Revision as of 00:42, 22 October 2021 (view source) FrancisGrace (Talk | contribs) ← Older edit		Latest revision as of 02:27, 22 October 2021 (view source) FrancisGrace (Talk | contribs)
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	disruption, resuspension and centrifugation, exhibiting high concentration.</p>		disruption, resuspension and centrifugation, exhibiting high concentration.</p>
−	<h2>Enzyme activity</h2>	+	<h3>Enzyme activity</h3>
	<p>As is seen in the SDS-PAGE results, after cytoclasis, RIAD-hydrophobin 4, RIDD-PETase, RIDD-MHETase all primarily		<p>As is seen in the SDS-PAGE results, after cytoclasis, RIAD-hydrophobin 4, 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		existed in the precipitate, while almost none were found in the lysis medium. It was known that most proteins in the
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	<p>Based on the results , we found that both RIDD-PETase and RIDD-MHETase have esterase activity. After 20 minutes of		<p>Based on the results , 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 405nm.</p>		reaction, each resulted in absorption peaks as high as 3.4873 and 2.7654 at 405nm.</p>
−	<h2>PET plastic sheet degradation test</h2>	+	<h3>PET plastic sheet degradation test</h3>
	<p>We obtained PET plastic sheet with 12% crystallinity ( scientific research only) from TJUSLS_China, and cut it into		<p>We obtained PET plastic sheet with 12% crystallinity ( scientific research only) from TJUSLS_China, and cut it into
	5mm*5mm fragments (0.07g per fragment). We incubated 1.5 mL of <i>E.coli BL21, E.coli BL21/pET28a-PD, E.coli		5mm*5mm fragments (0.07g per fragment). We incubated 1.5 mL of <i>E.coli BL21, E.coli BL21/pET28a-PD, E.coli
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	</p>		</p>
−	<h2>Scanning electron microscopy</h2>	+	<h3>Scanning electron microscopy</h3>
	<p>To further confirm the activity of the three-enzyme complex we constructed, we also selected PET plastic sheets in		<p>To further confirm the activity of the three-enzyme complex we constructed, we also selected PET plastic sheets in
	<i>E.coli BL21, E.coli BL21/pET28a-PD, E.coli BL21/pET28a-M, E.coli BL21/pET28a-PD-MD-hA</i> medium to be examined by		<i>E.coli BL21, E.coli BL21/pET28a-PD, E.coli BL21/pET28a-M, E.coli BL21/pET28a-PD-MD-hA</i> medium to be examined by

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Latest revision as of 02:27, 22 October 2021

PETase (PET-degrading enzyme, origin I. sakaiensis)

PETase is the PET(poly(ethylene terephtalate))-degrading enzyme first identified in Ideonella sakaiensis. This sequence is the E. coli K12 optimized DNA sequence for PETase.

This part includes a T7 promoter (BBa_I712074), RBS 34 (BBa_B0034), and PETase fused with a His-tag, for purification.

Showing our protein is inducibly expressed: SDS Page and Western blot

We can validate protein expression further with SDS Page and Western blot visualization. An SDS Page will further confirm by showing that a protein of the correct weight is being expressed. Additionally, a Western blot will show that our histag is functioning. The Western blot is more specific than an SDS Page because only the histagged protein will bind.

Before performing the SDS Page we grew up T7 lysY Iq cells containing a plasmid with PETase under control of the T7 promoter. Cells were induced with 0.4M, 0.1M, or 0M IPTG for either 6 hours or overnight, at either 15 or 30 degrees C. After expression, cells were then lysed according to our ultrasonication protocol, and supernatant and pellet were collected for the SDS Page gel. Supernatant is the soluble fraction of cell lysate, while pellet is the insoluble fraction. After collecting supernatant and pellet, we ran the SDS page, with an additional lysozyme control.
The following image highlights our significant result:


SDS Page shows protein band at correct size for PETase.
The red box shows an overexpressed protein between 20 and 25 kDa. PETase has a weight of 24 kDa, so the band is exactly where we would expect to see PETase. In the control that has not been induced (0M IPTG), we do not observe a band between 20 and 25 kDa. This experiment again demonstrates our system’s inducible control: the blue box shows there is no band in the absence of IPTG.
If you are wondering what the dark band below the PETase band is, it is lysozyme that we used in our cell lysis protocol. Lysozyme was run as a control, which you can see has a dark band in the same location.

For the SDS Page protocol, please refer to our experiments page. (http://2016.igem.org/Team:Harvard_BioDesign/Experiments)

An additional experiment we performed to confirm the presence of PETase was a Western Blot. While the SDS page can show a protein of the proper size is being expressed, a Western allows us to probe the identity of protein itself. To make PETase easily detectable via Western Blot, we designed our constructs (http://2016.igem.org/Team:Harvard_BioDesign/Design) to include a “His tag” which is targeted by a commercially available antibody. By running an SDS-PAGE as described above, we could separate all the proteins in the cell by size. Then we could transfer these proteins to a paper membrane and stain with a the commercially available anti-His antibody. Because our recombinant PETase protein is the only his-tagged protein in the cell, we would expect to see a signal only from the band which contains PETase. See details of this protocol on our experiments page (http://2016.igem.org/Team:Harvard_BioDesign/Experiments).
Here were our results:

Western Blot shows inducible expression of PETase and PETase-sfGFP fusion.

At 30 kD, we see a band which corresponds to the approximate expected size of PETase (28.6 kD). This band is strongest in lane 1, which contains lysate from our PETase construct that is unfused to GFP and induced with IPTG. Additionally, we see a band at 70 kD in lanes 2 and 3, which is the expected size of PETase-sfGFP fusion. Unexpectedly, we also see a PETase-sized band in lanes 2,3, and 4, which should only have a band at 70 kD for the PETase-sfGFP fusion. We hypothesize this is because the PETase GFP fusion is being degraded by proteases because it is so large. We hypothesize the smear we see between 70 kD and 30kD in lane 2 is because of the same phenomenon, such that the PETase-GFP fusion is being chopped into varying length fragments, each which has a histag and therefore shows up on the blot. Finally, we see no signal in lane 5, which was lysate from cells containing the same construct as lane 1, but uninduced. <p> In conclusion, this Western demonstrates that we have inducible control over PETase expression.

Exeter 2019 Characterisation

As part of the Exeter iGEM 2019 project we wanted to improve the stability of the PETase enzyme expressed in E.coli under the T7 promoter. A wide range of projects have been carried out looking at using directed evolution and rational design to improve the stability of the enzyme. We decided to undertake a two pronged approach to improving the enzyme stability. The first was to look at some mutations and combinations of mutations that have been made by groups previously to test their stability and effectiveness in breaking down PET fibres from clothes(Demonstrated in BBa_K3039003, BBa_K3039001). The second was to build ancestral reconstruction mutants that could potentially show an increased stability.(Demonstrated in BBa_K3039017, BBa_K3039018)

Purification graphs

Nickel Affinity Chromatography

Nickel affinity trace showing the elution of the protein from the column with an increase in Imidazole concentration

Size Exclusion Chromatography (Superdex-75)

Further purification of the enzyme by size exclusion chromatography using a calibrated Superdex-200 column. The large peak (2) at an elution volume of ~80 ml shows the monomeric form of the protein. A second smaller peak (1) ~67 ml corresponds to a higher oligomeric state of the protein (Potentially Dimeric) that will be further investigated as higher oligomeric states have been reported to show higher thermal stability

Esterase Activity

Activity was measured by spectrophotometrically flowing the hydrolysis of p-nitrophenyl acetate (pNPA) into acetate and p-nitrophenol. This was performed at room temperature in buffer containing 50 mM NaPhosphate buffer pH7.5, 100 mM NaCl. A range of substrate concentrations were tested and a blank used to subtract the auto-hydrolysis of the pNPA. The production of p-nitrophenol was measured at 405 nm.

The esterase activity assay shows the production of p-nitrophenol (A405nm) at different substrate concentrations

Thermal Stability Graphs

The specific activity of the enzyme at differing substrate concentrations

Thermal Stability

The thermostability of the enzyme was investigated incubating enzyme samples at a range of temperatures (20 °C - 90 °C) for one hour using the gradient function in a SensOQuest LabCycler (Geneflow) before samples are cooled to 4 °C and assayed for activity using the esterase assay method described previously.

The thermal stability assay shows the production of p-nitrophenol (A405nm) after the pre-incubation of the enzyme at increasing temperatures before the esterase assay was carried out.

Thermal Stability of Wild Type PETase Vs. BBa_K3039001 (SP1), BBa_K3039002 (SP2) and BBa_K3039003 (PTS)

Comparison of the thermal stability of the WTPETase with the intelligent design mutants Exeter 2019 have entered into the registry. The % activity of the enzymes compared to the activity at room temperature. WT PETase is most active at 40 °C before immediately falling off to 0% activity at 50 °C. PETase S212E_D186H_R280A (PTS) is also most active at 40 °C but is able to retain ~70 % activity at 50 °C before falling to 0% activity at 60 °C. SP1and SP2 although are not as active at the lower temperatures but SP1 is able to retain ~35 % activity at 50 °C before falling to 0% activity at 60 °C.

Thermal Stability of Wild Type PETase Vs. ANCESTRAL MUTANTS

Comparison of the thermal stability of the WTPETase with theAncestral reconstruction mutants Exeter 2019 have entered into the registry. The ancestral mutants were cloned and over expressed in E.coli and did show esterase activity. Although AR1 is unable to retain as high a level of activity at some of the lower temperatures AR1 is able to retain ~25 % activity at 50 °C

Improvement of BBa_K2010000 by iGEM19_HK_GTC

While the Wild Type PETase coded by this part has significant activity, it may not be high enough for industrial use. To improve the activity of the wild type, we used site-directed mutagenesis to create double mutants, and the most successful is W159H/S245I, encoded by BBa_K2982004

We analyze the rationale for PETase mutant design from previous studies done on the residue modification of this enzyme. A clear trend in most successful mutation attempts is that an increase in hydrophobicity or a binding site similar to T. fusca cutinase, which is narrower.

The mutation sites locate in substrate binding site, subsite II where three MHET moieties are bound through hydrophobic interaction.

In TfCut2, Histidine 169 residues and Isoleucine 253 are located at the corresponding positions of Trpytophan 159 and Serine 245 in subsite II of IsPETase. The resulting double mutant makes the substrate binding site, substrate II more cutinase-like and increases the hydrophobic property of the enzyme.

Then,we tested the mutant using an enzyme assay with p-nitrophenyl dodecanoate as the substrate. By comparing the optical densities of the reaction mixture with the mutant and the wild type PETase, we can see how the mutation affects activity.

Figure 1: Optical densities at 415nm for reaction mixtures with W159H/S245I and Wild Type PETase. It can be seen that W159H/S245I has a higher activity

Figure 2: Percentage increase of optical densities for reaction mixtures with W159H/S245I and Wild Type PETase

As shown in the data, the W159H/S245I double mutant exhibits a higher rate of percentage increase, and also higher overall increase in optical density at 415nm at all times. This shows that the activity of the mutant is undeniably higher than that of the wild type. This verifies that the mutant is an enhancement of the wild type, and thus, BBa_K2982004 is an improvement of BBa_K2010000.

[1]:Austin, H. P., Allen, M. D., Donohoe, B. S., Rorrer, N. A., Kearns, F. L., Silveira, R. L., . . Beckham, G. T. (2018). Characterization and engineering of a plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences, 115(19). doi:10.1073/pnas.1718804115

Improvement of BBa_K2010000 by TJUSLS_China

Here is TJUSLS_China(2021). We use PETase as our wild type as we want to improve the thermostability of PETase through mutations.

We choose five methods to improve the thermostability of PETase, salt bridges, hydrogen bonds, hydrophilic interaction, prolines, and disulfide bonds. We tried to find out unstable regions in PETase by reading literature as well as doing structural analysis and visual screening via bioinformatics tools.

Finally, we got 26 mutations which have 11-12 mutation sites each. And our best mutation-Super5 contains 11 mutation sites S214H-I168R-W159H-S188Q-R280A-A180I-G165A-Q119Y-L117F-T140D-S121E.

We use HPLC equipment to measure the peak area of the product of PET(MHET) of the reaction, in order to express the enzyme activity of PETase. For more information on the product of PET(MHET), please see our project introduction.

Figure 1. Enzyme activity determination, compared with wild type.

Well, the enzyme activity and thermostability of Super5 have greatly improved 163 times，compared with PETase.

If you want to know more detailed things about our project, please click this link to our parts page. Thanks a lot.

https://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2021&group=TJUSLS_China

DUT_China 2021 Contribution

New information learned from literature

According to a study by Son HF et al., the development of biodegradable plastics using cutinase and lipase has become an important research focus; however, these enzymes are limited by their low PET degradation ability and requirement of a considerably high temperature of approximately 70 °C to carry out their activity. Recently, a new bacterium, Ideonella sakaiensis, was isolated that was shown to effectively use PET as a carbon source. Importantly, PETase from I. sakaiensis (IsPETase) not only shows far higher degradation activity than any other enzyme known to be capable of degrading PET to date but also functions at a moderate temperature, indicating its environmental applicability. Nevertheless, the IsPETase activity for PET degradation is still too low for application to resolve the current environmental challenge. Some researchers use the structure information of IsPETase, several mutation studies have been attempted to improve the activity of IsPETase, and these variants were reported to have slightly higher enzyme activity in comparison to the wild type IsPETase. Although IsPETase exhibits the most powerful PET degradation activity among enzymes tested so far at moderate temperature, its enzyme activity and stability still limit its utilization for plastic waste treatment. Moreover, PETase needs to be secreted to carry out its function, which requires establishing conditions to allow for maintenance of its function under the harsh stresses of the extracellular environment. However, the stability of IsPETase is relatively low because I. sakaiensis only grows under mild conditions. Therefore, variants of IsPETase need to be designed with the characteristics of high stability to consequently retain enzymatic activity for a long period of time at a moderate temperature. Accordingly, on the basis of the reported crystal structure of IsPETase, they developed a rational protein engineering strategy to increase the thermal stability of the protein and successfully obtained IsPETase variants with remarkably enhanced thermal stability and highly improved PET degradation ability. This work can help to increase the possibility of achieving the complete biodegradation of PET under mild conditions.

[1] SON H F, CHO I J, JOO S, et al. Rational Protein Engineering of Thermo-Stable PETase from Ideonella sakaiensis for Highly Efficient PET Degradation [J]. ACS Catalysis, 2019, 9(4): 3519-26.

[2] YOSHIDA S, HIRAGA K, TAKEHANA T, et al. A bacterium that degrades and assimilates poly(ethylene terephthalate) [J]. Science, 2016, 351(6278): 1196-9.

[3] HAN X, LIU W D, HUANG J W, et al. Structural insight into catalytic mechanism of PET hydrolase [J]. Nature Communications, 2017, 8(

[4] JOO S, CHO I J, SEO H, et al. Structural insight into molecular mechanism of poly (ethylene terephthalate) degradation [J]. Nature Communications, 2018, 9(

New data collected from laboratory experiments

We use optimized PETase combined with a short peptide (BBa_K3898152) to construct a PET degrading enzyme complex (BBa_K3898166), which improves the degradation efficiency. The following is its introduction.

RIDD-PETase consists of two components RIDD and PETase. RIDD, as a part of skeleton-free protein, forms a three-enzyme complex of PETase, MHETase and hydrophobic protein by linking with RIDD and RADD parts of RIDD-MHETase and RIAD-hydrophobic protein as a bond. PETase is a polyethylene terephthalate degradation enzyme, and PETase (IsPETase) in Ideonella sakaiensis has the highest PET degradation activity under mild conditions among all PET degradation enzymes reported to date. We obtained by orthogenesis has a stabilized β6-β7 connecting loop and extended subsite IIc, IsPETase^{S121E D186H/R280A} variant variant (reference: Rational Protein Engineering of Thermo-Stable PETase from Ideonella sakaiensis for Highly Efficient PET Degradation,). Compared with IsPETase^WT, its T_m value increased by 8.81°C, and its PET degradation activity increased by 14-at 40°C, showing high thermal stability. The molecular weight of PETase is 32kD.

SDS-PAGE

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-hydrophobin 4 was not detectable on 10% separation gel.

Fig.1 SDS-PAGE results 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; Channel 4: 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.

Enzyme activity

As is seen in the SDS-PAGE results, after cytoclasis, RIAD-hydrophobin 4, 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 iGEM16_Harvard_BioDesign team and designed a set of methods suitable for our project (link to the experiment webpage). 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 405nm, the higher the yield of p-nitrophenol and the greater the enzyme activity.

Fig.2 Enzyme activity analysis of pNPB . PD: RIDD-PETase enzyme solution, ; MD: RIDD-MHETase enzyme solution; Blank: normal E.coli BL21's broken medium. (30 °C, pH=8.0,1000μM pNPB)

Based on the results , 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 405nm.

PET plastic sheet degradation test

We obtained PET plastic sheet with 12% crystallinity ( scientific research only) from TJUSLS_China, and cut it into 5mm*5mm fragments (0.07g 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 5mm*5mm 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 3).

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 4.

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.

Fig.3 Standard curve of TPA at OD240

Fig.4 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.

(a)	(b)
(c)	(d)

Fig.5 Scanning electron microscopy (a) PET plastic sheets treated in E.coli BL21. (b) PET plastic sheets treated in E.coli BL21/pET28a-PD. (c) PET plastic sheets treated in E.coli BL21/pET28a-M. (d) PET plastic sheets treated in E.coli BL21/pET28a-PD-MD-hA medium.

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.

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Sequence and Features BBa_K2010000 SequenceAndFeatures