advanced biotech 2014 a protein sensor for detecting single stranded dna by shania bitsoie and...
TRANSCRIPT
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Advanced Biotech
2014
A Protein Sensor For Detecting Single Stranded DNA
By Shania Bitsoie and Crystal Vejar
Antony Research GroupChemistry and Biochemistry Department
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Outline
0Objectives0What is SSB (Single Strand DNA Binding Protein). 0Our approach 0Experiments and results0Uses for biomedical research0Conclusion
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Objectives
0We want to detect single stranded DNA in solution.
0Being able to detect the presence of single stranded DNA and RNA viruses in body fluids.
Application
Experimental Objective
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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
nucleases0 Prevents premature annealing in DNA0 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.
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SSB binds to ssDNA
04 identical subunits (homotetramer)0 ssDNA wraps around the tetramer0Wraps like seams on a tennis ball
What do we want to do with this SSB protein?- In all of three days
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Our Goal
Make a fluorescent version of SSB. Hypothesis: Upon binding to ssDNA, the fluorescence will change
(hopefully)
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What is a fluorophore?
Where to attach the fluorophore on SSB?
A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation.
The fluorophore will bind to the cysteines.
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Experiment 1: Labeling SSB
0We added the Fluorescein to the protein and let it sit for about an hour
0Then we filtered it through a column containing small beads0The protein exited the column before the free Fluorescein0We ran a gel and determined that we correctly labeled 18.4%
of the protein
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0
0.2
0.4
0.6
0.8
1
1.2
1.4
40 80 120 160 200 240 280
Abs
Time (min)
Labeled Pf-SSB
Free Label
Experiment 2: Purification of Fluorescently labeled SSB
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SDS-PAGE gel to check labeled protein
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Experiment 3: SSB protein after purification
Coomassie Image: Stains all the protein
Fluorescence Image: Shows labelled-SSB
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Testing our hypothesis
Experiment: Add ssDNA to labeled-SSB and test if the fluorescence changes.
Make a fluorescent version of SSB. Hypothesis: Upon binding to ssDNA, the fluorescence will change
(hopefully)
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2 105
4 105
6 105
8 105
1 106
1 106
1 106
2 106
2 106
500 510 520 530 540 550
No DNA+DNA (dT)70
Emission Wavelength (nm)
Experiment 4: Fluorescence of SSB increases with ssDNA
Hypothesis: Upon binding to ssDNA, the fluorescence will change (hopefully) – IT WORKED!!
Fluorimeter
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0
5 104
1 105
1.5 105
2 105
0 0.5 1 1.5 2 2.5 3
[DNA]/[SSB]
+ssDNA
+dsDNA
Experiment 5: Can SSB selectively bind to ssDNA
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Summary of Results
In our research we:0Correctly labeled 18.4% of our protein0Measured fluorescence in SSB with and without DNA0Proved that SSB specifically binds to ssDNA
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Application in biomedical research
0Labeled SSB could be used to detect ssDNA in bodily fluids like blood.
0Many viruses have ssDNA or ssRNA.
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Acknowledgements