1. A Protein Sensor For Detecting Single Stranded DNA
Antony Research Group
Chemistry and Biochemistry Department
A Protein Sensor For Detecting Single Stranded DNA
By Shania Bitsoie and Crystal Vejar
Advanced Biotech
2014

2. Outline Objectives What is SSB (Single Strand DNA Binding Protein).
Our approach
Experiments and results
Uses for biomedical research
Conclusion

3. Experimental Objective
Objectives
Experimental Objective
We want to detect single stranded DNA in solution.
Being able to detect the presence of single stranded DNA and RNA viruses in body fluids.
Application

4. What is SSB?
Single Strand DNA binding protein is a protein that binds specifically to single stranded DNA
It is present in all organisms from bacteria to humans
Function:
It protects ssDNA from being digested by nucleases
Prevents premature annealing in DNA
Removes secondary structures from the DNA to allow enzymes to function efficiently
Its functions in many processes on the DNA in the cell: replication, recombination, and repair.

5. What do we want to do with this SSB protein?
SSB binds to ssDNA
4 identical subunits (homotetramer)
ssDNA wraps around the tetramer
Wraps like seams on a tennis ball
What do we want to do with this SSB protein?
- In all of three days 

6. Our Goal Make a fluorescent version of SSB.
Hypothesis: Upon binding to ssDNA, the fluorescence will change (hopefully)

7. Where to attach the fluorophore on SSB?
What is a fluorophore?
A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation.
Where to attach the fluorophore on SSB?
The fluorophore will bind to the cysteines.

8. Experiment 1: Labeling SSB
We added the Fluorescein to the protein and let it sit for about an hour
Then we filtered it through a column containing small beads
The protein exited the column before the free Fluorescein
We ran a gel and determined that we correctly labeled 18.4% of the protein

9. Experiment 2: Purification of Fluorescently labeled SSB
Pf-SSB
Free Label

10. SDS-PAGE gel to check labeled protein

11. Experiment 3: SSB protein after purification
Coomassie Image: Stains all the protein
Fluorescence Image: Shows labelled-SSB

12. Testing our hypothesis
Make a fluorescent version of SSB.
Hypothesis: Upon binding to ssDNA, the fluorescence will change (hopefully)
Experiment: Add ssDNA to labeled-SSB and test if the fluorescence changes.

13. Experiment 4: Fluorescence of SSB increases with ssDNA
Fluorimeter
Hypothesis: Upon binding to ssDNA, the fluorescence will change (hopefully) – IT WORKED!!

14. Experiment 5: Can SSB selectively bind to ssDNA

15. In our research we: Summary of Results
Correctly labeled 18.4% of our protein
Measured fluorescence in SSB with and without DNA
Proved that SSB specifically binds to ssDNA

16. Application in biomedical research
Labeled SSB could be used to detect ssDNA in bodily fluids like blood.
Many viruses have ssDNA or ssRNA.

17. Acknowledgements