iGEM 2022 — Team tamu

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Notebook

Notebook

* June
* July
* August

## June 1-7

In laboratory team training on specific procedures and operation of equipment.

## June 8-14

**Dr. Sun's Lab**

Performed PCR to amplify PETase, AIDA-I, YeeJ, and MHETase parts.

**Dr. Zhu's Lab**

Mutagenesis using PCR was started this week. We did this using two methods: error prone PCR and site directed mutagenesis.

## June 15-21

**Dr. Sun's Lab**

Gibson assembly of pET-24a backbone with PETase, AIDA-I, and YeeJ insert for intracellular PETase, YeeJ-PETase, and AIDA-I-PETase plasmids and similar MHETase plasmids.

**Dr. Zhu's Lab**

This week we focused on protein purification and colony PCR. In addition, a miniprep was attempted but failed. Meanwhile, we were continuing working on fluorescence and absorbance assays to detect enzyme activity. In order to do this, we created a culture plate using plates impregnated with BHTP. (shown below).

![project](https://static.igem.wiki/teams/4484/wiki/june21-2.png)

## June 22-30

**Dr. Zhus's Lab**

* FDBz was added to each sample in a well plate. The fluorescence was then measured periodically to measure the progress of the reaction. In addition, we used NEB5-alpha cells, and made them competent using CaCl2. Lastly, the minipreps are now turning out to be successful.
![project](https://static.igem.wiki/teams/4484/wiki/june24.png)* Furthermore, the first round of error prone PCR of PEtase was performed (shown above). Nanopore sequencing results showed that GFP instead of PETase was in the plasmid. One of the original colonies from the plate containing the desired plasmid showed fluorescent activity.

## July 1-7

**Dr. Sun's Lab**

* Transformed plasmids in *E. coli*(CD41) and plated on LB plates.
* After overnight growth, colonies were inoculated in LB medium and cultured overnight.
* Colony PCR used to confirm successful transformation.
* Enzyme expressions were induced with IPTG inducer.
* Cultures were collected for SDS-PAGE and western blot for protein expression level characterization.
* Bacteria were lysed and the intracellular enzymes were purified through his-tag purification columns.
![project](https://static.igem.wiki/teams/4484/wiki/july5-2.png)

**Dr. Zhu's Lab**

* This week we had promising results from the BHET plate cultures.The first plate (shown below) was spotted with pure PETase as a positive control (1) and uninduced *E. coli* as a negative control (5). The three other spots (2, 3, and 4) were induced surface display bacteria. All spots looked similar initially to the negative control. When the plate was washed with PBS to remove any cells, the negative control appeared clear, while every other spot remained cloudy. In addition to the BHET plate cultures, this week we started to perform Gibson Assembly.
* Additionally, the plasmid assembly was redone. We utilized a plasmid with PETase as a vector template, and three plasmids were sent in for sequencing along with a reference. The results were as follows: the first plasmid had a deletion, the second plasmid had an insertion early in the PETase sequence, and the third plasmid had the exact sequence as the reference sequence. Moreover, two different plasmids from a different "batch" were also sent for sequencing, which showed that both has no PETase insert and that the Gibson Assembly had failed due to a bad master mix.

![](https://static.igem.wiki/teams/4484/wiki/ecollie-1.png)

## July 8-15

**Dr. Sun's Lab**

* FDBz synthesis and purification

+ Mixed reactants followed by 24 hours of reaction under a nitrogen atmosphere.
+ Chromatography column loaded and run across 2 days while collecting fractions.
+ Fractions with only FDBz confirmed by thin liquid chromatography and rotovapped.
+ Powder scraped and allowed to dry in oven overnight.

* Began degradation activity assay for enzyme constructs.

+ Purified PETase and surface displayed samples placed in tubes with PET film piece.
+ MHETase samples placed in tubes with MHET solution.
+ Reaction proceeded at 25 C for the next week.

**Dr. Zhu's Lab**

The sequencing results of the first round of EP-PCR had arrived (shown below). The results were as follows: B1-1 had 7 point mutations and 3 amino acid changes. The B1-2 had 22 point mutations and mutiple stop codons. And lastly, B1-3 had 11 point mutations and 6 amino acid changes.
![project](https://static.igem.wiki/teams/4484/wiki/july8.png)

## July 16-23

**Dr. Sun's Lab**

* Took FDBz for H1 NMR measurement.

+ Results showed a matching structure for FDBz.
+ Small peak present at very high ppm indicating possible slight fluorescein contamination.

* Performed FDBz assay with purified PETase.

+ PETase concentration above 0.5 uM appeared to give nonlinear relation for 250 uM FDBz.
+ Sample range across 0.1 uM to 0.5 uM resulted in linear relation.
+ Repeated experiments gave corresponding results.

* Experiment to make BHET plates at final BHET concentrations of 5, 20, and 100 uM BHET.

+ Plates made using 0.5 M BHET in DMSO resulted in no visual turbidity.
+ Dilution of 1 M BHET in DMSO gave visual turbidity in higher concentration plate.
+ 100 uM BHET plate resulted in large crystal formation upon cooling.
+ 20 uM BHET plate gave consistent distribution of particles.

* Finished degradation assay and performed HPLC on solution.

+ Purified PETase showed formation of products while surface displayed PETase samples showed negligible activity.
+ Intracellular MHETase and surface displayed MHETase cell samples all showed activity. (Unable to confirm surface display of MHETase with constructs.)

**Dr. Zhu's Lab**

This week we again performed the protein purification of B1-1 and B1-3 in *E.coli* C41. These results showed a very low concentration for both enzymes (shown below). The B1-1 had a concentration of 2.88μM, and the B1-3 had a concentration of 16.9μM.
![project](https://static.igem.wiki/teams/4484/wiki/jul16.png)
Also, the EP-PCR sequencing results arrived and both bacthes were unfavorable. There were mutations and deletions in *ori* and *lacl* operons, and very few or silent mutations in PETase.

## July 24-31

**Dr. Sun's Lab**

* Performed BHET plate assay with purified PETase, MHETase, and negative control buffer.

+ PETase enzyme showed visual change with formation of cloudy circle while MHETase and buffer showed no change.

* BHET plate assay performed with cell samples of intracellular PETase, surface displayed PETase, intracellular MHETase, and unmodified *E. coli.*

+ Positive result for all PETase samples including intracellular and negative result for MHETase and *E. coli* samples.
+ Showed BHET plates can confirm PET degrading activity but cannot confirm surface display.

* FDBz assay with whole cell and lysate samples of PETase constructs.

+ Results show unexpected inverse trend of relative fluorescence with respect to cell concentration.
+ Mainly caused by increase in fluorescence over time in *E. coli* cell and lysate samples used as negative controls.

* Repetition of original PETase constructs with FAST-PETase

**Dr. Zhu's Lab**

Protein purification of B1-1 and B1-3 in  *E.coli* was performed again. However, this round we used a different lysis buffer by the name of BPER and EDTA. The results (shown below) revealed low concentrations for both enzymes again based on Nanodrop. The B1-1 had a concentration of 8.09μM, and the B1-3 had a concentration of 6.40μM. Lastly, the protein gel again showed now protein of interest.
![project](https://static.igem.wiki/teams/4484/wiki/july24.png)

## August 1-7

**Dr. Sun's Lab**

* Began degradation assay on FAST-PETase constructs.

+ Pure FAST-PETase plus surface display constructs loaded into tubes with PET film piece and reacted at 25 C for 1 week.

* BHET plate assay with FAST-PETase constructs.

+ Pure FAST-PETase shows rapid formation of cloudy zone then transition to clear zone with turbid ring at edges. Surface display constructs also show positive results.

* FDBz assay on pure FAST-PETase and PETase.

+ Results show significant increase in fluorescence with FAST-PETase compared to regular PETase. Show FDBz is capable of measuring differences in activity of PET degradation enzymes.

## August 8-15

**Dr. Sun's Lab**

* FAST-PETase degradation assay samples run in HPLC

+ AIDA-I-FAST-PETase and purified FAST-PETase show formation of products.
+ YeeJ-FAST-PETase shows no measurable product formation.
+ Confirmation of AIDA-I-FAST-PETase activity but autolysis of cells observed after several days of storage so surface display cannot be confirmed.

* Whole cell and lysate FAST-PETase samples in FDBz assay

+ Observed inverse trend in measured relative fluorescence with respect to cell concentration.
+ Negative control of *E. coli* whole cell and lysate showed growth over time in fluorescence comparable to active samples.

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Engineering Practices

Design & Build

The first step to our project was designing plasmids for the purpose of surface displaying PET degrading enzymes including PETase and MHETase. To achieve our goals, we designed recombinant proteins which linked transporter proteins to the degrading enzymes. We ordered the DNA parts and designed primers through benchling. We used the DNA template and primers to perform PCR to amplify each DNA part and Gibson assembly to connect the parts into each plasmid. These plasmids were then transformed into NEB5 alpha cells for cloning or BL21 E. coli for protein expression. Successful expression was confirmed using SDS PAGE and Western Blot.

Test

In order to test the effectiveness of the new enzymes, we used several efficient activity assays in the lab. The first was the BHET plate assay which involved using BHET, a material that would be degraded by PETase, on an agar plate to create a visual confirmation of activity. Figure 1 shows an example result for purified PETase, YeeJ-PETase, AIDA-I-PETase, and YeeJ-MHETase. The other activity assay involved placing the samples in a reaction vessel with a piece of PET for PETase or a solution of MHET for MHETase. After reacting for one full week, the samples are analyzed with HPLC to determine if the material was degraded or not.

![](https://static.igem.wiki/teams/4484/wiki/engr-img-1.png)

Figure 1. BHET plate showing results for (1) purified PETase, (4) YeeJ-PETase, (7) AIDA-I-PETase, and (19) YeeJ-MHETase.

![](https://static.igem.wiki/teams/4484/wiki/engr-img-2.jpg)

Figure 2. HPLC measurements for purified PETase and PETase constructs after 168 hours of reaction with PET film at 25 °C.

Learn & Redesign

From the tests mentioned above, we found generally that surface displayed MHETase was active while PETase was less certain. In the BHET plate assay, YeeJ-MHETase gave a negative result as expected and in the HPLC measurements the peak corresponding to MHET disappeared. YeeJ-PETase and AIDA-I-PETase showed the expected positive result in the BHET plate assay but showed almost no activity based on the HPLC measurements. We theorized that the surface display efficiency of the enzymes may be too low, resulting in activities that were difficult to measure. We capitalized on the recently revealed FAST-PETase, which showed higher activity that could potentially be more easily measured with our activity assays. As in the beginning of our project, this began a new process of designing plasmids and related primers that would be used to create the newly designed constructs that were used later in the project.

* [Design & Build](#design)
* [Test](#test)
* [Learn & Redesign](#learn)

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Experiments

Polymerase Chain Reaction (Q5 Polymerase)

**Materials:**

* Template DNA
* Forward and Reverse Primers
* Q5 Polymerase
* Deoxynucleotide Triphosphates (dNTPs)
* Nuclease free water
* PCR buffer

**Procedure:**

1. Create Master Mix using 0.4 μL dNTPs, 10 μL PCR buffer, 33.2 μL nuclease free water, and 0.5 μL polymerase, per sample. Add to each PCR tube.
2. Add 2.5 μL of appropriate forward and reverse primers to each sample.
3. Add appropriate template DNA to each sample.
4. Briefly centrifuge tubes so all liquid collects at the bottom of the tube.
5. Load into thermocycler.

* Annealing temperature is specific to each sample.
* Elongation time is 30 minutes per kbp.

Plasmid Extraction (ZymoPURE Miniprep)

**Materials:**

* ZymoPURE P1 (Red)
* ZymoPURE P2 (Green)
* ZymoPURE P3 (Yellow)
* ZymoPURE Binding Buffer
* ZymoPURE Wash 1
* ZymoPURE Wash 2
* ZymoPURE Elution Buffer

**Procedure:**

1. Centrifuge bacterial sample in a 1.5 mL tube, and discard supernatant.
2. Resuspend cell pellet using 250 μL ZymoPURE P1.
3. Add 250 μL ZymoPURE P2 and immediately mix by inversion. (8-10 times). Incubate at room temperature for 3 minutes.
4. Add 250 μL ZymoPURE P3 (Yellow) and mix by inversion. Invert an additional 5 times after sample turns yellow. Incubate on ice.
5. Centrifuge the lysate for 5 minutes at 16,000 x g.
6. Transfer exactly 600 μL of supernatant into a clean 1.5 mL tube, without disturbing yellow pellet.
7. At 260 μL of ZymoPURE binding buffer to cleared lysate and mix by vortexing.
8. Transfer mixture into a Zymo-Spin II column. Incubate at room temperature for 1 minute and centrifuge at 11,000 x g for 1 minute. Discard flow through.
9. Add 800 μL of ZymoPURE Wash 1 into the column and centrifuge at 11,000 x g for 1 minute. Discard Flow through.
10. Add 800 μL of ZymoPURE Wash 2 to the column and centrifuge at 11,000 x g for 1 minute. Discard flow through. Repeat with 200 μL of Wash 2.
11. Dry spin column at 11,000 x g for 2 minutes.
12. Transfer column to a new 1.5 mL tube. Add 25 μL ZymoPURE Elution Buffer to column matrix, and incubate at room temperature for 2 minutes. Centrifuge at 11,000 x g for 1 minute.
13. Analyze eluted sample using Nanodrop protocol.

Protein Purification

**Procedure:**

1. Create a 100 mL subculture of bacteria of interest.

* Materials: LB, antibiotics

1. Allow to grow to an OD600nm between 0.6 and 0.8.
2. Incubate on ice to stop growth until next step.

2. Induce subculture using IPTG.

* Materials: 1 M IPTG

1. Add IPTG to culture for a 0.1M concentration.
2. Incubate overnight at 16 °C and 200 RPM.

3. Spin down and add lysis buffer.

* Materials: Lysis buffer (50 mM HEPES, 300 mM NaCl, 10 mM imidazole, pH 8), Ultrapure (Milli-Q Water)

1. Spin at 4000 x g for 3 minutes at 4 °C.
2. Resuspend in 1 mL of ultrapure water (for large cultures).
3. Add 4 mL lysis buffer per 40 mL of culture.
4. Vortex and incubate on ice.

4. Sonicate.

* Place on ice.
* Amplitude: 20%
* Sonicate for 20 seconds, rest for 10 seconds. Repeat 9 times.

5. Centrifuge at 7197 x g for 1 hour at 4 °C.
6. Transfer to cold room.
7. Filter with MCE filter.
8. Add 1 mL of nickel beads solution, and nutate for 1 hr at 4 °C.
9. Wash and elute.

* Materials: DI water, gravity column, wash buffer (50 mM HEPES, 300 mM NaCl, 30 mM Imidazole, pH 8), elution buffer (50 mM HEPES, 300 mM NaCl, 250 mM Imidazole, pH 8), 30% ethanol, collection tubes

1. Load into gravity column. Allow to flow through and sample.
2. Wash using 12 mL of wash buffer. Sample.
3. Wash using 2 mL of wash buffer. Sample.
4. Elute using 12 mL of elution buffer. Sample.
5. Wash gravity column in RT. (Recycle nickel beads by suspending in 30% ethanol. Wash remaining column in DI water and store at 4 °C.)

10. Buffer Exchange.

* Materials: Amicon spin column, exchange buffer, DI water

1. Wash spin column DI water.
2. Transfer elution sample to spin column. (Cutoff depends on size of protein of interest. Save 300 microL for confirmation step.)
3. Spin at 4000 x g for 60 minutes at 4 °C for 0.2 mL
4. Add 12 mL of exchange buffer to column.
5. Repeat 3 and 4.

11. Measure concentration using Bradford protein assay kit (BioRad).
12. Make aliquots of purified protein and store at -80 °C.
13. Confirm protein using SDS-PAGE and, if needed, Western Blot.

SDS-PAGE

**Materials:**

* 1x SDS-PAGE Running Buffe
* Laemmli sample buffer
* Beta-mercaptoethanol
* Protein sample

**Procedure:**

1. Create SDS-PAGE gel (BioRad TGX Stain-Free™ Fast-Cast™ Acrylamide kit)
2. Prepare protein samples:

1. Combine 4.75 microL laemmli sample buffer, 0.25 μL beta-mercaptoethanol, and 5 microL protein sample in a PCR tube.
2. Heat sample at 90-100 °C for 5 minutes.

3. Load gel into electrophoresis cell. Remove comb, and fill chambers with running buffer.
4. Load samples into gel.
5. Run gel electrophoresis at 200V for 39 minutes.
6. Image gel using imager.

Western Blot

**Procedure:**

1. After SDS-PAGE is completed, electrophoretic transfer of proteins from SDS-PAGE gel to a PVDF membrane was done using BioRad Trans Blot turbo transfer system.
2. After successful transfer of proteins, place the blot in the blocking solution (5% milk in TBS-Tween) overnight at 4 °C.
3. Add 1 μl of Anti-6x His tag antibody (HRP conjugated) to 10 mL of blocking solution and incubate at room temperature for 1 hour.
4. Wash the membrane 3 times with TBS-Tween solution.
5. Add BioRad Clarity western ECL substrate and image using imager under chemiluminiscence and colorimetric modes.

DNA Electrophoresis

**Procedure:**

1. Create agarose gel.

* Materials: agarose, 1X TAE Buffer, SYBR Safe Gel Stain

1. Add 0.35 g of agarose and 35 mL of TAE buffer to a flask. Microwave on high for 1 minute.
2. Allow to cool until flask can be comfortably handled, but gel has not begun to solidify. Add 2 μL of SyberSafe dye. Mix.
3. Pour gel into mold. Cover to block out light and allow to solidify.

2. Prepare DNA samples with loading buffer, and add to wells in agarose gel.
3. Run electrophoresis. (Typically run at 120V for 20 minutes.)
4. Image gel using UV lamp or gel imagers.

Bacterial Transformation using Ca2+ Competent Cells

**Materials:**

* Competent cells
* Plasmid sample
* LB
* LB agar plate with ampicillin or carbenicillin

**Procedure:**

1. Add 1 μL of plasmid sample to a microtube. Gently add 100 μL of competent cells. Also create a sample with no plasmid added as a control.
2. Incubate for 30 minutes on ice. Flick tube every 10 minutes.
3. Heat shock for 1 minute at 42 °C. Place back on ice immediately.
4. Plate sample on LB agar plate with antibiotic added. Allow to grow overnight.
5. Select colonies to culture and test using colony PCR. Confirmed positive colonies may be used for further experiments.

PET Film Assay

**Procedure:**

1. Hole-punched amorphous PET films of 6mm diameter are used for PET film-based activity assays. Prior to use, the films are washed with 1% SDS, 100% DMSO, ultrapure water, and 95% ethanol.
2. These washed PET films are subsequently incubated with purified enzymes/E. coli whole cells in 500 μl reaction solution (50 mM Bicine-NaOH, pH 8.5) in a glass test tube at 25 °C accompanied by continuous shaking in an incubator.
3. For analysis, supernatant is withdrawn followed by addition of an equal volume of 100% methanol, heat treatment at 85 °C to quench enzymatic activity and centrifugation to separate *E. coli* whole cells.
4. The obtained supernatant is filtered using spin filters and used for HPLC analysis.

FDBz Assay

**Materials:**

* 0.01 M FDBz in DMSO
* Enzyme or cell sample (buffer enzyme stored in or wild type of cell sample)
* 10 mM Tris buffer pH 7.4
* Black bottom microplate
* Microplate reader
* Pipette
* 2.5 mL centrifuge tubes

**Procedure:**

1. Set microplate reader to incubate at 37 °C during preparation. Prepare to take regular fluorescence readings at excitation 488 and emission 523 for 3 hours every 30 minutes.
2. Calculate required volume of enzyme for 0.56 µM final concentration in 100 µL per replicate. Calculate volume of buffer by subtracting result form 100 µL.
3. In a separate tube for each sample, add required volume of buffer followed by enzyme and mix. Repeat for the corresponding negative control of enzyme storage buffer or wild type cell.
4. Transfer 97.5 µL of mixture into each microplate well.
5. Add 2.5 uL of FDBz mixture to each sample.
6. Place microplate into the reader and begin regular measurements.

BHET Assay

**Materials:**

* Petri dish
* Agar
* LB
* Water
* Antibiotic
* 1 M BHET in DMSO
* Enyme or cell sample
* If cell sample: bunsen burner, L shaped glass spreader, alcohol, PBS buffer

**Procedure:**

Plate Preparation:

1. Follow normal LB agar media preparation: Mix 2.5 g LB and 1.5 g agar with 200 mL water.
2. Autoclave and place in 55 °C water bath.
3. When cooled, add antibiotics to working concentration and 4 mL BHET solution to final concentration of 20 mM.
4. Pour approximately 10 mL into each petri dish and allow to solidify then store at 4 °C.

Assay:

1. Take plate from storage and allow to warm before making ~1 cm squares on bottom of plate.
2. Add 5 µL of enzyme or cell sample to each square and incubate at 30 °C up to overnight.
3. If cell sample: sterilize spreader by dipping into alcohol and carefully moving over flame of bunsen burner. Allow to cool for ~1 minute before pouring small amount of PBS buffer and spreading to remove top layer of bacteria.
4. Inspect for visual changes in each sample spot.

ZymoPURE DNA Gel Extraction

**Materials:**

* Gel from DNA Electrophoresis
* Agarose Dissolving Buffer (ADB)
* ZymoSpin Column and Collection Tube
* DNA Wash Buffer
* DNA Elution Buffer

**Procedure:**

1. Excise desired DNA fragment from agarose gel used in electrophoresis. Place gel fragment in 1.5 mL centrifuge tube.
2. Add ADB to centrifuge tube. Should be added in a 3:1 volume ADB to volume agarose gel ratio.
3. Incubate at 55 °C for 5-10 minutes, until gel fragment is completely dissolved.
4. Add 200 µL DNA wash buffer to the column. Centrifuge for 30 seconds. Discard flow through and repeat.
5. Dry spin at 12000 x g for 1 minute to remove residual wash buffer.
6. Transfer column to a new centrifuge tube. Add more than 6 microL DNA elution buffer directly to column matrix. Incubate for 5 minutes, then centrifuge for 30-60 seconds.
7. DNA now ready.

* [PCR](#exp1)
* [Plasmid Extraction](#exp2)
* [Protein Purification](#exp3)
* [SDS-PAGE](#exp4)
* [Western Blot](#exp5)
* [DNA Electrophoresis](#exp6)
* [Bacterial Transformation](#exp7)
* [PET Film Assay](#exp8)
* [FDBz Assay](#exp9)
* [BHET Plate Assay](#exp10)
* [Gel Extraction](#exp11)

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Results

Surface Display Constructs of PETase and MHETase

The first step was the production of plasmids encoding enzymes PETase (BBa\_K4484000) and MHETase (BBa\_K4484001). Additionally, surface displayed versions were designed by linking the transporter proteins AIDA-I (BBa\_K4484003) and YeeJ (BBa\_K4484007) to the desired enzyme sequences. Each of these plasmids were then transformed into *E. coli* C41(DE3) and protein expression were induced. SDS-PAGE and Western blot (His Tag targeting antibody) were used to confirm the presence of proteins at the expected size as in Figure 1. While issues appeared such as multiple bands which indicated the possibility of undesired protein truncation, the constructs were generally successful as evidenced by bands at expected sizes. Additionally, although none of the AIDA-I constructs showed a band on the Western blot (we are still trouble shooting on this topic), the AIDA-I-PETase constructs (BBa\_K4484005) later showed activity on the BHET plate and AIDA-I-FAST-PETase constructs (BBa\_K4484006) showed activity on a PET film, demonstrating the successful expression of functional proteins. The constructs YeeJ-FAST-PETase (BBa\_K4484010) and AIDA-I-FAST-PETase (BBa\_K448406) were also designed and expressed with similar results to these earlier constructs.

![](https://static.igem.wiki/teams/4484/wiki/results-img-1.jpg)

Figure 1. Example Western blot for various purified and surface displayed proteins.

Degradation Kinetic Assay

In order to verify the activity of our constructs, we used a common activity assay involving measuring the concentration of degradation products through HPLC after degrading PET/intermediates. For PETase, reaction solutions were made by mixing enzymes or whole cell samples in the reaction buffer with an amorphous PET film. In the case of MHETase, a solution of MHET was made as it is not active on PET on its own. The HPLC measurements of MHETase samples are shown in Figure 2. Each of the constructs shows a disappearance of the peak corresponding to MHET and appearance of a TPA peak, which implies the surface displayed enzymes are active. Meanwhile, *E. coli* cells expressing MHETase intracellularly also showed MHET degradation, giving the potential for using *E. coli* with intracellular MHETase for plastics degradations.

![](https://static.igem.wiki/teams/4484/wiki/results-img-2.jpg)

Figure 2. HPLC measurements of OD 20 whole cell MHETase constructs after 168 hours of reaction at 25 °C. Reaction medium is 250 μM MHET in 50 mM Bicine-NaOH pH 8.5. (All same scale)

For the assays of PETase, the HPLC measurements are shown in Figure 3. Both surface display constructs of PETase seem to show little activity compared to the purified enzyme. This was hypothesized to be from low surface display efficiency of enzymes resulting in negligible activity. New constructs with surface displayed FAST-PETase were made and expressed to evaluate if increasing the enzyme activity would allow for measurable HPLC results. The FAST-PETase measurements shown in Figure 4 display mixed results as YeeJ-PETase (BBa\_K4484008) shows negligible activity while AIDA-I-FAST-PETase shows measurable activity based on the appearance of peaks corresponding to degradation products TPA and MHET. The intensity is relatively lower compared to the purified enzyme which may indicate a relatively lower surface display efficiency limiting overall activity. However, in our AIDA-I-FAST-PETase samples stored at 4 °C, loss of cell viability was observed within 48 hours of expression. Hence, it is possible that the observed activity is because of cell lysates containing FAST-PETase. Future experiments will focus on ruling out this possibility.

![](https://static.igem.wiki/teams/4484/wiki/results-img-3.jpg)

Figure 3. HPLC measurements of pure PETase and OD 30 whole cell PETase constructs after reaction for 168 hours at 25 °C. Reaction medium is 50 mM Bicine-NaOH pH 8.5. (All same scale)

![](https://static.igem.wiki/teams/4484/wiki/results-img-4.jpg)

Figure 4. HPLC measurements of Pure FAST-PETase and OD 30 whole cell FAST-PETase constructs after 213 hours of reaction at 25 °C. Reaction medium is 50 mM Bicine-NaOH pH 8.5.

FDBz Fluorescence Assay

To begin developing our assay, we first had to synthesize the substrate material fluorescein dibenzoate (FDBz). Thin liquid chromatography was used to confirm product purity in the collected fractions which were dried by rotary evaporation. After product purification, it was analyzed by H1 NMR for which the results can be seen in Figure 5. Comparing literature available online as well as simulated NMR spectra, the spectra appears to align with expectations indicating the successful synthesis of FDBz. However, there are some questions of purity as the NMR spectra of FDBz and fluorescein are relatively close and some fluorescence was observed when the FDBz product is placed in aqueous solution, though it is unclear if this is due to fluorescein impurities or automatic hydrolysis.

![](https://static.igem.wiki/teams/4484/wiki/results-img-5.png)

Figure 5. H1 NMR for FDBz synthesis product.

**Tests with Pure Enzymes**

When evaluating the assays, we first used purified enzymes to evaluate the effectiveness of the test and various methods of analysis. Figure 6 below shows the result of one such assay with varying concentrations of PETase. Fluorescence values correspond to measurements taken at the end of three hours relative to negative controls. The approximately linear correlation between the fluorescence values and enzyme concentration confirms the ability to measure enzyme activity reliably.

![](https://static.igem.wiki/teams/4484/wiki/results-img-6.jpg)

Figure 6. Fluorescence relative to negative control across various purified PETase concentrations after 3 hours of reaction with 250 μM FDBz.

We also showed that the assay could display the increased efficiency of different enzymes using FAST-PETase, a modified PETase optimized using artificial intelligence. Figure 7 illustrates the significant increase in fluorescence of FAST-PETase compared to regular PETase which corresponds to the expected increase in activity.

![](https://static.igem.wiki/teams/4484/wiki/results-img-7.jpg)

Figure 7. Relative fluorescence of PETase and FAST-PETase after 3 hours of reaction with 250 μM FDBz (0.5 μM purified enzyme, 37°C)

**Tests with Surface Displayed Enyzmes**

After using purified enzymes, experiments with both surface displayed *E. coli* whole cell and PETase whole cell lysate samples (without purification) were performed. The results from these experiments typically looked similar to Figure 5, which shows an inverse relationship between fluorescence and cell concentration. This phenomenon is mostly caused by the unexpected rise in fluorescence with plain *E. coli* cells and lysate over time which is generally comparable with that of enzyme-containing samples. The reason behind this unexpected rise has not been explained and is hoped to be addressed in future work.

![](https://static.igem.wiki/teams/4484/wiki/results-img-8.jpg)

Figure 8. Fluorescence of PETase lysate at various concentrations (OD prior to cell lysis) after 3 hours of reaction with 250 μM FDBz, 37°C.

**BHET Plates**

BHET is a compound similar to PET that is a trimer made up of one TPA molecule and two ethylene glycol molecules. As such, PETase shows activity on this molecule while organisms lacking the enzymes will be unable to degrade it. Plates for qualitative evaluation of PET degrading activity were developed by creating a suspension of BHET in agar media. Early experiments evaluated varying concentrations of BHET to determine a standardized procedure. At high concentrations, the BHET would quickly form large crystal structures when cooling, which raised concerns about accuracy given the heterogeneous distribution. A BHET concentration of 20 mM was decided as the standard as it resulted in a uniform turbidity throughout the solution and plate. Next, we then began experiments to confirm the sensitivity of the assay by placing various purified enzymes and controls on the plate, for which an example result is shown in Figure 9. The BHET plates were considered successful as they showed visual changes in the spots of active enzymes and no visual change for enzymes inactive on PET. Further experiments investigated surface displayed enzymes on the plates, with an example shown in Figure 10. We were able to confirm that only PETase was able to degrade the BHET as evidenced by the change in appearance of the plate while MHETase and control portions showed no visual difference. These experiments also revealed that this assay could not distinguish between intracellular and surface displayed proteins as cells expressing intracellular PETase would create positive visual changes.

![](https://static.igem.wiki/teams/4484/wiki/results-img-9.jpg)

Figure 9. Example BHET plate assay containing purified PETase, FAST-PETase, and MHETase

![](https://static.igem.wiki/teams/4484/wiki/results-img-10.png)

Figure 10. Example BHET plate spots under 2x magnification showing (from left to right) purified PETase (1), YeeJ-PETase (4), AIDA-I-PETase (7), YeeJ-MHETase (19).

One final assay we performed was comparing the effectiveness of standard PETase against FAST-PETase. Multiple samples of both enzymes were placed on a 20 uM plate and recorded over approximately 30 minutes. The FAST-PETase quickly creates clear zones in the locations much more quickly than standard PETase, indicating its significantly higher efficiency as expected, though the activity generally cannot be quantified.

![](https://static.igem.wiki/teams/4484/wiki/results-img-11-pt1.png)
![](https://static.igem.wiki/teams/4484/wiki/results-img-11-pt2.png)

Figure 11. Before and after 30 minutes of reaction of purified FAST-PETase and regular PETase on a 20 mM BHET plate.

**Directed Evolution**

Another experiment performed was the mutation of the PETase insert sequence through error-prone PCR for the purpose of showing the ability to develop new enzymes with improved activity and thermal stability. Two mutant sequences were developed and expressed before being tested using a BHET plate assay. Based on the image in Figure 12, both mutants created no visual changes in the plate and as such appear to have caused a loss of function in the enzyme.

![](https://static.igem.wiki/teams/4484/wiki/results-img-12.jpg)

Figure 12. Mutant PETase enzymes on a BHET plate showing loss of function compared to the original PETase enzyme.

* [Surface Display Constructs](#abstract)
* [Degradation Kinetic Assay](#intro)
* [FDBz Fluorescence Assay](#discussion)

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