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Modularity of a carbon-fixing protein organelle

Modularity of a carbon-fixing protein organelle. Bonacci, Teng, Afonso, Niederholtmeyer, Grob, Silver, Savage PNAS - November 17, 2011 Shelley Ackerman & Donnie Turlington 20.385 Journal Club I. Types of Bacterial Microcompartments (BMCs). CB – CO 2 fixing carboxysomes

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Modularity of a carbon-fixing protein organelle

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  1. Modularity of a carbon-fixing protein organelle Bonacci, Teng, Afonso, Niederholtmeyer, Grob, Silver, Savage PNAS - November 17, 2011 Shelley Ackerman & Donnie Turlington 20.385 Journal Club I

  2. Types of Bacterial Microcompartments (BMCs) • CB – CO2 fixing carboxysomes • PDU – 1,2-propanediol utilization microcompartment • Ethanol-amine utilization microcompartment • BMC protein domains appear in about 20% of bacteria • Possibly a large and diverse group of BMCs we have not yet accounted for

  3. Wild Type System

  4. Their Original System • Goal: to present a genetic system for the expression of functional BMCs. • create CBs in E. Coli from H. neapolitanus • Show that synthetic CBs contain proper proteins • Modularize a CB system • 9 genes from H. neapolitanusand placed into E. Coli • Used IPTG inducible plasmid (pNS3)

  5. HnCB Expression Increases with Higher IPTG Concentrations 0 μM 50 μM High Induction 200 μM

  6. Functional Carboxysomes Assemble Correctly in vivo

  7. Carbon Fixation measured in 9-Gene System using Pentose Phosphate Pathway • Key Metabolites: • Ribulose 1,5-bisphosphate • Carbon dioxide • 3-phosphoglycerate

  8. HnCB Expression Results in Creation of Carbon Fixing Functional CBs

  9. Assessing & Redesigningthe System • Problems with 9-Gene System: • Error Rate in Assembly • Heavy IPTG Dependence • What’s Next? Bring in 10th Gene: CsoS1D • Goal: See how addition of protein affects CB function and structure

  10. pHnCBS12D Results in Smaller Error in Assembly than pHnCB Shell Defect Low-Density Lumen pHnCB Plasmid pHnCBS1D Plasmid

  11. HnCBS1D results in faster and higher levels of carbon fixation

  12. Carbon Fixation in 10-Gene System Verified in vitro • Recorded 2-phosphoglycerate dependent NADH oxidation in coupled enzyme reaction system • Created Michaelis-Menten Model • Similar KM values for pHnCB & pHnCBS1D • VMax two fold higher for pHnCB CB’s

  13. What was accomplished? • Successfully moved a CB • Proved that the CBs were still functioning • Modified and heavily improved operon • adding tenth gene, CsoS1D • This was only tested in vitro, not in vivo

  14. Significance • Modularity • standardized parts • compartmentalization • Importance of Carbon Fixation • Future applications • targeting different assembly systems • Improvements: • Optimization of the operon for specific hosts • Shell structure and permeability research • CsoS1D a minor component of shell structure • Possible Importance

  15. Future Applications • Plant adaptations • Biofuels • Molecular chassis • Nanotechnology • Future Research: • Identification of necessary genes • Improved understanding of shell formation • Development of cargo targeting system http://www.news.stanford.edu

  16. Works Cited • Talaro, Kathleen P., Foundations in Microbiology, sixth edition. 2008 McGraw-Hill Companies. • Bonacci, Walter, Teng, Pho K., Afonso, Bruno, Niederholtmeyer, Henrike, Grob, Patricia, Silver, Pamela A., Savage, David F. Modularity of a carbon-fixing protein organelle, PNAS,.Jan 10, 2012, Vol. 109, no.2.

  17. RuBisCo • Ribulose-1,5-bisphosphate carboxylaseoxygenase • Major part of the Calvin cycle • The rate of RuBisCO increases with increases in CO2 concentration (also affected by temperature, water, nitrogen availability, ect.) • Poor specificity for CO2 • Photosynthesis is single largest natural regulator of CO2 in our atmosphere, a good understanding is necessary for any climate impact studies. http://upload.wikimedia.org/wikipedia/commons/7/79/PDB_3rub_EBI.jpg

  18. Calvin-Benson-Basham cycle http://en.wikipedia.org/wiki/Calvin_cycle

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