Wetlab Protocol

1. SDS PAGE
   

   SDS-PAGE analysis was carried out based on Laemmli System (Laemmli, 1970). First, all of the reagents were mixed together in order to make stacking and separating gel. Bacterial culture was centrifuged at 14.000 g, 1 minutes, then the supernatant was discharged. Pellet cells were mixed with sample buffer then boiled at 100^oC for 5 minutes. 10 uL of samples applied to each wells. Electrophoresis perfomed in SDS-Glycine buffer at 130V constant until dye reached the bottom of the gel (Sambrook et al., 2003).



2. Ethylene Glycol Assay using Chromatic Acid
   1. Ethylene Glycol Standard Solution Procedure
      1. Ethylene Glycol concentration ≈ 0.5 M (31035 ppm) -> Dissolve 279 µL ethylene glycol in 10 mL deion water.
      2. Ethylene Glycol concentration ≈ 0.05 M (3103.5 ppm) -> Dissolve 300 µL ethylene glycol 0.5 M, then dilute until 3000 µL.
      3. Ethylene Glycol concentration ≈ 0.005 M (310.35 ppm) -> Dissolve 300 µL ethylene glycol 0.05 M, then dilute until 3000 µL.
      4. Ethylene Glycol concentration ≈ 0.0005 M (31.035 ppm) -> Dissolve 300 µL ethylene glycol 0.005 M, then dilute until 3000 µL.
      5. Ethylene Glycol concentration ≈ 0.00005 M (3.1035 ppm) -> Dissolve 300 µL ethylene glycol 0.0005 M, then dilute until 3000 µL.
   2. Chromic Acid Standard Solution Procedure
      1. Chromic acid ≈ 0.6 M (177000 ppm) -> Dissolve 1.77 gr K₂CrO₇ (Mr 294.18) with sulphate acid until the volume 10 mL.
      2. Do dilution of chromic acid same as ethylene glycol standard solution procedure.
   3. Ethylene Glycol Concentration Measurement through Spectrofotometri UV-Vis
      4 Cr₂O₇^2- + 3 C₂H₆O₂ + 32 H⁺ -> 8 Cr³⁺ + 3 C₂H₂O₄ + 22 H₂O
      1. Ethylene glycol 300 ppm -> Pipette 29 µL ethylene glycol solution 31.035 ppm then dilute it until 3 mL.
      2. Chromic acid 3.000 ppm -> Pipette 8.5 µL chromic acid solution 177.000 ppm then dilute it until 5 mL.
      3. Sulphate acid blank solution -> Enter 1 mL sulphate acid into cuvette, then search maximum wavelength for this solution.
      4. Chromic acid blank solution -> Enter 1 mL chromic acid 300 pm into cuvette, then search maximum wavelength for this solution.
      5. Add 600 µL chromic acid 300 ppm and 400 µL ethylene glycol, then measure the absorbance of this solution.
         The difference absorbance values between chromic acid were added with sulfuric acid and ethylene glycol can be used as a parameter to calculate the concentration of ethylene glycol in the sample.
   4. Qualitative and Quantitative Ethylene Glycol Measurement
      Qualitative Procedure:
      1. Add 0.25 mL sampel into microtube
      2. Add 0.5 mL Acetone and 0.1 mL Chromic acid (H₂CrO₄) into microtube
      3. Mix the mixture
      4. Incubate the mixture in waterbath at 65 - 700^oC for 10 minutes
         Positive result will show the color changes from orange to green
      Quantitative Procedure:
      1. Add 0.25 mL sampel into microtube
      2. Add 0.5 mL Acetone and 0.1 mL Chromic acid (H₂CrO₄) into microtube
      3. Mix the mixture
      4. Incubate the mixture in waterbath at 65 - 700^oC for 10 minutes
      5. Check the absorbance at A₄₄₆ nm


3. pNPP Assay
   

   Cutinase activity assays were performed using bacterial culture. The activity was determined using spectrophotometric assay at 410 nm with p-nitrophenyl palmitate (pNPP) as a substrate. pNPP was dissolved in acetonitrile at a concentration of 10 mM. Ethanol and 50 mM Tris-Cl buffer (pH 8.0) were added to a ratio of 1:4:95 (v/v/v) of acetonitrile/ ethanol/ buffer (Lee, 1999). 0.3 mL of the bacterial culture was reacted to the substrate solution (0.9 mL). Then, it was incubated at 25^oC for 15 minutes then centrifuge at 12,000g, 15 min, and 4^oC . Enzyme activity was measured by monitoring the change in absorbance at λ 410 nm that represents the amount of released p-nitrophenol (pNP). The activity of cutinase was determined based on the standard curve of p-nitrophenol. One unit of cutinase activity was defined as the amount of enzyme releasing 1 μmol pNP per minute under the assay conditions (Lee et al., 1999).



4. SEM (Scanning Electron Microscope)
   

   For biodegrading experiments, we used 3x5 cm² bottle plastics with average weight 0.05 g. Then, we washed it several times with water and ethanol. Plastic sample was applying just before we inoculated the bacteria on the medium. Bacterial culture were grown at 37^oC in Luria-Broth medium. After 3 days of incubation, we washed the plastic with water and ethanol then dried for measurement of the weight loss.

   Meanwhile, for UC Davis bacterial culture, we inoculated the bacteria in Luria-Broth medium supplemented with 170 g/ml cloramphenicol. After an absorbance A₆₀₀ nm of 0.6 was reached, we added 1% of arabinose and plastic sample. After 2 days of incubation, we washed the plastic with water and ethanol then dried for measurement of the weight loss. We used medium supplemented with antibiotics and plastic sample as control on this experiment.



5. References
   1. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
   2. Lee, Dong-Woo, Koh,Y.S.,Kim, K.J., Kim, B., et al. 1999. Isolation and characterization of a thermophilic lipase from Bacillus thermoleovorans ID-1, FEMS, Microbiology Letters. 179: 393-400.
   3. Sambrook, J. 2003. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory: Cold Spring Harbor.