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.
Schema on read is obsolete. Welcome metaprogramming..pdf
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
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.”