Isolation of PDIM

ISOLATION OF PDIM FROM M.MARINUM

Description: This will walk you through the entire PDIM isolation process. I recommend first attempting to isolate crude lipids from 1L of bacteria. You should be able to easily identify PDIM by analytical TLC. If successful I recommend doing preparative TLC on 20mg of crude lipid. You will most likely need 2-3 rounds of purification by preparative TLC until the compound looks relatively clean by proton NMR. Compare to the published NMR in the eLife paper. 

Preparative TLC is a challenging technique. It can be difficult to load a plate but you’ll get better with practice. Don’t give up. Importantly if for some reason you make a mistake you can always recover crude lipid from the plate. In fact, I recommend cutting off and extracting the non-PDIM fractions from your fist few plates to see how much PDIM you might have missed. 

Media Preparation.

If you’re going to be isolating a lot of PDIM, you’ll need a lot of media to grow up all that mycobacteria. I suggest using GAS medium instead of traditional 7H9 to save on costs:

Recipe (%weight/volume) for glycerol-alanine-salts (GAS) medium:

0.03% BactoCasitone (BD Science) 

0.005% ferric ammonium citrate (Sigma)

0.4% potassium phosphate dibasic anhydrous (VWR)

0.2% citric acid, anhydrous (VWR)

0.1% L-alanine (Sigma) 

0.12% magnesium chloride, heptahydrate (VWR)

0.06% potassium sulfate (VWR)

0.2% ammonium chloride (VWR)

1% glycerol.

18 mM sodium hydroxide

All in Milli-Q water pH 6.6

• Make in an autoclave ready and bacterial growth ready vessel. Such as an Erlenmeyer flask
• To make: Add salts to water (final volume minus one tenth) and stir. 
• Once salts dissolve add glycerol 
• Add concentrated reagent grade sodium hydroxide to get a final 18mM concentration. 
• Cover appropriately and autoclave using a normal short liquid run.
• If adding antibiotics, do so once the solution has cooled. 

Growth of M. marinum

Note: 0.05% Tween80 can be added during the 5 mL and 50 mL stationary cultures. This will make measuring the OD easier. Do not add Tween to your 1L culture. You will get more PDIM buildup on the surface of the bacteria in the absence of tween. 

Scrapings from frozen stocks of M. marinum were used to seed 5 mL stationary cultures in T25 tissue culture flasks and were grown at 33C until reaching OD600 of ~1.0. Stationary cultures are grown with the flasks on their side to maximize surface area. This may take a few days or up to a few weeks depending on how much bacteria you seed. 

Seed cultures were then split between two 50 mL cultures in T75 tissue culture flasks. These were also grown stationary at 33C until reaching OD600 of around 1.0. (~2-4 days). 

These two 50mL cultures are then used to seed a 1L culture. These are then grown with low shaking (<120 rpm in a typical bacterial shaker incubator), protect from light to prevent production of phytols which can be difficult to separate from PDIM (they are bright orange so you’ll see them). 

Bacteria are then pelleted by centrifugation 1000 – 10000 x g, 10 min across all speeds should give a good pellet. 

Next you want bacteria to be as dry as possible. If you have access to a lyophilizer in the appropriate biosafety level space then use it. If not, a good alternative is to blow the pellets dry with nitrogen, or even just letting the pellets sit over the weekend (or longer, PDIM is incredibly stable) exposed to air in a biosafety hood.  

If using a lyophilizer, freeze pellets in 50mL conicals, then attach a 0.2 micron filter to the top of the 50mL conical, then place this in the chamber for the lyophilizer and lyophilize overnight. 

Place dry bacterial pellet in an Erlenmyer flask. Add enough petroleum ether to cover pellet. I found that using a 250mL flask with around 50-100mL of peteroleum ether works. And stir for 1 hr at 23°C.

Allow bacteria to settle and decant petroleum ether into another glass vessel for temporary holding, leave behind as much of the pellet/sludge as possible. Add another volume of petroleum ether to pellet and extract again. Repeat as needed, I found 3x to be sufficient. Can always check progress via analytical TLC. 

The solvent extract is then passed through a 0.2 µm PTFE filter. Syringe filters work fine just mind the syringe you’re using. A lot of brands will lubricate their syringes with PDIM-similar fats. Give them a prerinse or two with pet ether. For best results use a glass filter with replaceable filter membranes. 

Filtered Crude lipids were concentrated under reduced pressure. I used a 500 mL round bottom with <300 mL pet ether on a rotovap. Pet ether is very volatile so start with low vacuum. Transfer lipids to tarred glass vial. Use dichloromethane or chloroform to transfer lipids/work with lipids. Dry in glass vial and record weight. 

Perform an analytical TLC. Dissolve crude lipid from 1L of bacteria in around 1 mL of DCM. Add a spot to the TLC plate (see eLife paper for plate type). Put in TLC chamber and run with 98:2 petroleum ether:ethyl acetate, run once or twice – see supplemental figure 1. 

Develop TLC: While holding the plate with long reach tweezers, spray with 10% CuSO4 in 1.3M phosphoric acid in water so just the surface is coated. Perform in hood with a tin-foil lined box as a backdrop. Then heat with a heat-gun, applying heat to the silicon side. You’ll get some nice colors showing up. PDIM will be the top band. 

Preparative TLC

Prepare chamber:

Make sure you have a large TLC glass chamber that can accommodate the entire plate (example link). Add around 50 mL of 98:2 petroleum ether: ethyl acetate and cover. 

Prepare plate:

Mark up an entire TLC plate, add a line across the plate 1 inch from the bottom. To this line add dashes 1 cm from each side.

Prepare Sample:

Add around 100-300 ul of dichloromethane to crude lipids. Use a flame drawn out pipet broken to allow a minimal amount of solvent to escape. Backfill your syringe twice with DCM to introduce vapor, then take up around 1/5^th of your sample. Using a consistent motion, slowly expel your sample while moving the pipet across the plate, following the lines you’ve drawn. Be sure to start and stop at the 1 cm dashes you’ve drawn. That 1 cm will allow for sample edge drift.

Run TLC

Add a line, let it dry, and repeat this process until your sample is gone. Add another 100ul of DCM to your sample and do it again, and then add another 100ul and do it again. Let the plate dry completely and then put into TLC chamber and cover. Run TLC until solvent edge is almost to plate edge. Remove plate and let dry. (A freshly dried plate can be held to the light, a slightly darker band can be seen where one expects PDIM. Try outlining with a pencil at this time.)

Using a plate cutter, cut either side of the plate at the 2 cm mark. Scar and break. Spray the broken off slides with CuSO4 development spray, and char to develop. Place developed sides next to plate. Mark dashes just above and below where PDIM should be, draw lines. Using a razor blade, scrap the silica off onto filter or paper. Then extract using DCM or 5% methanol in chloroform. You can recover solvent using a glass silica filter fitted onto a round bottom flask. Alternatively, you can use scintillation vial with a small piece of cotton upstream of an 18g needle. Spot extractions for an analytical to make sure you get all the PDIM. 

(Did your prespray outline match up with PDIM? Once you get good at this you can skip the side cut and spray.)

Dry and weigh lipid. Take an NMR! You’ll probably need to perform the preparative TLC 2x more to get pure sample.

Average PDIM isolated from M. marinum was 60 mg per 10 liters of culture. ¹H NMR (500 MHz, CDCl₃) δ 4.93–4.87 (m, 2H), 3.32 (s, 3H), 2.87–2.83 (m, 1H), 2.57–2.51 (m, 2H), 1.90–0.81 (m, 166H). ¹³C NMR (126 MHz, CDCl₃) δ 176.69, 176.63, 86.78, 86.73, 77.41, 77.37, 77.16, 76.90, 70.72, 70.67, 57.51, 45.56, 45.51, 41.41, 41.33, 38.56, 37.85, 37.73, 37.09, 36.77, 34.91, 34.18, 34.07, 32.83, 32.08, 30.24, 29.96, 29.87, 29.82, 29.75, 29.62, 29.53, 28.34, 27.64, 27.36, 27.25, 27.16, 27.12, 25.74, 25.35, 22.85, 22.74, 22.43, 20.81, 20.57, 20.52, 20.37, 20.27, 18.67, 18.58, 18.53, 14.80, 14.75, 14.28, 14.22, 10.26, 1.16. MALDI-TOF for: C82H162O5 Calc’d [M+Na⁺]=1250.23; found 1250.41. C84H166O5 Calc’d [M+Na⁺]=1278.26, found 1278.40. C86H170O5 Calc’d [M+Na⁺]=1306.29, found 1306.55.