Regulation of Superoxide Radicals in Escherichia coli Sara H. Schilling 2007.

Regulation of Superoxide Radicals in Escherichia coli

Sara H. Schilling2007

University of St. Thomas

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To learn more about the regulatory systems that protect E. coli bacteria

cells from harmful superoxide radicals

Overall Goal

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

DNA mRNA

Transcription

sodA

Gene Expression

DNA ProteinmRNA

Transcription Translation

sodA 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

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—TranscriptionDNA

PCR Purification

Transcription in Presence of the Three forms of Fur at Increasing Concentration

Negative Controls Constructed

mRNA

Methods—Reverse TranscriptionmRNA

Reverse Transcription

Negative Controls Constructed

cDNA

PCR

Amplified cDNA

Methods—Gel Electrophoresis

Photo by Author

Methods—Visualization



VersaDoc CameraPhoto by K. Shaeffer used with permission

Results—sodA 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

Results—sodA transcription with Fe+3Fur



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



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



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



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

Results—Negative 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)



Results—Negative 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




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




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)

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






4 L






8 L






1 L

Discussion—First Goal

• Hypothesis neither supported nor refuted

-sodA transcription in presence of Zn2Fur unsuccessful

• Zn1Fur most activated sodA transcription

To determine what form of Fur most activates 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

• Hypothesis correct

-Activation of sodA transcription did increase with Fur concentration

To determine the effect of Fur concentration on sodA transcription

Discussion—Third Goal

• Partially successful

-Negative control signaling present

-Cause of signaling determined to originate during process of RT-PCR

To eliminate and determine the cause of negative control signaling

Future Work—Third Goal

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

Discussion—Fourth Goal

• PCR product with 1 L of each sodA primer produced the best signaling– Amplification protocol was modified to

reflect the optimization

To optimize the Shaeffer PCR Protocol

Applications of Research

• Break down more harmful superoxide radicals



• Fur–sodA interaction may serve as model in human systems



• 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. Schilling2007

Regulation of Superoxide Radicals in Escherichia coli Sara H. Schilling 2007.

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