iGEM 2023 — Team aalto-helsinki

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# Notebook

### Calendar

[February](#feb)

[March](#mar)

[April](#apr)

[May](#may)

[June](#jun)

[July](#jul)

[August](#aug)

[September](#sep)

[October](#oct)

[November](#nov)

[December](#dec)

### February

* #### Week 6

  On February 7th, we had a team leader orientation, followed by a team orientation session
  on February 8th conducted over Zoom. Additionally, we organized a team hangout on February 9th,
  providing an opportunity for team members to connect and strengthen our relationships.
* #### Week 7

  During our recent discussion, we focused on generating topics for our upcoming meeting.
  We decided to divide the group into smaller teams based on individual interests in different iGEM tracks.
  These teams would research their chosen topics and present their findings to the larger group on Monday, February 20th.
  Looking ahead, we also recognized the importance of creating a schedule for the project and establishing clear goals and deadlines for each step.
* #### Week 8

  The subtrack teams started contacting last year’s members and organizing the workshops to clarify the different tasks and responsibilities.
  Each member had to do some research to get ideas for the topic of the project.

### March

* #### Week 9

  During this week, we engaged in an in-depth exploration of potential project tracks and reviewed the initial
  ideas that were contributed to the Flinga page. From these discussions, we identified several promising leads put forth by team members.
  These included:
  1. Investigation into the impact of unhealthy lifestyle choices on overall health and well-being.
  2. Exploring the development and implementation of rapid testing methods to enhance efficiency and accuracy in various domains.
  3. Advancements in enhancing food taste and additives to improve overall consumer experience and satisfaction.
  4. Utilizing synthetic biology techniques for food production, aiming to revolutionize the industry by developing innovative and sustainable solutions.
  After a voting process, two ideas were selected from each category for further consideration and exploration.
  Our team also worked together to update the Hilife proposal, making the necessary changes to keep it relevant and accurate.
  We also planned a workshop day to focus on human practices, where we can gain valuable insights for our project.
* #### Week 10

  This week we presented a few potential project ideas
  briefly. Potential ideas:
  1- Antibiotic treatments and gut microbiota disruption
  2- Sugar craving and addiction, leading to metabolic syndrome

  3- Optimization of the production process for a therapeutic
  protein
  4- Rapid testing and at-home STD tests
  5- Lab-on-a-chip, microfluidics for cheap diagnostic tests

  6- Biodiversity Loss and sustainable protein production
  7- Biological solution to plastic pollution
  8- Lack of diversity and taste in vegan food
  9- Energy generation from microbes
  10- Drug delivery enhancement
  11- Developing a biosensor for water system contamination
  detection
  Voting was conducted via Telegram with each person selecting
  their top 5 ideas.
* #### Week 11

  We focused on reviewing and refining the iGEM proposal during our progress review.
  To enhance our human practices section, we arranged a meeting with Zsófia, who participated in iGEM 2022.
  Zsófia's insights and recommendations were sought, and specific questions were prepared to facilitate productive discussions.
  In the meeting with our PIs, we discussed focusing on topics aligned with Aalto and the University of Helsinki’s
  research expertise. Good topics identified were: antibiotics (specifically antimicrobial peptides),
  a biological solution for plastic pollution, and improving the taste of vegan food.
  Markus approved these topics, considering available resources.
  We aimed to finalize the topic by next week through further research and contacting key individuals.
* #### Week 12

  This week, effective communication with faculties was established about salaries and contracts,
  fundraising efforts were organized, presentation materials were developed,
  and the process of creating a logo was initiated.
* #### Week 13

  This week, the team prepared presentation material for the deans of various faculties.
  Work was divided among team members. Productive brainstorming sessions were conducted to generate
  ideas and ultimately to reach a consensus on the logo design.
  Additionally, the team took the initiative to begin website development and activate social media efforts.

### April

* #### Week 14

  The aim was to present the project concepts to FIMAR, Onego Bio, and Rahul Mangayil.
  Collaboration with the ABOA iGEM team was also brought up.
  Individual meetings between the leaders and team members were planned for a later date.
  In terms of administrative tasks, a revised version of the sponsorship one-pager was to be created for distribution,
  and the iGEM registration deadline was noted.
  Contact with Heureka and finalizing the website, pending the logo, were also discussed.
  Finally, plans for team holidays and organizing fun activities were initiated.
  Importantly, we had a meeting with the School of Electrical Engineering at Aalto University regarding summer job positions.
* #### Week 15

  On April 10, 2023, small groups worked on presentations for the different topic proposals:
  Saving lives, one peptide at a time; From pollution to solution; Tasy, nutritious and sustainable.
  The objective was to present the proposals to our supervisors, get feedback and eventually vote to focus on our final project.

  During the meeting held on April 11, 2023, Sesilja Aranko and Ville Paavilainen discussed the three project proposals with us.
  Contacts were suggested, collaborations were considered, and the importance of defining the project story and ordering strains in advance was highlighted.
  Heli was assigned tasks related to team registration and access cards. The team also discussed sponsorships, DNA synthesis providers, and potential grant applications.

  We had a meeting with Nesli Sözer from VTT who suggested combining the food and plastic topic ideas into one! We loved it and decided to continue with this topic.

  The team discussed social media strategies and logo design,
  setting a deadline for the logo in the next meeting.
  Meetings were scheduled with the deans and Rahul Mangayil, and preparations were made for these interactions.
  Individual 30-minute meetings were organized with team members.
  Mentors were also discussed, considering their availability, and expertise, and addressing the team's weaknesses.
* #### Week 16

  The project, titled "PET-2-Protein: Revolutionizing
  Plastic Recycling and Protein Production," was discussed. Topics
  included the selection of plastic for degradation, the
  availability of enzymes such as PETase and MHETase, the use of
  HPLC for monitoring degradation progress, the potential use of
  scaffolds, and the feasibility of utilizing the end products as
  food for organisms. The team also addressed the possibility of
  combining the entire pipeline into a single system. The next
  steps included working on expression systems for PETase and
  MHETase, testing PET degradation using existing enzymes, and
  ordering the end products TPA and EG.
  On Saturday 22.4 Cleopatra attended Harvard OpenBio Team’s
  online “Future of Synthetic Biology Conference”. There were many
  speeches by esteemed guests, such as Pamela Silver, Ph.D who is
  one of the founders and on the Board of iGEM and Marc Facciotti
  who had mentored 11 iGEM teams in the States. During the
  conference, there were also two workshop slots. Cleopatra
  attended the "Reimagining A Century of Synthetic Biology: From
  GMOs to Cyborgs and Beyond!" and "Being a Leader in the Research
  World" workshops, both of which were thought-provoking.
  Lastly, we met with the School of Chemical Engineering and
  the School of Engineering of Aalto University regarding summer job positions.
* #### Week 17

  A meeting was held with Nesli Sözer and Kari Koivuranta from VTT.
  Points of discussion included UV pre-treatment of plastic bottles,
  usage of enzymes for degradation, and the potential of
  Pseudomonas putida for utilizing the end products.

  Additionally, we took part in a workshop hosted by Brendan Battersby
  at the Univeristy of Helsinki on the topic of mentoring, leadership and project management.
  From this meeting, we got in touch with Shameer Kodambiyakamenna, and Alesia Levanova, who
  would later act as an instructor for the team.

### May

* #### Week 18

  Social media and logo updates were shared.
  Collaboration efforts were initiated, and other iGEM teams were contacted.
  Decisions were made to prioritize collaboration and meeting with IBO Finland 2023 was scheduled.
  Search for relevant wet lab protocols was also initiated.
* #### Week 19

  This week we discussed and finalized our funding proposal to
  the Helsinki Institute of Life Science. Research on wet lab protocols continued.
  Furthermore, we had our first training at the School of Chemical Engineering, Aalto University,
  related to wet lab safety and other practicalities.
* #### Week 20

  This week our discussions focused on the Human Practices aspect of our project.
  Furthermore, we decided to propose hosting a collaborative educational workshop at Heureka
  with the ABOA iGEM team. Finally, we also had a social event at Aalto Design Factory.
* #### Week 21

  This week, we planned to meet with the Faculty of Agriculture and Forestry
  at the University of Helsinki to discuss our project.
  We also explored options for assistance with cloning and reached out to contacts for strains
  relevant to plastic degradation.
  We scheduled meetings with Thermo Fisher Scientific regarding a sponsorship deal,
  and with the previous Aalto-Helsinki iGEM team for advice.
  We also hosted a class for 6th graders at the Maunula Elementary School.
  Furthermore, we presented our project in Merja Penttilä's class at Aalto University.
  Lastly, we attended another mandatory laboratory safety session at Aalto University.

### June

* #### Week 22

  We had a meeting with the Faculty of Biological and Environmental Sciences from the University of Helsinki regarding summer job positions.
  We also had a meeting with Alesia Levanova where we discussed our initial insert designs and the cloning strategy.
  On the sponsorship side, we had meetings with Kemianteollisuus ry and Thermo Fisher Scientific.
  Work on the iGEM impact grant was begun. Lastly, this was our first week of full-time work.
* #### Week 23

  We took part in the laboratory orientation at the School of
  Chemical Engineering, prepared a presentation template, and
  finalized the project overview diagram. We had a fruitful
  discussion with the previous Aalto-Helsinki iGEM team.
  We had a valuable meeting with Associate Professor Stephen
  Techtmann from Michigan Technological University to discuss
  using FAST-PETase for plastic depolymerization. We explored
  strain development options for optimizing biomass production.
  We discussed the community-based degradation of mixed plastic
  waste and identified potential natural microorganisms for
  biomass recovery. Furthermore, we conducted training sessions
  for furnace use, reviewed protocols, wrote plastic and enzyme
  protocols, and compiled an order list.
* #### Week 24

  This week, our team made significant progress in our
  research and development endeavors. We began working on our
  biweekly newsletter, updated our website and confirmed the
  suitability of our parts for optimized protein expression. We
  also placed orders for the necessary primers and inserts.
  Furthermore, we engaged in collaborative discussions, submitted
  sponsorship applications, and held a meeting discussing the
  financial situation of the team. Additionally we designed,
  planned, and printed our team T-shirts at the Iso Omena library.
* #### Week 25

  Village selection for our project was made.
  In the dry lab, we ordered oligos, validated SpyCatcher/SpyTag designs, and integrated
  plastic degradation-related metabolic reactions. Updates were
  made to
  [aaltohelsinki.com](https://www.aaltohelsinki.com). We furthermore ordered primers for conducting colony PCR
  related to the SpyCatcher/SpyTag inserts.
* #### Week 26

  This week, we began working in the wet lab, completed ligation,
  and performed metabolic modeling in the dry lab. We had a
  productive meeting with Bioethicist Heikki Saxen, integrated
  his advice into our project, and developed bioethics guidelines
  based on his notes and the recommendations make by the 2022 UCopenhagen iGEM team.
  We published project updates on social media,
  finalized the project description, and maintained a daily lab
  diary. We contacted VTT and Kemianteollisuus, and secured
  collaboration with Terkko Health Hub. Additionally, we booked
  tickets for the upcoming Nordic iGEM Conference, and
  the European Meet-Up.

### July

* #### Week 27

  Last week, we achieved significant milestones in the lab,
  generating E. coli T7 electrocompetent cells and
  completing crucial experiments involving restriction enzyme
  digestion, gel purification, and ligation. Our metabolic
  modeling work showed potential growth without extra amino acids,
  with co-expression of the fucO and
  aldA genes playing a vital role in EG utilization.
  Additionally, we had a productive meeting with Solar Foods'
  Susanna Mäkinen and Juha-Pekka Pitkänen, discussing alternative food sources.
* #### Week 28

  This week, the wet lab successfully conducted cloning tasks,
  including restriction enzyme digestion, gel purification, and
  ligation. Additionally, we recultured clones from
  pET-28a-MHETase Top10 and pET-28a-PETase Top10 and produced
  E. coli Top10 electrocompetent cells. In the dry
  lab, we focused on metabolic modeling, evaluating co-cultures,
  and exploring the co-expression of fucO and
  aldA. Stakeholder feedback was incorporated, and
  we reached out to the Chief Engineer in Waste Management at
  the Department of Environment in Bangladesh. Progress was made
  on blog posts and graphic design, including the creation of
  comic strips.
* #### Week 29

  Last week, significant progress was made in both the wet and
  dry labs. In the wet lab, we successfully conducted
  electrotransformation of new vectors (pRSFDuet-1 and pET-28a)
  in Top10 cells and prepared media (LB and M9). Additionally,
  new FAST-PETase and MHETase inserts were ordered, and we
  designed
  fucO and aldA inserts for
  pRSFDuet-1. Translation rate issues were addressed, and
  protein folding on Puhti was set up, with SpyCatcher/SpyTag
  design optimization underway. In human practices, we had a
  productive meeting with stakeholders in Bangladesh. Graphic
  design also progressed, including the publication of a comic
  chapter, the completion of a brochure, the progress being made
  towards a project poster.

  We also had a valuable Zoom meeting with Ting Lu, receiving
  feedback on protein production and metabolic optimization for
  plastic degradation and bioconversion. He suggested
  bottleneck identification through HPLC analysis and conducting
  an adaptive lab evolution experiment.
* #### Week 30

  In the dry lab, the team discussed the feasibility of
  SpyTag/SpyCatcher interaction with Sesilja Aranko. Initial findings
  suggest a moderate interaction strength for the construct.
  Further analysis will precede any order, with no rush.

  With a focus on R. opacus due to Stephen's
  recommendation and literature, we're aiming to propose a
  growth hypothesis related to a specific pathway for our
  project presentation.

  Conducting BLAST searches for the tph operon and
  tpaK in R. opacus
  DSM 43205, we folded relevant ORFs from hits.

  Shifting to the wet lab, R. opacus and
  C. testosteroni were cultured in EG, TPA, and TPA
  + EG conditions, both individually and together. Broths and
  microbial stocks were duly prepared.

  Lastly, we attended the iGEM Nordic Conference in Odense hosted by the
  SDU-Denmark iGEM team over the weekend.

### August

* #### Week 31

  In the Viikki lab, we prepared M9 medium with both EG and TPA,
  and both broth and agar for the plates. We cultured
  R. opacus and C. testosteroni with
  M9 + PET monomers and left them for overnight incubation.
  The following day when
  starting bioconversion, we noticed that after centrifugation,
  there were no pellets from the overnight culture. We decided to
  leave them in incubation for the duration of our upcoming trip.
  Our team had a great opportunity to attend Junior Jam - the
  European Meet-up conference in Münster hosted by the UniMünster iGEM team over the weekend.
* #### Week 32

  This week, we were able to start bioconversion. Also,
  overnight culture of E.coli pSEVA-231 KanR was
  made and inserts IamB-MHETase-His-Stop and
  pelB-FAST-PETase-His-Stop were digested and ligated. In
  addition, we also did a transformation of pRSFDuet-1 with
  PETase and MHETase, and purified pSEVA-231.

  We investigated engineering MHETase in silico with
  MutCompute, which is a protein language model capable of
  giving predictions on potential residues to modify. The same
  model has also been used in recent literature concerning
  PETase variants.

  We had a meeting with this year’s KU Leuven iGEM team where
  we had a discussion related to the dry lab. We gave
  presentations about our project and iGEM at two different
  events. First we had a presentation at the Aalto Lab meeting
  where we were able to present our project to the people we
  had been working with. The second presentation was for the
  Finnish IBO team. We also got to know what IBO really is and
  after presentations we had some time to answer questions
  from high school students about iGEM and our fields of
  study.
* #### Week 33

  In the wet lab, R. opacus and
  C. testosteroni were grown in laboratory
  evolution for the HPLC analysis. Insert digestion, gel
  purification and ligation had to be repeated. This step was
  repeated because the previous time the inserts were not
  cleaned and did not run in the gel. We did some
  troubleshooting and tried some minor protocol changes to
  make the protocol. After working, we did transformation,
  plating and restreaking from those transformation plates.

  Meanwhile in the dry lab, primers for PCR were designed and
  ordered. We predicted ddG and selected the top 10 most
  negative candidates. We also used AlphaFold and looked into
  docking. We started to go through our survey data and cleaned
  up the data.

  This week we started to record our promotional video and the
  voice-overs that we were going to use in it.

  We also had a meeting with Finnish plastic agency called Suomen
  Uusiomuovi Oy. It was a great opportunity to talk and learn
  about plastic recycling in Finland and also hear their
  thoughts about our project.
* #### Week 34

  In the wet lab in Viikki, we got training for the use of
  HPLC, after which we got results from
  R. opacus and
  C. testosteroni bioconversion. The results were
  good for R. opacus but we decided to repeat
  the bioconversion to see if some modification could also
  give better results for C. testosteroni. We
  also received the PCR primers ordered previously and conducted
  PCR amplification of our PETase and MHETase inserts.

  The last biweekly newsletter for the summer was finished and
  sent out this week.
* #### Week 35

  In the wet lab, we noticed that
  P. putida can grow on M9+30mM of TPA and M9+10gr/l
  of EG. We got good results from the transformation and
  were finally able to send our pRSFDuet-1 FAST-PETase construct for
  sequencing.

  We worked on our wiki texts and started to transfer
  our survey data into a graphical form.

  We shot the clips for our promotional video all over the Aalto
  campus. We got some good video footage and began editing the video.

  We also organized the first Heureka workshop with the ABOA
  team this week. We were able to teach visitors about DNA, RNA,
  proteins and the field of synthetic biology in general.

### September

* #### Week 36

  We received promising results from the Sanger sequencing by Eurofins Genomics.
  The sequencing quality was excellent, and for one of the colonies,
  there was a perfect match between both reads. In the case of the
  other colony, both reads showed a single silent mutation, but it
  is worth noting that this mutation occurred in a specific peak
  with a relatively low intensity. This week in the wet lab we
  started a plastic degradation test and enzyme purification.
* #### Week 37

  The second Heureka workshop hosted together with the ABOA team
  was held this week. We introduced pipetting as a new element to
  the workshop, giving visitors hands-on experience on pipetting
  to a microplate. We had our meeting with the Science Basement
  and the ABOA team, during which we rehearsed and prepared for
  our upcoming presentation.
* #### Week 38

  This week in the laboratory, we conducted Bradford
  protein assays on R. opacus bioconversion
  samples. We also took steps to prepare our samples for
  SDS-PAGE analysis.

  We gave two presentations, one for Master's students and one
  for Bachelor's students. We aimed to introduce iGEM and our
  project while looking for potential members for next year's
  team.

  This week, we also began to shift our focus toward writing
  wiki texts.
* #### Week 39

  In the wet lab, we induced FAST-PETase expression by adding
  IPTG and attempted to confirm enzyme expression by SDS-PAGE
  analysis. Unfortunately, the result did not meet our
  expectations in terms of quality, so we decided to perform a
  second SDS-PAGE analysis.

  Additionally, on Saturday, we had the Science Basement talk in
  collaboration with the ABOA team at the Oodi Library in
  Helsinki.

  During our weekly meeting, we once again focused on wiki
  writing tasks and their deadlines.

### October

* #### Week 40

  We repeated the SDS-PAGE protocol, this time leaving it for
  two consecutive nights to destain. This time, the image of the
  gel showed some improvement. However, due to the absence of a
  control, we cannot definitively confirm that the observed band
  corresponds to our product.

  We also had another meeting with Nesli Sözer because we were
  finishing the experiments and we followed her advice on
  merging the two initial ideas, which were enzymatic plastic
  depolymerization and microbial protein production. We asked
  for her expertise in biotechnology and food production to
  discuss and refine our approach.

  This week, our goal was to complete all the wiki texts so that
  by the next week, they would be ready for publication on our
  wiki pages. After that our next step was to focus on the Grand
  Jamboree.
* #### Week 41

  This week was spent on moving all of the completed texts to the wiki before
  the freeze on the 12th of october. Additionally, the registry pages were double-checked and
  finalized, and the attribution and judging forms were filled.

### November

* #### Week 44

  Our team participated in the Grand Jamboree in Paris, where we engaged in different workshops and presentations.
  We had our project presentation as well as the judging session.
  In the finale, we were thrilled to be nominated for the Best Environment Project, Best Wiki, Best Integrated Human Practices, and Best Education and proudly secured a gold medal!
* #### Week 46

  We initiated the recruitment process for the Aalto-Helsinki 2024 team. The job description and application form were finalized,
  and we begun reaching out to Aalto University and the University of Helsinki to advertise our recruiting efforts.
  Furthermore, we have shared these updates on our social media platforms.

### December

* #### Week 50

  The wiki was proofread once more and updated with some mistakes fixed.
  Lastly, the achievement page was added before the final freeze day on the 14th of December.

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# Engineering

### Content

[Design 1](#design1)
[Build 1](#build1)
[Design 2](#design2)
[Build 2](#build2)
[Test](#test)
[Learn](#learn)

In the iGEM competition, and in the world of synthetic biology in
general, great emphasis is placed on following sound engineering
principles to iteratively improve a design. Often this process is
executed through a framework known as the design-build-test-learn cycle.
In the **design phase**, one defines the problem at hand and
proposes a design to solve it. Furthermore, one needs to define the
minimum requirements for a design to be acceptable as a solution. Next,
the design is implemented in the **build phase**. When the design has
been made into a real-world device, its function is tested in the
**test phase**. Last comes the **learn phase**. Here data
generated in the test phase is used to assess whether or not the
realized design meets the criteria set in the design phase. Often the
case is that the design has some flaws that can be improved on. This
leads back to updating the designs, closing the DBTL-cycle.

![](https://static.igem.wiki/teams/4701/wiki/new-cycle.png)

As illustrated above, going from designs to testing is often not a
trivial process in the world of synthetic biology. For us, the build
phase involved cloning, which proved difficult. This led us to iterate
between the design and build phases, incrementally making slight changes
to our designs and protocols in an attempt to troubleshoot issues with
cloning. While these troubleshooting cycles might be considered
DBTL-cycles themselves, our view is that the DBTL-cycle really is about
going from designs all the way to an implementation that can be tested
to assess whether the design criteria have been met. Thus in the text
below, we present one DBTL-cycle starting from our design and ending
with us testing the function of our part. We also outline some of the
iterative designing and troubleshooting that took place during our
engineering process.

For more detailed information, links are provided to our protocols, the
part pages, and to our modeling results.

### Producing enzymes active against PET

Arguably the most important subproject is the production of PETase and
MHETase, which together are capable of depolymerizing polyethylene
terephthalate (PET)1. While we aimed to produce the two
enzymes in separate hosts, the overall DBTL process was conducted in parallel
for both enzymes and thus they are described in tandem.

The cloning process required numerous attempts before being successful,
we thus outline multiple design-build iterations to highlight the most
prominent changes made during the cloning process, after which the final
results are presented.

#### Design 1 - Basic design

Ever since the initial discovery of IsPETase in 20161,
there have been a multitude of papers devoted to improving the
enzymatic capabilities of the enzyme through engineering. One of the
most recent and prominent ones is FAST-PETase, which has been shown to
exhibit superior capabilities compared to previous variants in terms
of depolymerization rate2. We thus chose it as the variant
we sought to produce. We chose E. coli as our expression
host as it is widely used and easily accessible. We also decided that
we would want to secrete the enzymes, and found that a variant of the
pelB signal sequence has been previously used to secrete IsPETase out
of E. coli cells3. We hypothesized that the
same signal sequence would similarly work for FAST-PETase.

As the activity of PETase is the bottleneck in the PET degradation
pathway, we decided to use the wild-type MHETase coding sequence as
the basis for our second part. As with PETase, we wanted to secrete
the enzyme, and while initially we planned to use the same pelB signal
sequence, we found literature suggesting it does not work with
MHETase4. Instead, we adopted the lamB signal sequence
which was shown to best secrete MHETase from E. coli.

As we wanted to eventually generate quantifiable data on the rate of
PET depolymerization by enzymes produced from our parts, we made sure
to choose a cloning strategy and a vector that would give our protein
a C-terminus His-tag for purification. pET-22b was chosen as the
expression vector as restriction cloning using NdeI and XhoI would
yield an open reading frame (ORF) with a C-terminus His-tag as shown
in Figure 1. Next, it was time to move into the wet lab.

![](https://static.igem.wiki/teams/4701/wiki/construct-1.png)
![](https://static.igem.wiki/teams/4701/wiki/construct-2.png)

Figure 1. Restriction cloning with NdeI and XhoI into pET-22b was
simulated using Geneious. Both ORFs begin with the signal sequence
and end in a His-tag. The coding sequences are under the
transcriptional control of an inducible T7lac promoter.

#### Build 1 - Disappearing vectors

We acquired pET-22b and pET-28a backbones, and while the latter was
not exactly compatible with our insert design, it proved useful in
troubleshooting our first issue.

While attempting to linearize pET-22b with NdeI and XhoI for ligation
with our inserts, we repeatedly ran into an issue where the plasmid
would not appear on our gels. Figure 2 shows that while insert
digestion and the digestion of our control vector pET-28a was
successful, no bands were observed for digested pET-22b vectors.

![](https://static.igem.wiki/teams/4701/wiki/lanes-1.png)

Figure 2. Lanes and expected product sizes. 1: undigested pET-22b
(5493 bp) 2,3,4: pET-22b digested with NdeI and XhoI (5367 bp) 5:
undigested pET-28a (5369 bp) 6,7,8: pET-28a digested with NdeI and
XhoI (5293 bp) 9: undigested FAST-PETase insert (924 bp) 10:
NdeI/XhoI digested FAST-PETase insert (861 bp) 11: undigested
MHETase insert (1908 bp) 12: NdeI/XhoI digested MHETase insert
(1845 bp)

While the restriction protocol for both plasmids was the same, they
were purified from different E. coli strains. pET-22b was
purified from BL21 (AI), while pET-28a was from TOP10. BL21 contains
the endA1 gene, which codes for a non-specific
periplasmic DNA endonuclease I, while in TOP10, this gene has been
removed. We hypothesized that our purified pET-22b samples were
contaminated with the endonucleases, which would activate when
digesting the vector at 37°C, shredding our plasmids. This would be
consistent with the gels as small DNA fragments would quickly move
through the gel. To test our hypothesis, we added an additional
washing step to our plasmid purification protocol.

Figure 3 shows that adding a washing step to our plasmid purification
protocol solved the issue of disappearing vectors, meaning that there
was most likely some type of contamination, possibly endonuclease I.

![](https://static.igem.wiki/teams/4701/wiki/lanes-2.png)

Figure 3. Lanes and expected product sizes. 1: washed and undigested
pET-22b (5493 bp) 2: washed and NdeI/XhoI digested pET-22b (5367
bp) 3: unwashed and NdeI/XhoI digested pET-22b (5367 bp) 4:
unwashed pET-22b incubated at 37°C for 30 minutes without
restriction enzymes (5493 bp).

Despite now having the linearized backbone, multiple ligation attempts
proved unsuccessful. We furthermore transformed the undigested pET-22b
vector purified from BL21 (AI) into TOP10 and saw no growth. This led
us to determine that the issue was most likely with the vector given
that we were able to clone our inserts into pET-28a, transform
successfully, and observe colonies with the same protocols. However, as
stated before, pET-28a was used simply as a control plasmid, as the
ORF formed with our cloning strategy was suboptimal in this vector.

It remains partially unclear to us why we had such issues with
pET-22b. The vector had been used in other projects before, and
supposedly had an insert cloned into the multiple cloning site (MCS)
using the same restriction enzymes we tried using. Even though
linearization was supposedly successful, it is curious that the 498 bp
insert does not show in our gel on lane 2 in Figure 3.

#### Design 2 - Updated design

After giving up on pET-22b, we got our hands on pRSFDuet-1. The second
MCS of this dual expression vector would be suitable for us as it
included the NdeI and XhoI sites. However, this backbone does not
include a His-tag downstream of the XhoI site, and as we had depleted
our stocks of the original inserts after many cloning attempts, we
decided to modify our design and order new inserts.

A His-tag and stop codon were added to both inserts. Before ordering
the updated inserts from IDT, we again simulated the restriction
cloning using Geneious as shown in Figure 4, and made sure translation
rates were sufficient using the RBS calculator5.

For a more detailed description of the design process refer to the
parts registry entries
[BBa\_K4701300](http://parts.igem.org/Part%3ABBa_K4701300)
and
[BBa\_K4701301](http://parts.igem.org/Part%3ABBa_K4701301)
for PETase and MHETase respectively, and our
[design page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D).

![](https://static.igem.wiki/teams/4701/wiki/construct-3.png)
![](https://static.igem.wiki/teams/4701/wiki/construct-4.png)

Figure 4. Restriction cloning with NdeI and XhoI into pRSFDuet-1
yields ORFs beginning with the desired signal sequences and ending
immediately after a C-terminus His-tag. Once again,
transcription can be controlled due to the presence of a T7 lac
promoter upstream of the MCS.

#### Build 2 - Successful transformation

After the new inserts arrived, we restricted both pRSFDuet-1 and the
inserts with NdeI and XhoI and ran a gel followed by gel
purification. Next, we transformed the ligated constructs by
electroporation into competent TOP10 E. coli. Unlike
during previous attempts, this time we actually saw a tiny number of
colonies on one of the plates with constructs containing our
FAST-PETase insert. We proceeded to restreak them and saw considerable
growth on kanamycin plates as shown in Figure 5. Unfortunately, none
of the plates with the MHETase construct had any colonies at this
point.

![](https://static.igem.wiki/teams/4701/wiki/growth-1.jpeg)

Figure 5. Growth on kanamycin plates after ligation of
FAST-PETase into pRSFDuet-1, and subsequent transformation into
TOP10 E. coli.

To gain confidence that the growing colonies indeed had our construct
in them, we took samples from some of the colonies and conducted
restriction analysis on purified plasmids as shown in Figure 6.

![](https://static.igem.wiki/teams/4701/wiki/lanes-3.png)

Figure 6. Plasmids purified from colonies 5, 6, 10, 11, and 15
(Figure 5.) were restricted with NdeI and XhoI and ran on a gel.
The expected sizes are 3775 bp for the pRSFDuet-1 backbone and
888 bp for the insert. As can be seen, the sizes are roughly
correct.

To confirm successful transformation, we purified plasmids from
colonies 5 and 11 (Figure 5) and sent them out to Eurofins Genomics
for Sanger sequencing with the DuetUP2 and T7term primers.

While we were waiting for the sequencing results, we decided to
adjust our cloning strategy once more, as we were still working on
completing the MHETase construct. We suspected that the largest reason
for our troubles with transformation efficiency was related to the low
DNA concentrations following gel purification, which were consistently
as low as 2-5ng/μl despite changing our gel purification protocol on
multiple occasions. Our new approach would be amplifying the inserts
by PCR and subsequently restricting right after PCR purification, only
running a control gel on some of the samples to retain high
concentrations. This way we could attempt skipping gel purification.

We designed and ordered PCR primers for our inserts. Next, we ran PCR
and successfully acquired concentrations above 300 ng/μl after PCR
purification. To confirm successful amplification, some of the PCR
product was run on a gel as shown in Figure 7 for MHETase.

![](https://static.igem.wiki/teams/4701/wiki/lanes-4.png)

Figure 7. Two lanes of MHETase PCR product, the gel indicates
successful amplification as the desirable product length of 1930
bp.

Meanwhile, the sequencing results from Eurofins Genomics confirmed
the successful cloning of the FAST-PETase insert. For colony 11, the
sequencing matched perfectly with our design as shown in Figure 8. For
colony 5, both reads called one silent mutation, although the
fluorescent peak was not strong, nevertheless, we proceeded forward
with colony 11. The construct from colony 11 was purified from the
TOP10 E. coli and transformed into NEB T7 Express
E. coli, which subsequently grew on kanamycin plates as
shown in Figure 9. Thus the build-phase for FAST-PETase was completed.

![](https://static.igem.wiki/teams/4701/wiki/construct-5.png)

Figure 8. A portion of the linearized pRSFDuet-1-FAST-PETase
construct aligned with two Sanger reads, one in the forward
direction from DuetUP2, and one in the reverse direction from
T7term. The match confirms the successful cloning of our FAST-PETase
insert into pRSFDuet-1.

![](https://static.igem.wiki/teams/4701/wiki/growth-2.jpeg)

Figure 9. NEB T7 Express E. coli with
pRSFDuet-1-FAST-PETase constructs growing on kanamycin plates.

We still needed to tackle cloning the MHETase insert. With our new
strategy skipping gel purification, we were able to finally get
colonies after transformation. Unfortunately, multiple restriction
analyses indicated that the insert was just under 1000 bp in length
while the desired length for the MHETase insert would have been 1872
bp. As the larger fragment was in each test the size of a correctly
linearized pRSFDuet-1 vector, we could rule out incorrect restriction.
We suspected that there may have been a mixup of the samples, as the
restriction analysis would be consistent with the FAST-PETase
construct. However, in the end, the reason for these results remained a
mystery for us.

#### Test

With the FAST-PETase construct built, we were then ready to move on to
the test phase. Here our goal would be to secrete the enzyme and attempt
depolymerizing PET. The first goal could be checked by inducing
protein expression with IPTG, followed by centrifugation and SDS-PAGE
analysis to check for correctly sized product in the supernatant. The
second goal can be studied by checking for difference in weight after
exposing PET to the enzyme, or by checking for the presence of PET
monomers by HPLC. Figure 10 shows the results of an SDS-PAGE run
following expression induction with IPTG.

![](https://static.igem.wiki/teams/4701/wiki/engineering-sds-page.png)

Figure 10. FAST-PETase expression was induced with IPTG, and
samples both from the supernatant and pellet were run on an
SDS-PAGE gel. The desired product size for our FAST-PETase product
is 30.731 kD, and while there is a faint line between the 25 kD
and 37 kD markers as denoted by the red arrow, the gel is
inconclusive. Running order: ladder, pellet 1 undiluted, 5x
diluted, 10x diluted, pellet 2 undiluted, 5x diluted, 10x diluted,
supernatant, supernatant.

#### Learn

As no strong band of the correct size was seen on the SDS-PAGE gel, the next
steps would include troubleshooting and possibly changing the design
of the inserts. The pelB signal sequence should secrete FAST-PETase
into the periplasmic space, where the leader sequence is subsequently
removed by a signal peptidase3. Although this still leaves
the outer membrane between the protein and the extracellular
environment, PET depolymerization with supernatant from
E. coli expressing IsPETase in this manner has been
previously reported3.

For a second engineering cycle, we might consider ditching the idea of
secreting the enzymes, instead opting for lysing the cells followed by
enzyme purification by affinity chromatography. However, this approach
is quite costly and does not scale up easily. Other possible
approaches include using a genetically modified strain of
E. coli that has a leaky outer membrane, or co-expressing
bacteriocin release proteins that disturb the outer
membrane6. Another study suggests that following the pelB
sequence with a short aspartate tag can allow proteins to be
secreted all the way across the outer membrane7. Lastly, we
could change the expression host from E. coli to a host
more readily capable of extracellular secretion. For example, IsPETase
has been successfully secreted with the native signal sequence from
Bacillus subtilis8.

### References

1. Yoshida S, Hiraga K, Takehana T, Taniguchi I, Yamaji H, Maeda Y,
   Toyohara K, Miyamoto K, Kimura Y, Oda K. A bacterium that degrades
   and assimilates poly(ethylene terephthalate). Science.
   2016;351(6278):1196–1199. doi:10.1126/science.aad6359
2. Lu H, Diaz DJ, Czarnecki NJ, Zhu C, Kim W, Shroff R, Acosta DJ,
   Alexander BR, Cole HO, Zhang Y, et al. Machine learning-aided
   engineering of hydrolases for PET depolymerization. Nature.
   2022;604(7907):662–667. doi:10.1038/s41586-022-04599-z
3. Shi L, Liu H, Gao S, Weng Y, Zhu L. Enhanced Extracellular
   Production of Is PETase in Escherichia coli via Engineering of the
   pelB Signal Peptide. Journal of Agricultural and Food Chemistry.
   2021;69(7):2245–2252. doi:10.1021/acs.jafc.0c07469
4. Sagong H-Y, Seo H, Kim T, Son HF, Joo S, Lee SH, Kim S, Woo J-S,
   Hwang SY, Kim K-J. Decomposition of the PET Film by MHETase Using
   Exo-PETase Function. ACS Catalysis. 2020;10(8):4805–4812.
   doi:10.1021/acscatal.9b05604
5. Salis HM, Mirsky EA, Voigt CA. Automated design of synthetic
   ribosome binding sites to control protein expression. Nature
   Biotechnology. 2009;27(10):946–950. doi:10.1038/nbt.1568
6. Kleiner‐Grote GRM, Risse JM, Friehs K. Secretion of recombinant
   proteins from E. coli. Engineering in Life Sciences.
   2018;18(8):532–550. doi:10.1002/elsc.201700200
7. Kim S-K, Min W-K, Park Y-C, Seo J-H. Application of repeated
   aspartate tags to improving extracellular production of
   Escherichia coli l-asparaginase isozyme II. Enzyme and Microbial
   Technology. 2015;79–80:49–54. doi:10.1016/j.enzmictec.2015.06.017
8. Huang X, Cao L, Qin Z, Li S, Kong W, Liu Y. Tat-Independent
   Secretion of Polyethylene Terephthalate Hydrolase PETase in
   Bacillus subtilis 168 Mediated by Its Native Signal Peptide.
   Journal of Agricultural and Food Chemistry.
   2018;66(50):13217–13227. doi:10.1021/acs.jafc.8b05038

{% endblock %}

{% extends "layout.html" %} {% block page\_content%}

# Experiments

### Content

[Enzyme production](#enzyme)
[Plastic depolymerization](#plastic)
[Biomass production](#microbial)
[References](#references)

On this page, you can read more about the experiments we carried out
throughout our project. We have divided the project into three different
parts: enzyme production, plastic depolymerization, and biomass
production. To see more detailed information about all the lab notes
from the experiments check out our lab book and
[protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D).

## Introduction

Since 2016, when Ideonella sakaiensis 201-F6, was isolated
from outside a bottle-recycling facility in Japan1, the
research in plastic degrading enzymes has been a hot topic. Current
plastic recycling methods are either physicochemical or thermal
treatments2. Their downfall is that they are energy-intensive
and often lead to the generation of secondary pollutants or toxic
byproducts. This is why enzymatic biodegradation has become an
attractive alternative. Among various types of plastics, polyethylene
terephthalate (PET) is one of the most commonly used plastics worldwide
and its accumulation in the environment is a global problem. PET is
mainly used for packaging, textiles, and bottles, and it can take
hundreds of years to degrade in the environment.
Ideonella sakaiensis 201-F6 can use PET as its major energy
and carbon source due to two native enzymes it produces, PETase and
MHETase1. PETase hydrolyzes PET into mono(2-hydroxyethyl)
terephthalate (MHET), while MHETase further cleaves MHET into
environmentally benign monomers, terephthalic acid (TPA), and ethylene
glycol (EG)1 as illustrated in Figure 1. This process is
known as enzymatic depolymerization, and it has several advantages over
conventional physical or chemical methods, such as lower energy
consumption, reduced greenhouse gas emissions, and higher selectivity.
However, enzymatic PET degradation still has many limitations, such as
slow processing times and low degradation rates, which limit upscaling
and achieving industrial levels2.

![](https://static.igem.wiki/teams/4701/wiki/fig1-result.jpg)

Figure 1. The PET depolymerization pathway.

Many naturally occurring microorganisms can depolymerize and biodegrade
Polyethylene terephthalate polymers. However, in nature, the degradation
rate is very slow. Enzymatic depolymerization can deconstruct plastics
into smaller aromatic building blocks, which microbes can break down
more rapidly. Thereby, the monomers obtained from the enzymatic
depolymerization of PET can be used as substrates for growing
microorganisms that can use them as carbon sources3. Several
bacteria and fungi have been reported to be able to metabolize TPA and
EG. When microbes metabolize waste products they break down the
particles into useful metabolites and other necessary compounds for
growth. When microbes grow they produce biomass with high protein
content. In recent years many microbe-based food products have appeared
into the market in an effort to have more sustainable alternatives to
meat. Single-cell proteins (SCP) are microbial cells, which can be used
as a source of nutrients for human or animal consumption. The protein
content of microbial biomass can vary depending on the strain, the
culture medium, and the growth conditions. The nutritional value and
safety of microbial biomass can be assessed by analyzing its amino acid
composition, essential fatty acids, vitamins, minerals, and potential
toxins or allergens.

## Enzyme production

#### Scientific background

Various enzymes exhibiting PET depolymerization activity have been
identified and investigated, like cutinases, PETase, MHETase, lipases,
and esterases4. The wild-type enzymes PETase and MHETase of
Ideonella sakaiensis 201-F6 display higher enzymatic
activity and specificity for PET than the other reported wild-type
enzymes at comparatively low temperatures (30–40 °C)4. In
efforts to make the enzymatic degradation of plastic more sustainable
and industrially applicable, there has been much focus on improving
the enzymatic activity and thermal stability of PETase. For this
purpose, FAST-PETase (Functional, Active, Stable, and Tolerant PETase)
was designed via machine learning-aided engineering5. It
contains five mutations compared to the wild type and shows improved
PET-hydrolytic activity in a wide range of temperatures and
pH5.

Extracellular secretion of PETase is also essential for the
application of this enzyme in PET degradation. Secretion greatly
facilitates downstream processing and significantly reduces the
production costs of recombinant proteins. There have been recent
studies where extracellular expression of PETase has been
reported4.

E. coli is a useful organism for recombinant protein
production due to its availability, easy cultivation, established
cloning techniques, and rapid protein expression. In bacteria,
two major export pathways are capable of transporting proteins
outside of the cell plasma membrane, the general secretion - Sec
pathway - and the twin-arginine translocation or Tat
pathway6. The activation of the protein export systems
requires a specific signal peptide to the amino-terminal end of the
desired target protein and the pathway6. In
E. coli, the translocation of proteins usually happens
via the Sec-dependent pathway, signal recognition particle (SRP)
pathway, or twin-arginine translocation (TAT) pathway, mediated by
N-terminal signal peptides4. Commonly used signal peptides
for the production of extracellular recombinant proteins in
E. coli include pelB, phoA, malE, or lamB. pelB has been
used in the pET-vector expression system4. pelB in
particular has been shown to be compatible with PETase4,
while lamB is suitable for the WT-MHETase construct.

#### The goal of the experiments

We wanted to design an iGEM-compatible recombinant system of
FAST-PETase and MHETase for E. coli that secretes the
enzymes. To build the expression strains, we did restriction cloning,
using the Xhol and Ndel restriction enzymes to cut both the gene
inserts and the pRSFDuet-1 vector. The ligated expression vector was
transferred first into cloning host E. coli TOP10 to
confirm the successful cloning, and later to expression host
E. coli T7. We had plans to further optimize the enzymes
by adding SpyCatcher/SpyTag domains into our products, thus binding
the secreted enzymes into a scaffold or to each other, further
optimizing enzyme activity. We wanted to optimize the expression
conditions and purify the enzymes by affinity chromatography using
His-tags and analyze the activity by measuring the release of TPA from
PET using HPLC.

#### The success of the experiments

We successfully designed the constructs for FAST-PETase and MHETase
based on recent literature. Both constructs were optimized for
translation and transcription by calculating the optimal sites for the
inserts and by removing rare codons. By adding the signal peptides
lamB and pelB respectively into our MHETase and FAST PETase constructs
as shown in Figure 2, we facilitated secretion into the culture media.
We successfully cloned FAST-PETase into both TOP10 and T7 expression
hosts. We then induced the expression of FAST-PETase by adding IPTG
and studied the expression of the enzyme by SDS-PAGE.

![](https://static.igem.wiki/teams/4701/wiki/fig2-result.png)

Figure 2. FAST-PETase and MHETase inserts. More detailed
illustrations are available on our
[design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D)
page.

For the MHETase, we never got the correct size bands in the restriction
analysis of the clones. We suspect that this may be due to a mix-up with
inserts. Unfortunately, we did not have the time or funds to purify our
enzymes or do activity analysis, due to many difficulties in the
cloning process. Due to the same problems, we also did not have time
to compare the effectiveness of SpyCather/SpyTag parts, even though they
were designed. Our
[engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)
page outlines the most central phases of our wet lab work.

## Plastic depolymerization

#### Scientific background

The depolymerization of PET by enzymes is a complex process that
depends on several factors, such as the type and form of the PET
plastic and its crystallinity, the enzyme concentration and ratio, the
reaction temperature and pH, and the presence of additives or
co-solvents7. PET polymers are large molecules that have a
crystalline structure. Often plastics do not have a homologous
structure, but a mix of regular crystals (crystalline region) and
irregular groups (amorphous region)7. This is what makes
them durable and flexible, but also hard to degrade or recycle.
PET-based plastics possess a high degree of crystallinity (30–50%),
which is one reason for the low rate of microbial degradation in
nature (~>50 years)7.

The enzymatic degradation of plastics occurs in two stages: 1)
adsorption of enzymes on the polymer surface, and 2) followed by
hydrolysis of the bonds7. The enzymes responsible for
breaking down PET are α/β-hydrolases7. They possess a
Nucleophil-His-Acid catalytic triad, in this case, Serine-His-Aspartame,
which efficiently catalyzes a variety of different
substrates7. The process of breaking down PET is initiated
by serine oxygen attacking the carbonyl carbon within the ester bonds
of PET7. The reaction is facilitated by the negatively
charged aspartate, which stabilizes the positively charged histidine
residue7. The charge transfer results in ester bond
hydrolysis by enabling serine to perform a nucleophilic attack,
leading to the breakdown of the ester bond7. PETase,
one of these enzymes, can specifically hydrolyze ester bonds found in
aromatic polyesters. It plays a crucial role in converting PET into
mono-2-hydroxyethyl terephthalate (MHET). On the other hand, MHETase
is responsible for breaking down MHET into terephthalic acid (TPA) and
ethylene glycol (EG).

#### The goal of the experiments

For this experiment, we wanted to use the purified enzymes to
depolymerize PET into TPA and EG. We wanted to test different types
and forms of PET (e.g., films, commercial packages, and untreated and
pretreated plastics) with different reaction parameters (e.g.,
temperature, pH, enzyme concentration) to determine the optimal
conditions for efficient depolymerization. To analyze the kinetics and
products of the enzymatic reaction we wanted to use HPLC to quantify
the produced monomers and demonstrate the degradation by taking
pictures using scanning electron microscopy (SEM).

#### The success of the experiments

As we did not have time to purify the enzyme, we attempted the
depolymerization without purification by adding different plastics
into the media to which our expression host had secreted the enzymes.
After a period of time, we measured the loss of mass.
Unfortunately, no reduction in mass was observed.

## Biomass production

#### Scientific background

Many naturally occurring microorganisms can depolymerize and
biodegrade polyethylene terephthalate polymers. From these, the most
notable regarding our project were Pseudomonas putida,
Rhodococcus opacus,
Comamonas testosteroni, and
Bacillus subtilis. They are all common soil microbes,
that have been documented to be able to use PET degradation monomers
as carbon sources8. Ethylene glycol has been shown to be
metabolized by various microbes through the sequential oxidation of EG
to glyoxylate (KEGG, R00476) via glycolaldehyde (KEGG, R01781) and
glycolate (KEGG, R01333) as intermediates8. TPA, in turn,
requires specific enzymes to turn it into protocatechuate, which is
then further changed to central carbon metabolism
intermediates8. Efficient metabolization of EG and TPA has
been achieved by laboratory evolution and by metabolic engineering
since natural uptake is quite conservative8.

#### The goal of the experiments

The goal of this section is to use the monomers obtained from
enzymatic depolymerization as substrates for growing microorganisms
that can produce protein-rich biomass. First, we wanted to know if the
strains selected actually could grow on minimal media with PET
monomers as a carbon source, and conduct laboratory evolution on the
microbes to get better growth. We wanted to evaluate the growth rate
and quality of the microbial biomass under different culture
conditions making pure cultures as well as growing the bacteria in
co-cultures to see if they have synergistic actions or whether they
would have competitive relations and inhibit the growth. We also
wanted to analyze the nutritional value and safety of the biomass for
potential applications as food or feed. We were also modeling possible
end products of metabolizing TPA and EG and considering genetical
engineering of E. coli and P. putida for
better TPA and EG uptake, respectively.

#### The success of the experiments

We selected P. putida, R. opacus, and
C. testosteroni as the strains of bacteria for
metabolizing TPA and EG. We found that P. putida KT2440
was the best candidate for our system, as they have high growth rates,
yields, and protein contents when cultured in M9 minimal medium
supplemented with TPA and EG. We analyzed the uptake of monomers by
performing HPLC. We also performed co-cultures of these strains to
enhance their synergistic interactions. We analyzed the protein
content by Bradford analysis, and we found that protein was generated
from biomass of R. opacus cultivated on the minimal media
containing TPA which accounted for 3.4 µg/ml.

However, we did not have enough guidance or resources to analyze other
compounds of the biomass, such as the amino acid composition,
essential fatty acids, vitamins, minerals, and potential toxins or
allergens of our biomass.

## References

1. Yoshida S, Hiraga K, Takehana T, Taniguchi I, Yamaji H, Maeda Y,
   Toyohara K, Miyamoto K, Kimura Y, Oda K. A bacterium that degrades
   and assimilates poly(ethylene terephthalate). Science.
   2016;351(6278):1196–1199. doi:10.1126/science.aad6359
2. Kushwaha A, Goswami L, Singhvi M, Kim BS. Biodegradation of
   poly(ethylene terephthalate): Mechanistic insights, advances, and
   future innovative strategies. Chemical Engineering Journal.
   2023;457:141230. doi:10.1016/j.cej.2022.141230
3. Schaerer LG, Wu R, Putman LI, Pearce JM, Lu T, Shonnard DR, Ong RG,
   Techtmann SM. Killing two birds with one stone: chemical and
   biological upcycling of polyethylene terephthalate plastics into
   food. Trends in Biotechnology. 2023;41(2):184–196.
   doi:10.1016/j.tibtech.2022.06.012
4. Shi L, Liu H, Gao S, Weng Y, Zhu L. Enhanced Extracellular
   Production of IsPETase in Escherichia coli via Engineering of the
   pelB Signal Peptide. Journal of Agricultural and Food Chemistry.
   2021;69(7):2245–2252. doi:10.1021/acs.jafc.0c07469
5. Lu H, Diaz DJ, Czarnecki NJ, Zhu C, Kim W, Shroff R, Acosta DJ,
   Alexander BR, Cole HO, Zhang Y, et al. Machine learning-aided
   engineering of hydrolases for PET depolymerization. Nature.
   2022;604(7907):662–667. doi:10.1038/s41586-022-04599-z
6. Freudl R. Signal peptides for recombinant protein secretion in
   bacterial expression systems. Microbial Cell Factories.
   2018;17(1):52. doi:10.1186/s12934-018-0901-3
7. Urbanek AK, Kosiorowska KE, Mirończuk AM. Current Knowledge on
   Polyethylene Terephthalate Degradation by Genetically Modified
   Microorganisms. Frontiers in Bioengineering and Biotechnology.
   2021;9:771133. doi:10.3389/fbioe.2021.771133
8. Tiso T, Winter B, Wei R, Hee J, de Witt J, Wierckx N, Quicker P,
   Bornscheuer UT, Bardow A, Nogales J, et al. The metabolic potential
   of plastics as biotechnological carbon sources – Review and targets
   for the future. Metabolic Engineering. 2022;71:77–98. (Substrates
   for Metabolic Engineering). doi:10.1016/j.ymben.2021.12.006

{% endblock %}

{% extends "layout.html" %} {% block page\_content%}

# Results

### Content

[Enzyme production](#enzyme)
[Plastic depolymerization](#plastic)
[Protein production](#protein)
[Future perspective](#future)

## Enzyme production

We successfully cloned the gene encoding for FAST-PETase into the
pRSFDuet-1 vector, using restriction digestion and ligation methods.
We verified the correct insertion of the gene by restriction analysis
and Sanger sequencing. We then transformed the recombinant plasmids
into an expression strain. The expression was induced via IPTG
and the pellet and supernatant of the expression were analyzed using
SDS-PAGE.

#### PCR

After multiple failed attempts at cloning, we had to perform PCR amplification for the MHETase insert.
It was first amplified via PCR using DreamTag polymerase (Thermo Fisher, #EP0703) and
purified using GeneJet Gel Extraction Kit by Thermo Scientific.
The purified PCR product was then analyzed by running electrophoresis
on a small amount with 1% agarose gel as shown in Figure 1.

![](https://static.igem.wiki/teams/4701/wiki/res1.png)

Figure 1. MHETase construct successfully amplified. The desirable
size of the construct is expected to be of 1930 bp.

#### PETase and MHETase transformation

The constructs for FAST-PETase and MHETase were introduced into
competent TOP10 E. coli via electroporation. The
transformed colonies for both enzymes showed growth in LB plates
supplemented with kanamycin as shown in Figure 2.

![](https://static.igem.wiki/teams/4701/wiki/ress2.png)

Figure 2. Growth of the E. coli cells transformed
with the constructs on the kanamycin-supplemented plate. Left:
MHETase constructs in TOP10, Right: FAST-PETase constructs in T7.

#### Restriction analysis

To confirm that the colonies were bearers of the correct construct, a
restriction analysis was carried out. The plasmid was first purified
with a GeneJET Plasmid Miniprep Kit (Thermo Fisher, #K0503). Next, the
plasmid was digested using the restriction enzymes NdeI and XhoI (both
Thermo Fisher, #ER0581 and #ER0692, respectively) (35 minutes 37ºC for
the reaction and 5 minutes 80ºC to stop the reaction). The products of
the restriction were analyzed by running electrophoresis in a 1%
agarose gel as shown in Figure 3.

![](https://static.igem.wiki/teams/4701/wiki/ress4.png)

Figure 3. Plasmids purified from the colonies restricted with the
NdeI and XhoI enzymes. Left: FAST-PETase-pRSFDuet1 restriction.
Right: MHETase-pRSFDuet1 restriction. The size of the pRSFDuet-1
backbone is expected to be 3775 bp and the size of the FAST-PETase
insert should be 888 bp, while MHETase should be 1872 bp. The
results of the left gel picture correspond to these sizes, but for
MHETase the insert size is too small.

#### Sequencing

After restriction analysis suggested that our FAST-PETase insert had
been successfully cloned into pRSFDuet-1, we purified products from
some of our colonies and sent them out to Eurofins Genomics for
sequencing. Using the Sanger sequencing results obtained by sequencing
with the DuetUP2, and T7term primers, we found that the sequence
within our multiple cloning site perfectly matched our expectations as
shown in Figure 4.

![](https://static.igem.wiki/teams/4701/wiki/res6.png)

Figure 4. MCS-2 of pRSFDuet-1 was sequenced once in both
directions, with both reads perfectly matching the sequence of
BBa\_K4701202 as denoted by the vertical red lines, and the
surrounding sequence from the backbone.

#### Enzyme expression and secretion

When we had the confirmation that the expression vectors were good, we
initiated protein expression by adding IPTG into the T7 liquid culture
on LB-broth with OD600 of 1. The expression lasted 24 hours until the
broth supernatant and pellet were collected. These were used to run an
SDS-PAGE shown in Figure 5.

![](https://static.igem.wiki/teams/4701/wiki/res7.jpg)

Figure 5. SDS-PAGE gel with pellet and supernatant samples after
24h protein expression. FAST-PETase should be 30.731 kDA. There is
a band around that size in each sample, including the supernatant,
which could indicate that the enzyme is being secreted into the
growth media. Though without a proper control, the result is
inconclusive.

## Plastic depolymerization

We performed an experiment of plastic depolymerization by adding
chemically and thermally pre-treated commercial plastic and gf-PET
film (GoodFellow, ES30-FM-000250) into the growth media of the
expression host. They were left at 37ºC for 5 days. The plastic
samples were weighted before and after incubation. Unfortunately, no
loss of mass was observed, most likely due to the issues we had with
enzyme secretion.

## Protein production

#### Growth experiments on TPA and EG

We successfully grew some common soil microbes on the M9 minimal
medium supplemented by 30 mM of TPA and on the M9 minimal medium
supplemented by 10 g/L of EG, as shown in Figure 6.
P. putida and R. opacus were growing on both
mediums. Meanwhile, C. testosteroni was only able to grow
on minimal media containing 30 mM of TPA. This result indicated that
TPA and EG obtained from enzymatic depolymerization could be utilized
as carbon sources for microbial growth.

![](https://static.igem.wiki/teams/4701/wiki/result-fig6a.png)

![](https://static.igem.wiki/teams/4701/wiki/result-fig6b.png)

Figure 6. Growth of P. putida,
R. opacus, and C. testosteroni in the
presence of PET monomers, respectively, from left to right. a) PET
+ 30 mM of TPA, b) PET + 10 g/L of EG.

To produce the protein-rich biomass, R. opacus, and
C. testosteroni were cultivated in the M9 broth medium
containing TPA and EG as seen in Figure 7. Due to the time
constraints, we were not able to execute similar experiments with
P. putida

![](https://static.igem.wiki/teams/4701/wiki/res10.jpg)

Figure 7. The biomass obtained from the microbes grown on the M9
broth medium supplemented by TPA and EG.

#### Metabolism and Bioconversion of PET monomers

Data obtained by HPLC analysis shows that PET monomers can be utilized
as a carbon source to produce biomass. This can be seen as the
decrease in TPA concentration over time in the biomass of
R. opacus as shown in Figure 8. On the other hand, TPA
was not metabolized by C. testosteroni. Due to the time
constraints, the metabolism of EG by the strains was not studied with
HPLC.

![](https://static.igem.wiki/teams/4701/wiki/res-tpa-uptake.png)

Figure 8. The metabolization of TPA by R. opacus and
C. testosteroni
was analyzed by HPLC.

#### Protein production

Lastly, we found that protein-rich biomass can be produced by growing
microbes with PET monomers as the primary carbon source. The protein
content of the resulting biomass was analyzed with a Bradford protein
assay. We found that R. opacus successfully produced
protein as shown in Figure 9. Even though we did not manage to analyze
the protein content of biomass produced by
C. testosteroni, the result from
R. opacus indicates that producing single-cell proteins
from PET monomers is a viable strategy.

![](https://static.igem.wiki/teams/4701/wiki/results-protein-c.png)

Figure 9. Protein produced by R. opacus  cultivated
on M9 minimal medium containing 30 mM of TPA was assessed using
Bradford analysis.

## Future perspective

Embarking on our iGEM journey, we knew that we had a very ambitious
project, with multiple stages which were not really the core knowledge
of any of our team members. However, as we were all interested and
motivated to prove that our concept could be viable, we put our best
effort towards it. Due to time constraints, our team was unable to
complete many of the experiments and protocols planned, so here is a
list of experiments our team would potentially work on in the future.

Firstly, we should work on MHETase cloning and expression, to improve
the degradation as PETase and MHETase work in synergy to completely
depolymerize PET into TPA and EG. After that, many different analyses
could be done to measure the enzyme activity and degradation rate.

In terms of protein production, we did not have time to engineer
P. putida or E. coli with our constructs. A
far future prospect would be to produce specific amino acids and
nutritional substances in the cells. In addition, a comprehensive
analysis of the biomass obtained needs to be carried out to ensure
that the protein produced is safe.

We see great prospects in the project if there was more time and
knowledge to execute all of our experiments. But hopefully, future iGEM
teams or even other research groups will be inspired to work on
similar projects.

{% endblock %}