TITLE: Quantitative detection of  vitamin B12 in algae by bioassay and ICP-MS/MS
AUTHORS: Sunnyjoy  Dupuis, Stefan Schmollinger, Sabeeha  S. Merchant

[DESCRIPTION]
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.

 

 


[STEPS]
SECTION: E. coli B12 Bioassay
1. 
 

SECTION: E. coli B12 Bioassay
2. Prepare cell culture fractions:
 

SECTION: E. coli B12 Bioassay
2.1. Collect 2 mL  culture into a 2 ml screw-cap tube. 
 


SECTION: E. coli B12 Bioassay
2.2. Centrifuge at >8000 rcf, 2 min. Quickly transfer  950 µL of the spent medium supernatant to each of two 1.5 ml screw-cap tubes without disturbing the pellet.

 

SECTION: E. coli B12 Bioassay
2.3. To wash the pellet, resuspend in 1 mL  0.85% NaCl, then centrifuge again at >8000 rcf, 2 min and discard the supernatant.

SECTION: E. coli B12 Bioassay
2.4. Resuspend the pellet in 1.9 mL  0.85% NaCl.
SECTION: E. coli B12 Bioassay
2.5. Boil both cell suspension and spent medium fractions at 100 °C for 10 min to extract B12 from the cells.
 

SECTION: E. coli B12 Bioassay
2.6. Centrifuge all samples at >8000 rcf, 2 min, 4 °C.
 


SECTION: E. coli B12 Bioassay
2.7. Transfer the supernatant into convenient portions (2 or more) for downstream testing. 
 


SECTION: E. coli B12 Bioassay
2.8. Flash freeze all samples in liquid nitrogen to store at -80 °C.

 

SECTION: E. coli B12 Bioassay
45. Prepare   (CNCbl) and   standards:

SECTION: E. coli B12 Bioassay
3. Prepare cyanocobalamin (CNCbl) and methionine standards:
SECTION: E. coli B12 Bioassay
3.1. 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.
 



SECTION: E. coli B12 Bioassay
3.2. Prepare a series of 1 mL methionine standards to 10X the target concentration in milliQ H2O.
 


SECTION: E. coli B12 Bioassay
3.3. Boil standards at 100 °C for 10 min.
 
  

SECTION: E. coli B12 Bioassay
3.4. Centrifuge the standards at >8000 rcf, 2 min, 4 °C.

SECTION: E. coli B12 Bioassay
3.5. Flash freeze the standards in liquid nitrogen to store at -80 °C.
SECTION: E. coli B12 Bioassay
4. Conduct the bioassay:
SECTION: E. coli B12 Bioassay
4.1. 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   for 1440 min to saturation. 
 


 

SECTION: E. coli B12 Bioassay
4.2. 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   for 1440 min to saturation.

 

SECTION: E. coli B12 Bioassay
4.3. 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)
 


SECTION: E. coli B12 Bioassay
4.4. Next, harvest 2 mL of each E. coli pre-culture in snap-cap tubes and pellet by centrifuging at 10000 rcf, 2 min. Discard the supernatant.


SECTION: E. coli B12 Bioassay
4.5. Resuspend in 1 mL sterile 0.85% NaCl, and repeat this wash twice more. 

SECTION: E. coli B12 Bioassay
4.6. Resuspend washed pellet in 1 mL 0.85% NaCl then measure OD600.

SECTION: E. coli B12 Bioassay
4.7. Use washed cells to inoculate the bioassay tubes at a starting OD600 of 0.01.
 

SECTION: E. coli B12 Bioassay
4.8.  Incubate cells at 37 °C   for 1440 min, then read final OD600. 

SECTION: E. coli B12 Bioassay
4.9. 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.
 

SECTION: Sample preparation for ICP-MS Detection of B12-Derived Cobalt
5. Collect and wash samples:
 

SECTION: Sample preparation for ICP-MS Detection of B12-Derived Cobalt
5.1. Collect 1x108 cells in a 50ml falcon tube. 


SECTION: Sample preparation for ICP-MS Detection of B12-Derived Cobalt
5.2. Centrifuge samples  3500 rpm, 3 min. Quickly transfer the supernatant to a clean tube for spent medium analysis. 

 

SECTION: Sample preparation for ICP-MS Detection of B12-Derived Cobalt
5.3. Resuspend the pellet in 5-10 mLof 1mM EDTA pH 8.0 (Washing Buffer 1), then fill up to 50 mL.

 

SECTION: Sample preparation for ICP-MS Detection of B12-Derived Cobalt
5.4. Repeat wash step: centrifuge samples  3500 rpm, 3 min. Quickly discard the supernatant, then resuspend pellet in  5-10 mLof Washing Buffer 1, and fill up to 50 mL.
SECTION: Sample preparation for ICP-MS Detection of B12-Derived Cobalt
5.5. Wash cells again: centrifuge samples  3500 rpm, 3 min. Quickly discard the supernatant, then resuspend pellet in  1 mLof 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.

SECTION: Sample preparation for ICP-MS Detection of B12-Derived Cobalt
5.6. Centrifuge samples  3500 rpm, 3 min. Quickly discard the supernatant, then resuspend the pellet  in 2-5 mL of Millipore water, then fill up to 10 mL of Millipore water.

SECTION: Sample preparation for ICP-MS Detection of B12-Derived Cobalt
5.7. Centrifuge samples  3500 rpm, 3 min. Quickly discard the supernatant. Then centrifuge samples  3500 rpm, 1 min, and remove the remaining supernatant completely using a filtered pipette tip.
SECTION: Sample preparation for ICP-MS Detection of B12-Derived Cobalt
5.8. Store the dry pellet and the spent medium at -20 °C until further processing.

SECTION: Sample preparation for ICP-MS Detection of B12-Derived Cobalt
6. Digest cell pellet samples:
SECTION: Sample preparation for ICP-MS Detection of B12-Derived Cobalt
6.1. Thaw pellet at 65 Room temperature, then centrifuge again 3500 rpm, 5 min to compact the pellet. 
SECTION: Sample preparation for ICP-MS Detection of B12-Derived Cobalt
6.2. 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).

 

SECTION: Sample preparation for ICP-MS Detection of B12-Derived Cobalt
6.3. Digest samples for 1440 min at 65 Room temperature, then incubate for 120 min at 65 °C.

SECTION: Sample preparation for ICP-MS Detection of B12-Derived Cobalt
6.4. Add 9.5 mL Millipore water for 2 % HNO3 final concentration. Vortex the sample thoroughly.
Samples are now ready for ICP-MS.

SECTION: Sample preparation for ICP-MS Detection of B12-Derived Cobalt
7. Prepare spent media samples: transfer 2 mL spent medium to a 15ml Falcon tube, and add 5 mLof 2.8% HNO3 for 2% final concentration. Spent media samples are now ready for ICP-MS.

SECTION: Sample preparation for ICP-MS Detection of B12-Derived Cobalt
8.