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Advanced Biotech 2014

Antony Research Group Chemistry and Biochemistry Department. A Protein Sensor For Detecting Single Stranded DNA By Shania Bitsoie and Crystal Vejar. Advanced Biotech 2014. Outline. Objectives What is SSB (Single Strand DNA Binding Protein). Our approach

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Advanced Biotech 2014

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

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

  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. Summary of Results In our research we: • 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

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