Experiments

General Protocols

1. LB broth and agar preparation

• To make liquid broth, we dissolved commercially available LB powder (Fisher Scientific) into ddH2O according to the manufacturer's recommended instructions.
• To make LB agar, we prepared the same amount of the LB powder as above and added sufficient agar powder (Fisher Scientific) to make a final concentration fo 1.5%.
• Mixtures were prepared in glass bottles with caps and autoclaved.
• When appropriate, antibiotics were added into the LB before bacterial culture. For LB agar, antibiotics were added prior to pour plate.
• Prepared media were stored in sealed plastic bags inside a 4C refrigerator until use.

2. Nitrate minimal salts (NMS) medium preparation

• Nitrate minimal salts medium were prepared according to the table below
• After all ingredients below were added, 700ml of water were added to dissolve, then water were added up to 1 liter. Medium were then autoclaved.

• Trace element solution (1X) were made according to the table below. All components were added to 700ml of water to dissolve then add water up to 1 liter. Solutions did not need to be sterilized.

• After the above were made and autoclaved, 10ml per liter of autoclave phosphate stock (see below) and 10ml per liter of filter-sterilized vitamin stock (see below) were added.
• Make 1 liter solution of the below stock. pH to 6.8 before autoclaving.

• Make 1 liter of the vitamin stock and sterilize by using a 0.2micron filter.

• Lastly, CuCl2-2H2O were added to a final concentration of 10micromolar (1.4mg/liter) to complete the medium.

3. MOPS buffer

• Ingredients below were added along with 800ml of dd water. pH were adjusted to 7.0 and then water were added up to 1 liter.

4. E. coli culturing

• E. coli strains were routinely streaked out from frozen glycerol stocks onto LB agar plates containing appropriate antibiotics (100 microgram per mil Ampicillin and/or 20microgram per mil chloramphenicol). Single colonies were picked with sterile tips or toothpicks and inoculated into LB broth containing appropriate antibiotics. Culture were grown at 37C in a shaking incubator (200 rpm).

5. Making E. coli competent cells

• TFB1 buffer were prepared by combining all ingredients below. pH were adjusted to 5.8 using 0.2% acetic acid. Buffer were filter sterilized and stored at room temperature.

• TFB2 buffer were prepared by combining all ingredients below. pH were adjusted to 6.5 using KOH.. Buffer were filter sterilized and stored at room temperature.

• E. coli strains were cultured according to above description until log phase growth, culture were kept on ice until use
• Cells were concentrated by centrifugation at 5000g for 10 min (at 4C). Supernatants were discarded and 100ml of TFB1 were added per 250mL of growth culture and resuspended.
• Mixture were incubated on ice for 5 min and then concentrated via centrifugation at 5000g for 5 min. Supernatant were discarded.
• 10ml of TFB2 were added to the cell pellet and gently resuspended. After incubating on ice for 60 minutes, 100 microliter of the mixture were aliquoted into pre-chilled 1.5mL microcentrifuge tube and snap freeze.
• Competent cells were stored at -80C freezer.

6. Plasmid transformation

• 2 microliter of purified plasmid DNA were added into 1 vial of competent cell containing up to 100 microliter. DNA were gently mixed with sterile pipet tips and the mixture were incubated on ice for 30 minutes.
• With the cap closed, the tube were dipped into water pre-heated to 42 degrees celsius for 1 min, and then immediately placed back on ice for 1 min.
• 500ml of LB broth were then added to the mixture and the tube were incubated at 37C with shaking (200rpm) for 1 hour.
• After centrifugation (10,000RPM for 1 min), 400 microliters of the supernatant were removed and the remaining suspension were plated into LB agar plates

7. Plasmid extraction

• Plasmid extraction were performed according to the instruction provided from the manufacture. We used the Thermo Scientific GeneJet Plasmid Miniprep Kit for all plasmid extraction.
• Briefly, E. coli strains carrying our plasmid were cultured in LB broth overnight and next day 2ml of the culture were collected by centrifugation at 10,000RPM for 1 min. Cell pellet were collected by discarding the supernatant.
• We then proceeded to extract the plasmid according to the provided instruction with the exception that at the end we eluted plasmid DNA using 50microliter of sterile water. DNA concentration were measured using the NanoDrop ND-1000 UV-Vis Spectrophotometer. DNA are stored at -20C.

8. SDS-Polyacrylamide Gel Electrophoresis

• To prepare a 10% resolving gel, we prepare 5ml of solution containing 1.3ml of 1.5M Tris-HCl at pH 8.8, 50 microliter of 10% w/v SDS, 1.275ml of 29:1 acrylamide mix, and add water up to 5mL.
• To polymerize the resolving gel, we added 4 microliter of TEMED and 50 microliter of 10% ammonium persulfate (APS). The resolving gel was loaded into a gel casting stand containing two glass cassettes. Sterile water were then added to overlay the resolving solution to prevent evaporation. We typically wait about 20 to 30 minutes for the gel to polymerize.
• To prepare the 5% stack gel, we prepared 2 ml of solution containing 0.25mL of 1.0M Tris-HCl at pH 6.8, 20 microliter of 10% w/v SDS, and 0.2475ml of 29:1 acrylamide mix. 2 microliter of TEMED and 20 microliter of 10% APS were then added to facilitate polymerization.
• The stacking solution were added on top of the polymerized resolving gel, a comb was placed over the top and we typically wait for 30 minutes for full polymerization.
• To prepare E. coli cell lysates for protein separation, 1ml of overnight bacterial culture would be concentrated via centrifugation at 10,000rpm for 1 min, the cell pellet would then be dissolved in 5 microliter of 5X SDS sample buffer containing 1M Tris-HCl at pH 8.0, 50% w/v glycerol, 10% w/v SDS, 1% w/v bromophencol blue, and 12.5 v/v of beta-mercaptaethanol to a final volume of 25 microliter (in PBS buffer). Protein samples would be heated at 97C for 20 minutes and then kept on ice.
• Next, 5 microliter of the mixture were loaded into the gel along with a pre-stained protein ladder (Fisher-Scientific). Electrophoresis were performed at 100 volts until the dye front has reached the bottom. Electrophoresis were performed with 1X running buffer (25mM Tris-base, 192mM glycine, 0.1% w/v SDS, pH 8.3) using the Bio-Rad Mini-Protean Tetra gel system.
• Protein bands were visualized by staining the gel in 100ml of the staining solution (to make 1 liter combine 1 g Coomassie R250 to 300ml methanol, 50ml acetic acid, and 650ml of water. The gel can be destained by soaking in destaining solution (10% acetic acid, 50% methanol, and 40% water) for at least 2 hours until the background are clear.  

Measurement of sMMO activity by coumarin

1. Culturing of M. trichosporium

• M. trichosporium were kindly provided by Dr. Babu Fathepure (OSU)
• M. trichosporium were inoculated into 30ml of NMS broth maintained in 125-ml serum vials. Syringe was inserted into the rubber septa to contain 50% methane and 50% air. The culture were incubated at 30C with shaking.
• 48 hours post-incubation, cell culture were harvested and washed 3 times with 20mM MOPS buffer by centrifugation at 5,000g for 5 min.
• After discarding supernatant, cells were resuspended in MOPS buffer containing 5 mm MgSO4 and 20mm sodium formate. Cell suspension were kept on ice.

2. Preparation of coumarin for enzyme activity

• To generate a standard curve, Coumarin purchased from Sigma Aldrich and dissolved in 20mM MOPS buffer at the following final concentration: 700mM, 70mM, 7mM, 0.7mM, 0.07mM, 0.007mM, and 0.0007mM.
• To measure sMMO activity, coumarin were prepared in final concentration of 0.5mM.

3. Enzyme activity detection

• 5ml of cell suspensions prepared in step 1 were mixed with 20mM sodium formate and appropriate volume of coumarin and sealed in glass serum vials for 1 hour at 30C with shaking.
• Reactions were stopped by filtering out the cells using 0.8 micron pore-size syringe filters followed by 0.2 micron pore-size syringe filters.
• Fluorescence were determined using the PTI QuantaMaster fluorescence spectrometer with excitation at 338nm and emission at 450nm.