Transcript PowerPoint - Breck School Science

Regulation of Superoxide
Radicals in Escherichia
coli
Sara H. Schilling
2007
University of St. Thomas
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Overall Goal
To learn more about the regulatory
systems that protect E. coli bacteria
cells from harmful superoxide radicals
www.science.howstuffworks.com
Why?
Information about protective
systems in E. coli can be applied
to understand similar systems in
humans
Superoxide Radicals in E. coli
Fe2+ + O2 
Superoxide Radicals in E. coli
Fe2+ + O2  Fe3+ + O2•
Radicals damage DNA, creating mutations
Breakdown of Superoxide
Radicals
SOD
2O2
•+
+
2H

Breakdown of Superoxide
Radicals
SOD
2O2
•+
+
2H
 H2O2 + O2
Gene Expression
DNA
sodA
Gene Expression
Transcription
DNA
sodA

mRNA
Gene Expression
Transcription
DNA
sodA

Translation
mRNA

Protein
SOD
Protein Regulation
sodA gene  SOD protein
Protein Regulation
Fur
sodA gene  SOD protein
Previous Research
• Fur activates sodA transcription (Schaeffer, 2006)
Previous Research
• Fur activates sodA transcription (Schaeffer, 2006)
Fur
sodA gene  MORE SOD protein
Previous Research
• Fur activates sodA transcription (Schaeffer, 2006)
Fur
sodA gene  MORE SOD protein
• Fur regulates sodA transcription when there are
Fe+2 and many superoxide radicals present
(Rollefson, et al. 2004)
Forms
of Fur Description
Zn2Fur Fur with zinc ions at each
binding site
Zn1Fur Fur with one zinc ion and one
open binding site
Fe3+Fur Fur with a zinc ion and a
ferric ion at the binding sites
Fe2+Fur Fur with a zinc ion and a
ferrous ion at the binding sites
Forms
of Fur Description
Zn2Fur Fur with zinc ions at each
binding site
Zn1Fur Fur with one zinc ion and one
open binding site
Fe3+Fur Fur with a zinc ion and a
ferric ion at the binding sites
Fe2+Fur Fur with a zinc ion and a
ferrous ion at the binding sites
Forms
of Fur Description
Zn2Fur Fur with zinc ions at each
binding site
Zn1Fur Fur with one zinc ion and one
open binding site
Fe3+Fur Fur with a zinc ion and a
ferric ion at the binding sites
Fe2+Fur Fur with a zinc ion and a
ferrous ion at the binding sites
Forms
of Fur Description
Zn2Fur Fur with zinc ions at each
binding site
Zn1Fur Fur with one zinc ion and one
open binding site
Fe3+Fur Fur with a zinc ion and a
ferric ion at the binding sites
Fe2+Fur Fur with a zinc ion and a
ferrous ion at the binding sites
Forms
of Fur Description
Zn2Fur Fur with zinc ions at each
binding site
Zn1Fur Fur with one zinc ion and one
open binding site
Fe3+Fur Fur with a zinc ion and a
ferric ion at the binding sites
Fe2+Fur Fur with a zinc ion and a
ferrous ion at the binding sites
First Goal
To compare activation of sodA transcription
in the presence of the three metal-ion
complexes of Fur:
• Zn1Fur
• Zn2Fur
• Fe3+Fur
First Hypothesis
Based on the research by Rollefson, et al.
(2004), I hypothesized that Zn2Fur would be
the metal-ion complex of Fur that most
activates sodA transcription
Second Goal
To determine the effect of Fur concentration
on activation of sodA transcription:
• 0 nM
• 50 nM
• 100 nM
• 150 nM
• 200 nM
Second Hypothesis
Based on research by Shaeffer (2006), I
hypothesized that increased Fur concentration
would increase activation of sodA transcription
Third Goal
To determine the root of and eliminate the
negative control signaling that was present
in the Schaeffer study
Third Goal
To determine the root of and eliminate the
negative control signaling that was present
in the Schaeffer study
Fourth Goal
To optimize DNA band signaling by
modifying the Schaeffer Protocols
Methods—PCR
Polymerase Chain Reaction
Diagramed used by permission from K. Shaeffer
Methods—Transcription
DNA
PCR
Purification
Transcription in Presence of the Three forms of Fur
at Increasing Concentration
Negative Controls Constructed
mRNA
Methods—Reverse Transcription
mRNA
Reverse Transcription
Negative Controls Constructed
cDNA
PCR
Amplified cDNA
Methods—Gel Electrophoresis
Photo by Author
Methods—Visualization
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VersaDoc Camera
Results—sodA transcription
of Zn1Fur
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Lane 1-2: sodA transcribed in absence of Zn1Fur,
Lane 3-4: sodA transcribed in presence of 50 nM
Zn1Fur; Lane 5-6: sodA transcribed in presence of
100 nM Zn1Fur, Lane 7-8: sodA transcribed in
presence of 150 nM Zn1Fur, Lane 9-10: sodA
transcribed in presence of 0 nM Zn1Fur
Results—sodA transcription
of Zn1Fur
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Lane 1-2: sodA transcribed in absence of Zn1Fur,
Lane 3-4: sodA transcribed in presence of 50 nM
Zn1Fur; Lane 5-6: sodA transcribed in presence of
100 nM Zn1Fur, Lane 7-8: sodA transcribed in
presence of 150 nM Zn1Fur, Lane 9-10: sodA
transcribed in presence of 0 nM Zn1Fur
Results—sodA transcription
with Fe+3Fur
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Lane 1-2: sodA transcribed in
absence of Fe3+Fur, Lane 3-4: sodA
transcribed in presence of 50 nM
Fe3+Fur; Lane 5-6: sodA transcribed
in presence of 100 nM Fe3+Fur, Lane
7-8: sodA transcribed in presence of
150 nM Fe3+Fur, Lane 9-10: sodA
transcribed in presence of 0 nM
Fe3+Fur
Results—sodA transcription
with Fe+3Fur
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Lane 1-2: sodA transcribed in
absence of Fe3+Fur, Lane 3-4: sodA
transcribed in presence of 50 nM
Fe3+Fur; Lane 5-6: sodA transcribed
in presence of 100 nM Fe3+Fur, Lane
7-8: sodA transcribed in presence of
150 nM Fe3+Fur, Lane 9-10: sodA
transcribed in presence of 0 nM
Fe3+Fur
Results—sodA Transcription
with Zn2Fur
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Lane 1-2: sodA transcribed in absence of
Zn2Fur, Lane 3-4: sodA transcribed in
presence of 50 nM Zn2Fur; Lane 5-6: sodA
transcribed in presence of 100 nM Zn2Fur,
Lane 7-8: sodA transcribed in presence of
150 nM Zn2Fur, Lane 9-10: sodA
transcribed in presence of 0 nM Zn2Fur
Results—Negative Controls
Initial Trial
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Lanes 1-3: positive controls, Lane 4:
negative control (without Master
Mix), Lane 5: negative control
(without RT primers), Lane 6: empty,
Lane 7: negative control (without
cDNA), Lanes 8-10: positive controls
Results—Negative Controls
Initial Trial
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No
cDNA
Lanes 1-3: positive controls, Lane 4:
negative control (without Master
Mix), Lane 5: negative control
(without RT primers), Lane 6: empty,
Lane 7: negative control (without
cDNA), Lanes 8-10: positive controls
Results—Negative Controls
Transcription Assay Components
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Lane 1-2: empty, Lane 3: DNase, Lane 4: RNA polymerase,
Lane 5: negative control (without DNA), Lane 6: RNase inhibitor,
Lane 7: empty, Lane 8: negative control (without cDNA)
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Lane 1: NTP-initiator mixture, Lane 2: RT primer #2, Lane
3: RT primer #3, Lane 4: negative control (without NTPinitiator mixture), Lane 5: negative control
(without mRNA), Lane 6: negative control (without
DNase), Lane 7: dNTP mixture,
Lane 8: positive control
Results—Negative Controls
Signaling Components Run with DNase
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Lane 1: positive control, Lane 2: empty, Lane 3:
RNase inhibitor incubated with DNase, Lane 4:
NTP-initiator mixture incubated with DNase,
Lane 5: 0.5 L RNA polymerase incubated with
DNase, Lane 6: 2.0 RNA polymerase incubated
with DNase, Lane 7: RNase inhibitor, NTPinitiator mixture, and RNA polymerase incubated
with DNase, Lane 8: DNA incubated with DNase
Results—Negative Controls
Signaling Components Run with DNase
Positive
Control
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Lane 1: positive control, Lane 2: empty, Lane 3:
RNase inhibitor incubated with DNase, Lane 4:
NTP-initiator mixture incubated with DNase,
Lane 5: 0.5 L RNA polymerase incubated with
DNase, Lane 6: 2.0 RNA polymerase incubated
with DNase, Lane 7: RNase inhibitor, NTPinitiator mixture, and RNA polymerase incubated
with DNase, Lane 8: DNA incubated with DNase
Results—Negative Controls
Constructed during RT-PCR
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Lane 1: positive control used in the negative
controls (originally run in Figure 9, Lane 1),
Lane 2: positive control (originally run in
Figure 4, Lane 2), Lane 3: negative control
(without mRNA, RT primers 2 and 3, reverse
transcriptase, and dNTP mixture), Lane 4:
negative control (without RT primers 2 and
3), Lane 5: negative control (without reverse
transcriptase), Lane 6: negative control
(without mRNA), Lane 7: negative control
(without dNTP mixture), Lane 8: negative
control (without cDNA), Lane 9: negative
control (without Master Mix), Lane 10:
negative control (without cDNA or RT
primers)
Results—Protocol Optimization
PCR Products with Different Concentrations of Primers
Lane 4: PCR product containing 4 L of sodA
primers; Lane 6: PCR product containing 1 L
of sodA primers; Lane 8: PCR product
containing 8 L sodA primers
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Results—Protocol Optimization
PCR Products with Different Concentrations of Primers
Lane 4: PCR product containing 4 L of sodA
primers; Lane 6: PCR product containing 1 L
of sodA primers; Lane 8: PCR product
containing 8 L sodA primers
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4 L
Results—Protocol Optimization
PCR Products with Different Concentrations of Primers
Lane 4: PCR product containing 4 L of sodA
primers; Lane 6: PCR product containing 1 L
of sodA primers; Lane 8: PCR product
containing 8 L sodA primers
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8 L
Results—Protocol Optimization
PCR Products with Different Concentrations of Primers
Lane 4: PCR product containing 4 L of sodA
primers; Lane 6: PCR product containing 1 L
of sodA primers; Lane 8: PCR product
containing 8 L sodA primers
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1 L
Discussion—First Goal
To determine what form of Fur most activates sodA transcription
• Hypothesis neither supported nor refuted
-sodA transcription in presence of Zn2Fur
unsuccessful
• Zn1Fur most activated sodA transcription
Future Work—First Goal
• Repeat sodA transcription in presence of
Zn2Fur
• Perform sodA transcription in the presence
of other metal-ion complexes of Fur
Discussion—Second Goal
To determine the effect of Fur concentration on sodA transcription
• Hypothesis correct
-Activation of sodA transcription did
increase with Fur concentration
Discussion—Third Goal
To eliminate and determine the cause of negative control signaling
• Partially successful
-Negative control signaling present
-Cause of signaling determined to originate
during process of RT-PCR
Future Work—Third Goal
• Determine what in RT-PCR is causing the
signaling
- Examine each component of the RT-PCR assay
Discussion—Fourth Goal
To optimize the Shaeffer PCR Protocol
• PCR product with 1 L of each sodA primer
produced the best signaling
– Amplification protocol was modified to
reflect the optimization
Applications of Research
• Break down more harmful superoxide
radicals
Applications of Research
• Break down more harmful superoxide
radicals
• Fur–sodA interaction may serve as model
in human systems
Applications of Research
• Break down more harmful superoxide
radicals
• Fur–sodA interaction may serve as model
in human systems
• May lead to synthesis of drugs that model
regulatory proteins and modify expression
of genes
Acknowledgements
• Dr. Kathy Olson
• University of St. Thomas Chemistry and
Biology Departments
• Mrs. Lois Fruen
• Dr. Jacob Miller
• Team Research
Regulation of Superoxide
Radicals in Escherichia
coli
Sara H. Schilling
2007