1st ITERATION
1st ITERATION – design:
In our 1st iteration design, RIAD or RIDD was fused to the N termini of hydrophobin 4 and PETase was linked
by a flexible linker (GGGGS)3, respectively. Moreover, RIDD was fused to the C termini of MHETase
by a
flexible linker (GGGGS)3. MHETase and PETase were linked (as shown in structure calculations) via
RIDD
peptides to accelerate MHET removement and reduce competitive inhibition to PETase. Meanwhile, short
peptides RIDD fused to enzymes can effectively avoid disturbances to enzymes active sites. As for prudential
and rational design, the multicomplex enzyme system we designed were evaluated via Molecular Dynamic
simulations (MD) to ensure the structure designing feasibilities. The binding stability of the components
was measured and the whole system was revealed to maintain its components binding stably while enzymes were
fixed in relative near positions to each other in liquid environment. Furthermore, analysis of the key
residues in enzymatic active sites and residues distances to Ca2+ ion in Ca2+ binding
site showed maintenance of structure stability (more details in Model). In order to purify the target protein by
affinity chromatography, we add a His-tag to the C-terminus of each protein.
Figure 1: Gene routes in 1st iteration
In the first iteration, we plan to independently verify that each target gene (part) is useful, which means
they were properly expressed and secreted into the culture medium . Therefore, we plan to construct three
plasmids with targeted gene and introduce them into three E. coli chassis cells, and analyze the
expressibility, secretability, and activity of three separate proteins in the three different engineered
bacteria.
1st ITERATION – build:
The three target genes were synthesized (GenScript). To obtain three different expression vectors, the
synthesized target genes were then cloned into three pET28a vectors, which were introduced into
E.coli BL21
by heat shock, respectively. After screening, we successfully obtained three engineered bacteria that can
recombinantly express the target protein. So we named these three kinds of engineering bacteria
BL21/pET28a-PD, E.coli BL21/pET28a-MD, E.coli BL21/pET28a-hA (E.coli BL21/pET28a-PD
means bacteria
containing pET28a-PETase-RIDD, E.coli BL21/pET28a-MD means bacteria containing
pET28a-MHETase-RIDD, while E.coli BL21/pET28a-hA means bacteria containing pET28a-hydrophobin4-RIAD).
1st ITERATION – test:
We obtained three engineered bacteria that can secrete RIDD-PETase, RIDD-MHETase, and RIAD-hydrophobin4
through the previous design and build process respectively. SDS-PAGE was shown as following. (Taking
RIDD-PETase as an example)
Figure 2: SDS-PAGE result. Channel 1: E.coli BL21/pET28a-PD's cell pellet (suspended
in 10 mM Tris-HCl, pH = 8.0); Channel 3: E.coli BL21/pET28a-PD's medium; Channel 5 and 7:
E.coli BL21/pET28a-PD's LB culture medium.
1st ITERATION – learn:
The results showed that the target protein was not detected in the the culture medium, while there are many
target proteins in the bacterial fragmentation, which indicates that our recombinant protein is not properly
secreted. However, our results contradicted with the previous report that PETase can be secreted through the
pelB signal peptide [1].
To find the reasons of this difference, we consulted with Dr. Feng Yanbin, an associate professor in DUT. He
is one of the specialists studying E. coli recombinant protein. Through discussion with Dr. Feng, we
learned
that the pelB signal peptide has strict requirements for the hydrophobicity of the first 20 amino acids at
its N-terminal. According to the literatures, the sequence following pelB is PETase [1],while in our
design it is the amino acid sequence of RIDD linked to pelB. Due to the significant difference between the
strong hydrophobicity of RIDD and the general hydrophobicity of the first 20 amino acids of PETase, PETase
might not be secreted in our study. Similarly, the hydrophobicity of RIAD and RIDD exceeds that of ordinary
proteins, pelB becomes ineffective and cannot function well. We took the suggestions from Dr. Feng and made
two proposals for follow-up work. One is to change the signal peptide. The other is to remove the signal
peptide once and for all, break the bacteria after expressing the recombinant protein and purify the
recombinant protein for further study.
The main goal of our design is based on the construction of multi-enzyme complexes, so the current priority
is to verify that the multi-enzyme complexes can be prepared by E. coli and are active. Therefore,
the
signal peptide from the plasmid was removed, and the bacteria were lysed to obtain the protein. The
subsequent researches were conducted using the enzyme complex. Aside of pelB, we have also researched
several other kinds of signal peptides, which is discussed in more details in the proof of concept.
2nd ITERATION
2nd ITERATION – design :
In the 2nd iteration, we learned from the failure of the signal peptide in the first iteration. To improve
the strategy, we plan to remove the signal peptide and construct three engineering bacteria to continue the
experiment, but in this iteration we only explore separately whether the three proteins can be expressed and
remain active in bacteria. The gene routes of this iteration are shown in Figure 3.
Figure 3: Gene routes in 2nd iteration
2nd ITERATION – build:
We used PCR to amplify each target gene along with its promoter, lac operator, terminator, and RBS. Then we
use overlapping PCR to remove the pelB sequence on all three genes (Figure 4).
Figure 4: Agarose gel electrophoresis diagram of each DNA fragment after removing pelB.
Channel 1: DNA
Marker(the label of each band is shown in the figures); Channel 2: RIDD-PETase; Channel 3:
RIAD-hydrophobin4 (with promoter, lac operator, terminator, and RBS); Channel 4: RIDD-MHETase ( with
promoter, lac operator, terminator, and RBS).
After obtaining the target gene sequence without pelB, we use seamless clone to construct vectors, and
transformed the three vectors into three E.coli BL21 by heat shock transformation. We cultivated
three kinds
of bacteria, inducing them with IPTG for 24 hours, and then ruptured the cells to obtain cell pellets and
medium.
2nd ITERATION – test :
SDS-PAGE
In the second project, we removed the signal peptide, and obtained three engineering bacteria that can
express RIDD-PETase, RIDD-MHETase, and RIAD-hydrophobin4, respectively. The results showed that
corresponding target proteins were present in the fragments of the three bacteria. However, due to its
smaller molecular weight (11 kDA), RIAD-hydrophobin4 was not detectable on 10% separation gel.
Figure 5: SDS-PAGE result s of PETase-RIDD and MHETase-RIDD expression.
Channel 1: No expected bands were found in the medium of E.coli BL21 without induced PETase
expression after resuspension and centrifugation; Channel 2: No expected bands were found in the precipitate
of E.coli BL21 expressing PETase without IPTG induction after centrifugation; M: marker;
Channel 3: There were expected bands in the medium of E.coli BL21 where RIDD-PETase expression was
induced following by cell disruption and resuspension, but the concentration was low; Channel 4: There were
significant correlation bands in the precipitate of induced RIDD-PETase expressing E.coli BL21 after
cell disruption, resuspension and centrifugation, exhibiting high concentration. Channel 5: There were
expected bands in the medium of E.coli BL21 where RIDD-MHETase expression was induced following by
cell disruption and resuspension, but the concentration was low; Channel 6: There were significant
correlation bands in the precipitate of induced RIDD-MHETase expressing E.coli BL21 after cell
disruption, resuspension and centrifugation, exhibiting high concentration.
Western blot
Take the supernatant for SDS-PAGE electrophoresis (70 V 30 min, 110 V 2 h);
After the electrophoresis, cut off the protein gel block, place it in a protein semi-dry transfer instrument,
and transfer the target protein to the PVDF membrane under the condition of 15 V 13 min; Put the PVDF
membrane into 50 g/L skimmed milk powder and leave it at room temperature for 2 hours (slowly shake); Use 20
g/L skimmed milk powder to dilute the anti-His mouse antibody at a ratio of 1:2000, and add an appropriate
amount of the diluted antibody. Submerge the PVDF membrane in a self-made closed bag containing PVDF
membrane, and incubate overnight at 4 °C (slowly shaking); Take out the PVDF membrane from the primary
antibody solution and place it in a petri dish, wash with TBST 5 times, each time 15 min; Use 20 g/L skimmed
milk powder to dilute goat anti-mouse IgG (called secondary antibody) at a ratio of 1:3000, and add an
appropriate amount of diluted secondary antibody to a self-made closed bag with PVDF membrane to submerge
the PVDF membrane and incubated at room temperature for 2 hours (slowly shake); Then, take out the PVDF
membrane from the secondary antibody solution and place it in a plate, wash five times with TBST, 15 minutes
each time; After washing, dry the PVDF membrane and drop it on the front. Add the prepared color developer
until it is completely covered, place it at room temperature for 2 minutes, place it in an ECL luminescence
imager for exposure and photographing, and record the result.
Figure 6: Western blot results
Enzyme activity
As is seen in the SDS-PAGE results, after cytoclasis, RIAD-hydrophobin4, RIDD-PETase, RIDD-MHETase all
primarily existed in the precipitate, while almost none were found in the lysis medium. It was known that
most proteins in the precipitate are in the form of inclusion bodies and not active. It is difficult to tell
whether the target proteins were present in the medium after the cell disruption by direct observation.
Therefore, we performed western-blot analysis. The results showed that there was still a small amount of
RIDD-PETase in the medium, indicating that instead of waiting for the results of renaturation experiment,
the medium can be used to determine the enzyme activity of RIDD-PETase, RIDD-MHETase as crude enzyme
solution.
Since it takes a long time to degrade PET plastics, it is impossible to effectively and scientifically
measure the enzyme activity of MHETase alone, so we referred to the method of iGEM 2016
Harvard_BioDesign team and
designed a set of methods suitable for our project. The enzyme activity
was measured using pNPB, a universal substrate for esterases. Under the reaction of pNPB esterase,
p-nitrophenol can be generated, which has a strong absorption peak at 405 nm. In a set period of time, the
higher the absorbance of the mixture of pNPB and enzyme solution is at 405 nm, the higher the yield of
p-nitrophenol and the greater the enzyme activity.
Figure 7: Enzyme activity analysis of pNPB. PD: RIDD-PETase enzyme solution, ; MD:
RIDD-MHETase enzyme
solution; Blank: normal E.coli BL21's broken medium.
Based on the results (Figure 5), we found that both RIDD-PETase and RIDD-MHETase have esterase activity.
After
20 minutes of reaction, each resulted in absorption peaks as high as 3.4873 and 2.7654 at 405 nm.
2nd ITERATION – learn:
The results showed that the three engineered bacteria we constructed could produce various target proteins
(Figure 5). The presence of protein can be detected in the medium by Western blot. After we added the
scaffold of RIDD to PETase and MHETase, the enzymes still exhibited esterase activity and the substrate pNPB
was degraded. This indicates that the addition of the short peptide RIDD is likely to have no effect on the
PET degradation activity of the enzyme. Because hydrophobin 4 itself does not have enzymatic activity, as
any ordinary hydrophobin, after adding hydrophobic RIAD, the hydrophobicity will not change
dramatically.
3rd ITERATION
3rd ITERATION – design
We are trying to construct a three-enzyme complex, so the secretion of the three target proteins is expected
to be close to or equal to 1:1:1. This can minimize mismatches (PD-PD-hA, MD-MD-hA), and it is also
beneficial to make full use of the produced protein to form a complex and avoid waste. To ensure that the
target proteins are produced at a ratio close to or equal to 1:1:1, we decided to use the same exact
promoter, lactose operon, RBS, and terminator to regulate the three target genes after discussion with Prof.
Zhu Zhiwei in DUT. Also, we decided to integrate PD, MD and hA into one plasmid (Figure 8).
Figure 8: Plasmid construction strategy.
3rd ITERATION – build
With the help of team Tianjin, who used the
CEPC method to connect the genes in the sequence of PD → MD → hA
(as is shown in Figure 6), we obtained a plasmid that can express all three proteins at the same time.
After the ligation product was transformed into E.coli DH5α, through colony PCR (Figure 7) and
restriction
enzyme digestion verification (Figure 8), we proved that the engineered bacteria containing the target
plasmid were obtained. We transformed the extracted plasmid into E.coli BL21 and then induced protein
expression with IPTG. The engineering bacteria was then lysed to obtain medium and cell pellet.
Figure 9: Colony PCR results. Channel 3 &4 are positive results, which means this bacteria
containing
pET28a-PD-MD-hA; Channel 1,2,5,6,7,&8: False positives.
Figure 10: Restriction enzyme digestion (XhoⅠ) verification of
pET28a-PD-MD-hA.
3rd ITERATION – test
SDS-PAGE
According to the first two rounds of engineering and the third round of design and construction, we have
obtained engineered bacteria that can theoretically secrete all three target proteins simultaneously. After
IPTG induction of these engineered bacteria, we obtained a large amount of E.coli
BL21/pET28a-PD-MD-hA,
and
the enzyme was extracted. However, due to the small molecular weight of RIAD-hydrophobin4, it can not be
found on SDS-PAGE. (As for the three protein moulecular weight, RIDD-PETase is 32 kDa, RIDD-MHETase is
68 kDa, RIAD-hydrophobin is 11 kDa.
Figure 11: SDS-PAGE results of the three enzyme complex expression (Channel 1: E.coli
BL21/pET28a-PD-MD-hA
medium; Channel 2: E.coli BL21/pET28a-PD-MD-hA precipitation resuspension)
Enzyme activity
The results of SDS-PAGE showed that there is still a large amount of target protein in the precipitates (only
RIDD-PETase and RIDD-MHETase). Based on the previous Western blot results, it was reasonable to speculate
that the target protein might be present in the cytolysis medium, so we again employed the pNPB method. The
enzyme activity was tested with the medium of E. coli BL21/pET28a-PD-MD-hA as crude enzyme
solution. To
control variables, the volume of crude enzyme solution in this experiment was the same as that used in the
previous determination of the RIDD-PETase crude enzyme solution and RIDD-MHETase crude enzyme solution. The
enzyme activity determination curve was shown in Figure 9.
It is not difficult to conclude that the slope of PD-MD-hA is higher than that of PD or MD, which indicates
that as a crude enzyme solution, the medium of E.coli BL21/pET28a-PD-MD-hA has esterase
activity. What's
more, the final concentration of p-nitrophenol was higher than that of the two esterases alone, which
suggested that the three-enzyme complex was successfully constructed.
Figure 12: Enzyme activity analysis of pNPB. PD: RIDD-PETase enzyme solution, abbreviated PD;
Dark grey: RIDD-MHETase enzyme solution, abbreviated PD; Pink: E.coli BL21/pET28a-PD-MD-hA
broken medium, abbreviated PD-MD-hA; Black: normal E.coli BL21's broken medium, abbreviated
blank.
PET plastic sheet degradation test
We obtained PET plastic sheet with 12% crystallinity (scientific research only) from TJUSLS_China, and cut
it into 5 mm*5 mm fragments (0.07 g per fragment). We incubated 1.5 mL of E.coli BL21, E.coli
BL21/pET28a-PD,
E.coli BL21/pET28a-M, E.coli BL21/pET28a-PD-MD-hA cell pellet and medium in several EP
tubes, and added three
5 mm*5 mm fragments, respectively. We then incubated the reaction mixture at 37 °C for 7 days. After 7 days,
the
degradation product, erephthalic acid (TPA), was detected by UV Spectrophotometry and thus determined PET
degradation efficiency.
In terms of measurement, we chose to use UV spectrophotometry to detect the output of TPA. Binding with
RIDD-PETase and RIDD-MHETase, PET will be decomposed with ethylene glycol (EG) and terephthalic acid (TPA)
as final product. Ethylene glycol is volatile, and the test results are not credible, so we decided to
detect TPA. Through previous literature research, we found that there are two mainstream detection methods
for TPA. One is to directly perform UV spectrophotometry on the sample. The increase in absorbance of the
reaction mixture in the ultraviolet region of the light spectrum (at 240 nm) indicates the release of
soluble TPA or its esters from an insoluble PET substrate. This compound shares an identical strong
absorbance peak around 240–244 nm with an identical extinction coefficient as all three compounds contain
the same number of carbonyl groups. The second is to adopt reverse-phase HPLC. Reverse-phase HPLC systems
have been widely used to analyze the products derived from the enzymatic hydrolysis of PET owing to their
powerful resolving capability and reproducibility. The different compounds produced by PET hydrolytic
enzymes (i.e., TPA, MHET, and BHET) can be efficiently separated on a C18 reverse-phase HPLC column: The
reaction mixture is loaded into a column equilibrated with a polar mobile phase and the concentration of the
organic solvent (acetonitrile).
Considering our experimental cycle, throughput, and laboratory conditions, we chose UV spectrophotometry to
detect TPA. Then we made the standard curve of TPA at OD240 (Figure 13).
The liquid obtained after incubation of the above eight samples was tested for TPA content, and the blank
absorption was subtracted. The data obtained is shown in Figure 10.
It can be seen from the concentration of TPA product that the concentration of TPA in PD-MD-hA's medium is
higher than that in PD's medium or MD's medium. This strongly supported the engineering success of our
three-enzyme complex construction.
Figure 13: Standard curve of TPA at OD240
Figure 14: The concentration of TPA product in each experiment group
Scanning electron microscopy
To further confirm the activity of the three-enzyme complex we constructed, we also selected PET plastic
sheets in E.coli BL21, E.coli BL21/pET28a-PD, E.coli BL21/pET28a-M, E.coli
BL21/pET28a-PD-MD-hA
medium to be
examined by scanning electron microscopy The results were consistent with expectations. The plastic sheet
treated with either E.coli BL21 medium or MD medium has almost no scratches or holes. The plastic
sheet
after PD treatment has some obvious scratches, while the plastic sheet after PD-MD-hA treatment showed
surface covered with scratches, and at the same time, densely packed with holes of various shapes. This
scanning electron microscopy further proved that the three-enzyme complex we constructed has a better PET
plastic degradation activity than single-enzyme degradation.
Figure 15: Scanning electron microscopy (a) PET plastic sheets treated in E.coli BL21.
(b) PET plastic sheets treated in E.coli BL21/pET28a-M. (c) PET plastic sheets
treated in E.coli BL21/pET28a-PD. (d)
PET plastic sheets treated in E.coli BL21/pET28a-PD-MD-hA medium.
3rd ITERATION – learn
The third engineering cycle is the last cycle of our main line of experimental tasks. Briefly, we
successfully constructed three-enzyme complex as expected, measured the complex's enzyme activity, and
performed scanning electron microscopy and TPA detection experiments. The degradation effect of the
three-enzyme complex on PET plastic was evaluated qualitatively and quantitatively.
Through the qualitative test of scanning electron microscopy, the enzyme complex created in our project has
better effect on PET plastic sheets than PETase alone. The use of PETase alone will only cause scratches on
the surface of the plastic sheet. However, our enzyme complex has exceeded PETase activity, causing more
significant scratches and on top of that, producing many deep holes. The results of scanning electron
microscopy also indirectly accounted for the role of hydrophobin 4 in the enzyme complex. With the presence
of hydrophobin 4, the enzyme complex can bind to the surface of the PET plastic sheet. Therefore, instead of
dispersing in water and induce reaction through random collisions, the enzyme is more likely to dig deeper
into the surface.
Through the quantitative test of TPA production detection, we found that the degradation effect of our enzyme
complex is better than PETase used alone, and the degradation efficiency has been increased by two
times.
Overall, we successfully constructed a three-enzyme complex, and confirmed its activity by analysis of
degradation effect and product yield . It will be promising for the subsequent enzyme complex to efficiently
degrade PET plastic and the industrialization of PET plastic bio recycling.
Reference
[1] Arshad A. Analytical methods for the investigation of polymer degradation. 2014.
