iGEM 2017 — Team ITB_Indonesia

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# Team:ITB Indonesia/Notebook

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# Notebook

# Notebook

***Protocols used in this study***

1. 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
     ![](https://static.igem.org/mediawiki/2017/2/25/RF_1.JPG)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
     ![](https://static.igem.org/mediawiki/2017/3/31/RF_2.JPG)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
     ![](https://static.igem.org/mediawiki/2017/e/ef/RF_6.JPG)7. Quick spin the PCR tube, the place on the thermal cycler
     8. Set the PCR cycle profile as follow and the run
     ![](https://static.igem.org/mediawiki/2017/4/4b/RF_8.JPG)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
     ![](https://static.igem.org/mediawiki/2017/6/6b/Restriction_1.JPG)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
2. 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.](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182663/)

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# Team:ITB Indonesia/Experiments

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# Experiments

# Experiments

Paragraph 1

Paragraph 2

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# Team:ITB Indonesia/Results

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# Results

# Results

Experiments to test our project were segmented into three different sections:

**PETase Activity Assay,
pSal Inducibility Assay, Biofilm Formation Assay**

**1. PETase Activity Assay**

The goal of this assay is to determine and measure the PET-degrading abilities of our parts. The first experiment is carried out using pNPB Assay. PETase activity was tested using pNPB (p-nitrophenyl butyrate) Assay which measures esterase activity. Trasformants of BBa\_K2378005 were grown for 4, 16, 24 hours in LB and tested against pNPB to determine their esterase activities, which were measured spectrophotometrically. As controls, we grew BL21 cells without plasmid with the same variations of age.

The results clearly showed that PETase activities were observed in all different variations of bacterial age, as the absorbance differences with their individual controls were significant. We can thus conclude that our designed PETase works as intended.

![](https://static.igem.org/mediawiki/parts/6/66/T--ITB_Indonesia--pNPBPETase.jpeg)
 *Figure 1. PETase Activity Assay (pNPB Assay) of BBa\_K2378005 Transformants*

The difference of PETase activity among the 3 varying culture ages were not significant. One major observation point is that in culture grown for only 4 hours, the initial activities were lower. This might be due to the that the initial concentration of available PETase was lower as well.

Aside from that, we also tested PETase activity by directly applying our PET-degrading bacteria into PET bottle fragments. After 2 days of incubation, the fragments were visualized using Scanning Electron Microscope (SEM) techniques.

The results showed that the surface of PET fragments inoculated by PETase-containing cultures are rougher and have more cracks. This is most likely due to the activity of PETase.

![](https://static.igem.org/mediawiki/parts/a/a0/T--ITB_Indonesia--SEMControl.jpeg)
 *Figure 2. SEM result of plastic fragment incubated with control BL21 cells bearing no plasmid*

![](https://static.igem.org/mediawiki/parts/7/72/T--ITB_Indonesia--SEMPETase.jpeg)
 *Figure 3. SEM result of plastic fragment incubated with BL21 cells transformed with BBa\_K2378005 shows rougher surface with more cracks*

![](https://static.igem.org/mediawiki/parts/c/c2/T--ITB_Indonesia--SEMNhaR.jpeg)
 *Figure 4. SEM result of plastic fragment incubated with BL21 cells transformed with BBa\_K2378006 shows rougher surface with more cracks*

Although cannot be determined precisely with the limited amount of data collected, image processing of SEM results to measure pore sizes suggest that pore sizes in plastic fragments incubated with PETase culture are relatively bigger than pore sizes in culture incubated with PETase cultures with enhanced biofilm production (facilitated by NhaR). This phenomenon might be due to the fact that biofilm matrix can hamper the transfer of enzymes due to its low permeability, as suggested by the model we developed.

**2. pSal Inducibility Assay**

ITB\_Indonesia iGEM team 2017 designed a PETase enzyme coding device consisting of NahR-pSal regulatory part which is commonly activated by salicylate induction. Beside salicylate, NahR-pSal regulatory system is also known to have a certain degree of responsiveness towards the presence of organic pollutants (OPs). This particular characteristic of NahR-pSal is utilized in iGEM 2017 Indonesia's project to detect the presence of plastic.

To test NahR-pSal regulatory system's inducibility, we induced the expression of PETase (measured using pNPB Assay) in two different ways; 1mM of salicylate, compared to direct use of organic pollutants from plastic surface as inducers.

The results showed that PETase expression can be induced using 1mM of salicylate. However, the the inducibility of NahR-pSal system using organic pollutants from plastic surface could not be determined due to the fact that although the expression PETase can be observed, the trend is not significantly different from uninduced culture. This is due to the fact that NahR-pSal regulatory system used might be leaky to a certain degree.

![](https://static.igem.org/mediawiki/parts/6/6b/T--ITB_Indonesia--psalpnpb.jpeg)
 *Figure 5. pSal-Regulated PETase Activity (pNPB Assay) of BBa K2378004 Transformants*

**3. Biofilm Formation Assay**

In order to adhere to plastic surfaces in the ocean, our genetically engineered bacteria need to be able to produce biofilm rapidly. We facilitate this ability by the means of NhaR gene. Expression of NhaR induces rapid production of biofilm. In order to observe the effect of NhaR in this part, we tested the rate of biofilm production of bacteria transformed with BBa\_K2378006 containing NhaR compared to control bacteria without NhaR.

The experiment was done by growing bacterial cultures in a 96-well microtiter plate, and measuring its biofilm formation after 6, 12, 18, 24 hours of incubation.

The result shows a more rapid biofilm production in cultures transformed with this part containing NhaR compared to its control without NhaR. We can thus conclude that the function of our designed NhaR gene to enhance biofilm production works as intended.

![](https://static.igem.org/mediawiki/parts/2/2c/T--ITB_Indonesia--NhaR.jpeg)
 *Figure 6. Quantification of biofilm production over 24 hours incubation of BBa\_K2378006 transformants (containing NhaR) compared to its control*

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