iGEM 2021 — Team WFLA_YK_PAO

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# Team:WFLA YK PAO/Notebook

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NOTEBOOK

August

**• Day2 8/7**

1. LB preparation

2. Transfer Top10 E. coli with IsPETase/MHETase containing pET28a into culture
medium

**• Day3 8/8**

1. Plasmid extraction

2. Enzyme cut extracted plasmids

3. Detect plasmids with gel electrophoresis

4. Transfer plasmids into BL21 E. coli

5. Transfer BL21 E. coli into culture medium

**• Day4 8/9**

1. Prepare seed broth

2. Prepare IPTG inducer

3. Transfer BL21 E. coli and seed broth into culture medium

4. Prepare for Buffer A and Buffer B

5. Test for optic density of the bacteria strain

6. Add inducer into the bacteria solution and induce the expression of IsPETase and
MHETase

**• Day5 8/10**

1. Adjust the pH of buffers

2. Test for optic density from the induced bacteria solution

3. Centrifugate the bacteria solution and preserve the rest of the bacteria

4. LB preparation

5. Add seed broth and induce bacteria solution into the shaking incubator

6. Test for optic density

7. Add IPTG inducer and put the solution in shaking incubator

8. Centrifugate to separate the bacteria

9. Break bacteria cell wall with ultrasound and preserve the proteins

10. Use nickle column purification to purify the expressed IsPETase proteins

**• Day6 8/12**

1. Use nickle column purification to purify the expressed MHETase proteins

2. Break uninduced bacteria cell wall with ultrasound and preserve the proteins

3. Prepare for SDS-page gel electrophoresis

4. Dye the gel and analyze the results

5. Overlapping PCR

a. Run PCR on IsPETase gene

b. Run PCR on MHETase gene

c. Run overlapping PCR on the products from the previous
two PCRs

![](https://static.igem.org/mediawiki/2021/1/13/T--WFLA_YK_PAO--notebook01.jpg)

**• Day7 8/13**

1. Agarose gel electrophoresis on the products from the
overlapping PCR

2. Observe and analyze the gel results

3. Redo PCR for IsPETase and MHETase gene

4. Redo SDS-page gel electrophoresis

5. Dye the gel and analyze the results

6. Redo agarose gel electrophoresis on the products from
the overlapping PCR

7. Recycle the IsPETase and MHETase gene segments from
the gel

8. Redo overlapping PCR step iii. to connect the two
genes

**• Day8 8/14**

1. Redo agarose gel electrophoresis on the products from
overlapping PCR

![](https://static.igem.org/mediawiki/2021/8/87/T--WFLA_YK_PAO--notebook02.jpg)

2. Recycle the connected gene segments from the gel

3. Enzyme cut the recycled gene segments

4. Run agarose gel electrophoresis on the cut genes

5. Recycle the cut gene segments from the gel

6. Connect the cut gene segments with cut pET28a

7. Induce the plasmid into competent cells

8. Add culture medium and centrifugate to preserve the
bacteria

9. Prepare seed broth

10. Test for enzyme activity

![](https://static.igem.org/mediawiki/2021/5/50/T--WFLA_YK_PAO--notebook03.jpg)

![](https://static.igem.org/mediawiki/2021/8/81/T--WFLA_YK_PAO--notebook04.jpg)

**• Day9 8/15**

1. Redo PCR to amplify the connected IsPETase-MHETase
gene segment

2. Run agarose gel electrophoresis on the connected gene
segments

3. Recycle the connected gene segments from the gel

4. Enzyme cut the connected gene segments

5. Run agarose gel electrophoresis on the cut gene
segments

6. Recycle the cut gene segments from the gel

7. Connect the cut gene segments with cut pET28a

8. Transfer the seed broth from 8/14 into the culture
medium

9. Centrifugate to preserve the bacteria

10. Culture the bacteria on a solid culture medium

**• Day10 8/16**

1. Tranformed the plasmid pET28a-IsPETase-MHETase into
E. coli BL21 and culture

2. Prepare Inducer and nickel column affinity
purification related buffer

**• Day11 8/17**

1. Colony PCR

![](https://static.igem.org/mediawiki/2021/d/d7/T--WFLA_YK_PAO--notebook05.jpg)

2. Protein induction and purification

September

1.Repeat transformation

2.Pilot expression

![](https://static.igem.org/mediawiki/2021/5/5d/T--WFLA_YK_PAO--notebook11.jpg)

3. Sample preparation for SDS PAGE

4. Run SDS PAGE for verifying the expression of our engineered strain

![](https://static.igem.org/mediawiki/2021/7/7e/T--WFLA_YK_PAO--notebook08.jpg)

5. Extract MHETase protein

![](https://static.igem.org/mediawiki/2021/7/7d/T--WFLA_YK_PAO--notebook12.jpg)

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# Team:WFLA YK PAO/Engineering

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Engineering

Background

The use of plastic (poly(ethylene terephthalate), PET) greatly facilitates human
life. However, because plastic products are difficult to degrade, the use of a large number of plastic
products eventually not only causes environmental pollution. At the same time, the daily intake of
microplastics by humans is gradually increasing, which damages human health. Study of a variety of model
organisms suggested that the microplastics ingested through diet are mainly concentrated in the digestive
tract. Therefore, the degradation of ingested microplastics through intestinal probiotics is an effective
way to prevent microplastics from spreading to other parts of the body and reduce the content of
microplastics in the intestine.

In 2016, the Science journal first reported the related enzyme (PETase) capable of
degrading PET. In 2018, the PNAS journal reported that MHETase can further degrade the degradation product
MHET into TPA (mono (2-hydroxyethyl) terephthalic acid) and EG (ethylene glycol). In 2021, Nature Catalysis
reported a high-efficiency enzyme (IsPETase) that degrades PET. So far, plastic products can be efficiently
degraded into recyclable monomers.

Design

We intended to express PET degrading enzymes (IsPETase, MHETase) probiotics
bacteria to reduce the content of human microplastics and improve human health.

Build

The genes IsPETase and MHETase were synthesized and inserted into pET28a vector to
obtain plasmids pET28a-IsPETase and pET28a-MHETase, respectively (Figure 1). A recombinant plasmid
pET28a-IsPETase-MHETase, which contains both genes, was also constructed.

In addition, IsPETase and MHETase genes were also cloned to the expression vector
pGEX-6P-1, which produce recombinant protein fusion with Glutathione-S-transferase (GST) tag (Figure 2).

![](https://static.igem.org/mediawiki/2021/b/b6/T--WFLA_YK_PAO--engineering01.jpg)

![](https://static.igem.org/mediawiki/2021/2/24/T--WFLA_YK_PAO--engineering02.jpg)
Figure 2. Schematic maps of pGEX-6P-1-IsPETase and pGEX-6P-1-MHETase.

Test

All the expression plasmids were transformed into Escherichia coli BL21(DE3).
Different expression conditions (temperature and inducer concentration) were tested to obtain soluble
enzymes. The samples of whole expression cell lysate, supernatant and pellet of cell lysate were analyzed
using SDS-PAGE and Western blot (only for his-tag proteins from pET28a vector) (Figure 2).

![](https://static.igem.org/mediawiki/2021/9/9c/T--WFLA_YK_PAO--engineering03.jpg)

Figure 2. SDS-PAGE and Western blot analysis of IsPETase from pET28a vector. Lane
1: Protein marker; Lane 2: Cell lysate without induction; Lane 3: Cell lysate with induction for 16h at 15
oC; Lane 4: Cell lysate with induction for 4 h at 37 oC; Lane 5: Supernatant of cell lysate without
induction; Lane 6: Supernatant of cell lysate with induction for 16h at 15 oC; Lane 7: Supernatant of cell
lysate with induction for 4 h at 37 oC; Lane 8: Pellet of cell lysate without induction; Lane 9: Pellet of
cell lysate with induction for 16h at 15 oC; Lane 10: Pellet of cell lysate with induction for 4 h at 37 oC;
The primary antibody for western blot is anti-His antibody.

The theoretical molecular weigh of the IsPETase is 35.13 kDa. As seen from the
SDS-PAGE and Western blot (Figure 2), IsPETase was predominantly expressed in insoluble form, and only
little soluble protein was produced at low temperature.

![](https://static.igem.org/mediawiki/2021/e/ec/T--WFLA_YK_PAO--engineering04.jpg)

Figure 3. SDS-PAGE and Western blot analysis of MHETase from pET28a vector. Lane 1:
Protein marker; Lane 2: Cell lysate without induction; Lane 3: Cell lysThe theoretical molecular weigh of
the MHETase is 65.17 kDa. As seen from the SDS-PAGE and Western blot (Figure 3), MHETase was also
predominantly expressed in insoluble form, and only little soluble protein was produced at low temperature.
ate with induction for 16h at 15 oC;
Lane 4: Cell lysate with induction for 4 h at 37 oC; Lane 5: Supernatant of cell lysate without induction;
Lane 6: Supernatant of cell lysate with induction for 16h at 15 oC; Lane 7: Supernatant of cell lysate with
induction for 4 h at 37 oC; Lane 8: Pellet of cell lysate without induction; Lane 9: Pellet of cell lysate
with induction for 16h at 15 oC; Lane 10: Pellet of cell lysate with induction for 4 h at 37 oC; The primary
antibody for western blot is anti-His antibody.

The supernatant of cell lysates with pET28a-IsPETase and pET28a-MHETase were
applied for enzymatic degradation of PET. As shown in Figure 4, no obvious change on the surface of PET
particles observed under light microscope.

![](https://static.igem.org/mediawiki/2021/a/a1/T--WFLA_YK_PAO--engineering05.jpg)

Figure 4. Microscope of the PET degradation by IsPETase and MHETase. left: blank;
right: IsPETase + MHETase.

![](https://static.igem.org/mediawiki/2021/0/0f/T--WFLA_YK_PAO--engineering06.jpg)
Figure 5. IsPETase and MHETase production from pGEX-6T-1 vector.

In order to improve the solubility of IsPETase and MHETase, we tried to expression
the genes using pGEX-6T-1 vector. The supernatant of cell lysate was purified by GST column. As shown in
Figure 5, bits of GST-MHETase was successfully yielded, and no GST-IsPETase was obtained.

MHETase Reactions were performed in triplicate over a 1 d time course at 30°C.
Reaction mixtures contain 50 nM recombinant GST-MHETase, 5 mM MHET in 90 mM NaCl, 10% (v/v) DMSO, 45 mM
sodium phosphate, pH 7.5. Reactions were terminated by adding an equal volume of methanol. The reactions
using inactive GST-MHETase (obtained by boiling for 10 min) were used as blank controls. The activity of
MHETase was indicated by the decline absorbances at a wavelength of 240 nm (Figure 6). The wavelength is the
specific absorption of MHET. As shown in figure 2, after 1 d reaction, MHET concentration was dropped by
31.4%.

![](https://static.igem.org/mediawiki/2021/a/aa/T--WFLA_YK_PAO--engineering07.jpg)
Figure 6. Detection of residual MHET by measuring the absorbance at 240 nm.

![](https://static.igem.org/mediawiki/2021/e/e5/T--WFLA_YK_PAO--engineering08.jpg)
Figure 2. MHETase activity assay

Learn

In this experiment, we successfully constructed IsPETase and MHETase expression
plasmids wit 6 x His tag and GST tag. Both IsPETase and MHETase were expressed. However, only little
recombinant enzymes were expressed in soluble form. The inactive enzyme hindered us to make a comprehensive
PET degradation experiment.

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# Team:WFLA YK PAO/Experiments

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Experiments

ATLATL Lab

**Bacteria culture medium preparation**

1. LB preparation

1. Add Water 100mL, NaCl 1g,
Tryptone 1g, Yeast Extraction 0.5g to a beaker

2. When preparing for solid
culture medium, add extra Agar 2.5g

2. Antibiotics solution

1. Add Water 2mL Kanamycin 0.1g
to a beaker

2. Add the solution to the
culture medium

**Plasmid extraction**

1. Prepare for buffer SP1, SP2,
and SP3, and wash solution

2. Add 500µl butter S to the
absorption column, centrifugate for 1 min at 12000g, remove the liquid in the collecting tube, put the
absorption column back in the collecting duct

3. Centrifugate 1.5-5ml
cultured bacteria solution, centrifugate at 8000g for 2 minutes, remove the supernatant

4. Add 250µl buffer SP1 to the
sediment to solve the bacteria

5. Add 250µl buffer SP2 and mix
the solution, then keep for 2-4 min at room temperature

6. Add 350µl buffer SP3 and mix
the solution

7. Centrifugate at 12000g for
5-10min, pour the supernatant into the absorption column, then centrifugate at 8000g for 30 s, remove the
liquid in the collecting tube

8. (Optional) add 500µl Buffer
DW1, centrifugate at 9000g for 30 s, remove the liquid in the collecting tube

9. Add 500µl Wash Solution,
centrifugate at 9000g for 30 s, remove the liquid in the collecting tube

10. Repeat step 9 once

11. Centrifugate an empty
absorption column at 9000g for 1min

12. Put the absorption column
in a clean 1.5ml centrifugation tube, add 50-100µl Elution Buffer, put at room temperature for 1 min,
centrifugate for 1 min, collect the DNA solution in the collecting tube

**Plasmid transfer**

1. Add plasmid solution (3 μL)
to competent cell

2. Ice-cool for 20min

3. Water bath at 42 centigrade
for 1 min

4. Ice-cool for 5 min

5. Shaking incubator for 45?
min

6. Centrifugate and keep the
pellet (containing bacteria)

**IPTG induction**

1. Spread plating BL21 with
transferred plasmid on the solid culture medium

2. Extracting bacteria from
solid culture medium to the liquid LB culture medium.

3. Put it in the shaking
incubator

4. Pour the over-night culture
solution (seed solution) into the new LB culture medium

5. Test optic density (OD) at
600nm of culture solution

6. Add IPTG (If OD600 ranges
from 0.6-0.8)

7. Centrifuge and keep the
supernate (containing proteins)

**SDS protein separation**

1. Aim: prepare the proteins
for SDS-PAGE electrophoresis

2. Add SDS solution to lyse
cell structure and denature proteins

3. Hot water-bath to accelerate
the reaction

4. Centrifugate and extract
supernatant to obtain proteins

**Nickel column purification**

Aim: to obtain pure IsPETase and MHPETase for further decomposition of plastics
Apparatus: Nickel column
Procedure:
a. Wash the nickel column with deionized water
b. Wash the nickel column with Buffer A
c. Add the sample to the nickel column using a syringe and filter and collect the drips with a tube.
d. Pour the liquid collected in the tube back into the nickel column
e. Add buffer A three times
f. Add Buffer B twice and collect the released protein with a tube

**Enzyme activity test**

Aim: examine the effectiveness of the chosen enzyme on decomposing plastics
Mix 300μL enzyme with one PET granule and 4.7mL glycine-NaOH. The bottle was put into the shakebed for one
night. Finally, we used a microscope to observe the degradation effect.

**SDS-page gel electrophoresis**

1. Make SDS-page gel

1. Add 2.7ml lower layer gel solution and lower layer gel buffer
2. Add 60µl coagulant
3. Pour into the mold and wait until it solidifies
4. After 5-10 min after the gel solidifies, remove the liquid above it
5. Add 0.75 ml upper layer gel solution and colored upper layer gel butter
6. Add 15µl coagulant
7. Pour into the mold and wait until it solidifies

2. Insert proteins into the gel

1. Mix 5µL of loading buffer with 10µl sample
2. Insert 10µL of sample in each hole and 5µl of the marker in the remaining two holes

**Overlapping PCR**

1. Use PCR to amplify copies of IsPETase.

1. Template: pET28a-IsPETase
2. Primers: IsPETase-F & Overlap-R

2. Use PCR to amplify copies of MHETase.

1. Template: pET28a-MHETase
2. Primers: Overlap-F & MHETase-R

3. Agarose gel electrophoresis + Gel DNA recovery

4. Use the overlap part to link the gene for IsPETase and MHETase & amplify copies of linked Is-MH

1. Template: pET28a-IsPETase & pET28a-MHETase
2. Primers: IsPETase-F & MHETase-R

**Agarose gel electrophoresis**

1. Make Agarose gel

1. 0.5g Agarose
2. 50ml TAE Buffer
3. Put in microwave for 1 minute
4. Add nucleic dye for 1 µL
5. pour into the mold and wait until it solidifies

2. Insert genes into the gel

1. Mix 10µL of loading buffer and the product of PCR
2. Insert 30µL of sample in each hole and 2µL of the marker in the remaining two holes.

**Enzyme cut and plasmid reconstruction**

1. Add EcoR1 and Not1 into the sample to cut the Is/MH sequence
2. Insert the Is/MH gene into the plasmid and connect the inserted gene with the vector gene using T4
ligase
3. Transfer the recombinant sequence to DH5α
4. Spread plating of bacteria on solid LB culture medium

**Test for enzyme activity**

1. Add 5 PET microparticles into the 50ml culture solution containing genetically modified bacteria
2. Put in the shaking incubator overnight
3. Observe the PET microparticles under 400x optical microscope

Xuhui Lab

**Plasmid extraction**

Aim: to obtain the pET28a plasmid with ISPETase and/or MHETase from the originally cultured strain, for
transfer into BL21(DE3) E.coli strain
Apparatus: Diamond plasmid kit
Bacteria strain: DH5α (TOP10) - a designed strain for amplifying a plasmid
Procedure:

1. Balance pH using buffer S, centrifugate to immerse
2. Add buffer SP1 (containing RNase) to already centrifugated cultured bacteria; mix thoroughly
3. Add an alkaline buffer SP2 to obtain DNA;; invert the centrifugation tube several times and wait for 2
minutes to complete reaction (destroy nucleoids); add buffer SP3 to balance pH for selection of
plasmid
4. Centrifugate to eliminate cell debris; add wash solution to purify by centrifugation (and let the sample
get rid of genome-related DNA (this step repeats for one time).
5. Add elution buffer, centrifugate to collect plasmids, refrigerate at -20 degrees celsius for storage

**Gel electrophoresis**

Aim: to identify whether the plasmid contains the wanted gene
Preparations: 1% agarose solution as gel immersed in TAE buffer (EB added to dye); keep pre-cut and cut
samples to identify the presence of plasmids and the wanted gene segment; EcoR I and Xho I enzymes are
selected to cut off the gene segment
Principles: DNA is negatively charged in dissolved form; they move across the same medium at different rates
given the same voltage, according to their relative molecular weights; linearized (enzyme-cut) samples move
faster than cyclic samples of the same length.

**Plasmid transfer**

Aim: to induce the E. coli BL21 to absorb in the engineered plasmid
Principles: transfer the plasmid into the bacteria and then prevent it from receiving other plasmids
Procedure:

1. Competence E. coli stored in -80 centigrade placed on ice
2. Add the plasmids and wait for 30 minutes for them to be absorbed
3. Hot activation at 42 centigrade for 90 seconds and immediately put on ice for 3 minutes to remove
competence
4. Culture the bacteria

**Ni column protein purification**

The aim of this procedure is to purify pet28a-IsPETase and pet28a-MHETase. The
basic principle behind this is that the Imidazole component in the buffers would competitively replace
His-tag on pET28a, causing the target proteins to be washed down for collection.
The following steps make up the whole procedure: First, Rinse the column with 10mL H2O. Second, equilibrate
the column with a 10ml Solution buffer. Then proceed to load the sample and Re-equilibrate the column.
After, load the 10ml wash buffer, collect the run-through. Finally, load 10ml Elution Buffer and collect
run-through. Rinse the column and keep the system with 6mL 20% ethanol under room temperature.

**Enzyme activity testing**

The aim of this step is to test the effect of IS-PETase&MH-PETase on microPET
degradation.
First, we mixed 10µg/mL enzyme with 5mg PET granule under 30 ℃. Then we proceeded to add 50mM Glycine buffer
(pH 9.0) and centrifuged the system under 1000 rpm. The system was then heated to 85℃ and kept for 10min.
Finally, we used a microscope to observe the degradation effect.

**EPCR**

This procedure is to amplify the IsPETase and MHETase DNA. The reaction system
consists of 50µL Primestar, 1µL cDNA, 1µL forward primer, 1µL reverse primer, 19µL ddH2O, and 3µL DMSO. The
whole PCR process is made up of three stages: DNA denaturation, primer annealing, and DNA synthesis. During
the first stage, the system was heated to 96℃ for 6min plus 10s to break the double-strand structure of the
target DNA strands. Then cooled down to 66℃ for 15s, the system entered the second stage during which
primers annealed to the single-stranded templates. Finally, the system was heated to 72℃ and kept for 90s,
during which DNA polymerase bind to the primers and started DNA synthesis. This whole process was repeated
for 35 cycles and finally extended for 90s under 72℃.

**T4 Ligase- enzyme ligation**

In the experiment of our group, the function of T4 is slightly different from the other group’s. They used
the T4 ligase enzyme to connect the target gene and the plasmid vector to obtain the recombinant vector.
However, we utilize the T4 ligase both to connect the gene of PETase and MHETase together and to link this
recombinant gene onto the plasmid vector. The material required for this step is:
-T4 DNA ligase 2µL
-DNA 0.03pmol：IsPETase: 0.02µg; MHETase: 0.04µg
-Vector 0.3pmol (0.106µg)
-ddH20 16µL (as the whole system is 20µL)
The solution with ligase should be put under 16 ℃ for 2 hours.

ATLATL Lab II

**Transformation**

1. Remove BL21(DE3) competent cell from -80oC and thaw on ice.
2. Add 100 ng plasmid DNA into BL21(DE3) strain and mix gently.
3. Incubate tube on ice for 30 minutes.
4. Heat shock tube at 42 oC for ~ 90 seconds without shaking.
5. Replace tube on ice for 3 minutes.
6. Add 100 μl room temperature LB medium.
7. Shake and incubate tube at ~ 200 rpm for 60 minutes at 37 oC.
8. Spread on LB agar plate containing 50 μg/ml kanamycin.
9. Incubate the plate upside down at 37 oC overnight.

**Pilot expression**

1. Pick three single, well-isolated colonies and inoculate it into 4 ml LB medium containing 50 μg/ml
kanamycin, respectively.
2. Incubate the cells in shaker at 37oC with shaking at 200 rpm.
3. When the OD600 value reaches 0.6 ~ 0.8, two of the three tubes are added IPTG with the final
concentration of 0.5 mM IPTG for induction at 15 oC for 16 h and at 37 oC for 4 h, respectively, and the
last one tube as the negative control.
4. Detect the protein expression and solubility using both SDS-PAGE and western blot.

**Sample preparation**

1. Harvest cell pellet from 450 μl culture, and lyse with 300 μl lysis Buffer (50 mM Tris, 150 mM NaCl, 5%
glycerol pH 8.0) using sonicator for 1 minutes.
2. Whole cell lysate: Mix 50 μl 5 x loading buffer to 100 μl cell lysate as the sample of whole cell lysate.
Heat the sample at 100 oC for 10 minutes, and centrifuge at 15,000 rpm for 5 minutes.
3. Supernatant and pellet of cell lysate: Centrifuge the remained 200 μl cell lysate at 15,000 rpm for 10
minutes, and collect the supernatant and pellet of cell lysate, respectively
4. Mix 90 μl 5 x loading buffer to 180 μl supernatant as the sample of supernatant of cell lysate. Resuspend
all of the precipitation with 150 μl 5 x loading buffer as the sample of pellet of cell lysate. Heat the
samples at 100 oC for 10 minutes, and centrifuge at 15,000 rpm for 5 minutes before loading into the gel.

**MHETase Enzyme Activity Test**

MHETase Reactions were performed in triplicate over a 1 d time course at 30°C. Reaction mixtures contain 50
nM recombinant GST-MHETase, 5 mM MHET in 90 mM NaCl, 10% (v/v) DMSO, 45 mM sodium phosphate, pH 7.5.
Reactions were terminated by adding an equal volume of methanol. The reactions using inactive GST-MHETase
(obtained by boiling for 10 min) were used as blank controls.
The activity of MHETase was indicated by the decline absorbances at a wavelength of 240 nm.

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# Team:WFLA YK PAO/Results

WFLA\_YK\_PAO

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Results

ATLATL

**1. PCR amplification results of individual gene segments**

![](https://static.igem.org/mediawiki/2021/7/74/T--WFLA_YK_PAO--results01.jpg)

In both pictures, we see a distinctive expression of IsPETase on the left and
MHETase on the right. From
the marker, we could judge that the band is IsPETase as it’s molecular weight is 897bp and MHETase is
2082bp.

**2. PCR amplification results of connected IsPETase-MHETase gene**

![](https://static.igem.org/mediawiki/2021/8/8f/T--WFLA_YK_PAO--results02.jpg)

The recombinant gene segment should be approximately 3000bp. Judging from the
marker, we could see the highlighted band is the recombinant sequence. There is also a left-over of MHETase
and IsPETase that haven’t been completely connected.

**3. Gel electrophoresis results on enzyme-cut IsPETase-MHETase gene and
pET28a**

![](https://static.igem.org/mediawiki/2021/5/51/T--WFLA_YK_PAO--results03.jpg)

Again, judging from the marker, we could see the Is-MH segment that has been cut
and also the vector DNA
that has been cut. There is also a remaining of uncut Is-MH segment.

**4. 1st SDS-PAGE results**

![](https://static.igem.org/mediawiki/2021/0/08/T--WFLA_YK_PAO--results04.jpg)

A wide band between 50 to 70 on the left gel standards matches the weight of
MHETase (61). This suggests
MHETase was successfully induced and expressed. However, at the 7th track, the pattern for purified MHETase
is very unclear, suggesting low solubility of MHETase.

A clear band between 25 and 35 on the right gel standards matches the weight of
IsPETase (31.5). This
suggests IsPETase was successfully induced and expressed. However, the location of the band is higher than
expected. This could be due to the small number of proteins added in the gel electrophoresis. Similar to
MHETase, there are very weak indications of IsPETase on the 3rd and 7th track, suggesting low solubility of
IsPETase.

**5. Microscopic image results**

![](https://static.igem.org/mediawiki/2021/a/aa/T--WFLA_YK_PAO--results05.jpg)

It was difficult to tell from the two photos how successful these enzymes were in
decomposing microplastic.
Comparing this microplastic to the control group, we can see that it appears to have some damage on its
edges, and even a few small holes. The decomposition impact, on the other hand, cannot be shown by this
metric. Our capacity to observe minute changes on the PET surface is limited by the modest magnification of
the light microscope.

Xuhui

**1. Result analysis of gel electrophoresis-Identifying target DNA**

![](https://static.igem.org/mediawiki/2021/5/5c/T--WFLA_YK_PAO--results06.jpg)

Two groups of recombinant IS-MH genes, separate IS and MH is examined in the gel
electrophoresis. The
actual molecular weight of separate IsPETase is about 790bp, which is different from the expected value of
897bp. The band of MH which is supposed to be 2082bp or the other two recombinant genes which are supposed
to be 3000bp could not be observed. This is because of the experimental conditions and operational problems.

**2. Result analysis of SDS-PAGE-Identifying purified protein**

![](https://static.igem.org/mediawiki/2021/a/ac/T--WFLA_YK_PAO--results07.jpg)

Following the order of precipitation, flow through, wash through eluted after 25 mM
imidazole, and 3
separate wash through after 250 mM imidazole to identify proteins for each IS and MH group. The standard
band of IS is about 31. At the 5th and 6th track, there is a sign that suggests the existence of protein,
but the indication of IS is about 27-28, which is still different from the standard molecular weight as it
is supposed to be in the middle of 25 and 35. Reasons include operation problems and experimental conditions
problems, such as the concentration of protein is too low. Also, during the first SDS Page, it is hard to
observe the MH protein, which shows the low solubility of MHPETase. After the second SDS-PAGE analysis and
added the precipitation of MH, this suggests that it formed the inclusion.

**3. Result analysis of the microscope picture- comparing enzyme activity of
IsPETase and MHETase to decompose micro plastic molecules.**

![](https://static.igem.org/mediawiki/2021/d/d7/T--WFLA_YK_PAO--results08.jpg)

left: blank
right: IsPETase+MHETase
The two pictures did not provide a clear indication of the true effectiveness of these two enzymes combined
to decompose microplastic. From the picture, we can observe that compared to the control group, the
microplastic in the group of two enzymes combined seems to have some damage on the edge, and even have some
small holes. However, this cannot be a significant indicator to prove the decomposition effect. One problem
with qualitative analysis was with the irregular shape of an individual microplastic molecule. As we did not
take microscope pictures of each individual molecule before they were put into the enzyme solution, we
cannot conduct a meaningful comparison because the comparison between molecules is not valid. Therefore,
this study requires more careful quantitative analysis to determine the actual enzyme activity and
decomposition effectiveness.

**4. Result analysis of the microscope picture- comparing the bacterial activity
of IsPETase and MHETase
to decompose micro plastic molecules.**

![](https://static.igem.org/mediawiki/2021/f/fd/T--WFLA_YK_PAO--results09.jpg)

leftmost: blank
middle: bacterial activity group 1
rightmost: bacterial activity group 2
Previously, we have added microplastic molecules into two solutions containing bacteria designed to
synthesize IsPETase and MHETase. After one day on the shaker, we want to compare the bacterial activity of
these bacteria on decomposing the microplastic. However, just by observation, it was hard to determine
whether the irregular edges can be attributed to the addition of these bacteria specifically. This study
requires quantitative analysis, such as using the liquid chromatogram, to compare the effectiveness of
decomposition in numerical terms.

ATLATL

**1. Pilot expression**

· IsPETase( ~35.13 kDa)\_BL21(DE3)

![](https://static.igem.org/mediawiki/2021/8/88/T--WFLA_YK_PAO--results10.jpg)
Fig.1 SDS-PAGE (left) and western blot (right) analysis for IsPETase cloned in pET28a and expressed in
BL21(DE3) strain.

Lane M1: Protein marker
Lane M2: Western blot marker
Lane PC1: BSA (1μg)
Lane PC2: BSA (2μg)
Lane NC: Cell lysate without induction
Lane 1: Cell lysate with induction for 16h at 15 oC
Lane 2: Cell lysate with induction for 4 h at 37 oC
Lane NC1: Supernatant of cell lysate without induction
Lane 3: Supernatant of cell lysate with induction for 16h at 15 oC
Lane 4: Supernatant of cell lysate with induction for 4 h at 37 oC
Lane NC2: Pellet of cell lysate without induction
Lane 5: Pellet of cell lysate with induction for 16h at 15 oC
Lane 6: Pellet of cell lysate with induction for 4 h at 37 oC
The primary antibody for western blot is anti-His antibody

· MHETase( ~65.17 kDa)\_BL21(DE3)

![](https://static.igem.org/mediawiki/2021/b/b3/T--WFLA_YK_PAO--results11.jpg)
Fig.2 SDS-PAGE (left) and western blot (right) analysis for MHETase cloned in pET28a and expressed in
BL21(DE3) strain.

Lane M1: Protein marker
Lane M2: Western blot marker
Lane PC1: BSA (1μg)
Lane PC2:BSA (2μg)
Lane NC: Cell lysate without induction
Lane 1: Cell lysate with induction for 16h at 15oC
Lane 2: Cell lysate with induction for 4 h at 37oC
Lane NC1: Supernatant of cell lysate without induction
Lane 3: Supernatant of cell lysate with induction for 16h at 15oC
Lane 4: Supernatant of cell lysate with induction for 4 h at 37oC
Lane NC2: Pellet of cell lysate without induction
Lane 5: Pellet of cell lysate with induction for 16h at 15oC
Lane 6: Pellet of cell lysate with induction for 4 h at 37oC
The primary antibody for western blot is anti-His antibody

**2. Protein production fusion with GST tag.**

![](https://static.igem.org/mediawiki/2021/4/41/T--WFLA_YK_PAO--results12.jpg)

Lane 1: MHETase Cell lysate without induction for 20 h at 16oC
Lane 2: MHETase Cell lysate with induction for 20 h at 16oC
Lane 3,4,5: GSH elution fractions of purification of lane 2 by GST-affinity chromatography
Lane 6: IsPETase Cell lysate without induction for 20 h at 16oC
Lane 7: IsPETase Cell lysate with induction for 20 h at 16oC
Lane 8,9,10: GSH elution fractions of purification of lane 7 by GST-affinity chromatography

**3. Conclusion and suggestion about protein express**

![](https://static.igem.org/mediawiki/2021/9/9c/T--WFLA_YK_PAO--results13.jpg)

**4. Enzyme Activity Tests**

The activity of MHETase was indicated by the decline absorbances at a wavelength of
240 nm (Figure 3). The
wavelength is the specific absorption of MHET. As shown in figure 2, after 1 d reaction, MHET concentration
was dropped by 31.4%.

![](https://static.igem.org/mediawiki/2021/e/ec/T--WFLA_YK_PAO--results14.jpg)
Figure 3. Detection of residual MHET by measuring the absorbance at 240 nm.

![](https://static.igem.org/mediawiki/2021/d/d1/T--WFLA_YK_PAO--results15.jpg)
Figure 4. MHETase activity assay

Future Plan

1. We have difficulties in extracting the protein IsPETase which seems insoluble.
In this case, we are thinking about to re-construct the plasmid which could be transformed as well as
expressed in other types of bacteria, like pseudomonas putida to further test the protein expression. In
addtion to this, once we could obtain the purified IsPETase would we further conduct the enzyme activity
tests to analyze the performance.

2. In future, we will consider the requirements of beverage in product preparation,
test the solubility in different common drinks, and further make sample beverage formula. It is very
important for the product development to consider the safety, stability and the storage problems.

3. As our product is designed for human beings, it would be necessary to simulate
the human gastrointestinal environment to test the effect.

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