• Add components to ddH₂O and adjust volume to 1 L.
• Autoclave for 20 min at 121°C.

• Add components to ddH₂O, adjust the pH to 7.0 and bring the volume to 987 mL.
• Autoclave for 20 min at 121°C.
• Add the following

The dehydrated medium is distributed by Laboratories Conda (Cat. 1542) and was prepared according to protocol.

Mineral medium with additon of yeast extract

• Add components to ddH₂O, adjust the pH to 7.5 and bring the volume to 1 L.
• 100 mM glycerine are added seperatly to the medium.

SL-4: Trace element solution (Pfenning and Lippert, 1966)

• Add components to ddH₂O, adjust the pH to 6.7 and bring the volume to 1 L.
• The solution is stored at 4°C and protected from light.

• Add components to ddH₂O, adjust the pH to 6.8-7.2 and bring the volume to 1 L.
• 50 mM glucose have to be added seperatly before the inoculation.

For preperation of competent cells and cloning, the E. coli strains DH5α and TOP10 are grown in LB medium at 37°C.

For expression studies of constructs in pBAD202, E. coli TOP cells are grown in LB medium supplemented with 1 mg/mL Vitamin B₁₂ and 50 µg/mL kanamycin.

For preperation of competent cells and cloning, R. platicola is grown in LB medium at its temperature optimum of 30°C.

For expression studies of constructs in pBAD202, R. planticola is grown under anaerobic conditions in MM medium supplemented with 50 µg/mL kanamycin.

For preperation of competent cells and cloning, the S. blattae is grown in LB medium at 37°C.

For expression studies of constructs in pBAD202, S. blattae was grown under anaerobic conditions in MM medium supplemented with 50 µg/mL kanamycin.

Material to prepare

• 2x 5 mL LB
• 250 mL LB in 1 L Erlenmeyer flask with chicane
• 500 mL sterile Millipore-H₂O at 4°C
• 50 mL sterile glycerol (10% (w/v)) at 4°C
• 30 sterile labeled Eppendorf cups at – 20°C or -80°C
• 1x sterile GS3 jar at 4°C
• 1x Aquatron at 30°C
• 1x Sorvall RC6 centrifuge at 4°C
• 1x Universal 320R centrifuge at 4°C
• 1 L liquid nitrogen

Day 1

• Inoculate 2x 5 mL LB (if required with antibiotics) with your strain of interest, either from cryo culture or from agar plate.
• Incubate overnight at 37°C and 150 rpm.

Day 2

• Inoculate 250 mL LB (without antibiotics) with 2% (5 mL) preculture.
• Incubate in the Aquatron at 30°C and 160 rpm until OD₆₀₀ of 0.5-0.8.
• Check the culture via microscope for contaminations.

All liquids and containers must be cooled on ice. The major task of cell preparation is the removal of salts. In case of some strains like pLys-strains, the pellets must be resuspended very carefully.

• Let the cells (OD₆₀₀ of 0.5-0.8) cool down in ice water for 10-20 min. All further steps are performed under cool conditions.
• Decant the cells in sterile GS3 jar and centrifuge at 4°C and 5000 rpm (4230 g) for 5-10 min.
• Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 250 mL sterile 4°C ddH₂O.
• Centrifuge the GS3 jar at 4°C and 5000 rpm (4230 g)for 5-10 min.
• Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 250 mL sterile 4°C ddH₂O.
• Centrifuge the GS3 jar at 4°C and 5000 rpm (4230 g)for 5-10 min.
• Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 10 mL sterile 4°C glycerol (10% (w/v)). Afterwards, transfer the culture into a 50 mL Falcon tube.
• Centrifuge the Falcon tube at 4°C and 6200 rpm (4230 g) for 5-10 min.
• Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 10 mL sterile 4°C glycerol (10% (w/v)).
• Centrifuge the Falcon tube at 4°C and 6200 rpm (4230 g) for 5-10 min.
• Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 500 µL sterile 4°C glycerol (10% (w/v)).
• Aliquot the cells into the Eppendorf cups (40 µL per Eppendorf cup). While aliquoting, filled Eppendorf cups must be frozen directly in liquid nitrogen.
• Store the filled Eppendorf cups in a cryobox at -80°C.

• Mix 50-200 ng plasmid DNA with 40 µL of competent cells (always defrost on ice!). Transfer attempt into precooled electroporation cuvette. Incubate on ice for 10 min.
• Important: DNA must be salt free! Use either directly in ddH₂O eluted plasmids, or (e.g. directly after ligation) desalt the DNA for 30 min on a Millipore filter (MF Membrane Filters).

• Electroporation: 2500 V for 2 mm cuvettes (1250 V for 1 mm cuvettes), 25 µF, 200 Ω, discharging time should be 3-5 msec. Contacts of the electroporation cuvette must be dry. Remove all air bubbles before electroporation.
• Immediately add 960 µL liquid LB (or, if available, SOC medium for a higher transformation efficiency) into the cuvette and transfer the content into a 2 ml Eppendorf cup.
• Incubate at 37°C and 150 rpm for 1 h. Fix tube in horizontal position with tape.
• Plate 100 µL of 10^-3 to 10^-6 dilutions on plates with selection pressure
• Incubate at 37°C overnight.

One Shot^®TOP10 Competent cells (ThermoFisher) were transformed following the respective protocol for chemical transformation:

• Add 3 µL of the TOPO^®Cloning Reaction into a vial of competent E. coli cells and mix gently by inverting.
• Incubate on ice for 5-30 min.
• Heat-shock the cells for 30 s at 42°C without shaking.
• Immediately transfer the tubes on ice.
• Add 250 µL of S.O.C. medium (RT), cap the tube tightly and shake it horizontally (200 rpm) at 37°C for 1 h.
• Spread 100-200 µL of different volumes or dilutions from each transformation on a prewarmed selective plate and incubate overnight at 37°C.
• Pick colonies for analysis.

Small scale preparation of plasmid DNA was performed using the NucleoSpin^® Plasmid kit distributed by Macherey-Nagel (REF 740588) according to protocol.

Phusion^® High-Fidelity DNA Polymerase is used for all standard PCR amplifications.

Pipetting scheme

• Mix all components, adding the DNA polymerase last and spin down.

Cycling

• To optimize the annealing temperature for each primer pair, a temperature gradient can be run.

Fusion PCR was performed according to Harrison et al.

Pipetting scheme

• Mix all components, adding the DNA polymerase last and spin down.

Cycling

• To optimize the annealing temperature for each primer pair, a temperature gradient can be run.

16S rDNA PCR was performed to check the identity of the bacterial strains we were working with. For this, colonies were picked and resuspended in 50 µL ddH₂O.

Pipetting scheme

• Mix all components, adding the DNA polymerase last and spin down.

Plasmid DNA and PCR products were digested according to FastDigest protocol (Thermo Scientific).

• Mix gently and spin down.
• Incubate at 37°C in a heat block or water thermostat for X min.
• Inactivate the enzyme by heating for X min at X°C.

Comment: Incubation time and inactivation time and temperature depend on the applied FastDigest enzyme and were done according to the respective protocol.

• If the FastDigest Green Buffer was used in the reaction, an aliquot of the reaction mixture can be loaded directly on an agarose gel.

MutB2 DNA was phosphorylated with T4 Polynucleotide Kinase (T4 PNK) according to protocol (NEB, #M0201).

• Prepare reaction mixture on ice.
• Incubate at 37°C for 30 min.
• Heat inactivate at 65°C for 20 min.

Ligations were either done with T4 DNA ligase distributed by Invitrogen (Cat. No. 15224-017) or by Thermo Scientific (#EL0014).

Invitrogen

• Prepare the following reaction mixture and spin briefly.

• Incubate at 16°C over night.
• Heat-inactivate at 65°C for 10 min.

Thermo Scientific

• Prepare the following reaction mixture and spin briefly.

• Incubate at RT for 1 h or at 16°C over night.
• Heat-inactivate at 65°C for 10 min.

Blunt end ligations were done with T4 DNA ligase distributed by Thermo Scientific (#EL0014).

• Prepare the following reaction mixture and spin briefly.

• Incubate at RT for 1 h.
• Heat-inactivate at 65°C for 10 min.

The D-TOPO^® cloning reaction of pBAD202 was performed according to protocol (Thermo Scientific, K420201). A 0.5:1 to 2:1 molar ratio of PCR product to vector was used.

• Mix reaction gently and incubate for 5 min at RT.
• Place the reaction on ice and proceed directly with the transformation of electrocompetent or chemically competent cells.

DNA purification after PCR and restriction was performed using different kits

• SureClean Plus distributed by Bioline (BIO-37047)
• DNA Clean & Concentrator™-25 distributed by Zymo Research (D4033)
• NucleoSpin^® Gel and PCR Clean-up distributed by Macherey-Nagel (REF740609)

The procedures were done according to the respective protocol.

For agarose gel electrophoresis, 1.0% agarose gels were prepared. For analytical purpose, the gels were run with the parameters U=90 V, I=400 mA, t=60 min. For preparative purpose, the gels were run with the parameters U=120 V, I=400 mA, t=35 min.
Afterwards, the gels were stained in ethidium bromide solution, and then analyzed by UV light.

Gel extractions of DNA were performed using the NucleoSpin^® Gel and PCR Clean-up kit distributed by Macherey-Nagel (REF740609).

In preperation for expression studies, the constructs in pBAD202 were transformed into E. coli TOP10, R. planticola or S. blattae cells. The respective cultivation conditions (medium, temperature) are listed in “Cultivation and Transformation”. The expression assay was done as described in the D-TOPO^® protocol.

• For each transformant, inoculate 2 mL of medium containing 50 µg/mL kanamycin with a single recombinant colony.
• Grow overnight with shaking (225-250 rpm) to OD₆₀₀ = 1-2.
• The next day, label five tubes for each transformant and add 10 mL medium (50 µg/mL kanamycin) to each tube.
• Inocculate each tube with 0.1 mL of the overnight culture.
• Grow the cultures with vigorous shaking to an OD₆₀₀= ~0.5.
• Prepare four 10-fold serial dilutions of 20% arabinose with sterile water (e.g 2%, 0.2%, 0.02%, and 0.002%).
• Remove a 1 mL aliquot of cells from each tube, centrifuge at maximum speed in a microcentrifuge for 30 s and aspirate the supernatant. Freeze the pellet at -20°C as a zero time point sample.
• Add arabinose to the five 9 mL cultures according to the following table.

• Grow with shaking for 4 h.
• Take 1 mL samples at 4 h and treat as described for the zero time point sample.

• Analyze the samples by suspending the pellets in 80 µL 1X SDS-PAGE sample buffer.
• Boil 5 min and centrifuge briefly.
• Load 5-10 µL of each sample on an SDS-PAGE gel and electrophorese.

• Harvest 500 mL of cells from an induction experiment by centrifugation and wash three times with 0.9% NaCl.
• Resuspend the pellet in 2 mL buffer P (pH 8.0) and incubate for 20-30 min on ice.

• Centrifuge for 5 min (14000 rpm, 4°C) and carefully remove the supernatant which contains the periplasm.
• Resuspend the pellet in 2 mL buffer P and incubate for 20-30 min on ice.
• After another centrifugation for 5 min (14000 rpm, 4°C) the supernatant with the spheroplasts can be carfully removed.
• The samples can either be stored at -20°C or directly used for analysis.

Acrylamid Stocksolution (AA)

• 40% Acrylamide 4x cryst.- Mix 37.5:1 (Biomol 54535; stored at 4°C)

Resolving gel Stocksolution (RG)

• Add components to ddH₂O, adjust the pH to 8.8 with HCl and bring the volume to 100 mL.
• Store at 4°C.

Stacking gel Stocksolution (SG)

For Western Blot an Anti-His antibody (Anti-His (C-term)/AP) was used.

The following solutions need to be prepared for the procedure.

10x TBS buffer

• Dissolve the components in ddH₂O, adjust the pH to 7.4 and bring the volume to 500 mL.
• Store at RT.

1x Transfer buffer

• Dissolve the components in ddH₂O and bring the volume to 1 L. The pH should be between 8.1 and 8.4.
• Store at RT.

10x TBS-Tween buffer

• Dissolve the components in ddH₂O, adjust the pH to 7.4 and bring the volume to 500 mL.
• Store at RT.

Blocking Solution

• Dissolve 2.5 g skim milk in 50 mL 1x TBS buffer.
• It has to be prepared freshly.

Phosphatase buffer

• Dissolve the components in ddH₂O, adjust the pH to 7.4 and bring the volume to 500 mL.
• Store at RT.

In preparation for the Western Blot, a SDS-PAGE with a Protein Ladder, which is compatible with Western Blot, has to be run.

• Prepare 6 gel sized Whatman paper and gel sized blot membrane by soaking them in 1x Transfer buffer.
• Set up the Western Blot. Place 3 moist Whatman papers on the anode (base of blotter), followed by the moist membrane, the unstained SDS-gel, 3 moist Whatman papers and the cathode (lid of blotter).
• Blot at 200 mA (1 gel) and constant 10 V for 1 h.
• Wash the membrane three times for 5 min with 1x TBS buffer on a rocker.
• Block with 50 mL 1x TBS buffer with 5% skim milk for 1 h or over night on a rocker.
• Wash three times for 15 min with 1x TBS buffer on a rocker.
• Incubate with antibody solution (14 mL 1x TBS-Tween buffer + 200 µL 1x TBS buffer/5% skim milk + 7 µL antibody) for 2 h on a rocker.
• Wash two times shortly with 1x TBS-Tween buffer.
• Wash once with 1x TBS-Tween buffer for 15 min on a rocker.
• Wash two times with 1x TBS-Tween buffer for 5 min on a rocker.
• Wash two times with 1x TBS buffer for 10 min on a rocker.
• Wash two times shortly in 1x phosphatase buffer.
• Place the membrane in staining solution (10ml 1x phosphatase buffer + 66µl NBT + 33µl BCIP), stain in the dark until bands become visible.
• Wash with H₂O to stop the reaction.
• Dry the membrane in Whatman paper and scan it.

The B₁₂ Plate Assay allows the detection of corrinoids in cell extracts or supernatants. It is based on the fact, that E. coli DSM 4261 relies on its B₁₂ dependent methionine synthase which is encoded by metH. This is caused by a point mutation in metE which encodes the B₁₂ independent methionine synthase. In a medium without methionine, E. coli DSM 4261 therefore relies on the presence of B₁₂.

Cultivation of E. coli DSM 4261

• 50 mL of Guttman medium are inoculated with a 5 mL preparatory overnight culture.
• The cells are harvested at an OD₆₀₀ of 0.2, washed three times with 20 mL 0.9% NaCl and afterwards resuspended in the same volume NaCl.

Preparation of samples

• After induction, the cells are centrifuged down, and washed three times with 0.1 M sodium chloride solution.:

• Add components to ddH2O, adjust the pH tp 7.0 and bring the volume to 1 L.
• Periplasm, cytoplasm and crude cell extracts from different expression studies are used as samples.

Assay

• 7 mL of the cell suspension, 50 mM and 0.2 g/L triphenyltetrazoliumchloride are added to 500 mL of the autoclaved mineral-agar (50°C).

• Add components to ddH₂O and bring the volume to 1 L.
• 4 sterile filter papers (Ø 0.9 cm, Fa. Schleicher & Schuell, Dassel) are laid onto the plates and loaded with 10-15 µL of a sample or a Vitamin B₁₂ calibration solution (0, 0.05, 0.25, 0.1, 5.0 μg/ml). Every sample or concentration is analyzed twice.
• The plates are incubated at 37°C for 16-24 h.

If the samples contained Vitamin B₁₂, the cells grow in a red zone around the filter paper. The size is proportional to the Vitamin ₁₂ concentration and the reduction of triphenyltetrazoliumchloride causes the red staining.

• To analyze the B₁₂ concentration in the samples, the diameter of the red zone is measured and compared to the calibrations.

To determine the corrinoid concentration in a sample, the corrinoids have to be converted into the soluble dicyano form and proteins and lipids have to be removed by precipitation and centrigugation.

• Incubate 1 mL of your sample with 0.2% (w/v) NaCN or KCN for 30 min at RT in the dark.
• Acidify the solution with pure acetic acid to a pH of 4.0.
• Boil the samples at 100°C for 20 min.
• Let the sample cool down and adjust the pH to 11.0 with KOH.
• Add 4-5 mg NaCN or KCN.
• Centriguge at 13,000 rpm for 30 min at RT.
• Measure a spectrum of the supernatant from 350 to 650 nm.

The concentration of Vitamin B₁₂ can be calculated with help of the molar extinction coefficient of dicyano cobalamine (Δε_580-640 = 7.3 mM^-1 · cm^-1) by determining the differences in the absorption at 580 and 650 nm.
Alternatively, the concentration of Vitamin B₁₂ can also be determined with the extinction coefficients of dicyano cobalamine at 580 nm (ε₅₄₀ = 7800 mM^-1 · cm^-1) and 367 nm (Δε₃₆₇ = 23800 mM^-1 · cm^-1) by calculating the average.
As a standard hydroxyl cobalamine and cyano cobalamine are used.

Bradford solution

• Mix 800 mL ddH₂O with 200 mL RotiQuant (5X) (Carl Roth).
• Store at 4°C protected from light.
• After preparing a new solution, the Bradford factor has to be determined by measuring a standard curve from protein standards with known concentrations.

Measuring protein sample

• Mix 20 µL of the sample with 1mL Bradford solution in a photometer cuvette. Prepare triplicates.
• Also prepare a blank by mixing 20 µL ddH₂O with 1 mL Bradford solution in a photometer cuvette.
• Incubate for 5 min at RT protected from light.
• Measure the absorbance at 595 nm against the black. It should lie between 0.1 and 0.6, otherwise the protein sample has to be diluted.

Calculation of protein concentration