Regulation of Superoxide Radicals in Escherichia coli

1
Regulation of Superoxide Radicals in Escherichia
coli

• Sara H. Schilling2007

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University of St. Thomas
3
Overall Goal

• To learn more about the regulatory systems that
  protect E. coli bacteria cells from harmful
  superoxide radicals

www.science.howstuffworks.com
4
Why?

• Information about protective systems in E.
  coli can be applied to understand similar systems
  in humans

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Superoxide Radicals in E. coli

• Fe2 O2 ?

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Superoxide Radicals in E. coli

• Fe2 O2 ? Fe3 O2
• Radicals damage DNA, creating mutations

7
Breakdown of Superoxide Radicals

• 
  SOD
• 2O2 2H ?

8
Breakdown of Superoxide Radicals

• 
  SOD
• 2O2 2H ? H2O2 O2

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Gene Expression

• DNA

sodA
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Gene Expression
Transcription

• DNA

?
mRNA
sodA
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Gene Expression
Transcription
Translation

• DNA

?
?
Protein
mRNA
sodA
SOD
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Protein Regulation

• sodA gene ? SOD protein

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Protein Regulation

• Fur
• sodA gene ? SOD protein

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Previous Research

• Fur activates sodA transcription (Schaeffer,
  2006)

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Previous Research

• Fur activates sodA transcription (Schaeffer,
  2006)
• Fur
• sodA gene ? MORE SOD protein

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Previous Research

• Fur activates sodA transcription (Schaeffer,
  2006)
• Fur
• sodA gene ? MORE SOD protein
• Fur regulates sodA transcription when there are
  Fe2 and many superoxide radicals present
• (Rollefson, et al. 2004)

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Forms of Fur Description
Zn2Fur Fur with zinc ions at each binding site
Zn1Fur Fur with one zinc ion and one open binding site
Fe3Fur Fur with a zinc ion and a ferric ion at the binding sites
Fe2Fur Fur with a zinc ion and a ferrous ion at the binding sites
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Forms of Fur Description
Zn2Fur Fur with zinc ions at each binding site
Zn1Fur Fur with one zinc ion and one open binding site
Fe3Fur Fur with a zinc ion and a ferric ion at the binding sites
Fe2Fur Fur with a zinc ion and a ferrous ion at the binding sites
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Forms of Fur Description
Zn2Fur Fur with zinc ions at each binding site
Zn1Fur Fur with one zinc ion and one open binding site
Fe3Fur Fur with a zinc ion and a ferric ion at the binding sites
Fe2Fur Fur with a zinc ion and a ferrous ion at the binding sites
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Forms of Fur Description
Zn2Fur Fur with zinc ions at each binding site
Zn1Fur Fur with one zinc ion and one open binding site
Fe3Fur Fur with a zinc ion and a ferric ion at the binding sites
Fe2Fur Fur with a zinc ion and a ferrous ion at the binding sites
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Forms of Fur Description
Zn2Fur Fur with zinc ions at each binding site
Zn1Fur Fur with one zinc ion and one open binding site
Fe3Fur Fur with a zinc ion and a ferric ion at the binding sites
Fe2Fur Fur with a zinc ion and a ferrous ion at the binding sites
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First Goal

• To compare activation of sodA transcription
  in the presence of the three metal-ion complexes
  of Fur
• Zn1Fur
• Zn2Fur
• Fe3Fur

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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

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Second Goal

• To determine the effect of Fur concentration
  on activation of sodA transcription
• 0 nM
• 50 nM
• 100 nM
• 150 nM
• 200 nM

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Second Hypothesis

• Based on research by Shaeffer (2006), I
  hypothesized that increased Fur concentration
  would increase activation of sodA transcription

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Third Goal

• To determine the root of and eliminate the
  negative control signaling that was present in
  the Schaeffer study

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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
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MethodsPCR
Polymerase Chain Reaction
Diagramed used by permission from K. Shaeffer
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MethodsTranscription

• DNA
• PCR
• 
  Purification
• Transcription in Presence of the
  Three forms of Fur
• at Increasing Concentration
• Negative Controls Constructed
• mRNA

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MethodsReverse Transcription

• mRNA
• Reverse Transcription
• Negative Controls Constructed
• cDNA
• PCR
• Amplified cDNA

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MethodsGel Electrophoresis
Photo by Author
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MethodsVisualization
Photo by K. Shaeffer used with permission
VersaDoc Camera
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ResultssodA transcription of Zn1Fur
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
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ResultssodA transcription of Zn1Fur
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
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ResultssodA transcription with Fe3Fur
Lane 1-2 sodA transcribed in absence of Fe3Fur,
Lane 3-4 sodA transcribed in presence of 50 nM
Fe3Fur Lane 5-6 sodA transcribed in presence
of 100 nM Fe3Fur, Lane 7-8 sodA transcribed in
presence of 150 nM Fe3Fur, Lane 9-10 sodA
transcribed in presence of 0 nM Fe3Fur
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ResultssodA transcription with Fe3Fur
Lane 1-2 sodA transcribed in absence of Fe3Fur,
Lane 3-4 sodA transcribed in presence of 50 nM
Fe3Fur Lane 5-6 sodA transcribed in presence
of 100 nM Fe3Fur, Lane 7-8 sodA transcribed in
presence of 150 nM Fe3Fur, Lane 9-10 sodA
transcribed in presence of 0 nM Fe3Fur
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ResultssodA Transcription with Zn2Fur
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
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ResultsNegative Controls Initial Trial
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
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ResultsNegative Controls Initial Trial
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
No cDNA
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ResultsNegative ControlsTranscription Assay
Components
Lane 1 NTP-initiator mixture, Lane 2 RT primer
2, Lane 3 RT primer 3, Lane 4 negative
control (without NTP-initiator mixture), Lane 5
negative control (without mRNA), Lane 6
negative control (without DNase), Lane 7 dNTP
mixture, Lane 8 positive control
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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ResultsNegative Controls
Signaling Components Run with DNase
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, NTP-initiator mixture,
and RNA polymerase incubated with DNase, Lane 8
DNA incubated with DNase
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ResultsNegative Controls
Signaling Components Run with DNase
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, NTP-initiator mixture,
and RNA polymerase incubated with DNase, Lane 8
DNA incubated with DNase
Positive Control
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ResultsNegative Controls
Constructed during RT-PCR
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)
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ResultsProtocol 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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ResultsProtocol 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
4 ?L
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ResultsProtocol 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
8 ?L
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ResultsProtocol 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
1 ?L
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DiscussionFirst 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

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Future WorkFirst Goal

• Repeat sodA transcription in presence of Zn2Fur
• Perform sodA transcription in the presence of
  other metal-ion complexes of Fur

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DiscussionSecond Goal
To determine the effect of Fur concentration on
sodA transcription

• Hypothesis correct
• -Activation of sodA transcription did increase
  with Fur concentration

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DiscussionThird 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

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Future WorkThird Goal

• Determine what in RT-PCR is causing the signaling
  
• - Examine each component of the RT-PCR assay

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DiscussionFourth 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

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Applications of Research

• Break down more harmful superoxide radicals

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Applications of Research

• Break down more harmful superoxide radicals
• FursodA interaction may serve as model in human
  systems

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Applications of Research

• Break down more harmful superoxide radicals
• FursodA interaction may serve as model in human
  systems
• May lead to synthesis of drugs that model
  regulatory proteins and modify expression of genes

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Acknowledgements

• Dr. Kathy Olson
• University of St. Thomas Chemistry and Biology
  Departments
• Mrs. Lois Fruen
• Dr. Jacob Miller
• Team Research

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Regulation of Superoxide Radicals in Escherichia
coli

• Sara H. Schilling2007