BACTOBLOOD

1. BACTOBLOOD Creating a Red Blood Cell Substitute

2. Artificial Blood Substitutes The Need Kristin’s making this slide tonightKristin’s making this slide tonight

3. Human Practice IP Considerations Kristin’s making this slide tonightKristin’s making this slide tonight

4. Human Practices IP Considerations

5. Human Practices IP Considerations

6. The Chassis

7. Expression of Human Hemoglobin

8. System Components In addition to constructing hemoglobin expression devices, we also constructed various devices that aid in increasing expression levels and maintaining functional hemoglobin. We made following devices in an attempt to “steal” the solutions that natural red blood use to address the problems that come with using hemoglobin as an oxygen carrier. Since we need to make loads of hemoglobin, we need loads of heme. Therefore we relieved the major bottlenecks in E. Coli’s natural heme biosynthesis pathway by supplementing those genes in our system. To prevent improper folding and to increase the yield of functional hemoglobin, we’ve included the natural chaperone, alpha hemoglobin stabilizing protein. To remove clear out damaging free radicals that come about during oxygen binding and unbinding, we’ve added the major antioxidant enzymes that are normally present in red blood cells. And to restore non-functional methemoglobin back to a functional state, we’ve imported human cytochrome b5 and cytochrome b5 reductase, which should do the trick. Just as these problems are solved in red blood cells, these problems would be solved in analogous ways for our bacto blood cell. In addition to constructing hemoglobin expression devices, we also constructed various devices that aid in increasing expression levels and maintaining functional hemoglobin. We made following devices in an attempt to “steal” the solutions that natural red blood use to address the problems that come with using hemoglobin as an oxygen carrier. Since we need to make loads of hemoglobin, we need loads of heme. Therefore we relieved the major bottlenecks in E. Coli’s natural heme biosynthesis pathway by supplementing those genes in our system. To prevent improper folding and to increase the yield of functional hemoglobin, we’ve included the natural chaperone, alpha hemoglobin stabilizing protein. To remove clear out damaging free radicals that come about during oxygen binding and unbinding, we’ve added the major antioxidant enzymes that are normally present in red blood cells. And to restore non-functional methemoglobin back to a functional state, we’ve imported human cytochrome b5 and cytochrome b5 reductase, which should do the trick. Just as these problems are solved in red blood cells, these problems would be solved in analogous ways for our bacto blood cell.

9. Freeze Drying Mention that it’s a high school projectMention that it’s a high school project

10. Freeze Drying

11. The Controller

12. The Controller

13. Controller Part Characterization

14. Copy Number Device Assays

15. Genetic Kill Switch Stops growth after hemoglobin has been made Stops growth after hemoglobin has been made

16. Kill Switch Growth Assays

17. Phenotype of Dead Cells

18. A Comprehensive System Emphasize why we should get best systemEmphasize why we should get best system

19. Acknowledgements The Arkin and Keasling Labs Kate Spohr, Kevin Costa and Gwyneth Terry SynBERC The Camille and Henry Dreyfus Foundation

23. Patent Timeline Kristin F.’s Question SlideKristin F.’s Question Slide

24. Swarming Assay Maybe use Austin’s old assay pictures instead – I think they may look betterMaybe use Austin’s old assay pictures instead – I think they may look better

25. Serum Survival Assay

26. Oxygen Transport Two important characteristics of oxygen transport are the partial pressure of oxygen at which half of the binding sites are filled, or the P50, and the concentration of functional oxygen carriers. Two important characteristics of oxygen transport are the partial pressure of oxygen at which half of the binding sites are filled, or the P50, and the concentration of functional oxygen carriers.

27. Oxygen Transport Basically, as you increase the concentration of functional oxygen carriers, or as you increase the P50 of the oxygen carrier, the amount of delivered oxygen will increase. How do we achieve both of these characteristics in our system? Well, the P50 is easy. To solve the problem of hemoglobin’s naturally low P50 values, we are simply using a hemoglobin mutant with two point mutations. The literature has shown that these mutations increase the P50 values to levels higher than natural red blood cells. Great, that’s done. Basically, as you increase the concentration of functional oxygen carriers, or as you increase the P50 of the oxygen carrier, the amount of delivered oxygen will increase. How do we achieve both of these characteristics in our system? Well, the P50 is easy. To solve the problem of hemoglobin’s naturally low P50 values, we are simply using a hemoglobin mutant with two point mutations. The literature has shown that these mutations increase the P50 values to levels higher than natural red blood cells. Great, that’s done.

28. Problems The other problems involve the process of oxygen binding and unbinding. (While animation is going) Every once in a while, and electron will spontaneously transfer from the heme to the oxygen and then unbind, resulting in a superoxide and methemoglobin. The superoxide will eventually result in free radicals, and the methemoglobin, it just doesn’t work anymore, it won’t bind oxygen. Both of which are not good. The other problems involve the process of oxygen binding and unbinding. (While animation is going) Every once in a while, and electron will spontaneously transfer from the heme to the oxygen and then unbind, resulting in a superoxide and methemoglobin. The superoxide will eventually result in free radicals, and the methemoglobin, it just doesn’t work anymore, it won’t bind oxygen. Both of which are not good.