Advanced Biotech 2014 A Protein Sensor For Detecting Single Stranded DNA By Shania Bitsoie and...
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Transcript of Advanced Biotech 2014 A Protein Sensor For Detecting Single Stranded DNA By Shania Bitsoie and...
Advanced Biotech
2014
A Protein Sensor For Detecting Single Stranded DNA
By Shania Bitsoie and Crystal Vejar
Antony Research GroupChemistry and Biochemistry Department
Outline
0Objectives0What is SSB (Single Strand DNA Binding Protein). 0Our approach 0Experiments and results0Uses for biomedical research0Conclusion
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
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.
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
Our Goal
Make a fluorescent version of SSB. Hypothesis: Upon binding to ssDNA, the fluorescence will change
(hopefully)
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.
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
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
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
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)
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
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
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
Application in biomedical research
0Labeled SSB could be used to detect ssDNA in bodily fluids like blood.
0Many viruses have ssDNA or ssRNA.
Acknowledgements