Antony Research Group
Chemistry and Biochemistry Department
Outline
0 Objectives
0 What is SSB (Single Strand DNA Binding Protein).
0 Our approach
0 Experiments and results
0 Uses for biomedical research
0 Conclusion
Objectives
Experimental Objective
0 We want to detect single stranded DNA in solution.
Application
0 Being able to detect the presence of single stranded
DNA and RNA viruses in body fluids.
What is SSB?
0 Single Strand DNA binding protein is a protein
that binds specifically to single stranded DNA
0 It is present in all organisms from bacteria to
humans
Function:
0 It protects ssDNA from being digested by
nucleases
0 Prevents premature annealing in DNA
0 Removes secondary structures from the DNA to
allow enzymes to function efficiently
0 Its functions in many processes on the DNA in the
cell: replication, recombination, and repair.
SSB binds to ssDNA
0 4 identical subunits (homotetramer)
0 ssDNA wraps around the tetramer
0 Wraps like seams on a tennis ball
What do we want to do with this SSB protein?
- In all of three days 
Our Goal
Make a fluorescent version of SSB.
Hypothesis: Upon binding to ssDNA,
the fluorescence will change
(hopefully)
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.
Experiment 1: Labeling SSB
0 We added the Fluorescein to the protein and let it sit for
about an hour
0 Then we filtered it through a column containing small beads
0 The protein exited the column before the free Fluorescein
0 We ran a gel and determined that we correctly labeled 18.4%
of the protein
Experiment 2: Purification of Fluorescently
labeled SSB
1.4
Abs
Labeled
Pf-SSB
1.2
1
0.8
Free Label
0.6
0.4
0.2
0
40
80
120 160 200 240 280
Time (min)
SDS-PAGE gel to check labeled protein
Experiment 3: SSB protein after purification
Coomassie Image: Stains all the protein
Fluorescence Image: Shows labelled-SSB
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.
Experiment 4: Fluorescence of SSB increases with
ssDNA
Fluorimeter
2 10
6
2 10
6
1 10
6
1 10
6
1 10
6
8 10
5
6 10
5
4 10
5
2 10
5
500
No DNA
+DNA (dT)70
510
520
530
540
Emission Wavelength (nm)
Hypothesis: Upon binding to ssDNA,
the fluorescence will change
(hopefully) – IT WORKED!!
550
Experiment 5: Can SSB selectively bind to ssDNA
2 10
5
1.5 10
5
1 10
5
5 10
4
+ssDNA
+dsDNA
0
0
0.5
1
1.5
[DNA]/[SSB]
2
2.5
3
Summary of Results
In our research we:
0 Correctly labeled 18.4% of our protein
0 Measured fluorescence in SSB with and without DNA
0 Proved that SSB specifically binds to ssDNA
Application in biomedical research
0 Labeled SSB could be used to detect ssDNA in bodily
fluids like blood.
0 Many viruses have ssDNA or ssRNA.
Acknowledgements