iGEM 2019 — Team Toronto

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Toronto/Notebook

[Our Project](https://2019.igem.org/Team%3AToronto/our-project)

[Wet Lab](https://2019.igem.org/Team%3AToronto/wetlab)

[Dry Lab](https://2019.igem.org/Team%3AToronto/drylab)

[PnP](https://2019.igem.org/Team%3AToronto/pnp)

[The Team](https://2019.igem.org/Team%3AToronto/Team)

# Experiments

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Our Project

---

Background
Design
Improvement
Attributions

Wet Lab

---

Experiments
Results
Notebook

Dry Lab

---

Model

PnP

---

Integrated Human Practices

Stock footage provided by Videvo: downloaded from https://www.videvo.net

iGEM Toronto | igemtoronto.ca


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Toronto/Experiments

[Our Project](https://2019.igem.org/Team%3AToronto/our-project)

[Wet Lab](https://2019.igem.org/Team%3AToronto/wetlab)

[Dry Lab](https://2019.igem.org/Team%3AToronto/drylab)

[PnP](https://2019.igem.org/Team%3AToronto/Human_Practices)

[The Team](https://2019.igem.org/Team%3AToronto/Team)

# Experiments

### Construct Design

---

In order to analyze the function of PETase mutants in-vitro, our team decided to use a pET22b plasmid vector. Since proper folding of PETase occurs in the E. coli periplasm, pET22b was chosen because it contains a pelB leader sequence (Singh et al. 2013), which allows for the periplasmic targeting of PETase, followed by signal cleavage via the periplasmic protein, signal peptidase (Singh et al. 2013). Furthermore, this vector allows for IPTG-induction of PETase production via the presence of the LacI promoter. PETase variants were synthesized and cloned into PET22b courtesy of GenScript.

### LOBSTR Cells

---

When conducting His-tagged purification of recombinant proteins from E.coli, naturally histidine rich proteins may be pulled down with the protein of interest. The most common of these proteins are arnA and slyD. Low Background Strain (LOBSTR) is a strain of E.coli similar to BL21. It grows at a similar rate to standard BL21, but contains mutants of slyD and arnA with lower affinity to Nickel-NTA resins. This allows for greater sample purity without the need of multiple purification steps, by eliminating arnA and slyD presence when eluting. (Andersen et al. 2013).

### Bacterial Chases

---

All of our plasmids were transformed into and purified from DH5alpha competent cells. For protein purification, transformation into LOBSTR cells was necessary to prevent the presence of unwanted proteins that have affinity to Nickel-NTA resins.

### Protein Purification

---

![](https://static.igem.org/mediawiki/2019/a/ad/T--Toronto--Experiment2.png)

We used immobilized metal-ion affinity chromatography to isolate our HIS-tagged protein of interest from LOBSTR cells. The Hexa-His-tag is a chain of six histidines that are attached to the C-terminus end of the our PETase enzyme. Histidine has an imidazole side chain that has high affinity for Ni^2+, hence Nickel-NTA resin is bound to the affinity chromatography column. The LOBSTR with all of the proteins is lysed by sonication and lysozyme, and spun-down to isolate all cellular proteins. Proteins are introduced into the column; the proteins of interest with the His-tag will bind to the Nickel-NTA resin. Washes with a slightly higher concentration of imidazole are completed to eliminate proteins not bound to Nickel-NTA. Finally, elutions are performed whereby a solution with excess imidazole is added to the column. This will complete with the His-tag to binding with the resin, dislodging the protein of interest and a relatively pure protein should be obtained in the end (Bornhorst, 2000).

### Quantification

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Following protein purification and SDS-PAGE, protein concentration was quantified by performing Bradford Assays in serial dilutions.

### PETase Activity Assays

---

To test the catalytic activity of our variants, a p-nitrophenyl butyrate assay was performed. This assay is designed to test for activity of certain hydrolases. The molecule consists of p-nitrophenol bonded to butyric acid in an ester linkage. Hydrolysis of the ester releases p-nitrophenolate (the alcohol is deprotonated), which is a chromophore than can be measured spectrophotometrically at 405 nm (Pliego et al., 2015). The p-nitrophenyl butyrate assay, which has been used to measure PETase activity in previous studies (Furukawa et al., 2018) was chosen because its structure is similar enough to PET subunits to be recognized by PETase and cleaved; thus, p-nitrophenolate production can be used as an indicator of enzymatic activity.

![](https://static.igem.org/mediawiki/2019/a/ad/T--Toronto--Experiment1.png)

### References

---

1.) Pliego, J., Mateos, J. C., Rodriguez, J., Valero, F., Baeza, M., Femat, R., Camacho, R., Sandoval, G., Herrera-López, E. J. (2015). Monitoring lipase/esterase activity by stopped flow in a sequential injection analysis system using p-nitrophenyl butyrate. Sensors (Basel, Switzerland), 15(2), 2798–2811. doi:10.3390/s150202798

2.) Furukawa, M., Kawakami, N., Oda, K., Miyamoto, K. (2018). Acceleration of Enzymatic Degradation of Poly(ethylene terephthalate) by Surface Coating with Anionic Surfactants. Chemsuschem, 11(23), 4018-4025. doi:10.1002/cssc.201802096

3) Andersen KR, Leksa NC, Schwartz TU. (2013). Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification. Proteins. 81(11):1857-61.

4) Seo, Hogyun, et al. “Production of Extracellular PETase from Ideonella Sakaiensis Using Sec-Dependent Signal Peptides in E. Coli.” Biochemical and Biophysical Research Communications, vol. 508, no. 1, 2019, pp. 250–255., doi:10.1016/j.bbrc.2018.11.087.

5) Singh, Pranveer, Likhesh Sharma, S. Rajendra Kulothungan, Bharat V. Adkar, Ravindra Singh Prajapati, P. Shaik Syed Ali, Beena Krishnan, and Raghavan Varadarajan. “Effect of Signal Peptide on Stability and Folding of Escherichia Coli Thioredoxin.” PLoS ONE8, no. 5 (July 2013). https://doi.org/10.1371/journal.pone.0063442.

6) Bornhorst, Joshua A., and Joseph J. Falke. “[16] Purification of Proteins Using Polyhistidine Affinity Tags.” Methods in Enzymology Applications of Chimeric Genes and Hybrid Proteins Part A: Gene Expression and Protein Purification, 2000, 245–54. https://doi.org/10.1016/s0076-6879(00)26058-8.

### Protocols

---

The following were the protocols used this year:

[Gibson Assembly](https://static.igem.org/mediawiki/2019/0/0c/T--Toronto--gibsonAssembly.pdf)

[Midi Plasmid Purification](https://static.igem.org/mediawiki/2019/1/10/T--Toronto--PlusmidPurification)

[Mini Plasmid Purification](https://static.igem.org/mediawiki/2019/3/39/T--Toronto--miniPlasmidPurification.pdf)

[Transformation](https://static.igem.org/mediawiki/2019/5/5a/T--Toronto--transformation.pdf)

[SDS Page](https://static.igem.org/mediawiki/2019/7/71/T--Toronto--SDSPAGE.pdf)

[Plate Preparation](https://static.igem.org/mediawiki/2019/7/74/T--Toronto--PlatePreparation.pdf)

[p-Nitrophenol Butyrate assay](https://static.igem.org/mediawiki/2019/9/9e/T--Toronto--pnitrophenolassay.pdf)

[Protein Purification](https://static.igem.org/mediawiki/2019/a/ac/T--Toronto--proteinPurification.pdf)

[Competent Cells](https://static.igem.org/mediawiki/2019/d/df/T--Toronto--calciumCompCells.pdf)

[Gel Extraction](https://static.igem.org/mediawiki/2019/e/e0/T--Toronto--gelExtraction.pdf)

[Bradford Assay](https://static.igem.org/mediawiki/2019/e/e5/T--Toronto--BradfordAssayp.pdf)

[Restriction Enzyme Digest](https://static.igem.org/mediawiki/2019/f/f3/T--Toronto--REdigest.pdf)

### Parts

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You can find the parts used in our project [[here]](https://2019.igem.org/Team%3AToronto/Parts)

Our Project

---

Background
Design
Improvement
Attributions

Wet Lab

---

Experiments
Results
Notebook

Dry Lab

---

Model

PnP

---

Integrated Human Practices

Stock footage provided by Videvo: downloaded from https://www.videvo.net

iGEM Toronto | igemtoronto.ca

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Toronto/Results

[Home](https://2019.igem.org/Team%3AToronto)

[Our Project](https://2019.igem.org/Team%3AToronto/Project)

[Wet Lab](https://2019.igem.org/Team%3AToronto/wetlab)

[Dry Lab](https://2019.igem.org/Team%3AToronto/drylab)

[PnP](https://2019.igem.org/Team%3AToronto/pnp)

[The Team](https://2019.igem.org/Team%3AToronto/Team)

# Results

### Overview

---

In the course of our research term, we managed to purify all six PETase variants (i.e. the mutant generated by Austin et al (2018): W159H S238F, PETase variant “Tianyu”, PETase variant “Dimi 1”, PETase variant “Dimi 2”, PETase variant “Dimi 3”, and PETase variant “Dimi 4”) using their C terminal hexahistidine tag using affinity purification. The catalytic activity of these variants was subsequently compared to wildtype PETase using a p-nitrophenol butyrate (pNPB) assay, which mimics the ester bond cleaved by PETase. It is frequently used in the literature as a substrate for assessing hydrolase activity, as the product of the reaction (p-nitrophenolate) conveniently fluoresces at 405 nm. A variety of substrate concentrations were used from 30 uM to 2500 uM, with controls for autohydrolysis of pNPB in solution. Prior to executing this assay, we endeavoured to improve the characterization of the wildtype PETase. We discovered that its cleavage of pNPB was optimal at pH 8.0-9.0, as opposed to "physiological pH" of 7.0. The comparison of all PETase variants was thus executed at pH 9.0. The assay temperature was also kept constant at 30 C, due to this temperature for PETase characterization used by Yoshida et al.
![](http://parts.igem.org/File:T--Toronto--pNPB-Hydrolysis-Wildtype_pH7-8-9.png)
![](https://static.igem.org/mediawiki/parts/c/cb/T--Toronto--pNPB-Hydrolysis-pH-9_Wildtype-All-Conc.png)
![](https://static.igem.org/mediawiki/parts/6/66/T--Toronto--pNPB-Hydrolysis-pH-8-Wildtype-All-Conc.png)
![](https://static.igem.org/mediawiki/parts/a/a7/T--Toronto--pNPB-Hydrolysis-pH-7_Wildtype-All-Conc.png)

Subsequently, a time-course (with a one minute kinetic interval for the first 10 minutes, followed by a five minute kinetic interval up to 30 minutes) was executed for all proteins. Due to the high catalytic activity of all of our variants in comparison to wildtype, the absorbance of the spectrophotometer was maxed out at ~15 minutes at substrate concentrations above 625 uM. We have thus chosen to show the linear portion of the graph (from 0-10 minutes) that was used in the experimental estimation of the [Km constants](https://static.igem.org/mediawiki/2019/5/54/T--Toronto--MichaelisMenten.pdf) for each protein.

![](https://static.igem.org/mediawiki/parts/c/cd/T--Toronto--pNPB-Hydrolysis-pH-9_All-PETase-Mutants.png)
From this graph, it is clear that all of our variants seem to present an improvement on the wildtype PETase, but do not match the remarkable activity of the 2018 mutant engineered by Austin et al: W159H S238F. Importantly, alternative assays will be required to follow up on this result. Running HPLC assays of the supernatant of purified PETases upon overnight incubation on PET film would result in the precise detection of the products of PET degradation (MHET, BHET, and TPA). This would rule out the possibility that we had pulled down nonspecific esterases in the E. coli genome capable of degrading pNPB. Another curiosity is the PETase variant "Tianyu", which was optimized for hydrophobicity and catalytic activity, using a machine learning algorithm. This resulted in a PETase that displayed very amino acid sequence conservation with the wildtype and an Instability Index of 50.08 (unstable) by Benchling's estimator. We were surprised to see the protein fold and apparently have activity comparable to one of our FuncLib variants, "Dimi 1", which had a much larger dataset for estimating thermostability.
![](https://static.igem.org/mediawiki/parts/c/cd/T--Toronto--pNPB-Hydrolysis-pH-9_All-PETase-Mutants.png)
![](https://static.igem.org/mediawiki/parts/0/0f/T--Toronto--pNPB-Hydrolysis-pH-9_Tianyu-All-Conc.png)

Our Project

---

Background
Design
Improvement
Attributions

Wet Lab

---

Experiments
Results
Notebook

Dry Lab

---

Model

PnP

---

Integrated Human Practices

Stock footage provided by Videvo: downloaded from https://www.videvo.net

iGEM Toronto | igemtoronto.ca