Protocol for Measuring Cyt c content of bacteria using diffuse reflectance

Authors: Cheryl Goldbeck, Caroline Ajo-Franklin
Last updated: 06/25/13
Goal: To quantitatively measure the cytochrome c content per intact cell from bacterial strains expressing cytochromes c using diffuse reflectance. At the end of this procedure, you should have a list of the cytochrome c content per cell for each strain.

Important notes: You need to be granted access to the 4th floor and be trained before you can use the diffuse reflectance system independently. Ask your supervisor to send a email to Jeff Urban (or the current Facility Director of the Inorganic floor) requesting access to the 4th floor diffuse reflectance system.

Critical Steps:

1. Keep the A600nm as measured by diffuse reflectance below 0.065 OD. In our diffuse reflectance system, the A600nm, which provides a measure of the scattering from cells, is linear with the cell concentration from A600nm = 0 – 0.065 OD.
2. Be sure to pipet the final bacterial solution up and down several times before adding it to the cuvette. These dense solutions will settle rapidly, so unless they are well mixed, you will add a different amount of cells than you intend.
3. Check that the added sodium dithionite fully reduces your samples. The cytochrome c peak at 552nm has a strong extinction coefficient when the heme contains Fe2+ (ε ~ 28,000 OD M-1 cm-1), but is much weaker (ε~3,000 OD M-1 cm-1) when the Fe is Fe3+. When grown in aerobic conditions, the cytochromes c are mostly in the oxidized Fe3+ state. Sodium dithionite reduces Fe3+ hemes to Fe2+ hemes, thus enabling quantitative measurement of the [cyt c]. Sodium dithionite degrades with exposure to moisture and air, so store the solid in sealed containers containing desiccant. Fully reduced MtrA has a background corrected A418nm/A552nm of ~0.4.
4. As much as possible, keep your loaded cuvette air bubble,smear, and debris free. We use the scattering at 600nm as an indication of the cell concentration. Anything that affects the scattering will alter our measure of the cell concentration.
5. Save each spectrum after acquiring it and save the project as a whole before exiting the software. The Indico Pro software is very prone to crashing. Do not click on any gray area or the software will crash.

Troubleshooting
Here are the [MtrC+MtrA] (uM/OD600) measured for all the strains in Goldbeck et al 2013. Your values should agree with these published values.DiffuseReflectanceData_Goldbeck2013

Safety Information:

This procedure has involves lots of pipetting. Be sure to follow good ergonomic practices. Specifically, take a 5 minute break every 20 minutes and adjust your workspace to minimize stress on your hands. And if you feel any discomfort, please let your supervisor know asap.

Waste Disposal:

• Decontaminate cultures by addition of bleach or wescodyne.
• Dispose of used cuvettes (coverslip + gasket) containing microbes  by putting them in the unregulated sharps containers (red bench top containers).

Materials Needed:

• 25 or 50 mL bacterial cultures expressing cytochromes c
• 25 or 50 mL bacterial culture of strain not expressing cytochromes c, e.g. MFe479 [BL21(DE3) pSB1ET2 & pACYC184] or MFe478 [C43(DE3) pSB1ET2 & pACYC184]
• 15 mL Falcon centrifuge tube, VWR catalog # 21008-940
• 1.5 mL eppendorf tubes
• 200 uL pcr tubes in a strip of 8
• 10 mM Hepes pH 7.5
• Secure-seal gaskets & port covers, 0.8mm deep x 22mm diameter,  Invitrogen #S24734
• 25x25mm glass coverslips #1.5, VWR catalog # 48366 249
• sodium dithionite, MW 174.1 g/mole, Sigma catalog # 157953-100G
• equine cytochrome c, MW 12,270 g/mole, CalBiochem catalog #250600
• USB Key for transfering data

Procedure:

Days 0-1.

1. Prepare cultures of cytochrome c expressing cells. See protocol: Cytochrome expression protocol in flasks.

Day 2.

Note: If you have a large number of samples, i.e. greater than 10, the sample preparation can be very time intensive. To make your day easier, pre-label centrifuge tubes and microfuge tubes, and pre-make the disposible cuvettes.

Prepare concentrated cell suspensions.

1. From each overnight culture, aliquot 10mL of culture into a labeled 15 mL centrifuge tube.

2. Pellet the cells by centrifuging the tubes at 4000 rpm for 10 min, 4C using the Beckmann centrifuge in 5208.

3. Carefully pour out or aspirate the excess media from the tube, leaving the cell pellet intact. Add 1 ml 10mM Hepes pH 7.5 to the pellet, and gently resuspend it by pipeting up and down using a pipetaid or pipetman. These are your original bacterial suspensions.

4. Label the end tubes of a strip of pcr tubes. Add 4uL of each cell suspension to each tube, followed by 196 uL 10 mM Hepes.  If you have many samples, use a multichannel pipet to add the 196 uL of Hepes buffer. These are your 1:50 dilutions.

5. Measure and record the A600nm of the 1:50 dilutions using the UV-Vis on Cheryl’s bench using the 100 uL cuvette.

6. Calculate the A600nm of your original bacterial suspension, assuming that the 1:50 dilution factor.Typically, the 1:50 dilution of cells have A600nm ~0.25-0.75OD, meaning that the 1 mL original bacterial suspensions have an A600nm in the range of 14 to 37 O.D.

Diluting cell suspensions to A600nm ~ 3-4 OD

7. Based on the calculated A600nm of the original bacterial suspension, calculate the amount of original bacterial suspension required to bring 1 mL of solution to A600nm = 3.0 OD.

8. Add 1 mL 10 mM Hepes buffer pH 7.5 to a 1.5 mL eppendorf tube. For each cell suspension, first remove a volume of Hepes buffer equal to the amount of cell suspension you are going to add. Then add the appropriate volume of cell suspension; this will ensure that the final volume is 1 mL. These are your corrected bacterial suspensions.

9. Add 135 uL 10 mM Hepes, pH 7.5 into another set of pcr strips. If you have many samples, use a multichannel pipet to do this. Add 15 uL of each corrected bacterial solution into these tubes.

10. Measure and record the A600nm of 100 uL of solution using the UV-VIS on Cheryl’s bench

11. Calculate the A600nm of the corrected bacterial suspension.

12. If the A600nm of the corrected bacterial solution is between 3-4OD, then it is your final bacterial suspension.

13. If the A600nm of the corrected bacterial solution is not between 3-4, then add additional 10 mM Hepes to adjust the A600nm of the corrected bacterial solution to an OD of 3.0-4.0, and re-measure the A600nm of a 1/10 dilution. Once the A600nm is 3-4OD, this is your final bacterial suspension.

14. Add freshly made 1 M sodium dithionite () in water to a final concentration of ~10 mM, e.g. ~10 uL to a 1 mL suspension,  to each final bacterial suspension.

Loading cell suspensions into disposable cuvettes.

15. Prepare enough disposable cuvettes for all your samples and a blank. Note: can be done in advance.

    1. The chambers for holding samples are invitrogen #S24734, these are 22 X 22 mm. These are sealed against a glass coverslip and used as disposible cuvettes. Measurements with known [cyt c] solutions show they have 0.65 mm pathlength.

    2. Wipe clean a 25 x 25 mm #1.5 glass coverslip with a Kimwipe. This step removes debris that make filling the cuvette more difficult.

    3. Remove the transparent plastic backing from the chamber and adhere it to the cleaned 25 x 25 mm #1.5 glass coverslip so that it forms a sealed chamber. (Note: either #1 or #1.5 coverslips work, but #1.5 coverslips are more sturdy.)

    4. Place the sealed chamber plastic side down on a Kimwipe. Use a pair of tweezers to gently seal the adhesive to the coverslip. This is now a disposable cuvette.

    5. Label the cuvette on the upside over the orange plastic with the sample number using a sharpie.

16. Set a pipetman to 400uL, and carefully load 10mM Hepes pH7.5 into a glass-attached chamber until the buffer comes out the opposite port.

17. Dab away any excess fluid with a kimwipe, and seal the holes in all the gaskets using either the Invitrogen-supplied adhesive port covers or ~2mm x 2mm squares of scotch tape.This is your blank.

18. Repeat loading procedure (steps 16 & 17) for each final bacterial suspension into a glass-attached chamber. These are your samples.

19. Put your samples in a plastic tray, then load that tray, a pair of tweezers, and a pair of gloves into a Nano-carrier. Use this to transport your samples to the 4th floor.

Measuring cytochrome c via diffuse reflectance.

20. The Maglite diffuse reflectance system is located on the 4th floor in Rm 4209.The 4th floor keeps their doors locked at all times, so you must be granted access to the 4th floor before you can do diffuse reflectance measurements by yourself.

21. Swap the fiber optic cords so as to connect to the Maglit attachment.

22. Open IndicoPro.

23. Click on New project button.

24. Set the signal averaging settings.

    1. From Spectrum select Spectrum then set the Sample count to 50.

    2. From Project select Instrument Control then set Display to 68 ms. Click ‘Set’ to set this value before exiting the window.

25. Wipe the glass and teflon surfaces clean with a kimwipe.

26. Place the blank, sealed port side up onto the glass plate, and center it over the bottom window just above the light source.

27. Place the black ring and white background on top of blank. Re-center the disposible cuvette so that no orange areas are over the bottom window

28. Turn on the light by pressing the button on front of the Maglite attachment. Check that the sample is illuminated by peeking around the edge of the central ring; you should see a orange glow.

29. Click the “baseline” button in the software.

30. Turn off the light by pressing the button on front of the Maglite attachment, then remove the blank.

31. To view absorbance choose Display –> Log 1/ T -> No Derivative. Also set the x-axis to 400-600nm and the y-axis to -0.05 to 0.1 OD.

32. Put a sample on the glass plate, then repeat steps 26-28.

33. Click on “Scan” button in software.

34. Visually inspect the spectrum to check that the A600nm< 0.065 OD and that the spectra looks like that of a reduced c-type cytochrome.

35. Turn off the light by pressing the button on front of the Maglite attachment, then remove your sample.

36. Important: you need to save each scan as you go. From the pull down menu, select Spectrum –> Save spectrum.

37. Repeat for each sample.

38. Save the project by selecting File -> Save Project.

39. Close the software.

40. Re-open the software and select Open Project. Open your project.

41. Select View, highlight all the files you want to export, and click open.

42. Choose Display –> Log 1/ T -> No Derivative to export absorbance data.

43. Select Spectrum -> Export Spectrum. Then select the columns button, and comma as the field delimiter. Make sure to also include the headers or the wavelengths will not show up in the exported file. Save this file to your USB key.

Analysis to determine the cytochrome c per cell
About the spectra: Reduced cytochromes c have a sharp absorption peak at 552 nm. However, at 552 nm there are contributions to the absorbance due to light scattering by the cell scatter in addition to the absorption of cytochromes c. To remove this unwanted background absorption, we subtract 1.04*A570nm from the A552nm.  The A570nm contains less than 5% of the peaks absorbance contributions from the cytochrome c peak, and the cell scattering at 552 nm is 1.04* the cell scattering at 570 nm (Goldbeck et al, ACS Synthetic Biology 2013).To monitor the cell concentration, we use scattering of light at 600nm (A600nm). Previous measurements (Goldbeck et al, ACS Synthetic Biology 2013) have shown that A600nm and the cell density are directly proportional (linear with a zero intercept) when the A600nm is less than 0.0650 OD.

1. Open Excel. Open the txt file in Excel.

Copy the absorbance at 552, 570 and 600nm for each sample.

2. By copying and transposing this data, create a table containing the strain descriptions alongside the values of A552nm, A570nm and A600nm.

3. Calculate the background corrected, A552nm/A600nm using the formula: A552nm,corr/A600nm = (A552nm – 1.04*A570nm)/A600nm. Note: this correction is for aerobically grown E. coli BL21(DE3) and C43(DE3) strains. If you are using a different cell type, you will need to assess the background absorbance at A552nm by measuring the A552nm for a series of different cell dilutions.

4. Calculate the concentration of cyt c.

   1. Using known [equine cyt c], we have shown that the corrected A552nnm (A552nm,corrected = A552nm – 1.04*A570nm) is linearly related  to the [cyt c]. Specifically, the A552nm,corrected is proportional to the extinction coefficient of the cyt c at 552 (ε; for equine cyt c this is 24300 OD*M-1*cm-1 at 552nm, the concentration of cyt c, and the effective pathlength(b’, which we have measured to be 0.08 cm): A552nm,corrected=ε*b’*[cyt c]

   2. Therefore, [cyt c] (uM/OD600) = (A552nm,corr/A600nm)/(ε*b’).

   3. This relationship only holds for low spin Fe2+.

5. Compare A552nm, norm across [IPTG].

6. An example of this file is saved in Google Docs as: DiffuseReflectanceAnalysis

Clean-up

1. Suspensions of living cells should be inactivated by the addition of 10% bleach for 15 minutes before disposal.

2. Place the used cuvettes in the red sharps containers.