Materials
Overnight culture of E. coli strain JM109 (recA^-) in 10 mL LB^Fe media
Overnight culture of E. coli strain IY12 (NADH/Q oxidoreductase) in 10 mL LB^Fe media
Overnight culture of E. coli strain IY13 (wt) in 10 mL LB^Fe media
10 mL sterile LB^Fe media in 50 mL flasks, prewarmed to 37 °C (5)
10 mL sterile LB^Fe media in 50 mL conical tube, RT (1)
10 mL sterile M9 salts in 50 mL conical tube, RT (1)
0.1 M KCN in water
0.1 M 2,2'-dipyridyl in ethanol
9.8 M (30%) Hydrogen Peroxide, on ICE
Pipettors (10 µL, 200 µL, 1000 µL)
Tips — sterilized (10µL, 200 µL, 1000 µL)
1.5 mL Eppendorf tubes (5)
50 mL orange capped conical tubes (2)
10x100 mm sterile test tubes (32)
Empty test tube racks (2)
LB bacterial plates (24)
Ethanol in 250 mL beaker with cell spreader
Bunsen burner
Shaking incubator set to 37 °C, 200 rpm
Vortex mixer
Personal computer with KaleidaGraph
Safety glasses

Introduction
Our experiments with purified DNA showed hydrogen peroxide can break DNA strands if NADH and Fe(III) are both present in the reaction mixture; no DNA strand breaks were detected with hydrogen peroxide alone. Perhaps a chemical species derived from hydrogen peroxide is the actual agent in DNA damage.

The most likely candidate for this agent in DNA damage is hydroxyl radical. Hydroxyl radical is highly reactive and is generated by single electron reduction of hydrogen peroxide (see Equation 1 from Lab 4 — Hydrogen peroxide treatment of living bacteria). The source of electrons to generate hydroxyl radical from hydrogen peroxide may be NADH. NADH is, however, a two electron carrier, and direct addition of both electrons from NADH to hydrogen peroxide would yield two harmless water molecules. If NADH first reduces Fe³⁺ to give Fe²⁺, a single electron carrier is generated. Fe²⁺ can then reduce hydrogen peroxide to produce hydroxyl radical (Eq. 1).

(Eq. 1)

To see if our model is consistent with oxidative DNA damage inside of cells, we will return to E. coli as our model organism for testing mechanisms of DNA damage in vivo. A quick review of electron transport and oxidative phosphorylation (Eqs. 2—3) will help understand our experimental design. Electrons derived from food are carried by NADH to the electron transport chain where enzyme complexes catalyze the stepwise transfer of electrons to intermediate carriers and the final electron acceptor, oxygen. Enzyme complexes of electron transport use the free energy of electron transfer to drive proton translocation across the inner mitochondrial (Eq. 2) or bacterial membrane (Eq. 3). The energy derived from food is thus captured as an electrochemical gradient that drives the phosphorylation of ADP to produce ATP.

(Eq. 2)

(Eq. 3)

NADH is a component of both our model for oxidative DNA damage (Eq. 1) and electron transport inside the cell (Eq. 3). If our model is correct, alterations in energy metabolism should have predictable effects on hydrogen peroxide's bactericide activity. In these experiments, we will probe the mechanism of hydrogen peroxide induced DNA damage by altering the energy balance and free metal concentration inside of living cells.

Safety

Be careful working with the Bunsen burner and ethanol. Secure long hair and loose clothing before using the flame when plating bacteria. Concentrated hydrogen peroxide can be irritating to the skin and eyes — wear gloves and eye protection. Potassium cyanide and 2,2'-dipyridyl are each toxic — wear gloves and handle with care.

Exercise 1 — Grow cultures of rapidly dividing bacteria for JM109, IY12, IY13

1. Guiness, Marty, and Zinc: Use a 1000 µL pipettor to inoculate 10 mL of sterile LB^Fe media with 200 µL of an overnight culture of JM109. Label this fresh culture with your team name and the name of the E. coli strain: JM109 (recA^—). Use sterile technique, flaming the mouth of each flask when opening and closing. The fresh media should be prewarmed to ensure that the bacteria start growing rapidly.
2. Quarks: Inoculate 10 mL of LB^Fe media with 400 µL of an overnight culture of IY13.
3. Quarks: Inoculate 10 mL of LB^Fe media with 400 µL of an overnight culture of IY12.
4. Continue growth at 37 °C while shaking at 200 rpm.

 

1. If your plates are stored in the refrigerator, remove and place them on the bench.

 

 
 
 

OVERVIEW: Matched cultures will be treated with either a control program or a test program prior to exposure to hydrogen peroxide at four concentrations (10 mM, 1 mM, 0.1 mM, and 0 mM) for 30 minutes. With this pairing of control and test, there will be 8 final cultures, 4 in the control set and 4 in the test set. Dilutions of these cultures will be plated to determine the fraction of cells surviving hydrogen peroxide exposure by our standard colony counting method. Each team will compare a different control and test program assigned in Table I. Work carefully; the whole class will depend on and write about results your team obtains.

Table I. Team assignments

Team	Control	Test
Guinness	water	3 mM KCN
Marty	food	no food
Quarks	IY13 (functional NADH dehydrogenases)	IY12 (impaired NADH dehydrogenases)
Zinc	ethanol	1 mM 2,2' dipyridyl

2. Label 10x100 sterile test tubes with final hydrogen peroxide concentrations (0 mM, 0.1 mM, 1 mM, 10 mM) for each treatment program — control or test.
3. Use the 1000 µL pipettor set to 900 µL to aliquot 900 µL of sterile water to each of 8 10x100 sterile test tubes. Label these as above and add the dilution label '1:10'.
4. Use the 1000 µL pipettor set to 990 µL to aliquot 990 µL of sterile water to each of 8 10x100 sterile test tubes. Label these as above and add the dilution label 1:10³
5. Use the 1000 µL pipettor set to 990 µL to aliquot 990 µL of sterile water to each of 8 10x100 sterile test tubes. Label these as above and add the dilution label 1:10⁵
6. Label 24 plates indicating the final hydrogen peroxide concentrations (0 mM, 0.1 mM, 1 mM, or 10 mM), the treatment program (control or test), and dilution factor (1:10, 1:10³, or 1:10⁵).

 

IMPORTANT NOTE! Plan your experiment so that the hydrogen peroxide reactions start very soon after preparing the dilutions of hydrogen peroxide.
 
1. Prepare 1.0 mL of 20 µg/mL catalase on ICE by diluting 200 µg/mL catalase (solution w from Lab 3) with 10 mM sodium phosphate (solution j from Lab 3).
2. Guiness, Quarks, and Zinc: Continue growth of the fresh cultures for 60 minutes.

Marty: Continue growth of the fresh culture for 45 minutes.
3. Guiness and Zinc:Divide the culture into two 5 mL volumes in two 50 mL tubes.
4. Follow the control and test programs indicated in Table II.

 

 

Table II. Treatment program

Team	Control	Test
Guinness	add 150 µL water, mix, and aliquot 900 µL to each of 4 tubes	add 150 µL 0.1 M KCN, mix, and aliquot 900 µL to each of 4 tubes
Marty	Aliquot 1000 µL of the fresh culture to each of 8 Eppendorf tubes. Centrifuge for 2 minutes at 6,000 rpm. Remove supn by pouring. Centrifuge again for 1 minute at 6,000 rpm. Carefully remove entire supn with a 200 µL pipettor.
	Add 1000 µL LBFe to each of 4 tubes.	Add 1000 µL M9 salts to each of 4 tubes.
	Set the pipettor to 900 µL. Resuspend each cell pellet and transfer to a sterile 10x100 mm tube.
Quarks	Aliquot 900 µL of the IY13 culture to each of 4 labeled sterile tubes	Aliquot 900 µL of the IY12 culture to each of 4 labeled sterile tubes
Zinc	Add 50 µL ethanol, mix, and aliquot 900 µL to each of 4 tubes	Add 50 µL of 0.1 M 2,2' dipyridyl

5. Place the 900 µL cultures in the shaking incubator for an additional 10 minutes.
6. Use sterile water to dilute 9.8 M hydrogen peroxide to prepare 1 mL of 100 mM hydrogen peroxide. Label and keep on ICE.
7. Use 1:10 dilutions in sterile water, to prepare 900 µL of 10 mM and 1 mM hydrogen peroxide. Label and keep on ICE. Reserve 900 µL of water as a 0 mM H₂O₂ control.

1. Bring all of the 900 µL cultures to your bench. Mark the time. Begin adding 100 µL aliquots from the 100 mM H₂O₂ dilution to control and test cultures. Mix each culture after each addition of hydrogen peroxide. Continue by adding 100 µL aliquots from the 10 mM H₂O₂ solution to control and test cultures, adding 100 µL aliquots from the 1 mM H₂O₂ solution to control and test cultures, and adding 100 µL water to control and test cultures. Mix each culture after each addition of hydrogen peroxide. Return the cultures to the shaking incubator.
2. After 30 minutes of exposure to hydrogen peroxide, bring the cultures back to your bench and stop each reaction by adding 100 µL of the 20 µg/mL catalase solution. Add the catalase in the same order and at the same pace as reactions were started.
3. For each hydrogen peroxide exposed bacterial culture, prepare a 1:10 dilution in water by transferring 100 µL of the exposed cells to the corresponding tube of 900 µL sterile water. Be sure to vortex each culture before removing the 100 µL volume.
4. Prepare 1:10³ dilutions of each cell suspension by transferring 10 µL of the 1:10 dilution to the corresponding tube of 990 µL sterile water. Be sure to vortex the 1:10 dilution before removing the 10 µL volume.
5. Prepare 1:10⁵ dilutions of each cell suspension by transferring 10 µL of the 1:10³ dilution to the corresponding tube of 990 µL sterile water. Be sure to vortex the 1:10³ dilution before removing the 10 µL volume.
6. Plate 100 µL of each bacterial cell dilution onto individual LB plates. Remember to vortex each cell suspension before removing the 100 µL aliquot to a plate. Also, cool the metal spreader before touching the cells.


TIME SAVING TIP: If you pipette the 100 µL volume to each plate of the 1:10, 1:10³, and 1:10⁵ dilution series, you can spread all three plates after sterilizing the spreader just one time. Be sure to spread the 1:10⁵ dilution first, then the 1:10³ dilution and finally the densest 1:10 cell suspension.
 

7. Place both sets of plates, inverted in a 37 °C incubator overnight. Quarks: Your colonies will be quite small even after 24 hours incubation. If convenient, please allow for a 40 hour incubation.

 

1. Record in your notebook, the number of colonies on each plate. Mark 'tmc' if there are too many colonies to count. Mark '0' if no colonies are seen.
2. Use the 0 mM control that did not get exposed to hydrogen peroxide to estimate the number of cells plated on each plate.
3. To calculate the fraction of cells that survived hydrogen peroxide treatment, f_survive, divide the number of viable colonies by the estimated number of cells plated.
4. Estimate the error for f_survive according to the formula we worked out for Lab 2 (Equation 3). We'll treat the uncertainty in volume transfers as negligible.


Uncertainty in f_survive = f_survive x (1/N + 1/N_0mM)^1/2 (Eq. 3)
 

5. Use Microsoft Word to prepare a table of f_survive for each treatment program. Be sure to identify the treatment program particular to your experiment as part of the column headings or as a footnote. Include the uncertainty in each reported value. Save the table to your team folder, make a backup to your team disk, and also upload the final version to Notes to Instructor on the Biol.3525 server. Team tables will be available through our website and in the Student Handouts folder on the Biol.3525 server.
6. Experiment with graphical representations of your data such as a bar graph with error bars. You may decide to focus on just one or two hydrogen peroxide concentrations that most clearly accentuate the effect of your treatment plan. Print a hardcopy of your plot for your notebook.
7. Save your plot both as a KaleidaGraph plot and as an exported PCT file. Remember to use the high resolution option for the exported PCT file in KaleidaGraph. Backup your KaleidaGraph plot and PCT file to your Zip disk and to your Biol.3525:Students' Stuff:Team folder and upload to Notes to Instructor on the Biology Courses Server.

Checklist of results to include in your Notes

• Table of colonies counted for each treatment plan, control and test
• Calculated fraction of bacteria surviving each treatment: f_survive
• Plot highlighting the effect of control and test treatment plans

• Concise Introduction that identifies the hypothesis or hypotheses being tested
• Brief description of Methods that is addressed to someone familiar with the techniques we used. Be sure to calculate final concentrations of reagents used.
• As part of the Methods section, include a gel image obtained in Lab 5 — purification of plasmid DNA that demonstrates the purity of your DNA.
• Results section describing what was observed in objective, concrete terms and which includes the following:

• Gel image showing reaction of plasmid DNA with hydrogen peroxide (Lab 6)
• Table or plot showing additives required to damage DNA in vitro (Lab 6)
• Table of colonies counted for each treatment plan in vivo (Lab 7)
• Table or plot highlighting effects of each treatment on hydrogen peroxide's ability to kill rapidly dividing bacteria (Lab 7).

• Discussion section which builds on the observations, offers an interpretation of the results in relationship to the hypothesis or hypotheses identified in the Introduction, and which should offer an underlying mechanism. It may be helpful to copy and paste Equation 1 and Equation 2 from this protocol into your discussion. An electronic version of the protocol is available from our website and from the Biol.3525 server. Of course you may chose create a drawing of your own creation that represents your interpretation of what is happening during hydrogen peroxide treatment of DNA and inside of living cells.