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Regulation of Superoxide Radicals in Escherichia coli

Regulation of Superoxide Radicals in Escherichia coli. Sara H. Schilling 2007. University of St. Thomas. Overall Goal. To learn more about the regulatory systems that protect E. coli bacteria cells from harmful superoxide radicals. www.science.howstuffworks.com. Why?.

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Regulation of Superoxide Radicals in Escherichia coli

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  1. Regulation of Superoxide Radicals in Escherichia coli Sara H. Schilling2007

  2. 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

  5. Superoxide Radicals in E. coli Fe2+ + O2

  6. 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

  9. Gene Expression DNA sodA

  10. Gene Expression Transcription DNA  mRNA sodA

  11. Gene Expression Transcription Translation DNA   mRNA Protein sodA SOD

  12. Protein Regulation sodA gene SOD protein

  13. Protein Regulation Fur sodA gene SOD protein

  14. Previous Research • Fur activates sodA transcription (Schaeffer, 2006)

  15. Previous Research • Fur activates sodA transcription (Schaeffer, 2006) Fur sodA geneMORE SOD protein

  16. 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)

  17. First Goal To compare activation of sodA transcription in the presence of the three metal-ion complexes of Fur: • Zn1Fur • Zn2Fur • Fe3+Fur

  18. 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

  19. Second Goal To determine the effect of Fur concentration on activation of sodA transcription: • 0 nM • 50 nM • 100 nM • 150 nM • 200 nM

  20. Second Hypothesis Based on research by Shaeffer (2006), I hypothesized thatincreased Fur concentration would increase activation of sodA transcription

  21. Third Goal To determine the root of and eliminate the negative control signaling that was present in the Schaeffer study

  22. Third Goal To determine the root of and eliminate the negative control signaling that was present in the Schaeffer study Fourth Goal Tooptimize DNA band signaling by modifying the Schaeffer Protocols

  23. Methods—PCR Polymerase Chain Reaction Diagramed used by permission from K. Shaeffer

  24. Methods—Transcription DNA PCR Purification Transcription in Presence of the Three forms of Fur at Increasing Concentration Negative Controls Constructed mRNA

  25. Methods—Reverse Transcription mRNA Reverse Transcription Negative Controls Constructed cDNA PCR Amplified cDNA

  26. Methods—Gel Electrophoresis Photo by Author

  27. Methods—Visualization Photo by K. Shaeffer used with permission VersaDoc Camera

  28. 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

  29. 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

  30. 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

  31. 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

  32. 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

  33. Results—Negative ControlsInitial 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

  34. Results—Negative ControlsInitial 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

  35. 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)

  36. 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

  37. 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 Positive Control

  38. Results—Negative 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)

  39. 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

  40. 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

  41. 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 8 L

  42. 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 1 L

  43. 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

  44. Future Work—First Goal • Repeat sodA transcription in presence of Zn2Fur • Perform sodA transcription in the presence of other metal-ion complexes of Fur

  45. Discussion—Second 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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