- Protocols -

Note: Neodymium (Nd) is used for all experiments, but the first four lanthanides (La, Ce, Pr, Nd) are expected to be able to be used effectively interchangeably for all protocols.

Swim Plates

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A widely used protocol in the literature for observing qualitatively whether a chemical acts as a chemoattractant or chemorepellent for a bacteria. This variant requires growth of bacteria, so metabolisable chemoattractants influence the result. We chose this protocol because it best mirrors the setup for our proof of concept.

A set of chemotaxis protocols can be found in this review by R. E. Parales and J. L. Ditty. Each has its own merits and flaws, and the correct protocol should be picked according to purpose.

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Attractant plugs

1. 2% w/v agar plates need to be prepared with the same composition as the respective swim plates (see below), but including 1mM attractant. If heat sensitive chemicals are required, 2X melted agar should be added to the components, instead of adding agar and then melting. The plates should have depth – we recommend 20mL media for most plates.
2. After setting, plugs are excised from these plates using a P1000 pipette tip. First, ethanol should be used to sterilise a pair of scissors, and the person's gloves. After the ethanol has evaporated, a few cm of the end of the pippette tip is cut off. Then, the other end (where the micropipette is usually inserted) is stabbed into the plate and twisted. A plug of agar should then be stuck in the tip, which can be pushed out into a swim plate using a thin glass rod, wooden applicator stick or similar.

One attractant plate can be used for about a hundred plugs; plates can be stored in the fridge.

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Swim plates

1. 0.3% w/v agar plates containing any necessary antibiotics or inducers need to be prepared. If heat-sensitive chemicals are required, 2X melted agar should be added to the components, instead of adding agar powder and then melting. The plates should have depth – we recommend 20mL media for most plates.
2. The agar needs to set slightly, so that it is viscous, after which a plug is added from an attractant plate. The plug should be placed about halfway between the centre of the plate and an edge. The area can be marked with a pen before adding the plug for ease of positioning.
3. Once the agar is set completely, it will still wobble, but the plug should not be able to change position in the plate.
4. The plate is inoculated in the centre and incubated until growth is visible.

If the chemical in the plug serves as an attractant, the resultant growth will be oblong towards the plug. If it does not, the growth will be radial. The ratio of the distance from 1) Inoculation point to closest point to plug; and 2) Inoculation point to furthest point from plug, can be used to quantify the strength of attraction.

Uptake Assay

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An assay of our own design that facilitates measurement of neodymium uptake by cell cultures. This assay can be standardised by cell density to measure neodymium concentration per cell.

1. Strains to be assayed are grown in media that does not contain Nd, until stationary. A WT control needs to be included.
2. Early in the day, the cultures are diluted to OD = 0.3, then grown with agitation at 30°C until the cells are in late exponential phase (OD = 0.6-0.9). This takes about 6 hours (M. extorquens has a doubling time of 3-4hrs, after its lag phase).
3. An appropriate amount of Nd is added (5ppm is good), and any required inducers (e.g. IPTG). The culture is then incubated at 15°C with agitation overnight.
4. Next morning, the cells are removed and centrifuged at 4000rpm for 10 minutes. The supernatant can be discarded and the pellet is resuspended in a volume of deionised water.
5. The washing step is repeated. OPTIONAL: The washing step can be repeated more times to remove all the Nd not inside the cells.
6. The washing step is repeated by resuspending in 1/100 of the volume. The sample needs to then be frozen.

The sample can now be assayed for Nd concentration. The cells need to be lysed prior to the assay if using quantification methods that cannot measure Nd inside of cells; or that do not liberate Nd from the cells normally.

Lanthanide Removal Assay

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An assay of our own design that measures the reduction in neodymium concentration in media across several growths. This assay is more sensitive than the uptake assay at lower concentrations of neodymium in starting media, but cannot be normalised by cell density.

1. 6ml media containing methanol or methanol and succinate are prepared. Any appropriate antibiotics, neodymium to the desired starting concentration (5ppm is good), and any necessary additional chemicals (e.g. 1mM IPTG if characterising an inducible strain) need to be added.
2. A 200µL aliquot needs to be frozen. This should have [Nd] = 5ppm. The aliquot is labeled with ‘0 previous growths’
3. The rest of the media is inoculated with the appropriate colony. A WT control should also be made.
4. The culture is grown at 30°C with agitation.
5. After approximately 3 days, or until cells are in stationary phase, the culture is centrifuged at 4000rpm for 10min, after which the supernatant is transferred to a new falcon tube.
6. A new 200µL aliquot is frozen, and the tube is labeled with the appropriate number of previous growths (initially, 1).
7. OPTIONAL: The supernatant should still contain bacteria, but it can be reinoculated with the same strain to ensure regrowth.
8. The growth, centrifugation, and aliquoting process is repeated until cells no longer grow (we have found this to occur after approx. 2-3 previous growths).

The aliquots can then be assayed for Nd concentration. Nd concentration should start at 5ppm before any growths, and decrease after each growth, as cells remove Nd from the medium.

Purification of the Ln-Dependent Methanol Dehydrogenase XoxF

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XoxF is a 65kDa protein that dimerises, which has several disulfide bonds. This renders it insoluble in basic protein expression systems. We recommend purifying it as described in J. Huang et al, although a thioredoxin fusion tag in an E. coli expression system may keep the protein soluble – we have not tested this.

No lanthanides should be included in the growth media or induction media, or else the protein will saturate before the assay begins. Trace lanthanide concentration about 133nM (19.2ppb) per 1mg/ml protein will also interfere with the sensitivity of the assay, and trace concentrations above 1µM (144ppb) per 1mg/ml protein will effectively prevent the assay from yielding any useful results.

Nd Quantification Assay using the Ln-Dependent Methanol Dehydrogenase XoxF

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The chemistry of this assay is well characterised in the literature, but we provide an adapted protocol for neodymium quantification. This assay requires the use of a spectrophotometer in a dark room, because one of the chemicals (PMS) is highly light sensitive.

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Stocks

300ml of assay buffer is made up in deionised H₂ and stored at room temperature.

• 100mM Tris-HCl pH 9 (or close)
• 45mM NH4Cl

Between 5ml and 15ml of 20X additional chemicals need to be made up in deionised H₂ individually.

• 3mM DCPIP (in the dark), stored at room temperature, in the dark.
• 20mM PMS (in the dark), stored at below -15°C, in the dark.
• 200mM Methanol, stored in a fume cupboard.

Between 5ml and 15ml of 1X protein cofactors need to be made up in deionised H₂O individually.

• 15µM Nd, storekd at room temperature.
• 18µM PQQ, unless using a PQQ synthesising expression system (e.g. expressing in M. extorquens), stored at 4°C.

As necessary, Master Mix is made in the dark and mixed by shaking. The Master Mix should be discarded after use instead of stored.

• 17 volumes assay buffer
• 1 volume 20X PMS
• 1 volume 20X Methanol
• 1 volume 20X DCPIP

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DCPIP calibration curve

In 1ml 10mm cuvettes, absorbance is recorded at 600nm of the following dilution series. Dilution is done with Master Mix (replacing the volume of DCPIP with assay buffer):

Volume Master Mix (µL)	Volume 20X DCPIP (µL)	[DCPIP] (µM)
950	50	150
960	40	120
970	30	90
980	20	60
990	10	30
1000	0	0 (BLANK)

Molar extinction coefficient can be calculated from this dilution curve. Existing values can be found here, as summarised by B. Jahn et al. We recommend making your own dilution curve, especially if measuring at different pHs or temperatures.

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Nd quantification calibration curve

1. A dilution series of Nd is created from 15µM (Stock) to 0µM in steps of 3µM, in deionised H₂O. 10µL will be needed per repeat at each concentration for a 1mg/ml protein preparation.
2. To a 1ml 10mm cuvette, 1000µL Master Mix is added, without DCPIP. This will serve as a blank.
3. For each Nd concentration, a 30µL protein mix is prepared, containing 10µL protein preparation at 1 mg/ml, 10µL 18µM PQQ and 10µL Nd at the appropriate dilution.
4. For each Nd concentration, 970µL Master Mix is added to a 1ml 10mm cuvette. When ready to start a new reaction, the absorbance of this cuvette is measured every 30 seconds for 2 minutes. This will record the background oxidation/degradation of DCPIP in the absence of protein. We have observed no meaningful change in absorbance in this period.
5. After these 2 minutes, the 30µL protein mix is added and mixed by pipetting up and down. Absorbance is recorded every 15 seconds for 3 minutes.
6. Absorbance is plotted over time to calculate rates. The rates should increase linearly with Nd concentration, such that a calibration curve of rate against Nd concentration can be plotted.

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Measuring samples

Samples need to be thoroughly homogenous, and if they come from M. extorquens all native XoxF needs to be inactivated. This can usually be achieved easily through sonication at high power, and filtering the mixture through a <10kDa filter.

Preparation of Electrocompetent Methylobacterium extorquens

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M. extorquens has no tools for making it competent, so electroporation is used to introduce vectors. The protocol consists of a set of washing and concentration steps.

Many thanks to the Tobias Erb group for providing this protocol.

1. An exponentially growing culture of M. extorquens is collected at OD=1.0 -1.5.
2. After 15 min incubation on ice, the culture is centrifuged at 4°C, 4000 rpm for 15 min.
3. Washing: The pellet is resuspended with 1 volume of cold sterile water, then centrifuged at 4°C, 4000 rpm for 15 min. The washing step is repeated with water.
4. Next, the pellet is resuspended with 1/2 volume of cold, sterile 10% glycerol solution, centrifuged at 4°C, 4000 rpm for 15 min.
5. Finally, the cells are resuspended in 1/100 volume of cold, sterile 10% glycerol solution. 50µl aliquots are frozen with liquid nitrogen and stored at -80°C. i.e. 1 mL for an original culture of 100 mL of media.

Electroporation of Methylobacterium extorquens

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M. extorquens has no tools for making it competent, so electroporation is used to introduce vectors.

Many thanks to the Tobias Erb group for providing this protocol.

1. Competent cells are thawed on ice.
2. ~500 ng of DNA is added (max. 2 µl of plasmid can be added) and mixed by flicking.
3. Cells are afterwards transferred to cold electroporation cuvettes (1mm gap; VWR 732-1135) while avoiding bubbles and on ice.
4. 1 mL of nutrient broth is transferred to a sterile 2mL Eppendorf tube and cooled on ice.
5. The cells are pulsed under the E. coli 1 preset program (1.8 kV, 5 ms time constant).
6. 1 mL of ice-cold nutrient broth is added immediately after, and cells are transferred to a cold 2 mL Eppendorf tube.
7. Following this, cells are incubataded shaking at 30°C for at least 2.5 hours.
8. Cells can now be spread on appropriate plates (e.g. C-source + Antibiotic*):
   • 1:1 - 150 µL directly from the Eppendorf tube
   • 1:10 dilution – 150µL
   • 10:1 concentrated – Eppendorf tube is spun down (~800 µL left) and everything is plated
9. Plates are incubated at 30°C until single colonies appear (5 days is common, can take longer).

Standard media for M. extorquens can be used as nutrient broth.

Media Preparation

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M. extorquens does not grow well in standard media (LB, blood agar, etc.) so a defined medium is used. Depending on purpose, succinate; methanol; or both together can be used as the carbon sources.

Many thanks to the Tobias Erb group for providing this media recipe.

The following solutions were prepared and autoclaved:

Solution name		Stock Solution	Final Media Concentration
Mineral Salts	NH4Cl	4.86g	1.62gL-1 | 30.29mM
	MgSO4•7H2O	0.60g	0.20gL-1 | 0.81mM
	Deionised Water	600mL
Buffer pH 6.7	K2HPO4	4.77g	1.59gL-1 | 9.13mM
	NaH2PO4•2H2O	5.40g	1.80gL-1 | 11.54mM
	Deionised Water	600mL
Succinate stock solution	Succinic acid disodium salt hexahydrate	41.65g	8.33gL-1 | 30.83mM
	Deionised Water	500mL
Water	Deionised Water	700mL

Agar: For plates, 2X Agar bottles (3g/100mL) or (15g/500mL) can be prepared depending on your needs.

The following solutions need to be prepared separately and filter sterilised:

Solution name		Stock Solution	Final Media Concentration
Iron solution (1000X)	Na2EDTA•2H2O	15.0g	15gL-1 | 10.79µM
	FeSO4•7H2O	3.0g	3gL-1 | 10.79µM
	MilliQ water (Add EDTA and FeSO4 to MilliQ and adjust pH to 4.0)	1000mL
Trace element solution (1000X)	ZnSO4•H2O	4.50g	4.50mgL-1 | 15.65 µM
	CoCl2•6H2O	3.00g	3.00mgL-1 | 12.61µM
	MnCl2	0.64g	0.64mgL-1 | 12.61µM
	H3BO3	1.00g	1.00mgL-1 | 16.71µM
	Na2MoO4•2H2O	0.40g	0.40mgL-1 | 16.17µM
	CuSO4•5H2O	0.30g	0.30mgL-1 | 1.20µM
	CaCl2•2H2O	3.00g	3.00mgL-1 | 20.41µM
	MilliQ water (add all trace elements to MilliQ and adjust pH to 1-2 until all salts are dissolved	1000mL

Media preparation for growth on Methanol

	1000mL	200mL
Buffer pH 7.1	300mL	60mL
Sterile water or 2X Agar	493mL	98.6 mL
Sterile MeOH	5mL	1mL
Iron solution	1mL	0.2mL
TE solution	1mL	0.2mL

Media preparation for growth on Succinate

	1000mL	200mL
Buffer pH 6.7	200mL	40mL
Mineral salts	200mL	40mL
Sterile water or 2X Agar	498mL	99.6mL
Succinate stock solution	100mL	20mL
Iron solution	1mL	0.2mL
TE solution	1mL	0.2mL

Media preparation for growth on MeOH AND Succinate: the same preparation as for succinate alone can be used with the addition of MeOH.

Antibiotic concentrations for M. extorquens AM1 (final concentrations)

• Rifampicin (in DMSO): 50µg•mL^-1
• Kanamycin (in H₂O): 50µg•mL^-1
• Tetracycline (in MeOH or 70% EtOH or H₂O): 10µg•mL^-1