Team:Exeter/Results

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  Bronze
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  Silver
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  Gold

Introduction

The green fluorescent protein (GFP) from Aequorea jellyfish is often used as a reporter molecule ¹. It is used in cellular based studies to determine how certain cellular processes work, and where in the cell they take place, this is possible because GFP can be attached to other proteins without significantly affecting their biological reactions and processes ^2,3. The iGEM distribution kit contains part BBa_I746909 which is a T7 (BBa_I719005) promoter driving expression of the superfolder variant (sGFP) (BBa_I746916), that folds efficiently when fused to poorly folded proteins ⁴.

The T7 promoter comes from the T7 bacteriophage and is a lac-promoter ⁵. Lac-promoters can be leaky, meaning that there is incomplete repression of gene expression. As we wanted to use the T7 promoter in our project, the main aim of these experiments was to determine how leaky the T7 promoter actually is by using the BBa_I746909 part from Cambridge 2008.
We decided to investigate the ‘leakiness’ of sGFP expression under control of the T7 promoter in three E. coli expression strains. E. coli BL21 is the most widely used strain of E. coli used for protein expression, however expression from a T7 promoter requires T7 polymerase which the wild type strain does not possess. Therefore, we should expect to see no sGFP expression from these cultures. E. coli BL21(DE3) carries the lambda prophage necessary for ITPG inducible expression from the T7 promoter (Novagen) ⁶. As stated above the T7 promoter is known to be leaky therefore, E. coli BL21(DE3)pLysS carries an additional plasmid that encodes T7 lysozyme (Novagen) ⁷. This is a natural inhibitor of T7 RNA polymerase that serves to repress basal expression of target genes under the control of the T7 promoter. In these cultures, we expect to see no sGFP expression before induction with IPTG

Experimental Data Collection

Plasmid pSB1C3-BBa_I746909 was extracted from the distribution kit and initially transformed into E. coli DH5α, for amplification, before transformation into E. coli BL21 and E. coli BL21(DE3). For transformation into E. coli BL21(DE3)pLysS, which is resistant to chloramphenicol, BBa_I746909 had to be cloned into an ampicillin resistant plasmid (pX1800) using restriction enzymes EcoRI and PstI (see protocols page). Cultures of E. coli carrying either pSB1C3-BBa_I746909 or pX1800-BBa_I746909 were grown in LB media with either 35μg/ml chloramphenicol or 100 μg/ml ampicillin as appropriate, in 96 well plates (200μl) at 37°C, 800 rpm. Cell growth was monitored by Optical Density (OD) at 600 nm (Tecan Infinite Plate reader) and calibrated using Cospheric monodisperse silica microspheres of a comparable size to E. coli (0.961 μm) (see Figure 1 below). Expression of sGFP was induced with 200μM of IPTG at an OD of 0.4. Protein expression was monitored by fluorescence (488 nm excitation, 520 nm emission) and calibrated using fluorescein isothiocyanate (see Figure 2 below). After 24 h growth sGFP expression in individual cells was monitored by fluorescence using a BD FACS AriaIII Flow Cytometer (excitation laser (blue) 488 nm, emission 530/30 nm). Molecules of equivalent soluble fluorophore was calculated from a standard curve generated by running Takara Clontech AcGFP Calibration Beads with the same settings (see Figure 3 below). In addition, cell morphology was investigated using an Image Stream Mark II (Amnis-Luminex Corp.) Imaging Flow Cytometer configured with Bright Field (white light), Side-Scatter (785 nm) and GFP (excitation laser 488 nm, emission 533/55 nm).

Calibration

Although the iGEM InterLab study was not run this year we wanted to ensure that any fluorescence data that we reported was presented as comparable units rather than arbitrary units (AU) ⁸. We therefore followed the 2018 iGEM InterLab plate reader protocol to convert OD measurements to estimated number of cells. We performed a serial dilution of a known concentration of silica beads, measured the OD and produced the calibration curve in Figure 1.

Figure 1: Graph showing optical density, measured at 600 nm, vs the number of silica beads. Measured in a 96 well plate with 200μl volumes. Error bars represent standard error of the mean, n = 4.

For bulk fluorescent measurements, again we followed 2018 iGEM InterLab plate reader protocol, by performing a serial dilution of a known concentration of fluorescein, measured fluorescence using the same excitation and emission wavelengths required by sGFP and produced the calibration curve in Figure 2.

Figure 2: Graph showing fluorescence, measured at 488 nm excitation wavelength, 520 nm emission wavelength, vs concentration of fluorescein. Measured in a 96 well plate with 200μl volumes. Error bars represent standard error of the mean, n = 4.

The two calibration curves were then used to calculate Molecules of Equivalent Soluble Fluorophore (MESF) of each culture. AcGFP Flow Cytometer Calibration beads consist of a mixture of six distinct populations that vary in the number of attached AcGFP molecules giving each population a distinct fluorescence.

Takara Clontech report a value for the corresponding Molecular Equivalent of Soluble Fluorophore (MESF) for each peak. The resulting histogram (Figure 3) was used to generate a standard curve of MESF vs fluorescence using the Flow Cytometer software, FlowJo.

Figure 3: Flow cytometer histogram of AcGFP calibration beads. Excitation with 488 nm laser (blue), emission 530/30 nm.

Results

Growth of E. coli BL21-pSB1C3-BBa_I746909,E. coli BL21(DE3)-pSB1C3-BBa_I746909 and E. coli BL21(DE3)pLysS-pX1800-BBa_I746909 was monitored over 24 h and OD data converted to estimated number of cells using the calibration curve above, Figure 4.

Figure 4: Calibrated growth curve data monitoring increase in number of estimated cells with time in cultures of: a) E. coli BL21-pSB1C3-BBa_I746909; b) E. coli BL21(DE3)-pSB1C3-BBa_I746909; and c) E. coli BL21(DE3)pLysS-pX1800-BBa_I746909. Measured in a 96 well plate with 200μl volumes. At OD = 0.4 sGFP expression was induced in half of the well cultures with 200μM of IPTG. Error bars represent standard error of the mean, n = 4.

Growth of uninduced cultures was consistent between strains and did not depend on type of antibiotic, with log phase lasting between 0 and 8 h. Addition of IPTG inhibited growth in all three cultures but more significantly in E. coli BL21(DE3)-pSB1C3-BBa_I746909 and E. coli BL21(DE3)pLysS-pX1800-BBa_I746909 where protein expression was expected. Expression of sGFP was monitored during culture growth in the same experiment as above. Data was calibrated using the standard curves and presented in Figure 5.

Figure 5: Fluorescence data monitoring increase in MESF with time in cultures of: a) E. coli BL21-pSB1C3-BBa_I746909; b) E. coli BL21(DE3)-pSB1C3-BBa_I746909; and c) E. coli BL21(DE3)pLysS-pX1800-BBa_I746909. Measured in a 96 well plate with 200μl volumes. At OD = 0.4 sGFP expression was induced in half of the well cultures with 200μM of IPTG. Error bars represent standard error of the mean, n = 4.

As expected, no sGFP expression was observed in cultures of E. coli BL21-pSB1C3-BBa_I746909 with or without addition of IPTG. Only after induction with IPTG was sGFP expression observed in cultures of E. coli BL21(DE3)-pSB1C3-BBa_I746909 and E. coli BL21(DE3)pLysS-pX1800-BBa_I746909 indicating that in this experiment there was no leakiness from the T7 promoter. Less sGFP expression was seen in cultures of E. coli BL21(DE3)pLysS-pX1800-BBa_I746909. Therefore, we concluded that for expression of the PETase and MHETase enzymes in our project, DE3 carrying E. coli strains would have sufficient control over expression from the T7 promoter and the additional pLysS was not required.
After 24 h, expression of sGFP in individual cells was determined via flow cytometry, Figure 6.

Figure 6: Overlaid flow cytometry histograms uninduced and induced cultures of: a) E. coli BL21-pSB1C3-BBa_I746909; b) E. coli BL21(DE3)-pSB1C3-BBa_I746909; and c) E. coli BL21(DE3)pLysS-pX1800-BBa_I746909 after 24 h growth.

Expression of sGFP, measured by fluorescence is only seen in IPTG induced cultures of E. coli BL21(DE3)-pSB1C3-BBa_I746909 and E. coli BL21(DE3)pLysS-pX1800-BBa_I746909, confirming the bulk fluorescence measurements. The lower expression seen in the bulk cultures of E. coli BL21(DE3)pLysS-pX1800-BBa_I746909 appear to arise from the fact that only a small population of cells are giving rise to the bulk fluorescent signal, rather than all cells having a lower fluorescence.

The geometric mean of MESF per cell for each population was calculated using the FlowJo flow cytometry software and presented in Figure 7.

Figure 7: MESF per cell for E .coli strains A) E. coli BL21-pSB1C3-BBa_I746909 induced, B) E. coli BL21-pSB1C3-BBa_I746909 uninduced, C) E. coli BL21(DE3)-pSB1C3-BBa_I746909 induced, D) E. coli BL21(DE3)-pSB1C3-BBa_I746909uninduced, E) E. coli BL21(DE3)pLysS-pX1800-BBa_I746909 induced, F) E. coli BL21(DE3)pLysS-pX1800-BBa_I746909 uninduced; Data gathered from the geometric mean of FACS plots.

The data from flow cytometry indicated that for over-expression and purification of our PETase and MHETase enzymes for use in our washing machine filter, pLysS containing E. coli strains would decrease the efficiency of production.

Finally, the lab we were working in has an Imaging Flow Cytometer, which gave us an excellent opportunity to look at the cell morphology during expression of sGFP, Figures 8-10.

Figure 8: Image: screen capture of an induced E. coli BL21 cell. Channel 2 (Ch02) shows sGFP fluorescence at 533/55 nm, Channel 4 (Ch04) shows the corresponding bright field image and Channel 6 (Ch06) shows side scatter.

Figure 9: Image: screen capture of an induced E. coli BL21(DE3) cell. Channel 2 (Ch02) shows sGFP fluorescence at 533/55 nm, Channel 4 (Ch04) shows the corresponding bright field image and Channel 6 (Ch06) shows side scatter. The additional feature in the bright field and side scatter images arises from a speed bead, used to calibrate the cytometer.

Figure 10: Image: screen capture of an induced E. coli BL21(DE3)pLysS cell. Channel 2 (Ch02) shows sGFP fluorescence at 533/55 nm, Channel 4 (Ch04) shows the corresponding bright field image and Channel 6 (Ch06) shows side scatter.

The images taken on Ch02 demonstrate that sGFP fluorescence is only detected in strains of E. coli carrying DE3. The bright field images (Ch04) are highly similar between the strains of E. coli indicating that the DE3 lysogen and pLysS plasmid do not significantly change cell morphology.

Conclusion

The results from this bronze medal study, investigating the ‘leakiness’ of the T7 promoter, show that in our lab expression from the T7 promoter is sufficiently controlled in strains of E. coli carrying DE3 only and therefore plasmids carrying our PETase and MHETase genes should not affect cell growth until expression of the enzymes is induced. In addition, strains carrying pLysS could reduce the overall expression of our enzymes, limiting the amount we could produce for our washing machine filter.

Silver >>

Introduction

As part of our filter system we wanted to completely degrade PET down to Terephthalic acid (TPA) Ethylene Glycol (EG). PETase enzymes that have the ability to degrade PET into BHET, MHET, TPA and EG have been previously introduced to the iGEM registry. As part of this project we wanted to introduce two BHETase enzymes that have been discovered (via the mutation of the MHETase enzyme) that can break down BHET into MHET, TPA and EG. As well as introducing a MHETase enzyme that would allow the complete breakdown of PET to its constitutive parts.

The enzymes PETase and MHETase were first discovered in Ideonella sakaiensis in 2016 by a group of researchers in Japan. These enzymes were found to degrade polyethylene terephthalate (PET) into its monomers, terephthalic acid (TPA) and ethylene glycol (EG). PETase degrades PET into Mono-(2-hydroxyethyl)terephthalic acid (MHET), Bis(2-Hydroxyethyl) terephthalate (BHET) and TPA, the main product being MHET. MHET is further degraded by MHETase into TPA and EG. We are aiming to use mutants of these enzymes to degrade the microfibres that are coming off clothing during washing cycles. The enzymes would be secreted into a filter that captures the microfibres. This sequence is the Escherichia coli K12 (E. coli K12) codon optimized DNA of the S416A_F424N mutant MHETase, with an attached His tag. The His tag was attached in order to more easily identify the enzymes. These mutations have been reported in past papers to increase the activity of MHETase.

The native predicted signal peptide (Met1-Ala19) was removed from the WT MHETase sequence (Palm et al 2019)⁹ and replaced with a start codon (Met), however all mutations are numbered according to the full-length WT sequence. The amino acid sequence was submitted to Twist Bioscience who codon optimised the sequence for E. coli, ensuring that there were no forbidden restriction sites, BsaI or SapI, to allow for potential TypeIIS assembly. The resulting CDS was synthesised and cloned, by Twist, into pET28. This added a 21 AA His-tag and thrombin cleavage site to the N-terminal of the protein, a T7 promoter and T7 terminator.

Experiments

The BHET and MHET were expressed in E. coli Arctic Express cells before being purified using Nickel affinity chromatography and size exclusion chromatography and then being checked for activity using a BHETase assay that was analysed using HPLC PROTOCOLS

Results

BHETase 1

Nickel Affinity Chromatography

Figure 1: Nickel affinity column trace taken during initial purification of the enzyme. The light blue line shows the change in imidazole concentration with increasing volume run through the column and the purple line shows the corresponding change in A280 of eluent from the column. The peak at 32 ml shows BHETase 1 elution.

BHETase 2

Figure 2: Nickel affinity column trace taken during initial purification of the enzyme. The light blue line shows the change in imidazole concentration with increasing volume run through the column and the purple line shows the corresponding change in A280 of eluent from the column. The peak at 28 ml shows BHETase 1 elution.

We were unable to further purify these enzymes using size exclusion chromatography. This could have been due to degradation of the protein sample.

BHETase Assay

BHETase 1 Assay

Figure 3: The concentration of the metabolites BHET, MHET and TPA in the assay solution after 24 hours with a change in enzyme concentration.

The assay shows the decrease in BHET concentration during the 24 hour BHETase assay. This BHET is being converted into MHET and a small amount of TPA as can be seen by the appearance of these compounds. We can also see from these assays that an increase in protein concentration corresponds to an increase in the amount of BHET used up as well as as increase in the amount of MHET and TPA produced.

BHETase 2 Assay

Figure 4: The concentration of the metabolites BHET, MHET and TPA in the assay solution after 24 hours with a change in enzyme concentration.

The assay shows the decrease in BHET concentration during the 24 hour BHETase assay. This BHET is being converted into MHET and a small amount of TPA as can be seen by the appearance of these compounds. We can also see from these assays that an increase in protein concentration corresponds to an increase in the amount of BHET used up as well as as increase in the amount of MHET and TPA produced.

Conclusions

The expression of both BHETase 1 and BHETase 2 enzymes was achieved using the E. coli Artic express cell line as can be seen from the Western Blot image above. Both proteins were able to be purified using Nickel affinity chromatography but their further purification by size exclusion chromatography was unsuccessful. This could have been due to some degradation that was seen in the original western blot. The Ni purified enzyme was used in activity assay using pure BHET purchased from Sigma Aldrich which showed that the enzyme was able degrade BHET to MHET over a 24 hour period. Unfortunately although the MHETase enzyme was expressed in small amounts (As seen in the Western Blot image) we were never able to obtain enough enzyme to carry out any enzymatic assays on.

Gold >>

Introduction

One of the Key goals of this project was to produce a PETase enzyme that was more thermal stable. 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. The second was to build ancestral reconstruction mutants^{10 11} that could potentially show an increased stability.

Rational Design

Part Name	Result Summary
PETase S121E_D186H_R280A	Enzyme expressed successfully Esterase assay confirms activity Microfibre degradation confirmed Increased thermal stability
PETase R280A	Enzyme expressed successfully Esterase assay confirms activity Microfibre degradation confirmed Worse specific activity compared to WT
PETase T88A_S121E_D186H_R280A	Enzyme expressed successfully Esterase assay confirms activity Microfibre degradation confirmed Worse specific activity compared to WT Increased thermal stability compared to WT
PETase T88A_S93M_S121E_W159F_D186H_R280A	Enzyme expressed successfully Esterase assay confirms activity Microfibre degradation confirmed Equivalent specific activity compared to WT Worse thermal stability compared to WT

Ancestral Mutants

Part Name	Result Summary
Ancestral PETase 1	Enzyme over-expressed successfully Esterase assay confirms activity Increased thermal stability compared to WT
Ancestral PETase 2	Enzyme over-expressed successfully Esterase assay confirms activity Decreased thermal stability compared to WT
Ancestral PETase 3	Enzyme expressed in low quantities Purification failed No assays possible
Ancestral PETase 4	Enzyme expressed in low quantities Purification of small quantity of enzyme Insufficient quantity for enzyme assays

Experiments

All of the PETase mutants from both the rational design and ancestral reconstruction were expressed in E. coli Arctic Express cells before being purified using Nickel affinity chromatography and size exclusion chromatography and then being checked for activity using an Esterase and PET fibre assay that was analysed using HPLC PROTOCOLS

Results

Wild Type PETase

Figure 1: Nickel affinity column trace taken during initial purification of the enzyme. The light blue line shows the change in imidazole concentration with increasing volume run through the column and the purple line shows the corresponding change in A280 of eluent from the column. There is a small peak at 48 ml showing protein elution.

Figure 2: Further purification of the enzyme by size exclusion chromatography using a calibrated Superdex-75 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

PETase S121E_D186H_R280A

Expression in E. coli

Figure 3: Western blot of the soluble fraction of Arctic Express strain showing expression of all mutants. The PageRuler Plus prestained protein ladder was used and labeled with the corresponding sizes. The negative control is labeled with 1. This part (PETase S121E_D186H_R280A) is labeled with 4. A clear band is visible with a size of about 30 kDa which is the size of PETase with the His tag attached to it.

Figure 4: Western blot of the soluble fraction of Rosetta Gami strain showing expression of all mutants. The PageRuler Plus prestained protein ladder was used and labeled with the corresponding sizes. The negative control is labeled with 1. This part (PETase S121E_D186H_R280A) is labeled with 4. A clear band is visible with a size of about 30 kDa which is the size of PETase with the His tag attached to it.

Purification graphs

Nickel Affinity Chromatography

Figure 5: Nickel affinity column trace taken during initial purification of the enzyme. The light blue line shows the change in imidazole concentration with increasing volume run through the column and the purple line shows the corresponding change in A280 of eluent from the column. The peak at 48 ml shows protein elution.

Size Exclusion Chromatography (Superdex-200)

Figure 6: Further purification of the enzyme by size exclusion chromatography using a calibrated Superdex-200 column. The large peak at an elution volume of ~95 ml shows the monomeric form of the protein.

Esterase Activity

Figure 7: The esterase activity assay shows the production of p-nitrophenol (A405nm) at different substrate concentrations.

Figure 8: The specific activity of the enzyme at differing substrate concentrations.

Thermal Stability Graphs

Thermal Stability

Figure 9: 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 BBa_K3039003 (PTS) Vs. Wild Type PETase

Figure 10: 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.

Fibre Assay Graphs

Figure 11: The breakdown of PET fibres harvested from our filter into its constitutive parts with a change in enzyme concentration over a 76 hour period.

BHET Assay

Figure 12: The breakdown of PET fibres harvested from our filter into BHET with a change in enzyme concentration over a 76 hour period.

MHET Assay

Figure 13: The breakdown of PET fibres harvested from our filter into MHET with a change in enzyme concentration over a 76 hour period.

TPA Assay

Figure 14: The breakdown of PET fibres harvested from our filter into TPA with a change in enzyme concentration over a 76 hour period.

Conclusion

The enzyme is over expressed and found to be active in both the esterase assay as well as the being able to break down PET fibres collected as part of the washing machine filter. The enzyme is more thermal stable than the WT PETase retaining ~70 % activity at 50 °C. The enzyme was also the most active having a specific activity 1.3 x that of the WT PETase

PETase R280A

Expression in E. coli

Figure 15: Western blot of the soluble fraction of Arctic Express strain showing expression of all mutants. The PageRuler Plus prestained protein ladder was used and labeled with the corresponding sizes. The negative control is labeled with 1. This part (PETase R280A) is labeled with 5. A clear band is visible with a size of about 30 kDa which is the size of PETase with the His tag attached to it.

Figure 16: Western blot of the soluble fraction of Rosetta Gami strain showing expression of all mutants. The PageRuler Plus prestained protein ladder was used and labeled with the corresponding sizes. The negative control is labeled with 1. This part (PETase R280A) is labeled with 5. A clear band is visible with a size of about 30 kDa which is the size of PETase with the His tag attached to it.

Protein Purification

Nickel column

Figure 17: Nickel affinity column trace taken during initial purification of the enzyme. The light blue line shows the change in imidazole concentration with increasing volume run through the column and the purple line shows the corresponding change in A280 of eluent from the column. The peak at 54 ml shows protein elution.

Size Exclusion Column (Superdex-75)

Figure 18: Further purification of the enzyme by size exclusion chromatography using a calibrated Superdex-75 column. While the trace is quite messy the largest peak with an elution volume of ~80 ml shows the monomeric form of the protein being eluted from the column.

Esterase Activity

Figure 19: The esterase activity assay shows the production of p-nitrophenol (A405nm) at different substrate concentrations

Figure 20: The specific activity of the enzyme at differing substrate concentrations

The enzyme is over expressed and found to be active in both the esterase assay as well as the being able to break down PET fibres collected as part of the washing machine filter. This enzyme was the worst expressing enzyme from the rational design section. The enzyme had a worst specific activity when compared to the WT PETase

PETase T88A_S121E_D186H_R280A

Expression in E. coli

Figure 21: Western blot of the soluble fraction of Arctic Express strain showing expression of all mutants. The PageRuler Plus prestained protein ladder was used and labeled with the corresponding sizes. The negative control is labeled with 1. This part (PETase T88A_S121E_D186H_R280A) is labeled with 2. A clear band is visible with a size of about 30 kDa which is the size of PETase with the His tag attached to it.

Figure 22: Western blot of the soluble fraction of Rosetta Gami strain showing expression of all mutants. The PageRuler Plus prestained protein ladder was used and labeled with the corresponding sizes. The negative control is labeled with 1. This part (PETase T88A_S121E_D186H_R280A) is labeled with 2. A clear band is visible with a size of about 30 kDa which is the size of PETase with the His tag attached to it.

Purification Graphs

Nickel Affinity Chromatography

Figure 23: Nickel affinity column trace taken during initial purification of the enzyme. The light blue line shows the change in imidazole concentration with increasing volume run through the column and the purple line shows the corresponding change in A280 of eluent from the column. The peak at 50 ml shows protein elution

Size Exclusion Chromatography (Superdex-200)

Figure 24: Further purification of the enzyme by size exclusion chromatography using a calibrated Superdex-200 column. The large peak at an elution volume of ~90 ml shows the monomeric form of the protein. There is a smaller peak at ~65 ml which could be a higher oligomeric state. This will be investigated further in the future as sometimes larger oligomeric states have been found to be more thermal stable.

Esterase Activity

Figure 25: The esterase activity assay shows the production of p-nitrophenol (A405nm) at different substrate concentrations

Figure 26: The specific activity of the enzyme at differing substrate concentrations

Thermal Stability Graphs

Thermal Stability

Figure 27: 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 BBa_K3039001 (SP1) Vs. Wild Type PETase

Figure 28: 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.

Fibre Assay Graphs

Figure 29: The breakdown of PET fibres harvested from our filter into its constitutive parts with a change in enzyme concentration over a 76 hour period.

BHET Assay

Figure 30: The breakdown of PET fibres harvested from our filter into BHET with a change in enzyme concentration over a 76 hour period.

MHET Assay

Figure 31: The breakdown of PET fibres harvested from our filter into MHET with a change in enzyme concentration over a 76 hour period.

TPA Assay

Figure 32: The breakdown of PET fibres harvested from our filter into TPA with a change in enzyme concentration over a 76 hour period.

Conclusions

The enzyme is over expressed and found to be active in both the esterase assay as well as the being able to break down PET fibres collected as part of the washing machine filter. PETase T88A_S121E_D186H_R280A is not as active at the lower temperatures but is able to retain ~35 % activity at 50 °C before falling to 0% activity at 60 °C. The enzyme had a lower specific activity when compared to the WT PETase.

PETase T88A_S93M_S121E_W159F_D186H_R280A

Expression in E. coli

Figure 33: Western blot of the soluble fraction of Arctic Express strain showing expression of all mutants. The PageRuler Plus prestained protein ladder was used and labeled with the corresponding sizes. The negative control is labeled with 1. This part (PETase T88A_S93M_S121E_W159F_D186H_R280A) is labeled with 3. A clear band is visible with a size of about 30 kDa which is the size of PETase with the His tag attached to it.

Figure 34: Western blot of the soluble fraction of Rosetta Gami strain showing expression of all mutants. The PageRuler Plus prestained protein ladder was used and labeled with the corresponding sizes. The negative control is labeled with 1. This part (PETase T88A_S93M_S121E_W159F_D186H_R280A) is labeled with 3. A clear band is visible with a size of about 30 kDa which is the size of PETase with the His tag attached to it.

Nickel Affinity Chromatography

Figure 35: Nickel affinity column trace taken during initial purification of the enzyme. The light blue line shows the change in imidazole concentration with increasing volume run through the column and the purple line shows the corresponding change in A280 of eluent from the column. The peak at 50 ml shows protein elution

Size Exclusion Chromatography (Superdex-200)

Figure 36: Further purification of the enzyme by size exclusion chromatography using a calibrated Superdex-200 column. The large peak at an elution volume of ~90 ml shows the monomeric form of the protein.

Esterase Activity

Figure 37: The esterase activity assay shows the production of p-nitrophenol (A405nm) at different substrate concentrations

Figure 38: The specific activity of the enzyme at differing substrate concentrations

Fibre Assay Graphs

Figure 39: The breakdown of PET fibres harvested from our filter into its constitutive parts with a change in enzyme concentration over a 76 hour period.

BHET Assay

Figure 40: The breakdown of PET fibres harvested from our filter into BHET with a change in enzyme concentration over a 76 hour period.

MHET Assay

Figure 41: The breakdown of PET fibres harvested from our filter into MHET with a change in enzyme concentration over a 76 hour period.

TPA Assay

Figure 42: The breakdown of PET fibres harvested from our filter into TPA with a change in enzyme concentration over a 76 hour period.

Conclusion

The enzyme is over expressed and found to be active in both the esterase assay as well as the being able to break down PET fibres collected as part of the washing machine filter. PETase T88A_S93M_S121E_D186H_R280A is not as active at the lower temperatures and is not as thermal stable as WT PETase. Although not as stable the enzyme is the only other rationally designed enzyme that has a comparable specific activity when compared to the WT PETase

Ancestral PETase 1

Expression in E. coli

Figure 43: Western blot of the soluble fraction of Arctic Express strain showing expression of all Ancestral reconstruction mutants. The PageRuler Plus prestained protein ladder was used and labelled with the corresponding sizes. The negative control is in lane 1. WT PETase is in lane 2. Ancestral Reconstruction Mutant 1 is in lane 3. Ancestral Reconstruction Mutant 2 is lane 4. Ancestral Reconstruction Mutant 3 is lane 5. Ancestral Reconstruction Mutant 4 is lane 6.

Purification graphs

Nickel Affinity Chromatography

Figure 44: Nickel affinity column trace taken during initial purification of the enzyme. The light blue line shows the change in imidazole concentration with increasing volume run through the column and the purple line shows the corresponding change in A280 of eluent from the column. The peak at 48 ml shows protein elution

Size Exclusion Chromatography (Superdex-75)

Figure 45: Further purification of the enzyme by size exclusion chromatography using a calibrated Superdex-75 column.

Esterase Activity

Figure 46: The esterase activity assay shows the production of p-nitrophenol (A405nm) at different substrate concentrations

Figure 47: The specific activity of the enzyme at differing substrate concentrations

Thermal Stability Graphs

Thermal Stability

Figure 48: 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 BBa_K3039017 (AP1) Vs. Wild Type PETase

Figure 49: 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. This is an improvement on the WT PETase which shows no activity at this temperature

Ancestral PETase 2

Expression of E. coli

Figure 50: Western blot of the soluble fraction of Arctic Express strain showing expression of all Ancestral reconstruction mutants. The PageRuler Plus prestained protein ladder was used and labelled with the corresponding sizes. The negative control is in lane 1. WT PETase is in lane 2. Ancestral Reconstruction Mutant 1 is in lane 3. Ancestral Reconstruction Mutant 2 is lane 4. Ancestral Reconstruction Mutant 3 is lane 5. Ancestral Reconstruction Mutant 4 is lane 6.

Purification graphs

Nickel Affinity Chromatography

Figure 51: Nickel affinity column trace taken during initial purification of the enzyme. The light blue line shows the change in imidazole concentration with increasing volume run through the column and the purple line shows the corresponding change in A280 of eluent from the column. The peak at 50 ml shows protein elution

Size Exclusion Chromatography (Superdex-75)

Figure 52: Further purification of the enzyme by size exclusion chromatography using a calibrated Superdex-75 column.

Esterase Activity

Figure 53: The esterase activity assay shows the production of p-nitrophenol (A405nm) at different substrate concentrations

Figure 54: The specific activity of the enzyme at differing substrate concentrations

Thermal Stability Graphs

Thermal Stability

Figure 55: 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 BBa_K3039018 (AP2) Vs. Wild Type PETase

Figure 56: Nickel affinity column trace taken during initial purification of the enzyme. The light blue line shows the change in imidazole concentration with increasing volume run through the column and the purple line shows the corresponding change in A280 of eluent from the column. The peak at 50 ml shows protein elution

Conclusion

The ancestral mutants were cloned and over expressed in E.coli and did show esterase activity. Unfortunately AR2 is not as thermal stable as the WT PETase. Although we did not see an improvement on the WT PETase his information can be fed back into the Ancestral Model and can be used in subsequent rounds of mutant design.

Ancestral PETase 3

Expression of E. coli

Figure 57: Western blot of the soluble fraction of Arctic Express strain showing expression of all Ancestral reconstruction mutants. The PageRuler Plus prestained protein ladder was used and labelled with the corresponding sizes. The negative control is in lane 1. WT PETase is in lane 2. Ancestral Reconstruction Mutant 1 is in lane 3. Ancestral Reconstruction Mutant 2 is lane 4. Ancestral Reconstruction Mutant 3 is lane 5. Ancestral Reconstruction Mutant 4 is lane 6.

Purification graphs

Nickel Affinity Chromatography

Figure 58: Nickel affinity column trace taken during initial purification of the enzyme. The light blue line shows the change in imidazole concentration with increasing volume run through the column and the purple line shows the corresponding change in A280 of eluent from the column. There is no peak showing protein elution

Although we were able to express soluble protein in E.coli we were unable to purify the protein or do any of the enzyme assays due to the low levels of expression.

Ancestral PETase 4

Expression of E. coli

Figure 59: Western blot of the soluble fraction of Arctic Express strain showing expression of all Ancestral reconstruction mutants. The PageRuler Plus prestained protein ladder was used and labelled with the corresponding sizes. The negative control is in lane 1. WT PETase is in lane 2. Ancestral Reconstruction Mutant 1 is in lane 3. Ancestral Reconstruction Mutant 2 is lane 4. Ancestral Reconstruction Mutant 3 is lane 5. Ancestral Reconstruction Mutant 4 is lane 6.

Purification graphs

Nickel Affinity Chromatography

Figure 60: Nickel affinity column trace taken during initial purification of the enzyme. The light blue line shows the change in imidazole concentration with increasing volume run through the column and the purple line shows the corresponding change in A280 of eluent from the column. There is a small peak at 50 ml showing protein elution

Although we were able to express soluble protein in E.coli and able to purify small amounts of the protein we were unable to obtain a sufficient amount to conduct any of the enzyme assays due to the low levels of expression.

4th one so this won't be seen unless another button is added.

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