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Bioengineering fluorescent protein tags from cyanobacteriochromes from T.elongatus: Protein Expression<br />CBST RESEARCH ...
T. elongatus BP-1<br /><ul><li>Origin: Japan in Beppu Hot Springs (BP-1)
Cyanobacterium
Group of photosynthetic bacteria
Prokaryotic
Chlorophyll a + phycobilin pigments
T. elongatusin particular
Genes make cyanobacteriochromes
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Monica's Barrett Presentation

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Monica's Barrett Presentation

  1. 1. Bioengineering fluorescent protein tags from cyanobacteriochromes from T.elongatus: Protein Expression<br />CBST RESEARCH PROJECT<br />SUMMER 2011<br />PRESENTED BY Monica bower<br />WITH SUPPORT FROM <br />DR. SUSAN C. SPILLER, <br />And members of the spiller lab<br />
  2. 2. T. elongatus BP-1<br /><ul><li>Origin: Japan in Beppu Hot Springs (BP-1)
  3. 3. Cyanobacterium
  4. 4. Group of photosynthetic bacteria
  5. 5. Prokaryotic
  6. 6. Chlorophyll a + phycobilin pigments
  7. 7. T. elongatusin particular
  8. 8. Genes make cyanobacteriochromes
  9. 9. Thermophilic
  10. 10. Photoreversibility
  11. 11. Cyanobacteriochromes – blue/green
  12. 12. Engineered cyanobacteriochromes – red fluorescent</li></li></ul><li>Imagine the recipient cells showing their actin or tubulincyto-skeletons in bright red!<br />
  13. 13. T. elongatus Genes<br />MINE!<br />
  14. 14. Two Plasmid System<br />pPL plasmid<br />Contains genetic information to make PCB<br />Hemoxygenase (Ho1)<br />Reductase (PcyA)<br />
  15. 15. LMG194 – PCB E. coli <br />(outer layer)<br />pPL plasmid<br />pBAD plasmid<br />KanR<br />Proteins<br />AmpR<br />Plates with colonies and both Ampicillin and Kanamycin<br />
  16. 16. 1 2 3 4<br />Photos: Tami Port, MS<br />Proteins<br />2 mL culture = RM broth (has glucose to turn off lac-operon) + billions of bacteria w/ plasmids <br />500 mL = LB broth + IPTG (turns on pPL plasmid) – after 1 hour: add L-ara (turns on pBAD plasmid)<br />50 mL = RM broth + ampicillin + kanamycin + bacteria<br />
  17. 17. Protein Expression<br />Blue?!<br />Can’t be 911T.<br />Yellow! Could be 911T…<br />Proteins<br />911: Photoreverses to appear yellow<br />Proteins<br />

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