In vivo metal transport assays

Gaudet Lab - Cobalt Uptake Assay Protocol

Rachelle Gaudet (protocol developed by Wilhelm Weihofen, Anne McCabe, Aaron Bozzi)

Cobalt assay buffer = 60 mM NaCl, 10 mM KCl, 0.5 mM MgCl₂, 50 mM HEPES pH 7.3, 0.216% glucose.

Day before assay: Start overnight cultures of bacteria transformed with the appropriate metal-transporter expression plasmid in appropriate antibiotic-containing medium (for us, LB-Amp (100 µg/mL)).

Day of assay: 

Inoculate LB-Amp cultures 1/50 from overnights and grow until cells reach an OD(600 nm) of approximately 0.6 (shaking at 225 rpm at 37°C—usually takes 2-2.5 hours).  Use at least 1 mL of media for every well I want to have cells for in the assay (so if you want 12 wells, grow cells in ~15 mL of media).

Induce cells with 1/100 volume of 10 mM IPTG (100 µM final concentration) and shake for an additional 60-75 minutes at 37°C.

Take cells out of warm room and measure OD₆₀₀ (usually OD = 0.8 to 1.4). Calculate the volume of cells you need (and normalize for different culture densities). Use the equivalent of 1 mL of OD = 1.0 of cells per well of a 96 well assay plate. 

Spin down cells (5-10 min, 2-3K rpm), remove media. 

Optional: wash cell pellets by resuspending in cobalt assay buffer and spinning down again.

Resuspend cells in (# of wells X 0.19 mL) cobalt assay buffer. 

Aliquot 190 µL cells per well to a 96 well clear round-bottom plate. 

Warm plate in warm room (37°C).

Add 10 µl of 20X metal (I use a multichannel) to cells and pipet up and down 8-12 times to mix.  For a time-course experiment, the 20X metal stock is 4 mM Co(NO₃)₂ [or 4 mM FeSO₄ + 4 mM ascorbic acid]. For a metal competition experiment, use 4 mM Co(NO₃)₂ mixed with a range of concentration of competing metals (CdCl₂, MnCl₂, ZnCl₂, CaCl₂, MgCl₂ – see our 2016 PNAS and 2019 eLife publications). 

For a competition experiment, just add all the cobalt/other metal mixtures at once and then incubate for 60 minutes at 37°C before quenching with an excess of EDTA (over total divalents— use 10 µL of 100 mM EDTA unless adding a ton of competing Mg²⁺ or Ca²⁺, then use more like 20 µL of 100 mM EDTA). 

For a time-course experiment, stagger the additions of metal and then quench with EDTA sequentially at the end. For example:

Note: For Fe²⁺ uptake, for which the Deinococcus radiodurans MntH transporter has faster kinetics, we typically use shorter time points: 0, 0.5, 1, 2, 4, 8, 15, and 30 min.

After quenching all the wells with EDTA, spin down plate for 5-6 min at 2K rpm. Remove supernatant, resuspend cells in 180 µL cobalt assay buffer by pipetting up and down with multichannel, spin down again.

Repeat for total of 3 washes (probably okay with just 1-2 washes though).

In the fume hood, resuspend cell pellets in 1% (NH₄)₂S (in cobalt assay buffer). Incubate plate at 37°C for 5 min.

Spin down plate for 6-7 min at 2K rpm, remove supernatant carefully. We usually seal the top of the plate with clear tape and then scan it from below. Do not invert the plate as the pellet will run/smear. 

Data processing and analysis:

Use ImageJ64 to quantify the relative darkness of the pellets (convert 600 ppi scan PNG image to 32 bit grayscale and invert so the dark pellets become white, then use the circle tool to measure the darkness of the pellet).

We subtract the no metal (0 min timepoint) darkness for each mutant from the remaining wells where metal was added, and we normalize the intermediate timepoints to %WT 60 minute darkness.