iGEM 2025 — Team fsu

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your daily activities, experiments, discussions, and decisions.{% endblock %}
{% block page\_content %}

## What Should this Page Contain?

---

* Record all activities in chronological order, with each entry clearly dated.
* Provide sufficient detail for each activity, including:

+ Purpose of the activity.
+ Methods and procedures used.
+ Results obtained.
+ Observations and notes.

* Document all experiments, protocols, data analysis, meetings, brainstorming sessions, and other project-related activities.
* Include photos, videos, and diagrams to illustrate your progress.
* Indicate which team members participated in each activity.

## Inspirations

---

* [2024 EPFL](https://2024.igem.wiki/epfl/notebook)
* [2024 JU-Krakow](https://2024.igem.wiki/ju-krakow/notebook)

↑
{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one
iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

* [Introduction](#introduction)
* [Ideation](#ideation)
  + [High-Level Design](#high-level-design-constraints)
  + [Design Specification](#design-specifications)
* [Engineering Cycle](#engineering-cycle)
  + [Ideation 1](#ideation1)
  - [Design](#design)
  - [Build](#build)
  - [Biological Devices](#biological-devices)
  - [Test](#test)
  - [Learn](#learn)+ [Ideation 2](#ideation2)
  - [Design](#design2)
  - [Build](#build2)
  - [Biological Devices](#biologicaldevices2)
  - [Test](#test2)
* [Future Cycles](#future-cycles)
* [Overall Conclusion](#overall-conclusion)
* [References](#references)

Introduction: Microplastics and the Small Intestine

Microplastics (MPs) are becoming a growing concern for environmentalists, with society’s integration of
single-use plastics into daily life directly resulting in an accumulation of MPs in ecosystems across
the world.
One such ecosystem is our own human bodies. MPs and their nano-scale counterparts, nanoplastics (NPs),
have been shown to accumulate in the liver, brain, and lungs, posing massive potential concerns for
human health
(Kim et. al, 2025). These health effects are being increasingly studied, and although the mechanisms and
severity
of MP accumulation are not yet fully understood, our team has approached the issue with the intent to
prevent any
potentially toxic side effects.

![fig1](https://static.igem.wiki/teams/5886/engineering/f1.webp)

Figure 1: Chemical Structure of Polystyrene Monomer

The primary bioengineering challenges with polymers such as polyethylene, polypropylene, and polystyrene
(PS) are their nonpolar structure and minimal biodegradability, causing them to be highly inert and
persistent in the environment (Cai et. al, 2023). However, developments in de novo peptide design and
phage display have increased the number of options for combatting MPs (Skariyachan et. al, 2022). As a
result, iGEM teams have engineered various bacterial chassis to secrete/display polyethylene
terephthalate hydrolases (PETases) and/or plastic binding peptides (PBPs).

The Design subteam’s preliminary research quickly led us to understand that MPs must travel to vital
organs
through the bloodstream. Most MPs reach the bloodstream by entering the body through oral ingestion,
travelling through the gastrointestinal tract, bypassing the intestinal barrier and finally infiltrating
the bloodstream (Li et. al, 2024). Considering this, our engineering team’s cycles were planned around
mitigating MP interaction with the intestinal mucosae to prevent endocytosis and phagocytosis, among
other things.

![fig2](https://static.igem.wiki/teams/5886/engineering/f2.webp)

Figure 2: Mechanisms of Absorption of Microplastics in the Small Intestine (Prata,
2023)

Our ideation process for combatting MP accumulation has ultimately culminated in our proposed engineered
probiotic,
**Plastipeutics**. Essentially, we’ve designed an Escherichia Coli Nissle 1917 (EnC) capable of
surface display of
fusion proteins individually composed of an anchor protein, a flexible linker protein, and a PBP. This
biological device not
only allows EnC to bind MPs, but to aggregate multiple of them around itself, increasing the MPs'
effective volume and limiting their capacity to pass through the intestinal epithelium.

![fig3](https://static.igem.wiki/teams/5886/engineering/f3.webp)

Figure 3: Graphic Design of Plastibinder Unit

*Created in https://BioRender.com*

High Level Design Ideation

Defining a High Level Design

In starting the design process, our team’s first initiative was to brainstorm high level designs,
or “big picture ideas,” for our solution. With the initial requirement that the small intestine
would be our point of intervention, we formulated three designs based on different synthetic biology
applications.

1. Engineering macrophages present in Peyer's Patches (a component of the enteric immune system), to produce MP-degrading enzymes for degrading phagocytosed MPs.
2. Engineering a probiotic bacteria to secrete MP-degrading enzymes into the mucosa/lumen.
3. Engineering a probiotic to secrete and/or display PBPs to bind and aggregate plastics.

The first option, engineering an immune cell, seemed fairly intuitive. M-cells, epithelial cells
foundational to the enteric immune system, are designed to transcytose antigens from the intestinal
lumen into Peyer’s patches to be degraded. Modelling our solution after the COVID-19 vaccine, we
theorized using a lipid nanoparticle to both transport mRNA safely through the GI tract and to
target the mRNA delivery to M-Cells, which are primarily present in the ileum.

![fig4](https://static.igem.wiki/teams/5886/engineering/f4.webp)

Figure 4: Pathway for Ingested Lipid Nanoparticle to Peyer's Patch

*Created in https://BioRender.com*

Alternatively, utilizing an engineered probiotic seemed viable, as it would allow us to address
bloodstream entrance points beyond M-cells. The initial concept involved engineering the probiotic
strain EnC to colonize the ileum and secrete PETases for degrading ambient MPs. We chose to model
this design around PETase because of its thorough usage in literature and iGEM. However, our third
design substitutes anchored PBPs in place of secreted PETase, making the EnC a microplastic binder
that could potentially aggregate multiple MPs together.

![fig5](https://static.igem.wiki/teams/5886/engineering/f5.webp)

Figure 5: Pathway for Probiotic to Ileum

*Created in https://BioRender.com*

With these initial designs established, our team began to break down our assumptions and develop
constraints. Many of our considerations were with respect to the environment of the small intestine.
For example, we wanted to ensure our solution functioned in the mucus-filled space and that it would
account for all the ways MPs move through the lumen. Also, we noted the need for an enteric coating
to bypass the stomach, and accounted for the ileum’s neutral pH (7.4) and human body temperature
(37°C) in our models. Furthermore, we factored in the size scales of MPs, NPs, peptides, bacteria,
epithelial cells, and the lumen into our modelling and decisions.

In fact, our research led us to understand that the size of MPs and NPs are integral to the nature of
their interaction with epithelial cells. For example, MPs larger than 150μm will not be
transcytosed, serving as an upper bound for our design, and a lower bound can be derived from the
fact that MPs smaller than 4μm are less likely to accumulate in tissue (Li et. al, 2024). Although
the mechanistic relationship between MPs and the intestinal epithelia is not fully understood,
compartmentalizing interactions such as tissue penetration, transcytosis, and endocytosis allowed us
to better understand what size ranges pose the largest threat.

With input from our advisors, our team decided to further develop the macrophage design. From our
interview with Jingjiao Guan, we determined that, to implement our solution, we’d need to ensure
that the mRNA vector wasn’t phagocytosed, PETase would be produced in a lysosome, PETase would
survive in the acidic lysosome, and PETase would function properly in the phagolysosome upon MP
phagocytosis.

![fig6](https://static.igem.wiki/teams/5886/engineering/f6.webp)

Figure 6: M-cell Modification Process

*Created in https://BioRender.com*

At this point, a couple of obstacles stood in our way. From our interview, we’d learned that
macrophages are notoriously challenging to transfect. Also, the mechanisms of phagocytosis are
highly complex, and attempting to express PETase in phagolysosomes would be challenging since
visualizing internal macrophage activity is very challenging. Considering these factors, our team
chose to pivot away from immune cell engineering.

With this, we began pursuing a probiotic capable of degrading microplastics because of the extensive
research done with PETase enzymes; we believed that attempting to apply PETase in intestinal
conditions would prove fruitful. However we later came upon a key flaw with this design: PET
catabolysis by most strains of PETase produces byproducts that are toxic to enterocytes (Syren et.
al, 2024). This dissuaded us from attempting any PETase-based solutions, as- unless our system was
capable of quickly degrading MPs into monomers- the potential toxicity would be even more harmful
than MPs themselves. With that said, made our final shift to a plastic-binding probiotic.

![fig7](https://static.igem.wiki/teams/5886/engineering/f7.webp)

Figure 7: Design Requirements for Degradation/Aggregation Probiotic

*Created in https://BioRender.com*

Specifying Our Chosen Design

With our overall design selected, our first specification was ensuring that our PBPs were capable of
functioning in the ileum. Our modelling team utilized a Propka model to ensure that our PBPs would
have the proper net charge at the pH of the ileum. We noticed that the Histag was actually the
limiting factor for suitable pH, which we can leverage in future designs where purification isn’t
necessary.

In selecting our chassis, we defaulted to EcN due to its overwhelming characterization among
probiotic bacteria strains. However, it’s important to note that EcN has limited secretion pathways,
which may necessitate our shift to another chassis later on (Lynch et. al, 2023).

In deciding the best MP to model our engineering decisions around, we initially thought of
polyethylene terephthalate since our degradation design centered around PETase. However, after
shifting to a PBP mechanism, our team realized that PS was advantageous because most research on the
health effects of MPs employs PS as the model particle. This means that, although the health
implications of PS are well understood, those results can’t be extrapolated to other MPs (Jasinski
et. al, 2024). Furthermore, we lacked the tools to create our own microplastics with micron-scale
precision, so we decided to purchase plastic microplasticles instead. Since PS MPs are much less
expensive than other polymers MPs, we were further compelled to model our processes after it.

Engineering Cycle

Iteration 1

Design

The goal of our first cycle was to prove that our PSBPs bind to MPs. To this end, we began by
searching for polystyrene binding peptides (PSBPs), identifying multiple broader categories in
literature including deactivated enzymes, nanobodies, and simple polypeptides. Deactivated enzymes
were more appealing when modelling around PET because we could utilize deactivated PETase, but since
PS was our new model MP, we strayed away from that option. Nanobodies were very compelling due to
their adaptability, but the lack of literature on PS-binding nanobodies dissuaded us from pursuing
them. Thus, we chose to employ polypeptides, as they allowed us more flexibility in design and
modelling.

For this cycle, we selected PSBPs
( [BBa\_25BXPW4W,](https://registry.igem.org/parts/bba-25bxpw4w) | [BBa\_254S02FL,](https://registry.igem.org/parts/bba-254s02fl) | [BBa\_2595L78Z,](https://registry.igem.org/parts/bba-2595l78z) | [BBa\_25UCSI3M,](https://registry.igem.org/parts/bba-25ucsi3m) | [BBa\_25HEIU8Z](https://registry.igem.org/parts/bba-25heiu8z) |)
that were computationally generated by Alshehri et al. (2025) and Dhoriyani et al. (2025), specifically
choosing ones with the highest binding affinity for PS based on their molecular dynamics simulations.
From there, our modelling team established a computational pipeline to increase our confidence in selected PSBPs:
The modeling team used Alphafold 3 to predict peptide folding structures, AutoDock Vina to estimate binding affinities
by performing molecular docking between our PSBPs and various polymers (PS, PET, PE,) and GROMACS to
observe peptide behavior in aqueous environments through molecular dynamics simulations.(See our Modelling Page for more!)

Beyond selecting PSBPs, we considered other coding sequences for the fusion protein, including
reporters, surface display/anchor proteins, and linker proteins. The anchor and linker proteins were
part of our high level design, but we realized that, for proving binding, they were not essential.
As a result, we omitted the anchor protein and, to see if the linker impacted binding/expression, we
created generator units with and without linkers. Our chosen linker (BBa\_K4586019) is a serine
glycine linker from the parts registry.

With our coding sequences selected, we designed a PSBP generator skeleton to insert them into for expression.
Our generator’s overall skeleton was guided by the NEB PURExpress® In Vitro Protein Synthesis
Instruction Manual’s template DNA sequence, as we planned to their in vitro and in vivo T7-based
protein expression systems.

![fig8](https://static.igem.wiki/teams/5886/engineering/f8.webp)

Figure 8: Required elements for template DNA (NEB, 2025, pg. 4)

Staying within the template DNA’s restrictions, we included a 5’ UTR
( [BBa\_K1758100](https://registry.igem.org/parts/bba-k1758100) |)
to maximize expression and a histag for purification. Finally, our coding sequences were too short
for the plasmid synthesis methods used by our DNA synthesis company, so we included DNA spacers to
meet the minimum base pair threshold. The final generator designs are shown below.

![fig9](https://static.igem.wiki/teams/5886/engineering/f9.webp)

Figure 9: SBOL Designs for PSBP Generator Units with and without Linker Protein

Build

We inserted our generator device into a Twist kanamycin resistance backbone
( [BBa\_25D1KRKB](https://registry.igem.org/parts/bba-25d1krkb) |)
which created our vector shown below (fig.10). The KanR gene was selected since past FSU iGEM teams used it for their
bacterial selection protocols.

![fig10](https://static.igem.wiki/teams/5886/engineering/f10-4.webp)

Figure 10: Snapgene Plasmid Map of BBa\_25BD5FF2

After ordering the synthetic plasmids from Twist Bioscience, we were informed that two of our three vectors failed
to synthesize. After receiving the full report from Twist, we realized the issue was that our 5’ UTR
had ten consecutive adenine repeats. Learning this, we researched 5’ UTR options and found that a
Poly-A spacer of five base pairs long would also be sufficient (Takahashi et. al, 2013) so we used a
truncated 5’ UTR ( [BBa\_2563ZJ4S](https://registry.igem.org/parts/bba-2563zj4s) |).
Furthermore, our PI advised us to add a spacer in front of our promoter so that if we were to have
our sequence synthesized linearly, then the promoter would have sufficient room to bind. As a result,
we employed the T7Max promoter (BBa\_K4207000), which has an adenine-rich region upstream of the promoter
to improve polymerase binding (Deich et. al, 2023). This resulted in our new generator skeleton
( [BBa\_252JX551](https://registry.igem.org/parts/bba-252jx551) |.)

Chronologically, this is when we shifted our focus from PET to PS. Also, this was when we chose to
order generators with and without linkers, which allowed our assays to test PSBP-linker fusion
proteins and pure PSBPs as experimental groups.

![fig11-1-2](https://static.igem.wiki/teams/5886/engineering/f11-1-2.webp)
![fig11-2-2](https://static.igem.wiki/teams/5886/engineering/f11-2-2.webp)

Figure 11: Snapgene Plasmid Maps for BBa\_258USD40 and BBa\_251QKY3O

The generators with and without linkers for five different PSBPs and a negative control were synthesized
by TWIST Bioscience. This left us with five PSBP generators, five PSBP + linker generators, our negative
control vector, and the PET binding peptide generator from our prior order. See our parts page for more
information!

Biological Devices

Our team attempted expression of 11 binding units, PET Binding Unit B and PS Binding Units A through PS Binding Units J.
We tried two methods of expression, referred to as plans A and B. Plan A involved using the NEBExpress Cell-free E. coli Protein Synthesis
System (NEB #E5360S), using our designed plasmids that contained the coding sequence to express PS Binding Unit A, C, E, F,
and G. We ran plan B in parallel, in which we transformed chemically competent T7 Express E. coli cells using PS Binding Unit
Generator A through PS Binding Unit Generator J (See the Parts section of our Wiki!) From these transformed cells, we plated
them on LB-KAN in 1:1 and 1:10 dilutions and allowed colonies to grow overnight at 37°C. The following morning, we moved the
plates to the fridge to prevent satellite colonies from growing. We then made 25mL overnight cultures in LB media supplemented
with Kanamycin. We took 5mL per construct of the overnight culture and made glycerol stocks for stable storage of transformed
T7 express cells for minipreps for more DNA for future use. The remaining 20mL was split in half for small scale expressions —
10mL for IPTG induction and 10mL for non-induced controls. The cell cultures were grown at 37° C until an optical density, OD600,
between 0.4-0.8 was reached. Then, IPTG or water (negative control) was added to reach a final concentration of 0.4 mM. Afterwards,
8mL of culture was centrifuged down in respective Eppendorf tubes to produce bacterial cell pellets which we then froze at -80° C,
for future use.

Test

When ready, the team defrosted the cell pellets, lysed them using sonication and ran the noninduced and induced cell cultures
in addition to the in-vitro expressed PS Binding Units A, C, E, F, and G in a 16% tricine gel. This gel percentage was chosen
due to this percentage having peptide resolution down to 1kDa. This gel was run for half the time to ensure the ~3kDa peptides
didn’t run off as the ladder only went down to 10kDa and was stained with a silver nitrate. Silver nitrate as a stain is very
sensitive as it can detect protein concentrations as low as 0.1 ng/band (Jiang et al). This was followed by Coomassie blue
dye to stain anything silver nitrate could not. The team observed highly visible bands in the 10-150kDa range, but the team
was unable to locate a band in the expected 2.5-3kDa range, which is a characteristic of our peptides.

As a result of this setback, we hypothesized that our peptide was aggregating to itself or other proteins, experienced
possible degradation, or it bound to the walls of the plastic pipettes. The result of our contribution, which verified
that the T7 Max promoter (BBa\_K4207000) works and does increase expression, made the possibility of our device not
expressing at unlikely. Therefore, to test our hypotheses of whether degradation or aggregation was the problem, the
team completed another 16% tricine gel running PS Binding Units A, H, and J in the gel. However, we decided to purify
the cell lysate and added it to our test. We ran this 16% tricine gel with non-purified and purified induced and non-induced
cells containing the DNA of the binding units mentioned above (fig.12,13), in addition to an anti-His western blot (fig.14) to confirm if
aggregation took place. In both tests we observed a strong ladder indicating a successful positive control, but we did
not observe any bands in the 2.5-3 kDa region where we expected our peptide.

![fig12](https://static.igem.wiki/teams/5886/engineering/f12.webp)
![fig13](https://static.igem.wiki/teams/5886/engineering/f13.webp)

Figure 12: 16% Tricine Gel with Silver Stain, High and Low Contrast

![fig14](https://static.igem.wiki/teams/5886/engineering/f14.webp)

Figure 13: 16% Tricine Gel with Silver and Coomassie Stain

![fig15](https://static.igem.wiki/teams/5886/engineering/f15.webp)

Figure 14: 16% Tricine Gel for anti-His Western Blot

![fig16-2](https://static.igem.wiki/teams/5886/engineering/f16-2.webp)

Figure 15: 16% Tricine Gel with Ponceau Stain

Learn

Our team faced the problem of no visible signs of expression in the induced, noninduced, and in-vitro lanes. We proceeded to
stain with a mixture of Coomassie and silver nitrate due to evidence suggesting that peptides or proteins found that are more
more basic tend to not be as efficiently stained [with silver nitrate] in comparison to acidic counterparts
(Chevallet et al., 2006).

At this point, our team developed two major hypotheses regarding our results of the tricine gel. We speculated that the
peptides were not expressing or that they were expressing but the peptides were aggregating, increasing their mass and
appearing in a band within the 10-150kDa range. Due to our confidence in our design, and the three different ways of
expression, we decided to investigate alternative ways to test our hypothesis that the peptide was expressing, but aggregating.
Working with our advisor, Alana Chang, we chose to test our hypothesis for peptide expression through an anti-His western blot,
due to the peptide having an engineered His-tag. Our design initially included this His-tag for the purpose of purification using
nickel columns. We then ran the tricine again but with purified versions (induced and noninduced cell cultures) of PS Binding
Units A, B, and H, instead of all 10 constructs. We decided to purify to attempt to remove undesired proteins in the cell lysate.
We chose PS Binding Units A, H, and J due to their more favorable absorbency values read at 595 nm (the wavelength for proteins)
from our Bradford assay.

Utilizing our glycerol stocks, we re-streaked PS Binding Units A, H, and J on LB-Kan plates followed by inoculating 20mL of LB
media supplemented with Kanamycin with single clones derived from the plates. As done previously, we divided the cell cultures
of PS Binding Units A, H, and J in half and induced half with IPTG and left the other half noninduced. Of these 2x10 mL of
induced/noninduced cells, we made 4 cell pellets: induced for whole cell lysate, induced for purification, noninduced for whole
cell lysate, non-induced for purification. To begin purification of the respective samples, we lysed the pellets using sonication
and used the NEBExpress Ni Spin Columns Quick Start Protocol (NEB #S1427) to purify the peptides. Using our purified strains of PS
Binding Units A, H, and J we ran another 16% tricine gel, transferred the peptides/proteins to a PVDF membrane and did an anti-His
western blot to attempt to see the presence of our peptides followed by ponceau staining the membrane to visualize all transferred
peptides. The lanes in figures 14 and 15 are of unpurified induced and noninduced whole cell lysates (BLUE), as well as purified induced and
noninduced samples (RED).

Our team observed no bands present upon anti-His western blot in any of the lanes (fig.14) for the 3 constructs hinting to failure of
expression of our peptides and not potential aggregation like we hypothesized from our initial silver/Coomassie-stained tricine gel.
The ladder was observed in this blot as the antibody was purified using a His tag. Upon ponceau staining, we observed stronger banding
in the whole cell lysate lanes (fig.15) which is expected as there should be more protein present in these lanes.

With these results, our team continued to question whether expression was successful and hypothesized that 1) expression
never occurred or 2) degradation of our peptides post-expression may be occurring or 3) expression is occurring, but the
peptides are being lost due to usage of plastic pipette tips and Eppendorf tubes, as the peptide was modelled to have high
binding affinity to plastics. All 3 hypotheses result in the absence of our peptides in the anti-His blot. With the various
unknowns of whether we experienced an expression issue, or our peptides binding to plastic equipment, the team decided to
alter the design with the addition of 2 fusion proteins comprised of Super Folding Green Fluorescent Protein (BBa-k5105008)
and Small Ubiquitin-like Modifier (BBa\_25USM2Y8) derived from Brachypodium distachyon, to increase the mass so we can run
SDS-PAGE gels instead of Tricine gels, which are more time-intensive and difficult, as well as to increase solubility
respectively.

Iteration 2

Design

Having determined that our peptides likely failed to express, our team chose to modify the designs to
maximize expression. With guidance from our advisor, Alana Chang, we added a bdSUMO tag
( [BBa\_25USM2Y8](https://registry.igem.org/parts/bba-25usm2y8) |)
to improve solubility and expression of PS Binding Peptide A ( [BBa\_25BXPW4W](https://registry.igem.org/parts/bba-25bxpw4w) |), PS Binding Peptide B ( [BBa\_254S02FL](https://registry.igem.org/parts/bba-254s02fl) |), and Peptide Binding Peptide D ( [BBa\_25UCSI3M](https://registry.igem.org/parts/bba-25ucsi3m) |). We also switched to a 12x his-tag
( [BBa\_25TQ8TGW](https://registry.igem.org/parts/bba-25tw8tgw) |)
based on protocols which she introduced to us. Finally, considering the success of our contribution
(See the Contribution page for more!) we chose to include a superfolding GFP reporter (BBa\_K5105008.)
With these changes, not only is expression more feasible, but we can also cleave the bdSUMO tag from the
C-terminus to have a functional binding unit which is referred to as PS Binding Unit K ( [BBa\_25X9JV6Y](https://registry.igem.org/parts/bba-25x9jv6y) |), PS Binding Unit L ( [BBa\_25UR4P3L](https://registry.igem.org/parts/bba-25ur4p3l) |), PS Binding Unit M ( [BBa\_25R9PXSD](https://registry.igem.org/parts/bba-25r9pxsd) |). The team decided on a linear design instead of circular DNA due to time constraints.

![fig16](https://static.igem.wiki/teams/5886/engineering/f16.webp)

Figure 16: Snapgene Linear Map for BBa\_25EYHQQP

Build

3 new designs of our biological devices were constructed: PS Binding Unit K Generator, PS Binding Unit L Generator, and PS Binding Unit M Generator. All of the devices were designed by our iGEM team and then synthesized by Twist Bioscience.

Biological Devices

Our team will attempt in-vitro expression of PS Binding Unit A, B, and D using the NEBExpress Cell-free E. coli Protein Synthesis System (NEB #E5360S). This will be followed by purification using NEBExpress Ni Spin Columns (NEB #S1427). We then will utilize the SpectraMax iD5 for a kinematic assay over 10 hours to verify expression. The results will be presented at the judging session.

Test

Testing will be continued after the wiki freeze with our new iteration of binding units. First, we plan to cleave the 12x his-tag and bdSUMO tag by using a SUMO protease solution, leaving a fusion protein composed of a glycine serine linker, a PS binding peptide, and the superfolded GFP. We had primarily considered centrifugation and vacuum filtration, with centrifugation affording more control while vacuum filtration was more cost and time-efficient. We ultimately chose to do the vacuum filtration for the binding assay. To seal the Buchner funnel and filter, pressure will be applied to the surface for 30 seconds while the vacuum is on. 250 µL of HEPEs buffer solution will be added to a 15mL test tube with 9.8 mL of DI water making approximately 10 mL of a buffer solution with a pH of 7.4. Separate 10 µL, 30 µL, 50 µL, 100 µL, and 150 µL peptide stock of each PS Binding Unit will be added to a 10 mL buffer solution to test binding characteristics. To detect the yield of the PS Binding Units that bound to the microplastics a Bradford assay will be used. The result of this test will be presented at the judging session if the build process of the PS Binding Units is successful. However, based on our modelling, the vacuum filtration binding assay shows promise. This is supported in section 8 of the modeling page, which created a Markov State Machine (MSM) model that computes the converged probability of the number of peptides bound at the surface interface given unfavourable probabilities of binding and unbinding and the total number of peptides.

Future Cycles

Once we’re able to consistently express our peptides and prove they bind to microplastics, future
engineering cycles would focus on working towards a functional, safe, and effective product. To realize
a functional product, we’d need to slowly build up to implementing our solution in the human small
intestine. This would involve proving that surface display of the fusion protein is occurring, testing
our biological system in ileum-like conditions, and finding how long and the concentration at which our
chassis can colonize the gut microbiome. Making our product safe would primarily involve adding a safety
module/kill switch to prevent the bacteria from developing antimicrobial resistance once excreted.
Increasing the efficacy of our product can be done in many ways. Accurately quantifying and
characterizing microplastics requires specific technology and skills our team lacks, which hinders our
ability to accurately interpret wet lab results for binding assays. In the dry lab, our modelling team
hopes to determine association and dissociation constants between our peptides and PS, ultimately
towards computationally modelling MP-PSBP binding dynamics in the gut and allowing further optimize our peptides characteristics.

Conclusion

"Plastipeutics" represents a novel approach to mitigating the internal threat of microplastic poisoning.
By engineering a probiotic to display PBPs, we aim to create a system that aggregates microplastics in
the gut to be excreted. Our first engineering cycle has successfully established that our
computationally derived peptides bind to microplastics. With a firm understanding of focuses for future
cycles, we are well-positioned to test and refine this system into a viable therapeutic solution.

References

Alshehri, A.S., Bergman, M.T., You, F. and Hall, C.K. (2024). Biophysics-Guided Uncertainty-Aware Deep
Learning Uncovers High-Affinity Plastic-Binding Peptides. Digital Discovery, [online] (2).
doi:https://doi.org/10.1039/d4dd00219a.

Chevallet, M., et al. (2006) Silver staining of proteins in polyacrylamide gels. PMC. Available at:
https://pmc.ncbi.nlm.nih.gov/articles/PMC1971133/ (Accessed: 3 October 2025)

Deich, C., Cash, B., Sato, W., Sharon, J., Aufdembrink, L., Gaut, N.J., Heili, J., Stokes, K., Engelhart,
A.E. and Adamala, K.P. (2023). T7Max Transcription System. Journal of Biological Engineering, 17(1).
doi:https://doi.org/10.1186/s13036-023-00323-1.

Dhoriyani, J., Bergman, M. T., Hall, C. K., & You, F. (2025). Integrating biophysical modeling, quantum
computing, and AI to discover plastic-binding peptides that combat microplastic pollution. PNAS nexus,
4(1), pgae572. https://doi.org/10.1093/pnasnexus/pgae572

Emenike, E.C., Okorie, C.J., Ojeyemi, T., Egbemhenghe, A., Iwuozor, K.O., Saliu, O.D., Okoro, H.K. and
Adeniyi, A.G. (2023). From oceans to dinner plates: The impact of microplastics on human health.
Heliyon, [online] 9(10), p.e20440. doi:https://doi.org/10.1016/j.heliyon.2023.e20440.

INSTRUCTION MANUAL PURExpress ® In Vitro Protein Synthesis. (2025). [online] NEB. Available at:
https://www.neb.com/en/-/media/nebus/files/manuals/manuale6800\_e3313\_e6840\_e6850.pdf?rev=60ba1e078b384d21bf0d5bbfd00495b9&hash=9D592FD6C6E23B68E8C5A4408E25B171

Jasinski, J., Matthias Völkl, Wilde, M.V., Valérie Jérôme, Fröhlich, T., Freitag, R. and Scheibel, T.
(2024). Influence of the polymer type of a microplastic challenge on the reaction of murine cells.
Journal of Hazardous Materials, 465, pp.133280–133280.
doi:https://doi.org/10.1016/j.jhazmat.2023.133280.

Jiang, Y., Zheng, L., Lin, L., Lin, S., Xu, K., Deng, S. & Zhang, Q. (2022) Modification in Silver Staining Procedure for
Enhanced Protein Staining. BioMed Research International, 2022, Article ID 6243971. Available at: https://pmc.ncbi.nlm.nih.gov/articles/PMC9122722/ ( Accessed: 4 October 2025)

Kim, D., Kim, D., Kim, H.-K., Jeon, E., Sung, M., Sung, S.-E., Choi, J.-H., Lee, Y., Kang, K.-K., Lee, S.
and Lee, S. (2025). Organ-specific accumulation and toxicity analysis of orally administered
polyethylene terephthalate microplastics. Scientific Reports, [online] 15(1).
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Li, Y., Chen, L., Zhou, N., Chen, Y., Ling, Z. and Xiang, P. (2024). Microplastics in the human body: A
comprehensive review of exposure, distribution, migration mechanisms, and toxicity. Science of The Total
Environment, [online] 946, pp.174215–174215. doi:https://doi.org/10.1016/j.scitotenv.2024.174215.

Lynch, J.P., González-Prieto, C., Reeves, A.Z., Bae, S., Powale, U., Godbole, N.P., Tremblay, J.M.,
Schmidt, F.I., Ploegh, H.L., Kansra, V., Glickman, J.N., Leong, J.M., Shoemaker, C.B., Garrett, W.S. and
Lesser, C.F. (2023). Engineered Escherichia coli for the in situ secretion of therapeutic nanobodies in
the gut. Cell Host & Microbe, 31(4). doi:https://doi.org/10.1016/j.chom.2023.03.007.

Prata, J. C. (2023) ‘Microplastics and human health: Integrating pharmacokinetics’, Critical Reviews in
Environmental Science and Technology, 53(16), pp. 1489–1511. doi: 10.1080/10643389.2023.2195798.

Skariyachan, S., Taskeen, N., Kishore, A.P. and Krishna, B.V. (2022). Recent advances in plastic
degradation – From microbial consortia-based methods to data sciences and computational biology driven
approaches. Journal of Hazardous Materials, 426, p.128086.
doi:https://doi.org/10.1016/j.jhazmat.2021.128086.

Takahashi, S., Furusawa, H., Ueda, T. and Okahata, Y. (2013). Translation Enhancer Improves the Ribosome
Liberation from Translation Initiation. Journal of the American Chemical Society, 135(35),
pp.13096–13106. doi:https://doi.org/10.1021/ja405967h.

Wikipedia contributors. (2025, September 2). Polystyrene. In Wikipedia, The Free Encyclopedia. Retrieved
20:32, September 22, 2025, from https://en.wikipedia.org/w/index.php?title=Polystyrene&oldid=1309133961

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microplastic particles to monomers in human serum by PETase. Faraday Discussions, [online] 252.
doi:https://doi.org/10.1039/d4fd00014e.

AI Statement: We’d like to acknowledge the use of Google’s Gemini in drafting of this page, although the
final product was composed by the FSU iGEM Engineering team.

 ↑

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
{% block page\_content %}

* [Introduction](#introduction)
* [Main 1](#main 1)
  + [Sub-main 1](#submain 1)
  + [Sub-main 2](#submain 2)
  + [Sub-main 3](#submain 3)
* [Main 2](#main 2)
* [Main 3](#main 3)

Introduction

"Sed ut perspiciatis unde omnis iste natus error sit voluptatem accusantium doloremque laudantium, totam rem aperiam, eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo. Nemo enim ipsam voluptatem quia voluptas sit aspernatur aut odit aut fugit, sed quia consequuntur magni dolores eos qui ratione voluptatem sequi nesciunt. Neque porro quisquam est, qui dolorem ipsum quia dolor sit amet, consectetur, adipisci velit, sed quia non numquam eius modi tempora incidunt ut labore et dolore magnam aliquam quaerat voluptatem. Ut enim ad minima veniam, quis nostrum exercitationem ullam corporis suscipit laboriosam, nisi ut aliquid ex ea commodi consequatur? Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur?"
"Sed ut perspiciatis unde omnis iste natus error sit voluptatem accusantium doloremque laudantium, totam rem aperiam, eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo. Nemo enim ipsam voluptatem quia voluptas sit aspernatur aut odit aut fugit, sed quia consequuntur magni dolores eos qui ratione voluptatem sequi nesciunt. Neque porro quisquam est, qui dolorem ipsum quia dolor sit amet, consectetur, adipisci velit, sed quia non numquam eius modi tempora incidunt ut labore et dolore magnam aliquam quaerat voluptatem. Ut enim ad minima veniam, quis nostrum exercitationem ullam corporis suscipit laboriosam, nisi ut aliquid ex ea commodi consequatur? Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur?"

Main 1

"Sed ut perspiciatis unde omnis iste natus error sit voluptatem accusantium doloremque laudantium, totam rem aperiam, eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo. Nemo enim ipsam voluptatem quia voluptas sit aspernatur aut odit aut fugit, sed quia consequuntur magni dolores eos qui ratione voluptatem sequi nesciunt. Neque porro quisquam est, qui dolorem ipsum quia dolor sit amet, consectetur, adipisci velit, sed quia non numquam eius modi tempora incidunt ut labore et dolore magnam aliquam quaerat voluptatem. Ut enim ad minima veniam, quis nostrum exercitationem ullam corporis suscipit laboriosam, nisi ut aliquid ex ea commodi consequatur? Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur?"
"Sed ut perspiciatis unde omnis iste natus error sit voluptatem accusantium doloremque laudantium, totam rem aperiam, eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo. Nemo enim ipsam voluptatem quia voluptas sit aspernatur aut odit aut fugit, sed quia consequuntur magni dolores eos qui ratione voluptatem sequi nesciunt. Neque porro quisquam est, qui dolorem ipsum quia dolor sit amet, consectetur, adipisci velit, sed quia non numquam eius modi tempora incidunt ut labore et dolore magnam aliquam quaerat voluptatem. Ut enim ad minima veniam, quis nostrum exercitationem ullam corporis suscipit laboriosam, nisi ut aliquid ex ea commodi consequatur? Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur?"

Sub-main 1

"Sed ut perspiciatis unde omnis iste natus error sit voluptatem accusantium doloremque laudantium, totam rem aperiam, eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo. Nemo enim ipsam voluptatem quia voluptas sit aspernatur aut odit aut fugit, sed quia consequuntur magni dolores eos qui ratione voluptatem sequi nesciunt. Neque porro quisquam est, qui dolorem ipsum quia dolor sit amet, consectetur, adipisci velit, sed quia non numquam eius modi tempora incidunt ut labore et dolore magnam aliquam quaerat voluptatem. Ut enim ad minima veniam, quis nostrum exercitationem ullam corporis suscipit laboriosam, nisi ut aliquid ex ea commodi consequatur? Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur?"
"Sed ut perspiciatis unde omnis iste natus error sit voluptatem accusantium doloremque laudantium, totam rem aperiam, eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo. Nemo enim ipsam voluptatem quia voluptas sit aspernatur aut odit aut fugit, sed quia consequuntur magni dolores eos qui ratione voluptatem sequi nesciunt. Neque porro quisquam est, qui dolorem ipsum quia dolor sit amet, consectetur, adipisci velit, sed quia non numquam eius modi tempora incidunt ut labore et dolore magnam aliquam quaerat voluptatem. Ut enim ad minima veniam, quis nostrum exercitationem ullam corporis suscipit laboriosam, nisi ut aliquid ex ea commodi consequatur? Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur?"

Sub-main 2

"Sed ut perspiciatis unde omnis iste natus error sit voluptatem accusantium doloremque laudantium, totam rem aperiam, eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo. Nemo enim ipsam voluptatem quia voluptas sit aspernatur aut odit aut fugit, sed quia consequuntur magni dolores eos qui ratione voluptatem sequi nesciunt. Neque porro quisquam est, qui dolorem ipsum quia dolor sit amet, consectetur, adipisci velit, sed quia non numquam eius modi tempora incidunt ut labore et dolore magnam aliquam quaerat voluptatem. Ut enim ad minima veniam, quis nostrum exercitationem ullam corporis suscipit laboriosam, nisi ut aliquid ex ea commodi consequatur? Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur?"
"Sed ut perspiciatis unde omnis iste natus error sit voluptatem accusantium doloremque laudantium, totam rem aperiam, eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo. Nemo enim ipsam voluptatem quia voluptas sit aspernatur aut odit aut fugit, sed quia consequuntur magni dolores eos qui ratione voluptatem sequi nesciunt. Neque porro quisquam est, qui dolorem ipsum quia dolor sit amet, consectetur, adipisci velit, sed quia non numquam eius modi tempora incidunt ut labore et dolore magnam aliquam quaerat voluptatem. Ut enim ad minima veniam, quis nostrum exercitationem ullam corporis suscipit laboriosam, nisi ut aliquid ex ea commodi consequatur? Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur?"

Sub-main 3

"Sed ut perspiciatis unde omnis iste natus error sit voluptatem accusantium doloremque laudantium, totam rem aperiam, eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo. Nemo enim ipsam voluptatem quia voluptas sit aspernatur aut odit aut fugit, sed quia consequuntur magni dolores eos qui ratione voluptatem sequi nesciunt. Neque porro quisquam est, qui dolorem ipsum quia dolor sit amet, consectetur, adipisci velit, sed quia non numquam eius modi tempora incidunt ut labore et dolore magnam aliquam quaerat voluptatem. Ut enim ad minima veniam, quis nostrum exercitationem ullam corporis suscipit laboriosam, nisi ut aliquid ex ea commodi consequatur? Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur?"
"Sed ut perspiciatis unde omnis iste natus error sit voluptatem accusantium doloremque laudantium, totam rem aperiam, eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo. Nemo enim ipsam voluptatem quia voluptas sit aspernatur aut odit aut fugit, sed quia consequuntur magni dolores eos qui ratione voluptatem sequi nesciunt. Neque porro quisquam est, qui dolorem ipsum quia dolor sit amet, consectetur, adipisci velit, sed quia non numquam eius modi tempora incidunt ut labore et dolore magnam aliquam quaerat voluptatem. Ut enim ad minima veniam, quis nostrum exercitationem ullam corporis suscipit laboriosam, nisi ut aliquid ex ea commodi consequatur? Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur?"

Main 2

"Sed ut perspiciatis unde omnis iste natus error sit voluptatem accusantium doloremque laudantium, totam rem aperiam, eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo. Nemo enim ipsam voluptatem quia voluptas sit aspernatur aut odit aut fugit, sed quia consequuntur magni dolores eos qui ratione voluptatem sequi nesciunt. Neque porro quisquam est, qui dolorem ipsum quia dolor sit amet, consectetur, adipisci velit, sed quia non numquam eius modi tempora incidunt ut labore et dolore magnam aliquam quaerat voluptatem. Ut enim ad minima veniam, quis nostrum exercitationem ullam corporis suscipit laboriosam, nisi ut aliquid ex ea commodi consequatur? Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur?"
"Sed ut perspiciatis unde omnis iste natus error sit voluptatem accusantium doloremque laudantium, totam rem aperiam, eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo. Nemo enim ipsam voluptatem quia voluptas sit aspernatur aut odit aut fugit, sed quia consequuntur magni dolores eos qui ratione voluptatem sequi nesciunt. Neque porro quisquam est, qui dolorem ipsum quia dolor sit amet, consectetur, adipisci velit, sed quia non numquam eius modi tempora incidunt ut labore et dolore magnam aliquam quaerat voluptatem. Ut enim ad minima veniam, quis nostrum exercitationem ullam corporis suscipit laboriosam, nisi ut aliquid ex ea commodi consequatur? Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur?"

Main 3

"Sed ut perspiciatis unde omnis iste natus error sit voluptatem accusantium doloremque laudantium, totam rem aperiam, eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo. Nemo enim ipsam voluptatem quia voluptas sit aspernatur aut odit aut fugit, sed quia consequuntur magni dolores eos qui ratione voluptatem sequi nesciunt. Neque porro quisquam est, qui dolorem ipsum quia dolor sit amet, consectetur, adipisci velit, sed quia non numquam eius modi tempora incidunt ut labore et dolore magnam aliquam quaerat voluptatem. Ut enim ad minima veniam, quis nostrum exercitationem ullam corporis suscipit laboriosam, nisi ut aliquid ex ea commodi consequatur? Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur?"
"Sed ut perspiciatis unde omnis iste natus error sit voluptatem accusantium doloremque laudantium, totam rem aperiam, eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo. Nemo enim ipsam voluptatem quia voluptas sit aspernatur aut odit aut fugit, sed quia consequuntur magni dolores eos qui ratione voluptatem sequi nesciunt. Neque porro quisquam est, qui dolorem ipsum quia dolor sit amet, consectetur, adipisci velit, sed quia non numquam eius modi tempora incidunt ut labore et dolore magnam aliquam quaerat voluptatem. Ut enim ad minima veniam, quis nostrum exercitationem ullam corporis suscipit laboriosam, nisi ut aliquid ex ea commodi consequatur? Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur?"

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{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}The results of our year-long effort :){% endblock %}
{% block page\_content %}

# Bronze

##### To achieve bronze, we...

* Completed the Wiki, Presentation Video, Judging Form, and Judging Session
* Submitted our Project Attributions, which describes what work everyone did on the team and everyone else who also contributed
* Submitted our 3 contributions to the iGEM Community (Visio SBOL Glyphs, Characterization of T7 Max Promoter, Sito)

# Silver

##### To achieve silver, we...

* Completed all the requirements for bronze
* Demonstrated that our project completed at least one engineering cycle (Design -> Build -> Test -> Learn)
* Conducted activities that have been determined to be responsible and good for the world (Human Practices)

# Gold

##### To achieve gold, we...

* Completed all the requirements for silver
* Impressed the judges with our work towards 3 special prizes of our choice: Human Practices, Modeling, and Entrepreneurship
* Conducted integrated human practices, including stakeholder engagement that influenced our project design and implementation
* Demonstrated high-level modeling for our project that informed our design and implementation
* Developed a high-level, comprehensive business plan and/or market analysis for our project

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{% endblock %}