iGEM 2019 — Team BUCT-China

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# Team:BUCT-China/Notebook

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# Notebook

## Electron Microscope 15 May.-19 May.

Surprisingly, we found some cracks on the surface of PE affected by the marine bacteria.

## Constract Gene Circle Map 19 May. -27 May.

Sequenced the entire genome of the marine bacteria.
Adopted gene annotation and predict gene function.
We successfully built the phylogenetic trees.

## GO Analysis 27 May.-10 Jun.

Through GO analysis, it could be speculated that the bacteria have a vigorous metabolic process and a large amount of enzyme production.

## Transduction Experiment 10 Jun.–23 Jun.

Plasmid synthesis and plasmid transformation.

The host cells:*E.coli*

## Verify The Degradation Ability 23 Jun. –15 Jul.

Mainly adopted the NMR and the chromatographic analysis
We have obtained about 18-20 carbon chains after the degradation.

## Biological Characterization 15 Jul. – 1 Aug.

We got three new parts and finished the biological characterization.

## Collaboration 1 Sept. – 7 Oct.

Communicated with the iGEMers in Lanzhou University. We finally reached an agreement in the cooperative proposal.

## Human Practices 8 Oct. – 19 Oct.

We visited waste treatment plant in Beijing to know the present situation of plastic waste disposal.
 Also we held some recycling activities in our school and made a presentation about our project, appealing for environmental protection.

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* [![北京化工大学](https://static.igem.org/mediawiki/2019/9/99/T--BUCT-China--member_buctlogo.png)](http://www.buct.edu.cn/)Copyright ©  2019  BUCT-China  All rights reserved.


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# Team:BUCT-China/Experiments

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# Experiments

## Experiments

Measure the growth curve of the cells

![](https://static.igem.org/mediawiki/2019/2/29/T--BUCT-China--experiment_icon2.png)

### Reagent

![](https://static.igem.org/mediawiki/2019/7/79/T--BUCT-China--experiment_icon1.png)

### Step

Take 25 sterile test tubes, mark them from 0 to 24.

   Incubate the seed solution for 12 h, transfer 5 ml of seed solution to 100 ml of the seed culture medium in a 5% inoculum, and shake the bacteria. The solution was evenly mixed, and 5 ml of the bacterial solution was transferred to 25 tubes.

   25 tubes were inoculated at 37°C, l80 rpm in a constant temperature shaker, and 1h as a cycle.The incubation time is set to 0, 1, 2, 3, 4, 5, 6. 7, 8-24h, the sample taken was stored at 4°C, and all samples were taken to determine the absorbance.

   Blank seed culture was measured as absorbance at 600 nm wavelength as an experimental control. value. Determine the measurement from the earliest taken out of the test tube, dilute the test tube with a large concentration of the bacterial solution in the medium, and record the dilution factor to ensure that the absorbance is between 0.1 and 0.65.

Test the optimum pH of the enzyme activity

![](https://static.igem.org/mediawiki/2019/2/29/T--BUCT-China--experiment_icon2.png)

### Reagent

Gly-HCl buffer: Prepare a Gly solution with a final concentration of 20 mM, and adjust the pH value with HCl to obtain a buffer of pH 1.0, 1.5, 2.0, 2.5, 3.0.

   NaAc-HAc buffer : A final concentration of 20 mM NaAc solution was prepared, and the pH was adjusted with glacial acetic acid to obtain a buffer having a pH value of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5.

![](https://static.igem.org/mediawiki/2019/7/79/T--BUCT-China--experiment_icon1.png)

### Step

Oxidase was separately added to a buffer solution, and the optimum reaction pH of oxidase was measured at 50℃.

Extract plasmid

![](https://static.igem.org/mediawiki/2019/2/29/T--BUCT-China--experiment_icon2.png)

### Reagent

50mMl glucose / 10mMl EDTA / 25mMl Tris-HCl, pH = 8.0

   0.2Ml NaOH / 1% SDS

   3Ml potassium acetate / 2Ml acetic acid

   Isopropyl alcohol, Anhydrous ethanol, 75% ethanol should be pre-cooled in -20°C refrigerator; phenol chloroform pre-stored in 4°Crefrigerator

   Shake bacteria, 2-3 ml corresponding resistant LB medium, cultured at 37°Covernight

![](https://static.igem.org/mediawiki/2019/7/79/T--BUCT-China--experiment_icon1.png)

### Step

Centrifuge the solution at 12000 rcf for 5 min, discard the supernatant, add 0.2 ml of solution I (already added RNase), mix well, add 0.25 ml of solution II, mix by inversion, place at room temperature for 5 min, add 0.4 ml of solution III After mixing, invert and centrifuge at 13000 rcf for 30 min

   Transfer the supernatant to a new 1.5 ml EP tube, add an equal volume of phenol chloroform mixture, and mix well. Centrifuge at 10000rcf for 10 min.

   Take the supernatant, transfer to a new EP tube, add an equal volume of pre-chilled isopropanol, mix well, and centrifuge at 13000 rcf for 6 min.

   Pour off the supernatant, use 1 ml of pre-cooled 75% ethanol, invert, and centrifuge at 13,000 rcf for 1 min.

   Pour off the supernatant and add 1 ml of pre-cooled 75% ethanol along the wall and pour directly.

   Add 1 ml of pre-cooled ethanol along the wall and pour directly. After depressing for 5 min, it was left at room temperature for 10 min. After the liquid in the tube is cleaned, 100 ul of water is added to the tube and incubated at 55°C for 5 min.

   The resulting plasmid solution can be stored at -20°C for long-term storage.

Oxicase enzyme activity with ABTS

![](https://static.igem.org/mediawiki/2019/2/29/T--BUCT-China--experiment_icon2.png)

### Reagents and instruments

Acetate-Sodium acetate Buffer 960l (20mM, pH4.5)

   ABTS9 (0.5M) 20ml

   Crude enzyme solution 20ml

   UV-2700 spectrophotometer

![](https://static.igem.org/mediawiki/2019/7/79/T--BUCT-China--experiment_icon1.png)

### Step

The reaction is carried out at 45°C, zero with buffer, The substrate was separately added to the substrate to self-decompose, and then the enzyme solution was added to measure the change in absorbance in one minute.

   The enzyme activity was calculated according to the formula U(/mg) = ΔA×V total×N / (ε×t ×V enzyme×d).

      △A: change in absorbance per unit time

      V total: total activity of enzyme activity measurement (mL)

      N: dilution factor of enzyme solution

      ε: molar absorptivity

      t: reaction time (min)

      V enzyme: amount of enzyme added in the reaction system ( mL)

      d: cuvette diameter (cm)

Plasmid transformation

![](https://static.igem.org/mediawiki/2019/2/29/T--BUCT-China--experiment_icon2.png)

### Reagent

LB medium

   Selective LB agar culture plate

   1‰ of antibiotics

   Host bacteria: Competent bacteria DH5αtreated with 100 mmol/L CaCl2

![](https://static.igem.org/mediawiki/2019/7/79/T--BUCT-China--experiment_icon1.png)

### Step

Take out the receptive state of large intestine and put it into the ice box at -80℃.

   Plasmid 10 (2) ul was added to the TOP10 (BL21) competent state and ice bath was performed for 30min.

   Heat excitation was performed at 42℃ and 90s.

   Place in the ice box and let stand for 5 minutes.

   Add 500ul LB medium to the centrifuge tube.

   Culture in a shaking table with 180rpm at 37℃ for 60min.

   Add 1‰ of antibiotics into LB tube and culture with 1% bacterial liquid for 12-14h.

SDS-PAGE

![](https://static.igem.org/mediawiki/2019/2/29/T--BUCT-China--experiment_icon2.png)

### Reagent

Standard protein mixture

   30% gel stock solution

   Separation gel buffer (1.5 mol/L)

   Concentrated gel buffer (0.5 mol/L)

   Electrode buffer (pH 8.3)

   10% SDS

   Mass concentration 10% ammonium persulfate (fresh formulation)

   Loading buffer

   0.25% Coomassie Brilliant Blue R-250 staining solution

   Decolorizing solution

   Protein sample of unknown molecular weight (1 mg/mL)

![](https://static.igem.org/mediawiki/2019/7/79/T--BUCT-China--experiment_icon1.png)

### Step

Loading plate

   Gel polymerization

      Configure 10% separation gel and 4.8% concentrated gel.

   Configure 10% separation gel and 4.8% concentrated gel.

      The sample used in this experiment is a 15 to 20 g standard protein sample solution, placed in a 0.5 mL centrifuge tube, and 15-20 l of the sample treatment solution is added. Treated in a 100°C water bath for 2 min, cooled to room temperature and set aside. A sample of 201 of a protein of unknown molecular weight was aspirated and treated according to the standard protein treatment method.

   Loading

      Add a sample to each sample tank in a micro-syringe, add 10 to 15 l each (containing 10 to 15 g of protein), and add 20 to 30 l to a dilute solution.

   Electrophoresis

      The current before the sample is injected is controlled at 15-20 mA, about 15-20 min; after the bromophenol blue indicator in the sample reaches the separation gel, the current rises to 30-45 mA, and the electrophoresis process keeps the current stable. When the bromophenol blue indicator migrates to a distance of 1 to 2 cm from the leading edge, electrophoresis is stopped for about 1-2 hours.

   Dyeing, bleaching

      The center of the indicator zone was inserted with copper wire as a marker, and stained in a large petri dish, stained with 0.25% Coomassie blue dye solution for 2 to 4 hours. Discard the staining solution, rinse the surface with distilled water several times, then add the decolorizing solution to diffuse and decolorize, often changing the decolorizing solution until the protein band is clear.

︽

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* [![北京化工大学](https://static.igem.org/mediawiki/2019/9/99/T--BUCT-China--member_buctlogo.png)](http://www.buct.edu.cn/)Copyright ©  2019  BUCT-China  All rights reserved.

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# Team:BUCT-China/Results

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# Results

## Parts 1:

At the beginning of the experiment, we wanted to verify that our idea, the possibility of plastic biodegradation by bacteria, was correct. So, SEM and FTIR technique (Fourier Transform infrared spectroscopy) were used.

![](https://static.igem.org/mediawiki/2019/b/b8/T--BUCT-China--results_test3.png)

Fig.1.SEM images of the degradation of PE under different conditions.Scale bar 2μm，MAG x30k.

（A）The image of the degradation of PE by ultraviolet light after 12 hours.

（B）The image of the degradation of PE by  *Microbulbifer hydrolyticus*  after 30 days.There are obvious cracks on the PE surface.

From the SEM images that the experimental group can clearly see the plastic crack. This shows the potential of this strain to degrade PE.

![](https://static.igem.org/mediawiki/2019/f/fa/T--BUCT-China--results_test.jpg)
![](https://static.igem.org/mediawiki/2019/8/83/T--BUCT-China--results_test2.jpg)

Fig.2.FTIR spectra of PE standards and the sample after reaction with bacteria.

(A) 2918.8- -CH3;2849.5- -CH2-;1466.0- -CH2-;722.6- -(CH2)N-,N>4; PE structure.

(B) Extra peaks in the range of 3000-3500,3392.2 -OH,3195.7-unsaturated and 1644.9-contain double bonds.

That means under the action of this bacteria, it does have a certain effect on plastic(PE) degradation.From the control group, the plastic is a straight carbon chain.In the experimental group, there have several extra small peaks.And indicate the insertion of oxygen atoms and the generation of double bonds.But the peaks intensity are low, which means that the PE is oxidized to a low degree.

What do these figures say?

After sem analysis, we could see cracks and other traces of degradation on the surface of the plastic film, so our idea was preliminarily proved to be correct. We suspect that enzyme systems in microbes greatly accelerate the degradation of plastics.In addition, we analyze the newly generated bonds and explain the mechanism based on this guess.

## Part 2:

After whole genome sequencing and COG gene annotation,we get the gene circle map.
After all CDs were classified by COG, in addition to the protein types necessary for life process , the protein with the highest expression was carbohydrate metabolic protein.We can infer that this bacteria has a good ability to use carbohydrates.

![](https://static.igem.org/mediawiki/2019/5/52/T--BUCT-China--results_part11.png)

Fig.3. genome sequencing

![](https://static.igem.org/mediawiki/2019/a/ac/T--BUCT-China--results_part13.png)

Fig.4.KEGG pathways

As the KEGG analysis shows, some proteins are relevant to the xenobiotics biodegradation and metabolism. So the bacteria is proved to have the capability to degrade some harmful foreign materials through its detoxification pathway. Basically, it is common to see various oxidase involved in the pathway of detoxification. So it could be speculated that the bacteria very likely have some special oxidase and degradation pathway which may also be effective in PE degradation.

After GO analysis, it was found that some proteins in the bacteria are special in the aspect of molecular function, particular in electron carrier activity, which further proved the existence of oxidase. Because oxidase is capable to catalyze the oxidation of substrate mainly by carrying electrons.

![](https://static.igem.org/mediawiki/2019/7/79/T--BUCT-China--results_part12.png)

Fig.5. Bar chart of GO

We found the oxidase gene exactly through gene sequencing and we registered it as a new biological parts.

## Part 3:

Future

We have determined the role of oxidase in the biodegradation process through data analysis, but the pathway after oxidase is not clear. Therefore, we will continue to find other important enzymes in the biodegradation process so that they can be optimized.

After the biodegradation of PE by the strain we used, we have obtained about 18-20 carbon chains. How to make them high value-added is the ultimate significance of the project.

* Part 1:
* Part 2:
* Part 3:

︽

* [![](https://static.igem.org/mediawiki/2019/8/8b/HQ_page_logo.jpg)](https://2019.igem.org/)
* [![](https://static.igem.org/mediawiki/2019/6/64/T--BUCT-China--buct_logo.png)](https://2019.igem.org/Team%3ABUCT-China)
* [![北京化工大学](https://static.igem.org/mediawiki/2019/9/99/T--BUCT-China--member_buctlogo.png)](http://www.buct.edu.cn/)Copyright ©  2019  BUCT-China  All rights reserved.