. 5. Methods: Protein Purification
  6. 6. Protein Purification: Microfluidizer <ul><li>Defrost pellets and resuspend in cell lysis buffer (in dark room) </li></ul><ul><li>Run machine and collect lysate </li></ul><ul><li>Centrifuge to remove waste </li></ul>Microfluidex LV1 Microfluidizer http://www.microfluidicscorp.com
  7. 7. Purification Column: Chitin Beads Credit to Spiller Lab for image
  8. 8. Purification Column
  9. 9. Results Obtained: Spectrophotometer
  10. 10. Results Obtained: Spectrophotometer
  11. 11. SDS-PAGE <ul><li>Separating protein bands using an electric current to determine protein purity </li></ul><ul><ul><li>Compare band lengths to ladder </li></ul></ul><ul><ul><li>Longer travel distance means a smaller protein </li></ul></ul>
  12. 12. SDS-PAGE http://upload.wikimedia.org/wikipedia/commons/7/75/SDS-PAGE_Acrylamide_gel.png - Modified Credit to Wikipedia Commons for image
  13. 13. SDS-PAGE Glass gel cassettes Electrode assembly Mini Tank Inner chamber Outer Chamber Protein Sample Lid Power Source Blue = Negative Red = Positive http://upload.wikimedia.org/wikipedia/commons/4/46/SDS-PAGE_Electrophoresis.png - Modified Credit to Wikipedia Commons for image
  14. 14. Results Obtained: SDS-PAGE Gel Credit to Spiller Lab for images
  15. 15. Future Directions <ul><li>Once we bioengineer the small, red fluorescent tag (SmuRF): </li></ul><ul><ul><li>Use tag to visualize lymphocyte cytoskeleton </li></ul></ul><ul><ul><li>Observe lymphocyte interactions with GFP labeled HIV to observe infection process </li></ul></ul>
  16. 16. Acknowledgements <ul><li>Dr. Susan Spiller </li></ul><ul><li>Rosa Meza-Acevedo, Kristen Beck, Monica Bower, Alex Magallan, Stefanie Yoshizuka, Mo Kaze, Ling Ou </li></ul><ul><li>Dr. Nathan Rockwell and Dr.Clark Lagarias at UC Davis </li></ul><ul><li>Dr. Ana Corbacho and Dr. Marco Molinaro of CBST </li></ul>

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