iGEM 2019 — Team Exeter

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# Team:Exeter/Notebook

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# Team:Exeter/Engineering

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* [Collaborations(current)](https://2019.igem.org/Team%3AExeter/Collaborations)

Engineering

[One](#s1)
[Two](#s2)
[Three](#s3)
[Four](#s4)

[back to top](#pageTitle)

# Engineering

## Section 1

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## Section 2

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## Section 3

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## Section 4

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# Team:Exeter/Experiments

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Protocols

[Transformation of *E.coli*](#s1)
[Type IIS Cloning](#s2)
[Preparation of Competent *E.coli* Cells](#s3)
[Buffers and LB](#s4)
[Expression of Cytosolic PETase and MHETase](#s5)
[Protein Purification](#s6)
[Esterase Assay](#s7)
[Thermal Stability Assay](#s8)
[PETase Assay with Fibres](#s9)
[BHET Assay](#s10)
[HPLC Assay](#s11)
[SDS-PAGE](#s12)
[Western Blot](#s13)

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# Protocols

![Protocols Icon](https://static.igem.org/mediawiki/2019/9/9c/T--Exeter--experiments_icon.png)

This page details the protocols followed in the **biological laboratory** to develop our final **PET degrading enzyme solution**. To see our results, check out our [Results page.](https://2019.igem.org/Team%3AExeter/Results)

## Transformation of *E.coli*

1. Defrost one 100 μL aliquot of *E.coli* competent cells per transformation, plus one extra as a control (use whole tube and do not re-freeze.)
2. Add 1-5μL of plasmid DNA (depending on concentration) to selected competent cells, mix by rolling Eppendorf between fingers.
3. Incubate on ice for 40 mins (allowing the DNA to bind to the cell walls.)
4. Heat shock in Eppendorf at 42°C for 2 minutes, initiating the movement of DNA into the cells.
5. Incubate on ice for 5 minutes.
6. Add 0.7mL of pre-warmed LB medium, incubate at 37°C, 200-220rpm for 60 minutes.
7. Spin down cells at 8000rpm for 5 minutes.
8. Remove 0.5mL of the supernatant.
9. Re-suspend cells in remaining supernatant.
10. Plate out 200μL on agar laced with 100μg/mL of ampicillin for IDT DNA and 50μg/mL of kanamycin for TWIST DNA.
11. Incubate at 37°C overnight.

## Type IIS Cloning

The **one pot cloning strategy** utilises the ability of a **type II restriction enzyme (Bsal)** to cut outside its **recognition sequence** thereby allowing the formation of a variety off different **4 bp overhangs**. Two Bsal are placed at the **5'** and **3'** end of each DNA segment in reverse orientation allowing **two DNA fragments** to be **ligated** seamlessly with the removal of the **Bsal recognition** sequence. To assemble a **complete gene** within a plasmid each DNA part(promoter, RBS, CDS, and terminator) are provided as **uncut plasmids** (entry vectors) and mixed with the **destination vector** in 'one-pot.' The addition of both Bsal and T4 DNA ligase allows for **repeat digestion-ligations cycles** to 'amplify' the desired product due to the removal of the Bsal sites in the final product.

For one pot cloning add all the regants together in their allocated volumes, then follow the reaction conditions.

**- Reagents**

* 20 fmol/μL Destination vector
* 10x Fast Digest Buffer
* 10 mM ATP
* 10U/μL Fast Digest BSal (Thermo)
* 5U/μL T4 DNA ligase (Thermo)

**- Final Reaction Volumes**

* 1μL of each entry vector
* 1μL destination vector
* 2μL buffer
* 1μL ATP
* 1μL Bsal
* 1μL T4 ligase
* Then make up with 20μL of water

**- Reaction conditions**

* 37°C for 2 minutes
* 22°C for 3 minutes
* Repeat above steps for 25-50 cycles
* 37°C for 5 minutes
* 22°C for 5 minutes
* 65°C for 10 minutes
* 10°C hold

## Preparation of Competent *E.coli* cells

1. Scrape a few cells from an *E.coli* glycerol stock (e.gDH5α), straight from the -80°C freezer onto a marked space on an LB-agar plate (no antibiotic!) Streak out three times with a wire and grow overnight at 37°C
2. Innoculate 5ml of LB, in a 50ml Falcon tube, with one colony and incubate overnight at 37°C,200rpm.
3. Innoculate 40ml of LB in a 350mL conical flask with 0.4mL of the overnight culture.
4. Grow at 37°C and 200rpm until the OD600 = 0.4-0.5
5. Transfer the culture to a 50mL Falcon tube and gravest by centrifugation at 5000rpm for 10 minutes at 4°C
6. Drain the pellet and re-suspend cells in 8mL of Transformation buffer, TF-1.
7. Place on ice for 15 minutes and then spin as above.
8. Thoroughly drain the pellet and re-suspend in 4mL of TF-2.
9. Create 100μL aliquots in 2mL Eppendorfs and immediately freeze in liquid nitrogen.
10. Store aliquots in a labelled box in -80° freezer.

## Buffers and LB

### Buffers

* Buffer A: 50 mM Tris, 0.5 mM NaCl, 20 mM imidazole, pH 7.5* Buffer B: 50 mM Tris, 0.5 M NaCl, 0.5 M imidazole, pH 7.5* GF Buffer: 100 mM NaPhosphate Buffer, 100 mM NaCl, pH 7.5

## Expression of Cytosolic PETase and BHETase

### Expression of Cytosolic PETase

1. Set overnights of transformed cells with the genes encoding for the PETase variants and incubate at 37°C, 220rpm.
2. Transfer 1 ml of the overnights into flasks containing 50 ml of LB media and the appropriate antibiotic and incubate at 37°C, 220rpm until OD 0.6-0.8.
3. Induce expression by adding 1µl IPTG / 1ml media and incubate overnight at 18o C, 160 rpm.
4. Centrifuge at 5000rpm for 10 minutes at 20°C.
5. Remove supernatant.
6. Resuspend pellets in 10ml buffer A and disrupt cells by sonication on ice.
7. Centrifuge at 12,000rpms for 20 minutes at 4°C.
8. Collect the supernatant.
9. Store in the cold room.

### Expression of Cytosolic BHETase

1. Set overnights of transformed cells with the genes encoding for the BHETase variants and incubate at 37°C, 220rpm.
2. Transfer 1 ml of the overnights into flasks containing 50ml of LB media and the appropriate antibiotic and incubate at 37°C, 220rpm until OD 0.6-0.8.
3. Induce expression by adding 1µl IPTG / 1ml media and incubate overnight at 18°C, 160rpm.
4. Centrifuge at 10,000g for 20 minutes at 4°C.
5. Remove supernatant.
6. Resuspend pellets in 10ml buffer A and disrupt cells by sonication on ice.
7. Centrifuge at 12,000rpm for 20 minutes at 4°C.
8. Collect the supernatant.
9. Store in the cold room.

## Protein Purification

1. The protein was purified using a 1 ml HisTrap FF crude column (GE Healthcare, Little Chalfont, England) using a gradient from 20 to 500 mM imidazole in 50 mM Tris-HCl pH 7.5, 0.5 mM NaCl.
2. The enzyme was then applied to a calibrated Superdex 75 HiLoad 16/60 gel filtration (GF) or Superdex 200 HiLoad 16/60 column (GE Healthcare, Little Chalfont, England) and eluted with one column volume of 50 mM NaPhosphate Buffer, 0.1 M NaCl, pH 7.5 at 1.0 ml min-1.

## Esterase Assay

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.

## Thermal Stability Assay

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.

## PETase Assay with Fibres

The PETase assay was carried out using fibres obtained from the washing machine and filter as part of this project. 35 µg of fibres was incubated in 500 µl of enzyme at a range of concentrations (50 to 2000 µM) in 50 mM Na Phosphate buffer pH 7.5 with 50 mM NaCl. The fibres were incubated with the enzyme solution for 76 hours before the reaction was terminated by heating at 80 °C for 15 mins. The reaction was then analysed by HPLC.

## BHET Assay

The assay enzyme activity reactions (100 µl) with 100 µg/ml substrate in 40 mM Na Phosphate buffer pH 7.5 with 50 mM NaCl and 20 % (v/v) DMSO were incubated at 25 °C with the enzyme at 3 different concentrations (250, 500 and 1000 µM) and the reaction incubated for 24 hours at 30 °C. The reaction is terminated by heating at 80 °C for 15 mins. BHET was purchased pure form Sigma Aldrich. The reaction was then analysed by HPLC.

## HPLC Assay

Samples (10 µl) was analysed using a high-performance liquid chromatography system (HPLC, Agilent 1200) using an Eclipse Plus C18 column (Agilent, UK). The mobile phase was 99.9 % Water with 0.1 % Formic Acid at a flow rate of 0.8 ml min-1 and the effluent monitored at 240 nm. The typical elution condition was 10 min with 20% - 80% acetonitrile. The amounts of products (BHET, MHET and TPA) were calculated by comparison to a standard curve. All samples were analysed in triplicate and the data averaged and standard errors calculated.

## SDS-Page

### Sample Preparation

1. Add 10 ul of sample into an Eppendorf tube.
2. Transfer 1 ml of the overnights into flasks containing 50ml of LB media and the appropriate antibiotic and incubate at 37°C, 220rpm until OD 0.6-0.8.
3. Add 10 ul Bolt LDS Sample Buffer (4X).
4. Add 4 ul Bolt/NuPAGE Reducing Agent (10X).
5. Make up to 40 ul using deionized water.
6. Heat samples at 70 degrees C for 10 min.

## Western Blot

### Transfer

1. Run SDS PAGE according the aforementioned method.
2. Soak PVDF membrane in methanol.
3. Add “1-step transfer buffer” in plastic gel box.
4. Place 2 filter papers in plastic gel box.
5. Place PVDF membrane in the box.
6. Place 2 more filter papers on top.
7. Make sure everything is soaked in transfer buffer.
8. Leave minimum of 5 minutes.

### Gel Sandwich

1. Make a gel sandwich on the fast transfer adding in this order:

* Filter paper
* Filter paper
* PVDF membrane
* Gel
* Filter paper
* Filter paper

2. Run program Mirella custom (25 V; 29 min; 1.3A)

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# Team:Exeter/Results

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* [Home (current)](https://2019.igem.org/Team%3AExeter)
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  [Modelling](https://2019.igem.org/Team%3AExeter/Model)
  [Design](https://2019.igem.org/Team%3AExeter/Design)
  [Safety](https://2019.igem.org/Team%3AExeter/Safety)
  [Lab Book](https://2019.igem.org/Team%3AExeter/Lab_Book)
  [Results](https://2019.igem.org/Team%3AExeter/Results)
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* Medals and Awards

  [Jamboree Awards](https://2019.igem.org/Team%3AExeter/Awards)
  [Medal Criteria](https://2019.igem.org/Team%3AExeter/Medals)
  [Entrepreneurship](https://2019.igem.org/Team%3AExeter/Entrepreneurship)
  [Hardware](https://2019.igem.org/Team%3AExeter/Hardware)
  [Integrated Human Practices](https://2019.igem.org/Team%3AExeter/Human_Practices)
  [Outreach and Engagement](https://2019.igem.org/Team%3AExeter/Public_Engagement)
* [Collaborations(current)](https://2019.igem.org/Team%3AExeter/Collaborations)

Results

* Bronze
* Silver
* Gold

# Bronze Results

![Results Icon](https://static.igem.org/mediawiki/2019/5/54/T--Exeter--iconresults.png)

[Introduction](#s1b)
[Experimental Data Collection](#s2b)
[Calibration](#s3b)
[Results](#s4b)
[Conclusion](#s5b)

## 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](http://parts.igem.org/Part%3ABBa_I746909) which is a T7 [(BBa\_I719005)](http://parts.igem.org/Part%3ABBa_I719005) promoter driving expression of the superfolder variant (sGFP) [(BBa\_I746916)](http://parts.igem.org/Part%3ABBa_I746916), that folds efficiently when fused to poorly folded proteins 4.

## 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.

![](https://static.igem.org/mediawiki/2019/4/4a/T--Exeter--OD1.jpg)

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.

[Silver >>](#very-top)

[Introduction](#s1s)
[Experiments](#s2s)
[Results](#s3s)
[Conclusions](#s4s)

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# Silver Results

## 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](https://2019.igem.org/Team%3AExeter/Experiments)

## Results

### BHETase 1

#### Nickel Affinity Chromatography

![](https://static.igem.org/mediawiki/2019/1/18/T--Exeter--BBa_K3039005_Ni.jpg)

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.

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

![](https://static.igem.org/mediawiki/2019/8/8e/T--Exeter--BHET1_assay.jpg)

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

## 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.

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