Part:BBa_K2010000
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.
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
Sequence and Features
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