Apr 21, 2022
Quantitative detection of vitamin B12 in algae by bioassay and ICP-MS/MS
- Sunnyjoy Dupuis1,
- Stefan Schmollinger1,
- Sabeeha S. Merchant1
- 1University of California, Berkeley
- Merchant Lab UC Berkeley
Protocol Citation: Sunnyjoy Dupuis, Stefan Schmollinger, Sabeeha S. Merchant 2022. Quantitative detection of vitamin B12 in algae by bioassay and ICP-MS/MS. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn7726v5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: February 03, 2022
Last Modified: July 07, 2023
Protocol Integer ID: 57727
Keywords: vitamin B12, cyanocobalamin, cobalt, ICPMS, bioassay, chlamydomonas
Funders Acknowledgements:
Sabeeha Merchant Symbiosis in Aquatic Systems Initiative Investigator Award
Grant ID: GBMF9203
Disclaimer
E. coli strains were generously provided by Michi Taga.
Abstract
This protocol describes two methods for determining the amount of vitamin B12 present in the spent medium and cell lysate of algae cultures. The first method is a bioassay, adapted from Mok, Hallberg, & Taga (2022), which estimates the B12 concentration in solution from the growth of a B12-requiring Escherichia coli mutant. The second method uses the direct detection of cobalt via Inductively Coupled Plasma Mass Spectrometry (ICP-MS/MS) as a proxy for vitamin B12. We describe the preparation of spent medium and cell extract fractions from the chlorophyte alga Chlamydomonas reinhardtii for each method, preparation of standard cyanocobalamin solutions, and the correlation between cobalt and cyanocobalamin in algal cells.
We thank Michi Taga and Alison Smith for their guidance in optimizing the bioassay for C. reinhardtii.
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Protocol materials
Cyanocobalamin
MethionineP212121
E. coli B12 Bioassay
E. coli B12 Bioassay
16m
16m
Note
This protocol makes use of the Escherichia coli ΔmetE and ΔmetEΔmetH mutant strains. MetE is the cobalamin-independent homocysteine transmethylase, and MetH is a cobalamin-dependent methionine synthase. The E. coli ΔmetE strain requires either methionine or vitamin B12 supplementation for growth. The ΔmetEΔmetH strain can only grow when provided methionine. Samples of interest are provided to both ΔmetE and ΔmetEΔmetH, and the growth of each is compared to that supported by either vitamin B12 standards or methionine standards, respectively. Thus B12 concentration in a solution can be measured and can be distinguished from methionine.
1w
Prepare cell culture fractions:
Note
The following procedure is effective for the preparation of spent medium and cell lysate from both the chlorophyte Chlamydomonas reinhardtii (both cell wall containing and cell wall reduced strains) as well as the α-proteobacteria Mesorhizobium loti and Sinorhizobium meliloti when grown in minimal media.
1h
Collect 2 mL culture into a 2 ml screw-cap tube.
Note
We use USA Scientific 2.0ml Self-Standing Grad Microcentrifuge Tubes, Catalogue Number 1420-9700.
Snap-cap tubes can be used if desired, but are prone to popping open during boiling (Step 2.5). If snap-cap tubes are used, secure lids with a special tube rack or a weight while boiling.
Centrifuge at >8000 rcf, 00:02:00 . Quickly transfer 950 µL of the spent medium supernatant to each of two 1.5 ml screw-cap tubes without disturbing the pellet.
Note
Work quickly to prevent cells from dispersing from the pellet and contaminating the supernatant fraction. This is especially important for motile strains.
2m
To wash the pellet, resuspend in 1 mL 0.85% NaCl, then centrifuge again at >8000 rcf, 00:02:00 and discard the supernatant.
2m
Resuspend the pellet in 1.9 mL 0.85% NaCl.
Boil both cell suspension and spent medium fractions at 100 °C for 00:10:00 to extract B12 from the cells.
10m
Centrifuge all samples at >8000 rcf, 4°C, 00:02:00 .
Note
This centrifugation step should be performed cold to decrease the temperature of the samples before further handling.
2m
Transfer the supernatant into convenient portions (2 or more) for downstream testing.
Note
This is to avoid repeated freeze-thaw of samples.
Flash freeze all samples in liquid nitrogen to store at -80 °C .
Note
Ensure tubes are sealed tightly to avoid sample loss during freezing or thawing.
Prepare cyanocobalamin (CNCbl) and methionine standards:
1h
Prepare a series of CNCbl standards up to 10X the target concentration in purified water (Millipore) by serial dilution. Aliquot at least 250 µL of each standard into screw-cap tubes.
Note
We have found serial dilutions to the following concentrations produces a useful range of 10X CNCbl standards: 900, 600, 400, 267, 178, 119, 79, 53, 35, 23 ng/L.
Standards can also be prepared in minimal media that were used for test samples if desired. However, this should not impact results, since B12 will be the limiting nutrient for E. coli growth during the bioassay.
Prepare a series of 1 mL methionine standards to 10X the target concentration in milliQ H2O.
Note
We have found serial dilutions of the following concentrations produces a useful range of 10X methionine standards: 300, 150, 75, 38, 19 mg/L
Boil standards at 100 °C for 00:10:00 .
Note
This ensures that any degradation of B12 or methionine that may occur in the samples also occurs in the standards. However, we have shown that boiling for 10 min does not significantly decrease the amount of CNCbl detected via this bioassay:
Boiling does not impact B12 detected by bioassay.
10m
Centrifuge the standards at >8000 rcf, 4°C, 00:02:00 .
2m
Flash freeze the standards in liquid nitrogen to store at -80 °C .
Conduct the bioassay:
4d
Inoculate starter cultures from single colonies of ΔmetE and ΔmetEΔmetH mutant strains from LB agar plates into2 mL of M9 + 0.2% glucose minimal medium with 1 mg/mL methionine. Grow at 37 °C 250 rpm for 24:00:00 to saturation.
Note
We use Falcon 14 mL Polystyrene Round-Bottom Tubes, Catalogue Number 352051 for all E. coli cultures.
Note
M9 recipe can be found here: http://cshprotocols.cshlp.org/content/2010/8/pdb.rec12295.short
1d
Generate pre-cultures by transferring 1% (vol/vol) of the saturated starter cultures into fresh 2 mL M9 + 0.2% glucose minimal medium with 1 mg/mL methionine and grow at 37 °C 250 rpm for 24:00:00 to saturation.
Note
Each bioassay culture will require ~20 uL of 2X pre-culture (Step 4.7), so plan the number of 2 mL pre-cultures you will need to provide sufficient inoculum for the bioassay.
1d
On the day of the assay, prepare bioassay cultures: add 1 mL of 2X M9 glucose, the desired amount of your sample or standard, and sterile Millipore water up to 2 mL final volume.
The following control and test cultures should be included:
- Sample to be inoculated with ΔmetE
- Sample to be inoculated with ΔmetEΔmetH
- M9 medium alone without B12 or methionine supplementation for each E. coli strain (important to test efficacy of E. coli innocula washing)
- B12 and methionine standard series for each E. coli strain (important to include with every bioassay you perform, as maximum OD600 of strains can vary slightly from assay to assay)
- Controls for the highest concentrations of B12 and methionine in the standard series, and for one unknown sample, that will not be inoculated with E. coli (important to test for contamination or failure to kill study species during sample preparation)
Note
Complete this step prior to harvesting E. coli pre-cultures.
Next, harvest 2 mL of each E. coli pre-culture in snap-cap tubes and pellet by centrifuging at 10000 rcf, 00:02:00 . Discard the supernatant.
2m
Resuspend in 1 mL sterile 0.85% NaCl, and repeat this wash twice more.
6m
Resuspend washed pellet in 1 mL 0.85% NaCl then measure OD600.
2m
Use washed cells to inoculate the bioassay tubes at a starting OD600 of 0.01.
Note
The volume of washed inocula needed per should be 10-30 uL.
20m
Incubate cells at 37 °C 250 rpm for 24:00:00 , then read final OD600.
1d
Establish a standard curve that correlates the provided B12 and methionine concentrations in the standards to the OD600 of each E. coli strain. Determine the amount of B12 and methionine in your unknown samples by comparing the OD600 reached by each strain to the standard curves.
Expected result
Example standard curves for cyanocobalamin and methionine.
Sample preparation for ICP-MS Detection of B12-Derived Cobalt
Sample preparation for ICP-MS Detection of B12-Derived Cobalt
1d 2h 21m
1d 2h 21m
Collect and wash samples:
Note
This protocol is effective for preparing spent media and cells of Chlamydomonas reinhardtii for ICP-MS/MS elemental analysis. The recommended cell densities are useful to ensure effective cell digestion and proper signal-to-noise for detecting elements of interest.
It is important to work in a clean environment free from dust, which could contaminate samples. All reagents should be prepared with trace-metal grade chemicals, water, and vessels. Avoid unnecessary exposure of samples, tubes, and pipettes to air.
If clean tubes and pipettes are not available, they can be prepared using the following steps:
- Soak pipettes and tubes in detergent overnight.
- Rinse thoroughly with de-ionized water.
- Submerge in 10 % nitric acid (normal grade) and soak for a week at 50 °C .
- Rinse thoroughly with Millipore grade water.
- Avoid unnecessary exposure of opened tubes to air.
Collect 1x108 cells in a 50ml falcon tube.
1m
Centrifuge samples 3500 rpm, 00:03:00 . Quickly transfer the supernatant to a clean tube for spent medium analysis.
Note
Work quickly to prevent cells from dispersing from the pellet and contaminating the supernatant fraction. This is especially important for motile strains.
3m
Resuspend the pellet in 5-10 mL of 1mM EDTA pH 8.0 (Washing Buffer 1), then fill up to 50 mL .
Note
Resuspend cell pellets by swirling or rotating the tube in a large radius with your arms, rather than pipetting or vortexing. This ensures cells will not be sheared during the wash steps.
2m
Repeat wash step: centrifuge samples 3500 rpm, 00:03:00 . Quickly discard the supernatant, then resuspend pellet in 5-10 mL of Washing Buffer 1, and fill up to 50 mL .
5m
Wash cells again: centrifuge samples 3500 rpm, 00:03:00 . Quickly discard the supernatant, then resuspend pellet in 1 mL of Washing Buffer 1, then transfer to a 15 ml Falcon tube. To ensure all cells have been collected, add 5 mL more Washing Buffer 1 to the 50 ml Falcon tube and transfer to the same 15 ml Falcon tube.
6m
Centrifuge samples 3500 rpm, 00:03:00 . Quickly discard the supernatant, then resuspend the pellet in 2-5 mL of Millipore water, then fill up to 10 mL of Millipore water.
5m
Centrifuge samples 3500 rpm, 00:03:00 . Quickly discard the supernatant. Then centrifuge samples 3500 rpm, 00:01:00 , and remove the remaining supernatant completely using a filtered pipette tip.
6m
Store the dry pellet and the spent medium at -20 °C until further processing.
Digest cell pellet samples:
Thaw pellet at Room temperature , then centrifuge again 3500 rpm, 00:05:00 to compact the pellet.
5m
Carefully overlay the pellet with 286 µL trace metal grade nitric acid (we use Fischer Chemical Trace Metal Grade Nitric Acid, Catalogue Number A509-P212).
Note
Take care to leave the compacted pellet undisturbed, as any cell material dissolving from the pellet will not be digested properly.
1m
Digest samples for 24:00:00 at Room temperature , then incubate for 02:00:00 at 65 °C .
1d 2h
Add 9.5 mL Millipore water for 2 % HNO3 final concentration. Vortex the sample thoroughly.
Samples are now ready for ICP-MS.
2m
Prepare spent media samples: transfer 2 mL spent medium to a 15ml Falcon tube, and add 5 mL of 2.8% HNO3 for 2% final concentration. Spent media samples are now ready for ICP-MS.
1m
(A) Chemical structure of cyanocobalamin showing coordinated cobalt atom. (B) Correlation between intracellular cobalt detected by ICP-MS, normalized per sulfur as a proxy for biomass, and intracellular B12 detected using the E. coli bioassay in C. reinhardtii.
Note that ICP-MS detection of vitamin B12 is only suitable for eukaryotic cells that are not supplied with any other source of cobalt in their growth medium.
Citations
Kenny C. Mok, Zachary F. Hallberg, Michiko E. Taga. Purification and detection of vitamin B12 analogs
https://doi.org/10.1016/bs.mie.2021.11.023Kirmiz N, Galindo K, Cross KL, Luna E, Rhoades N, Podar M, Flores GE. Comparative Genomics Guides Elucidation of Vitamin B12 Biosynthesis in Novel Human-Associated Akkermansia Strains.
https://doi.org/pii:e02117-19.10.1128/AEM.02117-19