Kennedy Lab Research
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This powerpoint outlines my work during my internship at the Buck Institute for Research on Aging. I have used this powerpoint (and similar variations) to present the findings to the rest of the Kennedy Lab, San Marin High School students and faculty, and to the Marin County Board of Education.
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Kennedy Lab Research
- 1. My Internship in the Kennedy Lab Studying calorie restriction and cell respiration in yeast cells—and how these factors affect the aging process Jason Freeberg Kennedy Lab The Buck Institute for Research on Aging March 12th to June 10th, 2013
- 2. Overview of Calorie Restriction It has been well documented that calorie restriction (CR) slows the aging process in many species of animals Results include resistance to oxidative stress, enhanced DNA repair, delayed onset of age-related diseases, even increased lifespan However, a mechanic understanding of this process is still unknown We know the results of CR, but we don’t know why this happens My section of the Kennedy Lab set out to test if and how cellular respiration is a factor in the calorie restriction process
- 3. Overview of Cellular Respiration
- 4. General Workflow Create the rho0 yeast cells Check for presence of mDNA Selective plating DAPI staining PCR test for mDNA Begin lifespan tests and calorie restriction Test for Sir2 expression in the cells Quantify the buildup of ERC’s
- 5. Generating Cells without Mitochondrial DNA Yeast cells lacking mitochondrial DNA (mDNA) are incapable of respiration, so this is how our lab was able to test the effects of respiration on calorie restricted cells Rho0 cells lack mDNA I replicate plated yeast cells on media treated with Ethidium Bromide (EtBr) to create rho0 cells The EtBr “tangles” with the mDNA, and the daughter cells will then lack the mDNA And bam! rho0 cells!
- 6. Testing for Presence of mDNA After the EtBr treatment, I created glucose and glycerol plates I split these down the middle, and Z-streaked the left sides with wild type yeast, and the right side with the rho0 yeast The wildtype yeast (i) easily grows on both mediums, while the rho0 yeast (ii) is unable to grow on glycerol The rho0 cells can’t respire, so growth on the glycerol is impossible
- 7. Testing for Presence of mDNA Alternatively, mDNA can be visualized through fluorescence staining and imaging microscopy DAPI is a fluorescent stain for the DNA in a cell, we can then image it under a microscope after we excite the DAPI molecule DAPI will show as a blue color DNA is present anywhere is a blue stain on the cells Specifically, the DAPI bonds to the DNA regions with high A-T concentration
- 8. DAPI Stains for mDNA Wildtype + for mDNA Rho0 strain - for mDNA
- 9. Testing for Presence of mDNA L 1 2 3 4 5 6 7 x 8 L: base-pair ladder 1-4: 2820 rho0 5-7: 2823 rho0 x: skipped, broken well 8: wildtype (+) PCR for mitochondrial DNA Should be no bands for rho0 because there is no mDNA to replicate
- 10. Testing for Presence of mDNA PCR for mitochondrial DNA Should be no bands for rho0 because there is no mDNA to replicate 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1-4: 2820 rho0 5-7: 2823 rho0 8: wildtype (+)
- 11. Testing for Chromosomal DNA 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 As a second control, I did PCR for a section of the yeast chromosomal DNA There should be bands for all samples, even the rho0 1-4: 2820 rho0 5-7: 2823 rho0 8: wildtype (+)
- 12. Conclusions from my Time at the Buck I created Rho0 cells by treatment with EtBr I checked the treatment using… Glucose and glycerol plating DAPI staining for DNA PCRs for mDNA The results show that the rho0 cells I made did lose their mDNA
- 13. Acknowledgements Dr. Scott Tsuchiyama, my mentor Dr. Julie Mangada, head of Education Outreach Dr. Brian Kennedy, Buck Inst. CEO and PI of the Kennedy Lab Camille Madfes, our School to Career Liaison Dr. Lafevre-Bernt, our Biotech II instructor
Editor's Notes
- “If we want to test respiration during calorie restriction, we need to make cells that can’t respire so we can make comparisons”
- “Another way I checked for mDNA was by staining it. When we add a chemical, DAPI, to the cells, it binds with the DNA. Then we can take it under a microscope, use a fluorescent light and the DAPI will light up blue.”