Notebook

Protocols used in this study

Basic Protocols
- Plasmid Isolation Using Alkaline Lysis Method
  1. Inoculate transformed cell into LB medium supplemented with appropriate antibiotics
  2. Incubate culture at 37˚C, 200 rpm for overnight
  3. Discard supernatan, and repeat the step until all culture is used
  4. Add 100 µl Alkaline Lysis Solution I (0.05 M glucose; 0.025 M Tris-Cl pH 8.0; 0.01 EDTA pH 8.0) and resuspend cell pellet completely
  5. Add 200 µl Alkaline Lysis Solution II (0.2 N NaOH; 1% SDS), and invert tube several times. The solution should turn into transparent white, indicating the cell is lysed
  6. Add 150 µl Alkaline Lysis Solution III (3M Pottasium Acetate pH 7.2; 11.5% Glacial Acetate), and invert tube several times. The chromosomal DNA should make white floating thread
  7. Incubate on ice for 3-5 minutes
  8. Centrifuge at full speed for 5 minutes. At this point, plasmid DNA is dissolved in the supernatan while chromosomal DNA turn into white precipitate on the bottom of the tube
  9. Transfer supernatan to a new 1.5 microtube, add 2X volumes of isopropanol/absolute ethanol to precipitate plasmid DNA
  10. Centrifuge at full speed for 5 minutes, and discard supernatan
  11. Add 500 µl 70% ethanol to precipitated DNA
  12. Centrifuge at full speed for 5 minutes, and discard supernatan
  13. Let the precipitated DNA dreid by putting the microtube upside-down on a piece of tissue paper
  14. Resuspend plasmid by adding 50 µl TE buffer/Nuclease Free Water
- Polymerase Chain Reaction for Restriction-Free Cloning
  1. Mix the following component in a PCR tube, for step I PCR
  2. Quick-spin the PCR tube, then place on the thermal cycler
  3. Set the PCR cycle profile as follow and then run as follows
  4. Confirm the amplicon by running the product on 1% agarose gel, 70V for 35 minutes
  5. If positive, continue to purified PCR product using GenepHlow Gel/PCR Purification Kit (Promega)
  6. Mix the following component in a PCR tube for step II PCR
  7. Quick spin the PCR tube, the place on the thermal cycler
  8. Set the PCR cycle profile as follow and the run
  9. Confirm the amplicon by running the PCR product on 1% agarose gel, 70V for 35 minutes
- Restriction
  1. Mix the following mixture in a PCR tube
  2. Quick spin the PCR tube, then incubate at 37˚C for 30 minutes-60 minutes
  3. Incubate at 80˚C for 10 minutes to deactivate enzyme
  4. Confirm product by running on 1% agarose gel, 70V for 35 minutes
- Purification using GenepHlow Gel/PCR Purification Kit (Promega)
- Chemically competent E. coli cell
  1. Inoculate 5 ml glycerol stock of E. coli to 10 ml of LB medium
  2. Incubate culture at 37˚C, 200 rpm for overnight
  3. Transfer 1% of the overnight-grown cell into a new LB medium
  4. Incubate culture at 37˚C, 200 rpm until it reaches OD 0.3-0.4
  5. Aliquote culture into 1.5 ml microtube
  6. Centrifuge at 3.0000 xg, 4˚C for 10 minutes, discard the supernatant
  7. Add 480 µl cold CCMB buffer, and resuspend the cell pellet
  8. Incubate on ice for 20 minutes
  9. Centrifuge at 3.0000 xg, 4˚C for 10 minutes, discard the supernatant
  10. Add 60 µl cold CCMB buffer, and resuspend the cell pellet
  11. Incubate on ice for 20 minutes
  12. Store at -80˚C until use
- Heat-shock transformation
  1. Incubate competent E. coli cell on ice for 10 minutes
  2. Add 5-10 µl plasmid into 60 µl of competent E.coli cell, place on ice for 30 minutes
  3. Incubate on 42˚C for 90 seconds for heat shock treatment
  4. Place back on ice and incubate for 2 minutes
  5. Add 600 µl of LB medium and resuspend
  6. Incubate culture at 37˚C, 200 rpm for 3 hours
  7. Centrifuge at 11.ooo xg for 1 minutes, and discard 550 µl of the supernatant
  8. Resuspend the cell pellet with the remaining supernatan
  9. Inoculate 50 µl of transformed E. coli cell on LB agar medium supplemented with appropriate antibiotics using spread technique
  10. Incubate at 37˚C overnight
Specialized Protocols
- pNPB Assay:
  
  PETase activity were determined using this assay. The assay is adapted from 2012 UC Davis iGEM’s LC Cutinase pnPB assay. p-nitrophenyl butyrate (pNPB) is a substrate that contains an ester bond resembling the ester bond broken in PET degradation. PETase are expected to break the ester bond. The resulting product emits a wavelength of 405nm. pNPB 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. 0.3 mL of the bacterial culture was reacted to the 0.9 mL substrate solution. Then, it was incubated at 25oC for 15 minutes and centrifuge at 12,000g, 15 min, and 4oC . Enzyme activity was measured by monitoring the change in absorbance 405nm.
- Red Fluorescence Protein Assay:
  
  The test will determine the effect of IPTG induction and incubation time regarding red color absorbance from E.coli BL21, Top10 and DH5α. These bacteria has already been transformed with RFP containing plasmid (BBa_J04450). Each of the bacteria strain was given two variations, induced with IPTG and not induced. This test was repeated three times for each variation. The red color absorbance was measure during 40 hours of incubation time at 588 nm.
- SEM (Scanning Electron Microscope):
  
  For biodegrading experiments, we used 1x1 cm2 bottle plastics with uniform average weight. The plastic sample were washed with water and ethanol. The plastic sample were mixed with Luria-Broth Medium supplemented with kanamycin. The bacteria containing PETase enzyme was the inoculated on to the medium and grown at 37oC. After 3 days of incubation, we washed the plastic with water and ethanol then dried for measurement with SEM.
- Microtiter Biofilm Assay:
  
  Adapted from this protocol.

Team:ITB Indonesia/Notebook

Notebook

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