iGEM 2024 — Team kcis-xiugang-taipei

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Previous Week
Next Week
Next Week

Today marked our first day in the lab, and it felt completely different from any other lab experience we've had before.

Our lecturer, Ms. Ching, introduced us to the fundamentals of molecular biology, including DNA replication and retro-transcription. Having some prior knowledge of these topics helped us feel prepared for what lies ahead this year.

The rest of the session was to perform a bacterial transformation experiment on yeast competent cells. This was our first-ever molecular biology experiment, making it a truly new and exciting experience for us.

![lab-01](https://static.igem.wiki/teams/5094/notebook/lab-01.jpg)

Our other lecturer: Ms. Parker Gave us an introduction to the field of synthetic biology.

Lecture notes:

* Synthetic biology purpose: Synthetic biology has the potential to modify species purposely for our benefits.
* You can only clone genes downstream if the promoter: get several different plasmids to start design genetic modification.
* Bacteria selection plate→inoculate→LB-Amp liquid culture: to filter out the bacteria that failed antibiotic gene transformation.
* ampicillin: bacterial antibiotic we just used
* Sanitize tools with bunsen burner
* Incubation: to create an environment for bacteria to grow.
* phenol/chloroform: a substance that removes the cell membrane and irrelevant proteins.

Day 2 experiment: dividing plasmid from whole cell. We are doing this to extract the plasmids of our bacteria colonies we made last week.

Day 2 Experiment conclusion:

* We use a base solution (NaOH solution) to remove the cell membrane and use phenol/chloroform to remove irrelevant proteins and rna.
* Proteins are broken by NaOA

![Chairman Lee](https://static.igem.wiki/teams/5094/notebook/experiment-06.jpg)

Today the senior team from last year demonstrated the ways of Gel electrophoresis and PCR, two of the very important techniques we will be frequently using afterwards.

When they're done we kept on with the plasmid extraction protocol we were doing last week.

Today is the day when Ms. Parker announced the importance of notebooks, we will be taking notes on every IGEM activities. This notebook is essential for the judging in IGEM competition.

![exp-08](https://static.igem.wiki/teams/5094/notebook/experiment-14.jpg)

We were making a slides presentation of previous iGEM teams' project. This presentation gives us a better idea of IGEM competition and a big idea of our future path towards the IGEM Jamboree.

Later on, we when back to continue the experiment from last week, we measured the concentration of our plasmids in a spectrometer.

Today, we have a lecture on how PCR works and some PCR terms.

![exp-04](https://static.igem.wiki/teams/5094/notebook/experiment-04.jpg)

some terms we have learned:

1. Denaturation: the reaction that splits a dna double helix into two single strands.
2. Annealing: after the double helix is splitted, we add chosen primers with the single strands, and then, we lower the temperature for the primer to attach. (lowered temperature=50~65 c)
3. Extension: let the dna polymerase attach to complete the strands. (Dna polymerase is thermal bacteria’s Dna polymerase.)

After the lecture, we did some experiments on yeast genomic DNA extraction

In yeast genomic DNA extraction experiment, we are not just extracting the wild genome of yeast, instead, we cloned our desired gene with amino acid lysis synthesizing DNA, therefore, if we were to culture our partially-transformed colonies on a plate lacking amino acid lysis, after a few days of growth, all that is left on the plate will be guaranteed to carry our desired gene.

Today we executed another important synthetic biology technique, PCR. we did PCR on the yeast genomic DNA we have extracted last week. After PCR, we were starting have a sense of the principle of synthetic biology.

PCR is the technique of amplifying a strain of gene x34000000000 times so that it will be easier to perform other analysis later.

![exp-15](https://static.igem.wiki/teams/5094/notebook/experiment-15.jpg)

Today we learned how to use NCBI and snapgene. Other than that, we finished the PCR protocol by running our data on NCBI.

Today, we learned how to do double enzyme digestion and understand the science behind restriction enzyme cut sites.

#### PCR

1. Use the GDNA (genomic DNA) previous made that are 100 ng/μl
2. Create master mix of 8 serves, (1 μl XMAI forward primer, 1 μl KPMII reverse primer , 8.5 μl dH2O, 12.5 μl 2x PCR)/ per serve
3. Further steps in protocol

Today we learned primer design using the software: Primer-C.

Such a website provides great convenience for people from students to biology experts, especially for students competing for competitions like iGEM.

Today we cloned of gene into pGal promoter, the pGal promoter is for yeast, we will be using T7 promoter for E.coli.

We also learned about the exclusiveness of promoters between species.

We want to multiply the amount of plasmids for each colony in order to see our ligation results, and keep the success samples to let it grow.

1. Separate a new agar plate with a marker into two sides
2. Use the pipet tip to touch a colony on the plate we did before, then spread them on the other agar plate. (to spread to bacteria)
3. Release the tip in an eppendorf tube containing the LB-AMP
4. Label number associated with the agar plate number
5. Repeat step 3~4
6. Further steps in protocol

![exp-01](https://static.igem.wiki/teams/5094/notebook/experiment-01.jpg)

Day off!

Day off!

We have learnt basic training of synthetic biology including:

* Plasmid extraction
* Bacterial transformation
* PCR
* Gel electrophoresis
* Double enzyme digestion
* Primer C
* T4 ligation
* And more

All of which we did many times repeatedly until we all thoroughly understand every step of the process.

![exp-09](https://static.igem.wiki/teams/5094/notebook/experiment-09.jpg)

Prior to the long vacation, our whole team has divided into three groups which came up with three independent project ideas. At the end the PET degradation team won the vote, the content of the other groups are also very interesting, one of them is about human health, achieved through a complicated procedure; while the other one is about modifying a type of tea plant and extracting's content to suppress dust mites.

The big thing of this month is bring up the progression of each group over the winter-break. We presented our progress and findings by groups (Wiki, Human-Practices and wetlab )

Winter break till Feb 22

Winter break till Feb 22

Winter break till Feb 22

Winter break till Feb 22

Winter break till Feb 22

Winter break till Feb 22

Winter break till Feb 22

Our team project starts today!

We need pRSET A plasmid to clone the PETase gene for in vitro protein purification (purify protein into the tube). Despite being recently introduced to our project, Thanks to all the effort into training us, most of us can now properly follow protocols and understand molecular biology terms.

For the experiment part, we purified PETase protein with pRSET-A plasmid for in vitro purification. This process is more complicated than a lot of other experiments, it's because it involves tools and methods that are mew.(metal beads and resin)

Last week was just the preparation for protein purification, although there are only two steps remaining, it was finished successfully. After that we briefly discussed about our team aims and visions, Ms. Ching introduced the idea of site-directed-mutagenesis, the idea is to alter three amino acids of our PETase gene by altering three nucleotides of the gene, doing so will change the physical structure of our PETase and therefore theoretically enhance its ability of degrading PET.

We talked a little more about Site-directed mutagenesis, since all of us are still waiting for the materials for the next experiment. Hope the shipping can arrive next week.

While the human practice group rechecked contact with experts and other iGEM teams, they also confirmed schedules.

The wiki team started their search for good looking templates for our web page.

![exp-05](https://static.igem.wiki/teams/5094/notebook/experiment-05.jpg)

We did plasmid extraction fot pRSET-B plasmids, today is also the day when the wetlab people planned site-directed mutagenesis. The three groups came up with different project ideas, and we chose the PET degrading one. As a class, we also voted for the web page template we liked out of all the avaliable templates the wiki team has found, they all looked nice.

To take note, Ms. Ching confirmed that our pRSET-B plasmid, although time consuming, we are not going to use it in our actual product.

The HP team planned, checked and posted posts on our team's instagram page. (planned with art team)

Today we once again did double enzyme digestion for pRSET-A plasmid, this has to be the most familiarized protocols we have ever done. Nothing went wrong except for me and Joseph losing our tubes.

For the HP team, OUR FIRST INSTAGRAM POSTS ARE OUT !!

Follow our Instagram @igem.kcisxiugangtaipei

Today we checked the wave lengths of our PET products, that pretty much all we did today.

Anyhow, we have the rest of the day to ourselves, to do our individually assigned work. We did paper works in the "experiments" document, and that's where the outline of our design cycle goes.

![exp-13](https://static.igem.wiki/teams/5094/notebook/experiment-13.jpg)

Today all of us made three sections of the plate covered with transformed or not transformed colonies of T7-PET pRSET A plasmid, so that we can select the successful ones next week.

The Wiki team made the outline of what our web page should look like, progress for them is really starting to take shape. Ethan also did research on a data related software called: Matlab

The HP team posted something about the "Aquatic Animal Day", 12 posts on total until now. Good work for them also.

We did have several T7-PET pRSETA plasmid bacterial candidate colonies

Today,(from the plates which we made last week) we will also need to do selections of pGal-PET plasmid. The process was very straight forward, we just picked out successful colonies from the plate like candidates and put them in a new one.

![exp-12](https://static.igem.wiki/teams/5094/notebook/experiment-12.jpg)

We also started the same thing we did last week for another gene: pGal-PET

The wiki team did the layout for their websites.

The HP team started gathering information about team members by making surveys, they gathered information like personalized profile and quotes.

We the wetlab team picked out pGal-PET candidate colonies from last week and once again made a lot of plates.

HP: Messaged teams in taiwan through instagram for collaborations:

Meeting summary

* Survey: Are people interested in our final product, is the product going to be useful, household use possibilities
* Survey for experts: Landfill or industrial usage, price point at which the product could be sold, expert opinion on household use
* Contacting experts: in waste recycling, microplastics, government, groups working with plastic cleanup, etc.
* Collaborations: reaching out to Taiwanese teams, contacting slack chat HK group about the event they are planning to hold
* SDG experts: Iris Qui from last year
* Education: could do an event for the 7th graders this year during summer
* Science Fair: Use it as a survey opportunity to promote iGEM, get data from students on what they are interested in, and if we are to hold events in school, what they would like to do for tutorial sessions
* IHP: Put together a template of how we could introduce our project to specialists with possible questions, then we can edit it for specific people
* Proposal website: could hold an interview with the people, or collab for an instagram post

Mean While the wiki team starts filling out content for our web page.

Day off

Today we did plasmid extraction for triple mutation PETase. The concept of mutation in the protein's genomic sequence to directly affect the efficiency of an enzyme although isn't new to me, it is one of the thing that make me appreciate the extraordinary capacity of synthetic biology. If we really think about it, we are most likely currently living in the "early years" of synthetic biology.

HP group progress: Meeting notes:

Likou seminar (8/24-25)

* Present project (day 1 poster present)
* Day 2 is the actual seminar

Presentation to econ. or business teachers (6/20)

* Focused on product and SDGs

To do

* Short summary of the science fair and what we did for wiki page
* Instagram update on science fair

![lab-02](https://static.igem.wiki/teams/5094/notebook/lab-02.jpg)

While the wetlab and most of the wiki team is still unavaliable. the HP team finalized preparation for science fair.

Three groups are doing three different protocols for today, shown below.

Group1: do bacterial transformation for IsT7-PETase
S121E/D186H/R280A
, and IspGal-PETase
S121E/D186H/R280A

Group 2: set up 2X PCR for bacterial colonies candidates containing IsT7ase-PET
S121E/D186H/R280A
(set up #1-20)

Group 3: Do pGal1,10-*Is*PETase
S121E/D186H/R280A
plasmid extraction from bacterial colonies

All three experiments are consecutive so we did one group after another. I'm from group 1 so I did bacterial transformation, personally, I think bacterial transformation was the easiest protocol out of the three because there were much less step to cover.

For the HP group, They have sent out the "iGEM survey for teachers and students" and finished science fair posters.

Day off!

Day off!

Day off!

Day off!

Day off!

Day off!

![exp-16](https://static.igem.wiki/teams/5094/notebook/experiment-16.jpg)

This Morning we worked on the design cycle, Wetlab members made more LB and PET pellets and the PET pellets part, the process was easy but painstakingly long.

The wiki team applied our team info to the template for our website. Also as a team we all work on the video script for a little, someone even brought a funny looking costume he brought online.

Ms. Ching also talked about the western blot protocol we're gonna do two days later.

Today we voted for what our team hoodie would look like, the two options were a sea otter icon and a badge with a whale, both of the accompanied with some plastic trash, the team settled with the sea otter icon.

Today we did western blot for all the protein products we have, in other words, all the mutations that successful.

Today (2024-8-24) is the first day of the TGem (Taiwan iGem) seminar the, the first day was just like any other typical "first days", we spent most of our times on introductions and experience sharing, regardless of the lengthiness of procedural events, we still managed to catch some time for our local teams. The next day was the day the teams presented their projects.

![tgem-03](https://static.igem.wiki/teams/5094/notebook/tgem-03.jpg)

This is our team poster

![poster-01](https://static.igem.wiki/teams/5094/notebook/poster-01.jpg)

Most of us are down to the final touch, some of us are working on our group presentation while others are doing functional assay.

Wiki: The wiki team are simplifying our wiki code to make them more accessible.

HP: The HP team is rather busy around this time, as for today, they:

* contact experts from difference fields.
* made surveys.
* organized spreadsheets and graphs of their surveyed data.

Wetlab: We did time course.

This week was the last chance to touch anything about the team, the finale.

Unsurprisingly, the wiki team worked on debugging the website and the wet lab worked on the final organizations of our current data, whilst making up for missing information. The debugging of the website was a lot tougher than we anticipated, it was almost as if the bugs were arbitrary. Bugs are discovered one after another.

Today we tried to find the physical docking site of PETase but to no avail, this was part of the personnel professor's request, however, the professor's request wasn't very specific so we had a hard time maneuvering the enzyme software, there's much to learn.

Today we finally had some progress with the docking, turns out, we should have applied our binding ligand with a PET polymer with three or more monomers, this checks out in almost every reference paper.
This was such a whoopsy.

To make matters worse, we needed more time to generate an enzyme model with our current mutations. If we wanted to include this information, this step had to be complete because we could make sure whether the mutation would alter its conditions drastically or not.

Today the wet lab members did bacterial transformation, looking forward! Meanwhile the wiki team did more uploading and debugging almost finished the page.

wiki freeze!

Prepare for iGEM Jamboree

Prepare for iGEM Jamboree

iGEM Jamboree!!!

#### Explore

* [![eng-button](https://static.igem.wiki/teams/5094/button-link/wheel.gif)

  ### Parts Collection

  Explore parts](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts-collection%27%29%20%7D%7D)
* [![imp-button](https://static.igem.wiki/teams/5094/button-link/experiment-buttome.gif)

  ### Experiments

  View our experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)
* [![team-button](https://static.igem.wiki/teams/5094/nav-buttom/teanmhans.gif)

  ### Sustainability

  Learn more](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27sustainable%27%29%20%7D%7D)
* [![safety-button](https://static.igem.wiki/teams/5094/button-link/smle.gif)

  ### Education

  Our programs](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27education%27%29%20%7D%7D)
* [![des-button](https://static.igem.wiki/teams/5094/button-link/rectangle.gif)

  ### Project Description

  Read more](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)

#

##### Engineering

* [Initial design](#first)
* [Experimental design](#second)
* [Future Directions](#third)
* [Citations](#fourth)

## Initial design

### How can we solve the problem of plastic pollution impacting the environment and humans?

Our team was inspired to tackle plastic pollution after we learnt that plastics could contribute to endocrine disruption. After some research, we decided to focus on PET. We found that a popular solution was recycling because it prevents waste from entering the environment. However most recycling results in pollution of its own, and has other problems like scalability. We decided to focus on biorecycling, as the use of enzymes has fewer environmental impacts, and can produce the monomers of PET. These monomers can then be extracted and used for future PET production instead of raw materials, resulting in a circular economy for PET.

However, the PET biorecycling industry is mostly done on a small scale, and our interview with Georg Snyman found a large problem with public buy-in to recycling programs. We decided to focus on investigating ways to increase the enzymes efficiency and production, and therefore, the efficiency of the industry so that it could become more economically viable and contribute better to the downstream problem of plastic pollution prevention.

### What genes could we use to achieve PET degradation?

In order to achieve this aim we researched different PET enzymes. We initially focused on waxworms due to an article we had found on them producing an enzyme to degrade PET, but soon found that it was infeasible due to the withdrawal of the gene sequence from publication. Consequently, the team searched for PETase enzymes in various microorganisms, including Ideonella sakaiensis bacteria, which had the entire PETase gene sequence published.

PET plastic degradation pathway is well-studied, and according to several studies by Japanese research groups, Ideonella sakaiensis bacteria contain two enzymes that relate to our project: PETase and MHETase. PETase breaks down the PET polymer to form MHET, TPA, and BHET, which MHETase further degrades into TPA and ethylene glycol.

A study conducted by Japanese researchers in 2016 demonstrated that *Is*PETase has a higher tolerance to environmental changes, such as variations in pH and temperature while maintaining its catalytic activity. The researchers compared *Is*PETase with different types of enzymes also degrading PET plastic from other microorganisms, such as fungal and bacterial cutinases at a reaction temperature of around 40 °C. Among these, *Is*PETase exhibited the highest PET plastic degradation efficiency (Yoshida et al, 2021)

Our team also checked previous iGEM teams' projects related to PET plastic degradation, and the 2018 Yale University iGEM team. “Making bacteria eat plastic" got the team’s attention (Yale iGEM). The Yale iGEM team also used the PETase gene for their project.

After many studies and experiments, several characteristics make *Is*PETase a promising candidate for the degradation and recycling of PET at higher tolerance to environmental changes. *Is*PETase has been extensively studied, and its entire gene sequence has been published. Our team can use this information to identify the *Is*PETase gene and request its synthesis from Mission Biotech company.

### How can we increase the efficiency of biorecycling to make it a more viable solution to plastic pollution?

In order to enhance the PETase activity we did further research to see if we could focus on specific mutations.

Japanese scientists did investigations and showed that by altering 3 amino acids, serine 121 switched to glutamic acid, aspartic acid 186 switched to histidine, and arginine 280 switched to alanine, at the catalytic site on the PETase enzyme, (*Is*PETaseS121E/D186H/R280A), enhances the PETase activity compared to the wild-type PETase. Several papers demonstrated that different amino acid mutations on the *Is*PETase enzyme enhanced catalytic activity(Sevilla et al, 2023).

This mutated enzyme not only improves PETase activity but also retains catalytic efficiency at higher temperatures, accelerating PET plastic degradation (Brett et al, 2021; Maity et al, 2021; Son et al, 2019)

Our team wanted to upgrade the PET plastic degradation project to make an enhanced PETase enzyme with 3 amino acid mutations that we found to be promising while doing research on PET degradation utilizing a strain of Ideonella sakaiensis 201-F6, *Is*PETaseS121E/D186H/R280A. Our team also upgraded the original Yale project using bacteria for induction only.

![igem-10](https://static.igem.wiki/teams/5094/experiment/igem-10.png)

### How could we increase the amount of enzyme being produced?

We not only researched potentially more efficient enzymes but also ways to increase enzyme production. We found that scientists engineered yeast Gal1, and Gal10 promoter into a single Gal1,10 promoter on a plasmid for synthetic cloning that switches on in the presence of galactose and switches off in the presence of glucose. Research showed that the pGal1,10 promoter for the yeast strain, Saccharomyces cerevisiae, can induce 20-30-fold induction in the presence of galactose (Rajeshkannan et al, 2022). Therefore, our cloned *Is*PETase (BBa\_K5094002) along with several mutations, expressed downstream of the pGal1,10 promoter (BBa\_K5094001), could result in a 20-30 fold induction in the presence of galactose.

For galactose-inducing pGal1,10 promoter, this system is sufficient/efficient to induce 20-30 fold of the gene expression, which is one of the major benefits of using yeast our team designed. Our team did yeast transformation plate selection, and only yeast containing our pGal1, 10 promoter plasmid with G418 resistant gene can survive (more detail is on the cloning design cycle). However, if the team integrates the *Is*PETase gene in the yeast genome, respectively, G418 will not be needed for the selection anymore.

#### Can we isolate the enzyme to create a product?

![Protein Purification](https://static.igem.wiki/teams/5094/experiment/engineering-1.png)

We researched the idea of being able to extract the enzyme to be able to sell it to industry. The plasmid our team used for making bio-bricks was the pRSET A, a type of common prokaryotic expression plasmid, designed for high-level prokaryotic expression due to the T7 promoter. It encoded an N-terminal 34aa precursor peptide with a 6-histidine tag sequence to bind to the nickel-resin column for the in vitro protein purification. The bio-bricks our team designed were T7-*Is*PETase, T7-*Is*PETaseThr116A, T7-*Is*PETaseThr116A/k259Glu, T7-*Is*PETaseThr116A/M154Thr, which T7 RNA polymerase specifically recognizes this promoter in bacterial competent cells of BL21(DE3) and our team expressed those variable *Is*PETase enzymes downstream of the T7 promoter in the presence of the inducer isopropyl β-D-1-thiogalactopyranoside (IPTG), respectively. This would allow the PETase to be extracted.

## Experimental design

### How can we create the desired mutations on the PETase

To verify our approach of generating pGal1,10-*Is*PETase (BBa\_K5094010) mutations, the team cloned the wild-type *Is*PETase gene downstream of the pGal1,10 promoter plasmid (the same as the T7 promoter). Our team used this pGal1,10 promoter driving *Is*PETase gene in vivo to leverage that yeast-expressed under pGal1,10 inducible promoter can generate 20-30 fold induction in the presence of galactose(Rajeshkannan et al, 2022).

Here's the procedure our team did to generate several mutations in the *Is*PETase gene:

1. Cloning: The wild-type *Is*PETase gene was cloned downstream of the pGal1,10 promoter plasmid.
2. Site-Directed Mutagenesis: our team performed site-directed mutagenesis to introduce the desired mutations (S121E, D186H, R280A). Primers containing the mutant nucleotides were designed for this purpose, however, our team designed 18-20bp of complementary sequence on either side of the desired mutation (usually 1-3 mismatched bases), which should be sufficient for the primers to anneal to the pGal1,10-*Is*PETase plasmid during the PCR reaction.
   PCR: PCR was performed using the pGal1,10-*Is*PETase plasmid as the DNA template.
   DpnI Digestion: The PCR product using primers containing S121E mutation was treated with DpnI enzyme to digest the parental DNA template that contains methylation markers. The new mutant plasmid, pGal 1,10-*Is*PETaseS121E, remained undigested due to the lack of methylation.
   Bacterial Transformation: The mutated plasmid was transformed into bacteria to generate more copies of the pGal 1,10-*Is*PETaseS121E plasmid.
3. Repeating Mutagenesis: The team repeated the site-directed mutagenesis twice to introduce the additional mutations, with the aim of creating the enhanced *Is*PETaseS121E/D186H/R280A enzyme downstream of the pGal1,10 promoter.

Our team also did the same procedure to create the T7- *Is*PETaseS121E/D186H/R280A enzyme. After site-directed mutagenesis, our team sent out those mutant plasmids to sequence.

### What problems did our team struggle with after doing site-directed mutagenesis to generate several mutant *Is*PETase enzymes downstream of either the T7 promoter or the pGal1,10 promoter?

When we received our sequencing, it showed that our desired mutations had not been successful. With Dr. HsiaoFen Han’s help, we noticed the *Is*PETase gene sequence contained multiple DNA fragments with repeat regions that our team didn’t expect while designing those primers, leading to challenges for the site-directed mutagenesis technique. The primers containing mutated nucleotides that our team desired to switch specific amino acids targeted incorrect amino acids due to these repeats so our team created T7-*Is*PETase (BBa\_K5094006), T7-*Is*PETaseThr116A (BBa\_K5094007), T7-*Is*PETaseThr116A/k259Glu (BBa\_K5094008), T7-*Is*PETaseThr116A/M154Thr (BBa\_K5094009) instead. For the pGal1,10 promoter, our team didn’t make any mutations on the *Is*PETase gene, so the team has pGal1,10-*Is*PETase (BBa\_K5094010), and pGal1,10-eGFP used as the control (BBa\_K418008).

Our team still decided to use the *Is*PETase mutations downstream of the T7 promoter to do a functional assay because the mutations are also involved in the PETase catalytic activity. Other publications showed altered several amino acids in the *Is*PETase gene, to enhance the enzyme activity, our team predicted the mutations created by accident might also affect the catalytic activity (Sevilla et al, 2023).

We also decided to continue with tests for the pGal1,10-*Is*PETase, which the sequencing showed was successfully cloned, with pGal1,10-eGFP used as the control(BBa\_K418008) to determine if the promoter was, in fact, able to increase expression of the enzyme.

### What could our team do to fix the random mutations created on the *Is*PETase gene via the site-directed mutagenesis technique?

The team used pGal1,10-*Is*PETase plasmid as a DNA template to make mutations but failed due to the large size of the pGal1,10-*Is*PETase plasmid (~7kb). Several DNA fragments being repeated in the *Is*PETase gene sequence hindered the site-directed mutagenesis technique to alter the specific amino acids our team desired. Dr. HsiaoFen Han suggested that one way our team could do this is to directly add the mutations to the *Is*PETase gene sequence and order the synthesized mutant gene from the Mission Biotech company. After that, the team could do the traditional cloning as the team did with the wild-type *Is*PETase gene.

The team could also optimize several primer sets of annealing conditions further for site-directed mutagenesis.

### What functional assays did the team perform to determine functionality for the yeast containing cloned genes downstream of the pGal1,10 promoter and DE3BL21 bacteria containing cloned genes downstream of the T7 promoter?

In order to determine if the plasmids were functioning within the E. coli and yeast we designed various functional assays, our team transformed the T7-*Is*PETase plasmid into DE3BL21 bacteria along with several respective mutations of the *Is*PETase plasmids, and induced the expression in the presence of IPTG. The pGal1,10-*Is*PETase plasmid was transformed into Saccharomyces cerevisiae yeast strain, as was the pGal1,10-eGFP as a control, and expression was induced in the presence of galactose to generate more *Is*PETase catalytic activity.

#### mRNA Production

For the functional assays, in order to determine if the mRNA is being produced, the team did RT-qPCR to detect *Is*PETase mRNA level after being induced by galactose for the pGal1,10 promoter in yeast.

To manipulate our team’s wild-type of the *Is*PETase enzyme with the control eGFP cloned downstream of the pGal1, 10 promoter, respectively, yeast containing pGal1,10-eGFP was the control, and yeast containing pGal1,10- *Is*PETase was the team’s experimental sample. The samples of the yeast, each containing two different composite parts, respectively, were induced in the presence of the galactose at 30’, 60’, 90’, and 120’, and the whole cell RNA extracted samples were operated before the RT-qPCR technique was performed.

The team conducted RT-qPCR to assess the induction of *Is*PETase mRNA levels downstream of the pGal1,10 promoter in yeast treated with 2% galactose. We used pGal1,10-eGFP as a control and measured eGFP mRNA induction at 0, 30, 60, 90, and 120 minutes. We observed a peak eGFP mRNA induction of approximately 8-fold at 120 minutes in the presence of 2% galactose compared to the 0-minute sample without galactose.

However, there was no significant induction of *Is*PETase mRNA across the 2-hour time course.

To further investigate the induction pattern of *Is*PETase mRNA, the team plans to collect additional time-course samples beyond the initial 2 hours of galactose treatment.

#### Protein Expression

![engineering-2](https://static.igem.wiki/teams/5094/experiment/experiments-2.png)

To verify whether several different IsPETse mutants cloned downstream of the T7 promoter, were been manipulated to generate protein, the team set up the SDS page and western blot experiments, our team cultured 50ml of DE3BL21 bacteria cells containing T7-*Is*PETase and various mutation plasmids in LB broth medium until OD~0.2, respectively. Then cell culture was split into half (25mls) and induced at 0.5mM of IPTG at 37 degrees for 6 hours and the other half (25mls) as uninduced at 37 degrees for 6 hours as controls.

Our team cultured 50ml of DE3BL21 bacteria cells containing T7-*Is*PETase and the various mutated plasmids in LB broth medium until eac reached OD~0.2, respectively. The cell culture was then split into half (25mls) and induced at 0.5mM of IPTG at 37°C for 6 hours and the other half (25mls) was uninduced at 37°Cfor 6 hours as controls.

The team did whole cell protein extraction via the 95°C boiling method in protein extraction lysis buffer and ran samples on an an 18% SDS page gel to show a stronger band of the *Is*PETase along with various mutantions of *Is*PETase at the correct protein size in the presence of IPTG.

After conducting 18% SDS-PAGE gel analysis, our team proceeded with Western blotting to assess the expression of the *Is*PETase protein. We cloned the *Is*PETase gene downstream of a 6-his tag, which resulted in the expressed protein also having a his-tag at its N-terminal.

For the western blot, we utilized a his-tag primary antibody and an HRP-conjugated anti-mouse secondary antibody to detect *Is*PETase expression for various samples. However, we did not detect any distinct bands of the *Is*PETase protein expression, even in the presence of IPTG.

Moving forward, we need to optimize the Western blot conditions, as this procedure involves multiple steps and two antibody incubations. Additionally, the use of an incorrect antibody dilution may have contributed to the lack of strong *Is*PETase expression bands.

#### PET Degradation

The final more direct functional assay is to determine the efficiency of PET degradation through co-culture of the same amounts of PET film with either DE3BL21 bacteria containing T7-*Is*PETase plasmids along with various mutations on *Is*PETase enzyme, respectively in the presence of IPTG to induce T7 promoter in DE3BL21 as experimental samples, lack of IPTG as controls. The team used a Nabi machine, which detects 240 nm, to detect the amount of TPA product generated daily for the co-culture experiment. The same experiment was performed with yeast containing the pGal1,10-*Is*PETase plasmid, along with the pGal1, 10-eGFP as a control, in the presence of galactose as experimental samples, and in the glucose as controls.

After doing the PET co-culture functional assay, we did not find any pattern in the spectrometer results. Peaks were low (0.001-0.04 OD only) and we didn’t see the peaks appearing at the same wavelength (232-248 nm all had peaks). After discussing with our instructor, we concluded that these reasons may have caused it: First, our UV-visible spectrometer isn’t as accurate as the university ones, so the peaks might shift and cause the inconsistent wavelengths we observed. Secondly, the reason for the weak signal might be caused by too little products being made, so with Professor Ng’s suggestion, we decided to do a p-NPB assay first to ensure our plasmid does have activity, then use PET powder to co-culture to increase the surface area, therefore, increase the reaction rate (UCM team also suggested us if we couldn’t buy the powder, freeze our PET film then grind it, it would be easier). Professor Ng also suggested we use crude protein (whole cell extract) to do our experiments so the enzyme diffusing in and out of the cell wouldn’t cause another variable.

After closely analyzing the results, we also believe technical errors caused the inconsistent data. Every time a particular teammate collected the data, the absorbance was always higher. It was concluded that she shook the flasks more vigorously than the other teammates before taking the samples. To fix this problem, our team will keep the same member on all sample collections.

## Future Directions

### How can we use the his-tag for enzyme extraction?

Besides in vivo experiments, our team also designed in vitro protein purification to extract the *Is*PETase enzyme and the several respective amino acid mutations of the *Is*PETase enzymes. Downstream of the T7 promoter is engineered to contain a 6-histidine tag (6-his tag). Mission Biotech confirmed that this had been done successfully when we sent the plasmids for sequencing.

Once our team finished cloning *Is*PETase gene downstream of the 6-his tag, along with the several amino acid mutants of the *Is*PETase enzymes, the team will transform them into BL21DE3 bacteria to express the enzymes in the presence of IPTG.

The wild-type and different mutations of the *Is*PETase enzymes containing the 6-his tag should be in the protein extract after the chemical process to break BL21DE3 bacterial cells. We will then run the protein extract through the Ni+-resin column, which can bind to the 6-his tag on the wild-type PETase and the several amino acid mutants of the *Is*PETase enzymes. Then several chemical buffers will be used to wash the column containing the samples to wash away unbound/unwanted proteins respectively, until the elution buffer is used to purify out each of the respecitive enzymes our team desires to the tubes.

## Citations

Brott, S., Pfaff, L., Schuricht, J., Schwarz, J., Böttcher, D., Christoffel, Wei, R., & Bornscheuer, U. T. (2021). Engineering and evaluation of thermostable *Is*PETase variants for PET degradation. *Engineering in Life Sciences*, 22(3-4), 192–203.

Maity, W., Maity, S., & Bera, S. (2021). Emerging Roles of PETase and MHETase in the Biodegradation of Plastic Wastes.

Rajeshkannan, Mahilkar, A., & Saini, S. (2022). GAL Regulon in the Yeast S. cerevisiae is Highly Evolvable via Acquisition in the Coding Regions of the Regulatory Elements of the Network. *Frontiers in Molecular Biosciences*, 9. https://doi.org/10.3389/fmolb.2022.801011

Sevilla, M. E., Garcia, M. D., Perez-Castillo, Y., Armijos-Jaramillo, V., Casado, S., Vizuete, K., Debut, A., & Cerda-Mejía, L. (2023). Degradation of PET Bottles by an Engineered Ideonella sakaiensis PETase. *Polymers*, 15(7), 1779.

Son, H. F., Cho, I. J., Joo, S., Seo, H., Sagong, H.-Y., Choi, S. Y., Lee, S. Y., & Kim, K.-J. (2019). Rational Protein Engineering of Thermo-Stable PETase from Ideonella sakaiensis for Highly Efficient PET Degradation. *ACS Publications*.

Yoshida, S., Hiraga, K., Taniguchi, I., & Oda, K. (2021). Ideonella sakaiensis, PETase, and MHETase: From identification of microbial PET degradation to enzyme characterization. *Methods in Enzymology*, 648, 187–205.

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#

##### Experiments

* [Experiment Aims](#first)
* [Choosing Target Genes](#second)
* [Creating our Genetically Engineered Composition Parts](#third)
* [Checking Our Products/Functional Assays](#fourth)
* [Future Experiments to Extend this Project](#fifth)
* [Protocols](#sixth)
* [Citations](#seventh)

## Experiment Aims

Plastic, being the cheapest and thus most commonly used packaging materials, poses a grave environmental threat when its disposal is not properly managed. Our team’s aim is to create a genetically engineered mutant *Is*PETase that is capable of degrading PET plastic more efficiently than current industry methods, as well as more compatible to function at a wider range of conditions. We believe this would encourage adoption of bio recycling and reduce the amount of plastics and microplastics that are in the ocean.

## Choosing Target Genes

### PETase

PETase is an enzyme that belongs to the esterase class of enzymes. It catalyzes the breakdown of polyethylene terephthalate (PET) plastic through hydrolysis, resulting in the formation of monomeric mono-2-hydroxyethyl terephthalate (MHET), BHET (bis(2-hydroxyethyl) terephthalate), and terephthalic acid (TPA), opening up possibilities for biological PET recycling and degradation. The most popular PETase enzyme was identified in 2016 from a strain of bacteria called Ideonella sakaiensis 201-F6, found in sludge samples collected near a PET bottle recycling site in Japan (Yoshida,2021). *Is*PETase studied by Japanese groups for several years showed that it has more tolerance for catalytic activities to degrade PET plastic in high temperatures, and acidic environments (Yoshida,2021). *Is*PETase has been confirmed to have the highest PET degradation activity of all PET-degrading enzymes from different microorganisms, as evidenced by its structure and chemical bonding (Khairul et al, 2022). One of the key factors to this is the enzyme's structure, in which *Is*PETase has a broader and more open active-site cleft compared to other enzymes like cutinases, which allows it to accommodate PET molecules more effectively. The wider substrate-binding pocket of the PETase is critical for PET hydrolysis, which is the major process of breaking down plastics into monomeric blocks. Furthermore, *Is*PETase possesses two disulfide bonds in its active site, contributing to its efficiency in PET substrate binding. After many studies and experiments, *Is*PETase is found to be a promising candidate for PET degradation at both ideal temperatures and room temperature.

Therefore, the team chose *Is*PETase to synthetically biodegrade PET plastic.

### T7 Promoter and Antibiotic Resistant Gene

The T7 promoter is a sequence of DNA used to regulate the gene expression of recombinant proteins in various applications (Tabor, 2021). The T7 RNA polymerase specifically recognizes T7 promoter in the bacterial competent cells of BL21(DE3)pLys. This allows a high efficiency of RNA transcription of the gene cloned downstream of the T7 promoter, when the promoter is induced by isopropyl β-D-Thiogalactoside (IPTG). It also has a ribosome binding site for high-efficiency protein expression.

The T7 promoter plasmid also contains an N-terminal 34aa precursor peptide with a histidine tag sequence, 6-his tag, to bind to the nickel-resin column for the in vitro protein purification to extract the proteins our team is interested in (T7 Promoter System, 2016). The ampicillin-resistant gene is also synthetic and cloned into the T7 promoter plasmid for bacterial transformation selection on LB-Amp plates.

### pGal 1, 10 Plasmid and Antibiotic Resistant Genes

In yeast, the promoters of Gal1 and Gal 10 are named after the genes they regulate, GAL1 and GAL10, which encode enzymes involved in the conversion of galactose into glucose-1-phosphate, a form that can be further metabolized by Saccharomyces cerevisiae, yeast cells(O’Connor, 2014). Scientists engineered yeast Gal1 and Gal 10 promoters to create a Gal 1,10 promoter on the plasmid. This plasmid can be switched on in the presence of galactose, and switched off in the presence of glucose in the medium. The benefit of using the pGal1,10 promoter is to regulate the cloned gene’s expression by adding either glucose or galactose into the yeast medium to turn off or on the gene transcription. Our team will adopt this pGal promoter mainly due to its high expression of the genes (20-30 fold induction) related to this project cloned downstream of the pGal promoter in the presence of galactose for the in vivo protein expression in yeast (Rajeshkannan et al, 2022).

The pGal1,10-SPT5-Streptavidin binding protein (SBP) plasmid, containing yeast origin of replication, 2u, and also bacteria origin of replication, was sponsored by Dr. Tien-Hsien Chang at the Genomics Research Center of Academia Sinica, in Taipei. The reason our team is using this pGal1,10 promoter plasmid is due to the benefit of making biobricks in bacteria and performing the in vivo protein functional assay in saccharomyces yeast. There are two resistant genes on this plasmid, one is the Geneticin (G418) resistance gene which can be used to select yeast colonies on YPD-G418 plates since G418 blocks the protein translation process. Only yeast containing our pGal1,10 plasmid can survive on YPD-G418 plates ; the other is ampicillin, which shows no effect on yeast, but can be used to do bacterial colony selection on LB-Amp plates.

### *Is*PETaseS121E/D186H/R280A Mutations

Japanese scientists did further investigations to show that after changing 3 amino acids at the catalytic site on the PETase enzyme, (*Is*PETase S121E/D186H/R280A), the mutant enzyme enhances the PETase activity compared to the wild-type PETase, and also can have catalytic activity at higher temperatures to degrade PET plastic products faster (Brett et al, 2021; Maity et al, 2021; Son et al, 2019). The 3 amino acid mutations our team aimed to create will widen the substrate binding cleft, allowing the catalytic activities to perform easier and faster, enhancing the overall efficiency of PET product recycling (Office U.S.G.A, 2022). Therefore, our team’s will focus on enhancing the PETase enzyme via site-directed mutagenesis technique to create (*Is*PETaseS121E/D186H/R280A) for PET plastic degradation

### Design Goal

Our team’s final goal is to focus on enhancing the PETase enzyme activity via site-directed mutagenesis technique to create (*Is*PETaseS121E/D186H/R280A) for more efficient PET plastic degradation. We will create two versions, one with a pGal1,10 promoter to increase enzyme production, for yeast, and one with a T7 promoter with a his-tag to allow for in vitro protein purification, in E. coli.

![fig1](https://static.igem.wiki/teams/5094/experiment/fig1.png)

Fig 1: pGal 1,10-*Is*PETase target plasmids

![fig1](https://static.igem.wiki/teams/5094/experiment/fig2.png)

Fig 2: T7-*Is*PETase target plasmids

​​

## Creating our Genetically Engineered Composition Parts

### Making T7-*Is*PETase plasmid

![making-t7-<i>Is</i>PETase-plasmid](https://static.igem.wiki/teams/5094/experiment/fig-1-making-t7-ispetase-plasmid.jpg)

Fig. 3: Making T7-*Is*PETase plasmid diagram

[#### PCR via PET synthesized from a biotech company](https://static.igem.wiki/teams/5094/protocol/2x-pcr-mix-protocol.pdf)

We synthesized the IsPET gene via PCR protocol. We designed the forward primer flanked with the BamHI enzyme recognition site and the reverse primer flanked with the HindIII enzyme recognition site. Our team purchased the synthesized *Is*PETase gene from the Mission Biotech Company as the DNA template, and a PCR experiment was performed to amplify the *Is*PETase gene flanked with BamHI and HindIII enzyme digestion sites at both ends of the gene.

[#### Double enzyme digestions on PCR products](https://static.igem.wiki/teams/5094/protocol/neb-enzyme-digestion-protocol.pdf)

We performed double enzyme digestion using the BamHI and HindIII enzymes simultaneously on the *Is*PETase gene PCR product and the T7 promoter plasmid, which generates compatible sticky ends on both the *Is*PETase gene PCR product and the plasmid, allowing them to be ligated together.

[#### T4 ligation](https://static.igem.wiki/teams/5094/protocol/t4-ligation.pdf)

We used T4 DNA ligase to ligate the digested *Is*PETase gene PCR product and the digested T7 Promoter plasmid together. The T4 ligase catalyzes the formation of phosphodiester bonds between the DNA fragments, joining them together to form the T7-*Is*PETase plasmid.

[#### Bacterial transformation](https://static.igem.wiki/teams/5094/protocol/bacterial-transformation-heat-shock-protocol.pdf)

We transformed the ligated T7-*Is*PETase plasmid into DH5ɑ bacterial competent cells, and did LB-amp plate selection. The bacterial colonies grew on the LB-amp plates indicating that they contain our team’s cloned plasmids. This was done by heat-shocking the bacterial competent cells with the cloned plasmids and then allowing them to recover and grow on LB-amp selected plates.

We then inoculated those bacterial colonies on LB-amp plates and broth. This allowed the bacteria to grow and multiply, amplifying the cloned DNA in our team’s T7-*Is*PETase plasmid. We then plated the transformed bacteria on LB-AMP plates and broth. The ampicillin in the plates selected bacteria that have taken up the plasmid, as the plasmid contained an ampicillin resistance gene. Our team picked those single bacterial colonies and stored them in 25% glycerol stocks at -80 °C.

[#### BACTERIAL PLASMID EXTRACTION](https://static.igem.wiki/teams/5094/protocol/bacterial-plasmid-extraction.pdf)

After bacterial inoculation, we did bacterial plasmid extraction: our team used a TENs buffer, which contained a more basic solution (0.1M NaOH/0.5% SDS in TE, PH=8), and 10% SDS to break down bacterial cell membranes. Then, we added 3M sodium acetate (NaOAc) to neutralize the solution to precipitate down debris. Then, we used RNase to break down the remaining RNA in the tube and incubated them at 37°C for at least 20 minutes. Next, we used phenol-chloroform and separated the proteins and debris from our plasmids. Finally, we added ethanol and sodium acetate to precipitate the plasmid at the bottom of the tube and poured out the supernatant to obtain our pure, desired plasmid.

[#### PCR Verification](https://static.igem.wiki/teams/5094/experiment/2x-pcr-mix-protocol.pdf)

We did PCR again to confirm bacterial colonies containing our team’s T7-*Is*PETase plasmid: After selecting colonies on the LB-AMP plates, we performed PCR using primers specific to the cloned *Is*PETase gene to confirm the presence of the desired insert in the plasmid. This helped verify the success of the cloning process (amplification of the targeted DNA segment).

[#### Proof of Concept](https://static.igem.wiki/teams/5094/experiment/gel-electrolysis.pdf)

![fig4](https://static.igem.wiki/teams/5094/parts/2024-09-26-155801.png)

Fig. 4: *Is*PETase full length product, 893bp.

After the team amplified *Is*PETase with two enzyme cut sites, BamHI1 and HindIII, flanked on the two sides via primers designed through PCR. After PCR, the team ran a sample through 1% agarose gel, *Is*PETase PCR product showed a single distinct DNA band with a length of 893 base pairs+20 bp of enzyme cut sites designed on primers and showed a nice single band data after PCR amplification. The DNA fragment corresponded with DNA ladders according to the gel electrophoresis.

![fig5](https://static.igem.wiki/teams/5094/parts/2024-09-25-122245.png)

Fig. 5: *Is*PETase PCR product

After cloning *Is*PETase downstream of the T7 promoter plasmid, bacterial transformation was done, and bacterial colonies were grown on the LB-Amp selection plates. The colonies were directly inoculated to perform the PCR technique via the *Is*PETase primer set. In Fig2, the 1% agarose gel showed that bacterial colonies # 1, 2, and 5 had a distinct *Is*PETase single PCR product band, which indicated that the bacterial colonies contained T7-*Is*PETase

### Making the pGal 1,10-*Is*PETase Plasmid

![making-<i>Is</i>PETase-on-pgal-1-10-plasmid](https://static.igem.wiki/teams/5094/experiment/fig-4-making-ispetase-on-pgal-1-10-plasmid.jpg)

Fig. 6: Making the pGal1,10-*Is*PETase plasmid

For making pGal 1,10-*Is*PETase, we performed similar experiments as that of making T7-*Is*PETase. However, when performing double enzyme digestion, we used XmaI and SpeI double enzymes, instead of BamHI and HindIII enzymes used during double enzyme digestion for T7-*Is*PETase. For the rest of the procedure, the making of pGal 1,10-*Is*PETase was identical to that of the experiments done when making T7-*Is*PETase.

#### Proof of Concepts

After our experiments, we did PCR again to confirm the yeast contained our team’s pGal1,10-*Is*PETase plasmid:

![fig7](https://static.igem.wiki/teams/5094/experiment/fig7.png)

Fig.7: pGal 1,10-*Is*PETase full-length product, 893bp

In order to amplify the *Is*PETase with two enzyme cut sites, XmaI and SpeI, flanked on the two sides via primers designed, the team performed PCR. After PCR, the team ran a sample through 1% agarose gel, *Is*PETase PCR product showed a single distinct DNA band with a length of 893 base pairs+20 bp of enzyme cut sites designed on primers and showed a nice single band data after PCR amplification. The DNA fragment corresponds with DNA ladders according to the gel electrophoresis.

![fig8](https://static.igem.wiki/teams/5094/experiment/fig8.png)

Fig. 8: pGal1,10-*Is*PETase PCR product

After cloning *Is*PETase downstream of the pGal1,10 promoter plasmid, bacterial transformation was done, and bacterial colonies were grown on the LB-Amp selection plates. The colonies were directly inoculated to perform the PCR technique via the *Is*PETase primer set. In Fig4, the 1% agarose gel showed that bacterial colonies #5 and 6 had a distinct *Is*PETase single PCR product band, which indicated that the bacterial colonies contained pGal 1,10-*Is*PETase

[#### Mutating the Plasmids - Site-Directed Mutagenesis](https://static.igem.wiki/teams/5094/protocol/site-directed-mutagenesis.pdf)
![site-directed-mutagenesis Image](https://static.igem.wiki/teams/5094/experiment/fig-7-site-directed-mutagenesis.jpg)

Fig. 9: T7-*Is*PETaseS121E Plasmid Creation Diagram

#### Amplification of mutant DNA

To mutate our *Is*PETase, we designed point mutations on the forward and reverse primers, changing *Is*PETase serine 121 to glutamic acid. Next, we performed PCR amplification: for the PCR, prepare the T7-*Is*PETase a DNA template, pfu DNA polymerase buffer, pfu DNA polymerase, dNTP mix, and the mutated forward and reverse primers. The mutated plasmid was amplified after PCR. Thus, we obtained copies of the mutated plasmid.

#### Degradation of parental DNA

After the PCR, we separated the mutated plasmid, T7-*Is*PETaseS121E plasmid, from the parent plasmid by adding 1μl of DpnI to the PCR reaction at 37 °C for 1 hour. This allowed the DpnI to digest the parental DNA, which contains methylation markers, leaving only the T7-*Is*PETaseS121E plasmid.

#### Bacterial Transformation into E. coli

We transformed the mutated plasmid into DH5α bacteria competent cells via 42 °Cheat shock protocol on LB-amp selection plates. Then, we prepared it for LB-AMP plate selection, and the resulting bacteria were the E. coli that contained the T7-*Is*PETaseS121E plasmid. By performing bacterial transformation, we obtained more single bacterial colonies that contained the T7-*Is*PETaseS121E plasmid.

#### Inoculate bacterial colonies

We inoculated several bacterial colonies on LB-amp plates and broth; by doing this, we could select the colonies that have the T7-*Is*PETaseS121E plasmid with an amp-resistant gene. We plated the transformed bacteria on LB-AMP plates and broth. The ampicillin in the plates selected the bacteria that have taken up the T7-*Is*PETaseS121E plasmid, as the plasmid contains an ampicillin resistance gene, killing the bacterial colonies that do not contain the plasmid. That confirmed that our bacterial colonies contained the T7-*Is*PETaseS121E plasmid.

#### Bacterial plasmid extraction

After we got more copies of bacteria cells containing the mutated plasmid, we used the bacterial plasmid extraction protocol to extract the mutated plasmid from the bacteria cells. Our team used TENs buffer that contained a more basic solution and 10% SDS to break down bacterial cell membranes and then 3M NaOAC pH5.2 was added to neutralize the solution to precipitate down protein and membrane debris. RNase was used to break down all of the RNA in the tube and incubate at 37 °C for 20-30 mins. We then used phenol-chloroform and separated the proteins and debris from our plasmids. After extracting the top layer of the remaining mixture, we added ethanol and 3M sodium acetate to precipitate the plasmid at the bottom of the tube. In this stage, the ethanol interacted with the plasmids and helped the plasmids to precipitate at the bottom of the tube. We then spun down the mixture and discarded the supernatant, leaving only the pellet. Again, we added ethanol to clean up the chemicals attached to the plasmids. Finally, we spun down the mixture and discarded the supernatant, and the pure, desired plasmid pellet will be at the bottom of the tube. We then resuspended it with dH₂O. We repeated site-directed mutagenesis two more times to get triple mutations on the *Is*PETase.

#### Checking the sequence

After completing each site-directed mutagenesis, we sent our mutant plasmids to Mission Biotech to check our mutation results.

The results showed that our team failed to create the desired mutations. No mutations were on the pGal1,10-*Is*PETase (BBa\_K5094010), however it confirmed we had achieved the pGal1,10-*Is*PETase (BBa\_K5094010) sequence in our plasmid.

Similarly the desired mutations were also not found on the T7-*Is*PETase (BBa\_K5094006). However we had created new mutations: T7-*Is*PETaseThr116A(BBa\_K5094007), T7-*Is*PETaseThr116A/k259Glu (BBa\_K5094009), and T7-*Is*PETaseThr116A/M154Thr (BBa\_K5094008) instead.

Nevertheless, we decided to continue the project, as other publications showed alternative mutations in the *Is*PETase gene which also enhanced *Is*PETase enzyme activity(Sevilla et al, 2023). Therefore, our new mutations may also affect the catalytic activity of the *Is*PETase. The pGal1,10 promoter plasmid we engineered with *Is*PETase should have a 20-30 fold increase in gene expression of the gene cloned downstream of the promoter, when induced by galactose, which is a valuable design (O’Connor).

Based on the promise of these factors, we decided to continue to determine proof of concept and designed functional assay experiments that would test the functionality of our new mutations in our bacteria and the wild type pGal1,10-*Is*PETase.

#### Proof of Concept

![fig10](https://static.igem.wiki/teams/5094/experiment/fig10.png)

Fig. 10 Before sending out sequencing to show the mutations of the *Is*PETase team expected, our team ran the plasmids on 1% agarose gel. Lane 2 is pGal1,10-*Is*PETase, lane 3 is pGal1,10-*Is*PETase S121E, lane 4 is pGal1,10-*Is*PETase S121E/D186H and lane 5 is pGal1,10-*Is*PETase S121E/D186H/R280A.

## Checking Our Products/Functional Assays

[### RT-qPCR](https://static.igem.wiki/teams/5094/protocol/rt-qpcr.pdf)
![fig9](https://static.igem.wiki/teams/5094/experiment/fig-9.png)

Fig. 11: RT-qPCR Time Course Sample Collection Experiment Design

After site-directed mutagenesis, the team couldn’t make any mutations in *Is*PETase. However, the team decided to use the pGal1,10-eGFP composite part created by the 2022 KCIS team as a control and test if our wild-type of pGal1,10-*Is*PETase produces our desired enzyme. To manipulate our team’s wild-type of the *Is*PETase enzyme along with the control eGFP cloned downstream of the pGal1, 10 promoter, we transformed the BBa\_K418008, which contained pGal1,10-eGFP created by the 2022 KCIS team, and the BBa\_K5094010, which contained BBa\_K5094001K+BBa\_K5094002 (pGal1,10- *Is*PETase), into wild-type Saccharomyces Yeast Strain, BY4741.

The function of each cloned gene to generate these 2 composite parts is as follows:

1. BBa\_K418008 created by the 2022 KCIS team was transformed into BY4741 to make the BY4741 which contains BBa\_K418008, and was used as a control yeast strain in our project. The purpose of using this composite part was to determine the specification. The team wants to verify the gene downstream of the pGal1,10 promoter can be induced in the presence of galactose, but eGFP protein can't degrade PET plastic for the co-culture experiments later on.
2. BBa\_K5094010 contained BBa\_K5094001K+BBa\_K5094002 (pGal1,10-*Is*PETase) was transformed into BY4741 to make the BY4741 containing BBa\_K5094010 as the experimental yeast strain in our project. BBa\_K5094010 contains a wild-type of the *Is*PETase encodes for the PETase enzyme specifically degrading PET plastic. The team wants to verify the gene downstream of the pGal1,10 promoter can be induced in the presence of galactose, *Is*PETase protein.

To further verify whether our team’s composite parts produce mRNA, RT-qPCR technique was operated to detect the mRNA induction of eGFP in BY4741 containing BBa\_K418008 as a control, the mRNA induction of *Is*PETase in BY4741 containing BBa\_K5094010 as an experimental sample, via time course sample collection, 0min, 30min, 60min, 90min, 120min in the presence of the 2%YP-galactose medium.

The BY4741 containing the two different composite parts was grown in 150ml of 2%YP-glucose-200 μg/ml G418 until OD600 ~0.4 of the 40 ml of yeast culture medium and taken out as 0 min as a control. Without galactose, the pGal1,10 promoter would not express the downstream genes’ mRNA.

The rest of the culture samples were collected at 5000 rpm for 5 mins and we washed the culture samples with dH₂O twice to eliminate glucose. We then transferred the 2 composite parts of the yeast culture samples to 200 ml of 2%YP-galactose-200 μg/ml G418, for which our team took out 40 ml of yeast culture medium samples at different time courses in the presence of galactose.

[### SDS Gel Electrophoresis and Western Blot](https://static.igem.wiki/teams/5094/protocol/western-blot.pdf)
![fig12](https://static.igem.wiki/teams/5094/experiment/fig-10.png)

Fig. 12: IPTG Induction Experiment

Before setting up the SDS page and western blot experiments, our team cultured 50 ml of DE3BL21 bacteria cells which contained T7-*Is*PETase and our mutated plasmids in the LB broth medium until OD~0.2. Then cell culture was split into one half (25mls) that contained 0.5mM of IPTG at 37 °C for 6 hours and the other half (25mls) that did not have IPTG at 37°C for 6 hours as controls (Mühlmann et al, 2017; Namdev et al, 2019).

To verify whether our composite parts—BBa\_K 5094006 containing BBa\_K5094000+ BBa\_K5094002 (T7-*Is*PETase), BBa\_K5094007 containing BBa\_K5094000+BBa\_K 5094003(T7-*Is*PETase Thr116A), BBa\_K5094009 containing BBa\_K5094000+BBa\_ K5094005 (T7-*Is*PETaseThr116Ala/Lys259Glu), and BBa\_K5094008 containing BBa\_K5094000+BBa\_K5094004 (T7-*Is*PETaseThr116Ala/Met154Thr )—can express the proteins our team desires in DE3BL21 bacteria, the team did whole cell protein extraction of the DE3BL21 bacteria after being induced by IPTG, via the 95-degree boiling method in protein extract lysis buffer protocol. According to the data shown on BBa\_K2010000 from the Harvard 2017 team, the *Is*PETase protein was in the soluble condition, and the supernatants were collected for a SDS page and Western blot (Team:Harvard, 2017).

The team performed the SDS page first. We ran an 18% SDS-PAGE gel to confirm whether the whole cell extract contained all proteins, as well as to look for a stronger band of the *Is*PETase along with various mutants of *Is*PETase protein, approximately 30 kDa in the presence of IPTG.

After 18% SDS-PAGE gel data, our team performed the western blot technique to determine the specific *Is*PETase protein expression. The team cloned the *Is*PETase gene downstream of the 6-his tag. Once the *Is*PETase protein is expressed, it contains a his-tag at the N-terminal of the *Is*PETase, so the his-tag antibody could be used for the western blot experiment.

[### Co-Culture Experiment](https://static.igem.wiki/teams/5094/protocol/co-culture-protocol.pdf)
![fig11](https://static.igem.wiki/teams/5094/experiment/fig-11.png)

Fig.13: Co-Culture Experiments Design

The more direct functional assay is to co-culture the same amount of PET film with either DE3BL21 bacteria containing T7-*Is*PETase plasmid and our mutations on *Is*PETase enzyme, respectively in the presence of ITPG to induce T7 promoter in DE3BL21 as experimental samples, lack of ITPG as controls. The team used a Nabi machine with 240 wavelengths to detect the amount of TPA, which is the product of PET plastic after degradation, generated daily for the co-culture experiment(Terephthalic acid, 2023). The same experiment was performed with yeast containing the pGal1,10-*Is*PETase plasmid, along with various mutations on *Is*PETase, in the presence of galactose as experimental samples, in the glucose as controls.

## Future experiments to extend this project:

### For the in vitro functional assay:

In addition to in vivo experiments, our team will extend this project to perform in vitro protein purification to isolate the *Is*PETase enzyme and its various *Is*PETase enzyme mutants. The *Is*PETase gene and its mutants are cloned downstream of a T7 promoter engineered with a 6-histidine (6-his) tag. These constructs are then transformed into BL21(DE3) bacteria, and enzyme expression is induced using IPTG.

Following cell lysis, the protein extracts containing the 6-his-tagged wild-type and mutant *Is*PETase enzymes are passed through a Ni²⁺-resin column, which selectively binds to the 6-his tag. The column is then washed with several buffers to remove unbound and unwanted proteins. Finally, an elution buffer is used to purify the desired enzymes, which are collected in separate tubes for further analysis. The team will use *Is*PETase enzyme and mutant *Is*PETase enzymes to perform the enzyme-abstract “p-Nitrophenyl Esters Assay”, which PETase can be tested using p-nitrophenyl butyrate, which releases p-nitrophenol upon hydrolysis by PETase, which can be quantified spectrophotometrically by measuring the absorbance at 405 nm.

### Making MHETase biobricks:

The MHET produced from PET degradation by PETase can be further broken down into terephthalic acid (TPA) and ethylene glycol (EG) by MHETase. Our team's future work will focus on designing MHETase biobricks to overexpress this enzyme, aiming to enhance the efficiency of degrading PET plastic into its monomers. This approach will allow for a more complete and efficient breakdown of PET waste, ultimately converting it into reusable building blocks for new plastic production or other industrial applications.

## Protocols

* [Bacterial Transformation Heat Shock](https://static.igem.wiki/teams/5094/protocol/bacterial-transformation-heat-shock-protocol.pdf)
* [Co-Culture Experiments Protocol](https://static.igem.wiki/teams/5094/protocol/co-culture-protocol.pdf)
* [Gel Electrolysis](https://static.igem.wiki/teams/5094/protocol/gel-electrolysis.pdf)
* [NEB Enzyme Digestion Protocol](https://static.igem.wiki/teams/5094/protocol/neb-enzyme-digestion-protocol.pdf)
* [RT-qPCR](https://static.igem.wiki/teams/5094/protocol/rt-qpcr.pdf)
* [Site-Directed Mutagenesis](https://static.igem.wiki/teams/5094/protocol/site-directed-mutagenesis.pdf)
* [T4 Ligation](https://static.igem.wiki/teams/5094/protocol/t4-ligation.pdf)
* [Western Blot](https://static.igem.wiki/teams/5094/protocol/western-blot.pdf)
* [Yeast Plasmid Transformation Protocol](https://static.igem.wiki/teams/5094/protocol/yeast-plasmid-transformation-protocol.pdf)
* [Bacterial Plasmid Extraction Protocol](https://static.igem.wiki/teams/5094/protocol/bacterial-plasmid-extraction.pdf)
* [2X PCR Mix Protocol](https://static.igem.wiki/teams/5094/protocol/2x-pcr-mix-protocol.pdf)

## Citations

Brott, S., Pfaff, L., Schuricht, J., Schwarz, J., Böttcher, D., Christoffel, Wei, R., & Bornscheuer, U. T. (2021). Engineering and evaluation of thermostable IsPETase variants for PET degradation. *Engineering in Life Sciences*, 22(3-4), 192–203. https://doi.org/10.1002/elsc.202100105

Khairul Anuar, N. F. S., Huyop, F., Ur-Rehman, G., Abdullah, F., Normi, Y. M., Sabullah, M. K., & Abdul Wahab, R. (2022). An Overview into Polyethylene Terephthalate (PET) Hydrolases and Efforts in Tailoring Enzymes for Improved Plastic Degradation. *International journal of molecular sciences*, 23(20), 12644. https://doi.org/10.3390/ijms232012644

Maity, W., Maity, S., Bera, S. (2021). Emerging Roles of PETase and MHETase in the Biodegradation of Plastic Wastes https://doi.org/10.1007/s12010-021-03562-4

Mühlmann, M., Forsten, E., Noack, S., & Büchs, J. (2017). Optimizing recombinant protein expression via automated induction profiling in microtiter plates at different temperatures. *Microbial cell factories*, 16(1), 220. https://doi.org/10.1186/s12934-017-0832-4

Namdev, P., Dar, H. Y., Srivastava, R. K., Mondal, R., & Anupam, R. (2019). Induction of T7 Promoter at Higher Temperatures May Be Counterproductive. *Indian journal of clinical biochemistry : IJCB*, 34(3), 357–360.

O’Connor CM. 13.1: Regulation of the GAL1 promoter. (2014). *California: Libretexts*. https://bio.libretexts.org/Bookshelves/Cell\_and\_Molecular\_Biology/Book%3A\_
Investigations\_in\_Molecular\_Cell\_Biology\_(O%27Connor)/13%3A\_Protein\_overex
pression/13.01%3A\_Regulation\_of\_the\_GAL1\_promoter.

Office, U. S. G. A. (2022). Science & Tech Spotlight: Biorecycling of Plastics. *www.gao.gov*. https://www.gao.gov/products/gao-23-106261

Rajeshkannan, Mahilkar, A., & Saini, S. (2022). GAL Regulon in the Yeast S. cerevisiae is Highly Evolvable via Acquisition in the Coding Regions of the Regulatory Elements of the Network. *Frontiers in Molecular Biosciences*, 9. https://doi.org/10.3389/fmolb.2022.801011

Sevilla, M. E., Garcia, M. D., Perez-Castillo, Y., Armijos-Jaramillo, V., Casado, S., Vizuete, K., Debut, A., & Cerda-Mejía, L. (2023). Degradation of PET Bottles by an Engineered Ideonella sakaiensis PETase. *Polymers*, 15(7), 1779. https://doi.org/10.3390/polym15071779

Tabor S. (2001). Expression using the T7 RNA polymerase/promoter system. *Current protocols in molecular biology, Chapter 16, Unit16.2*. https://doi.org/10.1002/0471142727.mb1602s11

Team:Harvard - 2017.igem.org. (2017). *Igem.org*. https://2017.igem.org/Team:Harvard

Terephthalic acid. (2023). *Nist.gov; National Institute of Standards and Technology*. https://webbook.nist.gov/cgi/cbook.cgi?ID=C100210&Mask=400

T7 Promoter System. (2016). *Sigmaaldrich.com*. https://doi.org/Dogri

Son, H. F., Cho, I. J., Joo, S., Seo, H., Sagong, H.-Y., Choi, S. Y., Lee, S. Y., & Kim, K.-J. (2019). Rational Protein Engineering of Thermo-Stable PETase from Ideonella sakaiensis for Highly Efficient PET Degradation. *ACS Publications*. https://doi.org/10.1021/acscatal.9b00568

‌Yoshida, S., Hiraga, K., Taniguchi, I., & Oda, K. (2021). Ideonella sakaiensis, PETase, and MHETase: From identification of microbial PET degradation to enzyme characterization. *Methods in enzymology*, 648, 187–205. https://doi.org/10.1016/bs.mie.2020.12.007

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#

##### Result

* [Confirmation of Each Cloning Step](#first)
* [Making several mutated *Is*PETase enzymes via site-directed mutagenesis](#second)
* [Sequencing Data](#third)
* [Proof of Concept](#fourth)
* [Future Experiments for Functional Assay](#fifth)
* [Future Experiments to Extend This Project](#sixth)
* [Citations](#seventh)

## Confirmation of each cloning step

### *Is*PETase for T7 promoter cloning

Our team performed the PCR technique to amplify *Is*PETase using the *Is*PETase gene synthesized by the Mission Biotech company as the DNA template and then confirmed the step's success by observing the right size bands of the *Is*PETase gene PCR products on 1% agarose gel in Figure 1. The team designed the forward and reverse primer set flanked with two enzyme cut sites, BamHI1 and HindIII to generate the composite part, BBa\_K5094006 (T7-*Is*PETase).

![](https://static.igem.wiki/teams/5094/parts/resulltttt.jpg)

Fig 1:BBa\_K5094002,IsPETase full length product, 873bp

After the team amplified *Is*PETase with two enzyme cut sites, BamHI1 and HindIII, flanked on the two sides via primers designed through PCR. After PCR, the team ran a sample through 1% agarose gel, *Is*PETase PCR product showed a single distinct DNA band with a length of 873 base pairs+20 bp of enzyme cut sites designed on primers and Figure 3 showed a nice single band data after PCR amplification. The DNA fragment corresponds with DNA ladders according to the gel electrophoresis.

### T7-*Is*PETase

The team performed bacterial transformation on the LB-Amp selection plates after finishing the cloning steps and inoculated several bacterial colonies to conduct the PCR technique to prove that our team had successfully created a composite part, BBa\_K 5094006 (T7-*Is*PETase) in Figure 2.

![](https://static.igem.wiki/teams/5094/parts/2024-09-25-122245.png)

Fig2: After bacterial transformation
for T7-*Is*PETase cloning, bacteria colonies
picked up directly to do *Is*PETase PCR.3
colonies on lane 2,3,4 had BBa\_K5094006
Composite part (full-length *Is*PETase PCR
product)

After cloning *Is*PETase downstream of the T7 promoter plasmid, bacterial transformation was done, and bacterial colonies were grown on the LB-Amp selection plates. The colonies were directly inoculated to perform the PCR technique via the *Is*PETase primer set. In Figure 2, the 1% agarose gel showed that bacterial colonies # 1, 2, and 5 had a distinct *Is*PETase single PCR product band, which indicated that the bacterial colonies contained our team’s composite part, BBa\_K5094006 (T7-*Is*PETase).

### *Is*PETase for pGal1, 10 promoter cloning

Our team not only cloned *Is*PETase downstream of the T7 promoter plasmid but also pGal1,10 plasmid. The team performed PCR technique to amplify *Is*PETase using the *Is*PETase gene synthesized by the Mission Biotech company as the DNA template and then confirmed the step's success by observing the right size bands of the *Is*PETase gene PCR products on 1% agarose gel in Figure 3. The team designed the forward and reverse primer set flanked with two enzyme cut sites, XmaI and SpeI to generate the composite part, BBa\_K5094010 (pGal1,10-*Is*PETase).

![](https://static.igem.wiki/teams/5094/parts/screenshot-2024-09-26-at-4-17-08-pm.png)

Fig3: BBa\_K5094002 *Is*PETase full-length product, 873bp

After the team amplified *Is*PETase with two enzyme cut sites, XmaI and SpeI, flanked on the two sides via primers designed through PCR. After PCR, the team ran a sample through 1% agarose gel, *Is*PETase PCR product showed a single distinct DNA band with a length of 873 base pairs+20 bp of enzyme cut sites designed on primers and showed a nice single band data after PCR amplification. The DNA fragment corresponds with DNA ladders according to the gel electrophoresis.

### pGal1,10-*Is*PETase

The team performed bacterial transformation on the LB-Amp selection plates after finishing the cloning steps and inoculated several bacterial colonies to conduct the PCR technique to prove that our team had successfully created a composite part, BBa\_K5094010 (pGal1,10-*Is*PETase) in Figure 4.

![](https://static.igem.wiki/teams/5094/parts/2024-09-25-170617.png)

Fig4: After bacterial transformation for pGal-*Is*PETase cloning, bacteria colonies were picked up directly to do *Is*PETase PCR. Lane 2 was *Is*PETase PCR product control. 2 colonies on lanes 5 and 6 had BBa\_K5094010 composite part(full-length *Is*PETase PCR product)

After cloning *Is*PETase downstream of the pGal1,10 promoter plasmid, bacterial transformation was done, and bacterial colonies were grown on the LB-Amp selection plates. The colonies were directly inoculated to perform the PCR technique via the *Is*PETase primer set. In Figure 4, the 1% agarose gel showed that bacterial colonies #5 and 6 had a distinct *Is*PETase single PCR product band, which indicated that the bacterial colonies contained our team’s composite part, BBa\_K5094010 (pGal1,10-*Is*PETase).

## Making several mutant *Is*PETase enzymes via site-directed mutagenesis

### T7-*Is*PETase and several T7-*Is*PETase mutations

Our team's original idea was to make single, double, and triple mutants of the *Is*PETase downstream of the T7 promoter, and the pGal1,10 promoter respectively. Those mutations were (*Is*PETase S121E, *Is*PETase S121E/D186H, and *Is*PETase S121E/D186H/R280A), since
Japanese scientists showed that changing 3 amino acids at the catalytic site on the *Is*PETase enzyme enhances the PETase activity compared to the wild-type PETase, and also can have catalytic activity at higher temperatures to degrade PET plastic products faster (Son et al paper, and 2021 PET degradation pathway and ref45 on the 2021 paper). To reach the goal of the team’s idea, site-directed mutagenesis was performed, and the desirable mutated amino acid was designed on forward and reverse primers (detailed in the experimental design cycle). Repeat site-directed mutagenesis two more times gave the team the double and triple mutant of the *Is*PETase. Before sending out for the sequence, the plasmids were run on 1% of the agarose gel, and after receiving the sequence data, the team created alternative *Is*PETase mutant constructs: T7-*Is*PETase as a wild-type, T7-*Is*PETase Thr116Ala, T7-*Is*PETase Thr116Ala/M154Thr, and T7-*Is*PETaseThr116Ala/K259Glu on Figure 5.

![](https://static.igem.wiki/teams/5094/parts/2024-09-25-123529.png)

Fig5: After sequencing, the mutations of the *Is*PETase
the team created: lane 1 was a 1kb DNA ladder, and lane 2
was T7-*Is*PETase, lane 3 was T7-*Is*PETaseThr116Ala,
lane 4 was T7-*Is*PETaseThr116Ala/M154Thr,and lane 5 was
T7-*Is*PETaseThr116Ala/K259Glu. The plasmids’ conformations
were supercoiled, open-circular, and linear on the gel.

After the team did site-directed mutagenesis to make mutations of the *Is*PETase, the plasmids were run on 1% agarose gel to show in Figure 5 that the team did have the T7 promoter *Is*PETase, and after sending out sequencing at the Mission Biotech company, the team created T7-*Is*PETase Thr116Ala, T7-*Is*PETase Thr116Ala/M154Thr, and T7-*Is*PETaseThr116Ala/K259Glu.
The plasmids’ conformations were supercoiled, open-circular, and linear on the gel.

### pGal1,10-*Is*PETase

The pGal1,10 promoter allows for highly efficient expression of our team’s cloned *Is*PETase gene by adding galactose to the yeast medium for about 20-30-fold induction (2022). Our team utilized the pGal1, 10 promoter in yeast to determine if the *Is*PETase gene, along with variable mutations of the *Is*PETase gene cloned downstream of the pGal1,10 promoter, respectively, exhibit different PET plastic degradation levels in the co-culture functional assay.

To reach the goal of the team’s idea mentioned above, site-directed mutagenesis was performed, and the desirable mutated amino acid was designed on forward and reverse primers (detailed in the experimental design cycle). Repeat site-directed mutagenesis two more times gave the team the double and triple mutations of the *Is*PETase. Before sending out for the sequence, the plasmids were run on 1% of the agarose gel, and after receiving the sequence data, the team realized that we only have pGal1,10- *Is*PETase plasmid in Fig6, which showed 1% of the agarose gel contained the plasmids, which were all pGal1,10- *Is*PETase plasmid without any mutations.

![](https://static.igem.wiki/teams/5094/results/001.png)

Fig6: After sequencing, the team only created
pGal1,10-*Is*PETase plasmid (lanes2,3,4), and
lane1 is a 1kb DNA ladder.

After the team did site-directed mutagenesis to make mutations of the *Is*PETase, the plasmids were run on 1% agarose gel to show the team did have the T7 promoter *Is*PETase, and after sending out sequencing at the Mission Biotech company, the team created pGal1,10-*Is*PETase plasmid on lanes2,3, and 4. The plasmids’ conformations were supercoiled, open-circular, and linear on the gel.

## Sequencing data

The *Is*PETase gene sequence suppressed our team with unexpected challenges during the site-directed mutagenesis technique due to multiple DNA fragments containing repeat regions that we hadn’t anticipated when designing the mutations. The primers, intended to switch specific amino acids by adding mutations to the primers, targeting incorrect amino acids because of these repeats. As a result, our team created alternative *Is*PETase mutant constructs: T7-*Is*PETase as a wild-type (BBa\_K5094006), T7-*Is*PETase Thr116Ala(BBa\_K5094007), T7-*Is*PETase Thr116Ala/M154Thr(BBa\_K5094008), and T7-*Is*PETaseThr116Ala/K259Glu (BBa\_K5094009) for DE3BL21 bacteria. pGal1,10-IsPETse (BBa\_K5094010) for the yeast. Despite these setbacks, our team decided to use the *Is*PETase mutations downstream of the T7 promoter for further experiments. This decision was based on previous publications demonstrating how altering several amino acids in the *Is*PETase gene could enhance enzyme activity. We hypothesize that the unexpected mutations the team created might also enhance the enzyme’s catalytic activity (more detail in part section).

## Proof of Concept

### RT-qPCR time course data for Galactose Induction

To further verify whether our team’s composite parts were biological function, RT-qPCR technique was operated to detect the mRNA induction of eGFP in BY4741 containing BBa\_K418008 created by the 2022 KCIS team as a control, the mRNA induction of *Is*PETase in BY4741 containing BBa\_K5094010 as an experimental sample, via timecourse sample collection, 0min, 30min, 60min,90min, 120min in the presence of the 2%YP-galactose medium. The BY4741 contained two different composite parts, respectively, growing in 150ml of 2%YP-glucose-200ug/ml G418, until OD600 ~0.4, and then 40 ml of yeast culture medium was taken out as 0 min as a control. Without galactose, the pGal1,10 promoter would not be induced to express downstream of genes’ mRNA, and the rest of the culture samples were collected, respectively, at 5000rpm for 5 mins and washing the culture samples with dH2O twice to eliminate glucose. Then transferring the 2 composite parts of the yeast culture samples to 200ml of 2%YP-galactose-200ug/ml G418, our team took out 40ml of yeast culture medium samples at, 30’,60’, 90’, and 120’ in the presence of galactose.

### RT-qPCR technique was performed to show the cloned genes downstream of the pGal1,10 promoter were induced in the presence of the galactose

To manipulate our team’s wild-type of the *Is*PETase enzyme with the control eGFP cloned downstream of the pGal1, 10 promoter, respectively. BBa\_K418008 contained pGal1,10-eGFP, BBa\_K5094010 contained pGal1,10- *Is*PETase as the team’s experimental sample, were then transformed into wild-type Saccharomyces Yeast Strain, BY4741, respectively. After collecting different time course samples, the whole cell RNA extracted was operated before the RT-qPCR technique was performed.

![pcr1](https://static.igem.wiki/teams/5094/parts/screenshot-2024-09-26-at-3-59-26-pm.png)

*Fig 7a: The mRNA induction of eGFP showed strong manipulation, above 5-fold induction, in the presence of galactose at 60 mins, and the maximum of 7.5-fold eGFP mRNA induction at 120 mins in the presence of galactose.*

![](https://static.igem.wiki/teams/5094/parts/2024-09-25-170901.png)

*Fig 7b: The mRNA induction of *Is*PETase didn’t exhibit significant induction at different time courses in the presence of galactose compared to the 0’ sample with glucose only.*

To detect the *Is*PETase mRNA induction, our team has designed a pair of *Is*PETase primers within the gene and a common primer set of ACT1 to detect ACT1 mRNA induction as an internal control. BY4741 yeast containing BBa\_K418008 (pGal1,10-eGFP), as an experimental control, did show significant eGFP mRNA induction approximately 4-5 fold at 30’,60’, and 90’ in the presence of the galactose compared to the 0’ sample without galactose. The maximum eGFP mRNA induction was around 8-fold at 120’ in the presence of the galactose compared to the sample at 0’ without galactose. Overall, Figure 7a, indicated the team’s experimental design was working properly to use 2% galactose for the pGal1,10 promoter induction.

In the Figure 7b, BY4741 yeast contained BBa\_K5094010 (pGal1,10-*Is*PETase) showed no significant *Is*PETase mRNA induction at different time course samples in the presence of the galactose compared to the 0’ sample without galactose in Figure 7b. The data indicated that the *Is*PETase cloned downstream of the pGal1,10 promoter was not induced within 2 hours in the presence of the galactose. For the future experiment, the team will extend the time course sample collection after 2 hours to detect the *Is*PETase mRNA induction. Commonly, different genes cloned downstream of the pGal1, 10 promoter have different mRNA induction timecourse patterns in the presence of the galactose, and our team clearly showed the experimental design was working in Figure 7a, To further determine an IsPETse mRNA induction timecourse pattern, the team will collect multiple time-course samples in the presence of the galactose behind 2 hours.

### SDS gel electrophoresis

Before setting up the SDS page and western blot experiments, our team cultured 50ml of DE3BL21 bacteria cells containing T7-*Is*PETase and various mutation plasmids in LB broth medium until OD~0.2, respectively. Then cell culture was split into half (25mls) and induced at 0.5mM of IPTG at 37 degrees for 6 hours and the other half (25mls) as uninduced at 37 degrees for 6 hours as controls(Mühlmann et al, 2017; Namdev et al, 2019).

To verify whether those composite parts, BBa\_K5094006 containing BBa\_K5094000- BBa\_K5094002(T7-*Is*PETase), BBa\_K5094007 containing BBa\_K5094000- BBa\_K5094003 (T7-*Is*PETase Thr116Ala), and BBa\_K5094008 containing BBa\_K5094000- BBa\_K5094004 (T7-*Is*PETaseThr116Ala/Met154Thr), and BBa\_K5094009 containing BBa\_K5094000-BBa\_K5094005 (T7-*Is*PETaseThr116Ala/k259Glu ), can express the proteins our team desires, the team did the whole cell protein extract via the 95-degree boiling method in protein extract lysis buffer protocol, according to the data shown on BBa\_K2010000 from the Harvard 2017 team, the *Is*PETase protein was in the soluble condition, and the supernatants will be collected for the SDS page and Western blot. The team performed an 18% SDS page first. To confirm whether the whole cell extract contained all proteins and also see a stronger band of the *Is*PETase along with various mutants of *Is*PETase at the correct protein size in the presence of IPTG, an 18% SDS page image was exhibited in Fig 8.

![](https://static.igem.wiki/teams/5094/parts/2024-09-25-123936.png)

Fig 8: The 18% SDS-PAGE gel showed the team successfully extracted the whole protectin extract. Lane 1 is a protein-size ladder. Lane 2 is T7-*Is*PETase (-)IPTG,and lane 3 is T7-*Is*PETase (+0.5mM)IPTG.Lane 4 is T7-*Is*PETaseThr116A (-)IPTG, and lane 5 is T7-*Is*PETaseThr116A (+0.5mM)IPTG. Lane 6 is T7-*Is*PETaseThr116A/k259Glu (-)IPTG, and lane 7 is T7-*Is*PETaseThr116A/k259Glu (+0.5mM)IPTG. Lane 8 is T7-*Is*PETaseThr116A/M154Thr (-)IPTG, and lane 9 is T7-*Is*PETaseThr116A/M154Thr (+0.5mM)IPTG. *Is*PETase protein has about 30 kDa molecular weight based on the Harvard 2016 team’s part information.

After the team did the whole cell protein extract with all of the samples (detailed in Fig 8), we ran the protein extract samples on 18% SDS-PAGE gel to show that the team successfully retained the whole protein extract from each sample. In Figure 8, the team labeled the *Is*PETase protein approximately 30 kDa molecular weight on 18% SDS-PAGE gel. Figure 8, also showed one strong band around 30 kDa in lane 7, T7-*Is*PETaseThr116A/k259Glu (+0.5mM)IPTG. However, the team wanted to determine the specific *Is*PETase protein expression at different samples, so we will perform the Western Blot analysis.

### Co-culture with PET film experiments

The more direct functional assay was to co-culture the same amount/size of PET film with either DE3BL21 bacteria containing T7-*Is*PETase plasmid along with various mutations on *Is*PETase enzyme, respectively in the presence of 0.5mM IPTG to induce T7 promoter in DE3BL21 at 37 degrees as experimental samples, lack of IPTG as controls. The team used a Nabi machine with a “ 240nm” wavelength to detect the total TPA product generated after the PET plastic degradation daily at 9 am and 3 pm for the co-culture experiment. The same experiment was performed with BY4741 yeast strain containing the pGal1,10-*Is*PETase plasmid as an experimental sample, along with pGal1,10-eGFP as a control sample in the presence of galactose, samples in the glucose as comparison group without inducing the pGal1,10 promoter.

The more direct functional assay was to co-culture the same amount/size of PET film with either DE3BL21 bacteria containing T7-*Is*PETase plasmid along with various mutations on *Is*PETase enzyme, respectively in the presence of 0.5mM IPTG to induce T7 promoter in DE3BL21 at 37 degrees as experimental samples, lack of IPTG as controls. The team used a Nabi machine with a “ 240nm” wavelength to detect the total TPA product generated after the PET plastic degradation daily at 9 am and 3 pm for the co-culture experiment. The same experiment was performed with BY4741 yeast strain containing the pGal1,10-*Is*PETase plasmid as an experimental sample, along with pGal1,10-eGFP as a control sample in the presence of galactose, samples in the glucose as comparison group without inducing the pGal1,10 promoter.

![](https://static.igem.wiki/teams/5094/parts/2024-09-25-123219.png)

*Fig 9a: The co-cultured experiment data didn’t show any clear patterns of the TPA product increasing in the DE3 bacteria containing T7-*Is*PETase along with several *Is*PETase mutants, respectively, in the presence of the IPTG during the time course experiment. For the experimental controls, DE3 bacteria only, along with several *Is*PETase mutants that lack IPTG still also showed TPA product generation.*

Our team, originally, was expecting to see an increased trend of TPA product generated in the DE3 bacteria containing T7-*Is*PETase along with several *Is*PETase mutants, respectively, after being induced by the IPTG compared to the control samples lack of IPTG, so the team designed the co-cultured experiment. The team added the same amount/shape of the PET film in each co-culture sample medium. To detect the TPA product, the wavelength 240 nm was used to detect the TPA product(Terephthalic acid, 2023). However, in Figure 9a, for the experimental controls, DE3 bacteria only, along with several *Is*PETase mutants without IPTG generated TPA product, indicated that the culture mediums might have some materials which can also be detected at 240 nm wavelength. For the experimental samples, the DE3 bacteria containing T7-*Is*PETase along with several *Is*PETase mutants, respectively, with and without IPTG also had unclear TPA product increase patterns.

Overall, there was no significant increase/any pattern TPA product in the PET-film co-cultured with the DE3 bacteria containing T7-*Is*PETase, along with several *Is*PETase mutants, respectively, at the time course data in the presence of IPTG compared to the ones without IPTG.

![](https://static.igem.wiki/teams/5094/parts/2024-09-25-171008.png)

*Fig 9b: The co-cultured experiment data didn’t show a clear pattern of the TPA product increasing in the BY4741 yeast strain containing pGal1,10-*Is*PETase in the presence of galactose during the time course experiment. The experimental control, BY4741 yeast strain containing pGal1,10-eGFP, either in glucose or galactose, should not show any TPA product increase due to lack of the *Is*PETase. However, the team did detect a small amount of TPA products in both co-cultured mediums. A similar unclear pattern of TPA products was observed in BY4741 containing pGal1,10-*Is*PETase either in glucose or galactose.*

Our team, originally, was expecting to see an increased trend of TPA product generated in the BY4741 yeast strain containing pGal1,10-*Is*PETase after being induced by the 2% galactose compared to the control sample in the 2%glucose, so the team designed the co-cultured experiment. The team added the same amount/shape of the PET film in each co-culture sample medium. To detect TPA product, the team used the wavelength 240nm to detect the TPA product(Terephthalic acid, 2023) However, in Figure 9b, the experimental control, BY4741 containing pGal1,10-eGFP, either in glucose or galactose, should not show any TPA product increase due to lack of the *Is*PETase. However, the team did detect a small amount of TPA products in both co-cultured mediums.

There was no significant increase of the TPA in the PET-film co-cultured with the yeast containing pGal1,10-*Is*PETase at the time course data in the presence of galactose compared to the one in the glucose. In the final time course, the team did see the TPA was increased in the presence of the galactose, however, the experiment was only done once, and repeating experiments will need to be done. At the same time, our team will also try to smash PET film into powder for the co-culture experiment.

## Future experiments for functional assay

The team has successfully created various mutant *Is*PETase enzymes and the wild-type *Is*PETase enzyme cloned downstream of the T7 promoter, respectively, as well as the wild-type *Is*PETase cloned downstream of the pGal1,10 promoter, which is an impressive accomplishment for a high school student team given the complexity of these tasks. The team has also performed RT-qPCR, Western Blot, and co-culture functional assays to evaluate enzyme expression and activity. However, these assays will require further optimization to produce clear and reliable data, enabling more precise insights into the enzyme's performance.

Here are the ideas the team will use to optimize these functional assays:

### Optimize RT-qPCR

The team conducted RT-qPCR to assess the induction of *Is*PETase mRNA levels downstream of the pGal1,10 promoter in yeast treated with 2% galactose. We used pGal1,10-eGFP as a control and measured eGFP mRNA induction at 0, 30, 60, 90, and 120 minutes. We observed a peak eGFP mRNA induction of approximately 8-fold at 120 minutes in the presence of 2% galactose compared to the 0-minute sample without galactose. However, there was no significant induction of *Is*PETase mRNA across the 2-hour time course. To further investigate the induction pattern of *Is*PETase mRNA, the team plans to collect additional time-course samples beyond the initial 2 hours of galactose treatment.

### Optimize Western Blot

After conducting 18% SDS-PAGE gel analysis, our team proceeded with Western blotting to assess the expression of the *Is*PETase protein. We cloned the *Is*PETase gene downstream of a 6-his tag, which resulted in the expressed protein also having a his-tag at its N-terminal. For the Western blot, we utilized a his-tag primary antibody and an HRP-conjugated anti-mouse secondary antibody to detect *Is*PETase expression for various samples (as indicated in Fig. 8). However, we did not detect any distinct bands of the *Is*PETase protein expression, even in the presence of IPTG. Moving forward, we need to optimize the Western blot conditions, as this procedure involves multiple steps and two antibody incubations. Additionally, the use of an incorrect antibody dilution may have contributed to the lack of strong *Is*PETase expression bands.

### Optimize co-culture functional assay

The team cut PET film into uniform circular pieces, adding 15 pieces to each sample for the co-culture assay. However, the results did not meet our expectations. Following an online meeting with our collaborators, it was suggested that we grind the PET into a powder for the co-culture experiment. We plan to implement this recommendation in our next trial.

## Future experiments to extend this project

### For the in vitro functional assay

In addition to in vivo experiments, our team will extend this project to perform in vitro protein purification to isolate the *Is*PETase enzyme and its various *Is*PETase enzyme mutants. The *Is*PETase gene and its mutants are cloned downstream of a T7 promoter engineered with a 6-histidine (6-his) tag. These constructs are then transformed into BL21(DE3) bacteria, and enzyme expression is induced using IPTG.

Following cell lysis, the protein extracts containing the 6-his-tagged wild-type and mutant *Is*PETase enzymes are passed through a Ni²⁺-resin column, which selectively binds to the 6-his tag. The column is then washed with several buffers to remove unbound and unwanted proteins. Finally, an elution buffer is used to purify the desired enzymes, which are collected in separate tubes for further analysis. The team will use *Is*PETase enzyme and mutant *Is*PETase enzymes to perform the enzyme-abstract “p-Nitrophenyl Esters Assay”, which PETase can be tested using p-nitrophenyl butyrate, which releases p-nitrophenol upon hydrolysis by PETase, which can be quantified spectrophotometrically by measuring the absorbance at 405 nm.

### Making MHETase biobricks

The MHET produced from PET degradation by PETase can be further broken down into terephthalic acid (TPA) and ethylene glycol (EG) by MHETase. Our team's future work will focus on designing MHETase biobricks to overexpress this enzyme, aiming to enhance the efficiency of degrading PET plastic into its monomers. This approach will allow for a more complete and efficient breakdown of PET waste, ultimately converting it into reusable building blocks for new plastic production or other industrial applications.

## Citations

Brott, S., Pfaff, L., Schuricht, J., Schwarz, J., Böttcher, D., Christoffel, Wei, R., & Bornscheuer, U. T. (2021). Engineering and evaluation of thermostable *Is*PETase variants for PET degradation. *Engineering in Life Sciences*, 22(3-4), 192–203.

Maity, W., Maity, S., & Bera, S. (2021). Emerging Roles of PETase and MHETase in the Biodegradation of Plastic Wastes.

Mühlmann, M., Forsten, E., Noack, S., & Büchs, J. (2017). Optimizing recombinant protein expression via automated induction profiling in microtiter plates at different temperatures. *Microbial Cell Factories*, 16(1), 220.

Namdev, P., Dar, H. Y., Srivastava, R. K., Mondal, R., & Anupam, R. (2019). Induction of T7 Promoter at Higher Temperatures May Be Counterproductive. *Indian Journal of Clinical Biochemistry*: IJCB, 34(3), 357–360.

Rajeshkannan, Mahilkar, A., & Saini, S. (2022). GAL Regulon in the Yeast S. cerevisiae is Highly Evolvable via Acquisition in the Coding Regions of the Regulatory Elements of the Network. *Frontiers in Molecular Biosciences*, 9.

Son, H. F., Cho, I. J., Joo, S., Seo, H., Sagong, H.-Y., Choi, S. Y., Lee, S. Y., & Kim, K.-J. (2019). Rational Protein Engineering of Thermo-Stable PETase from Ideonella sakaiensis for Highly Efficient PET Degradation. *ACS Publications*.

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