iGEM 2023 — Team unilausanne

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{% block title %}Notebook{% endblock %}
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[Cloning lab book](#section-cloning)
[Gene delection lab book](#section-gene)
[Biofilm lab book](#section-biofilm)
[Microplastics lab book](#section-microplastics)
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# Biofilm notebook

---

We divided our laboratory work into four different notebooks; cloning, microplastics, gene deletion and biofilm growth + measurement.
This approach allowed us to keep an accurate and detailed record of each aspect of our research.
In each of them, we illustrate the findings, procedures and results associated with each chapter of our work.

* ## Cloning lab book

* ## Gene delection lab book

* ## Biofilm lab book

* ## Microplastics lab book

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{% block title %}Engineering Success{% endblock %}
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[Overview](#section-overview)
[Rubber-binding domain](#section-rbd)

[Overview](#section-rbd-overview)
[Design](#rbd-design)
[Build](#rbd-build)
[Test](#rbd-test)
[Learn](#rbd-learn)
[Second cycle: Test](#rbd-test2)
[Second cycle: Learn](#rbd-learn2)

[Functionalization of the Curli fibers with plastic-binding peptides](#section-peptide)

[Overview](#peptide-overview)
[Design](#peptide-design)
[Build](#peptide-build)
[Test](#peptide-test)
[Learn](#peptide-learn)
[Second cycle: Design](#peptide-design2)
[Second cycle: Build](#peptide-test2)
[Second cycle: Test](#peptide-test2)
[Second cycle: Learn](#peptide-learn2)

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# Overview

---

The objective of our project is to find a way to prevent microplastics from continuing to accumulate
in the environment, as there are currently no effective solutions to this problem, we decided to develop
a biofilm capable of trapping plastic particles. The main objective was to overexpress the Curli fibers which
are the main protein component of the biofilm and then add Plastic-specific peptides and a Rubber-binding domain
to the Curli fibers.

To achieve our goal, we had to engineer and construct several elements, but also create new protocols that would allow us
to verify the identity of our constructs.

In these sections we are going to present how we went through the design-build-test-learn cycles:

* [Addition of the Rubber-binding domain](#section-rbd)
* [Addition of the plastic-binding peptides](#section-pbp)

# Rubber-binding domain

---

## Overview

![](https://static.igem.wiki/teams/4685/wiki/engineering/rubber-eng.png)

**Figure 1:** Schematic representation of the engineering cycle for the Rubber-binding domain.

After discussion with experts, we learned that particles from car tyres can also cause
serious damage to aquatic ecosystems (see section Integrated human practices).
To tackle this problem we decided to expand our project, by functionalizing our
biofilm so that it would be able to capture particles from car tyres.

## Design

![function of rbd](https://static.igem.wiki/teams/4685/wiki/results/results/results/results/rubber-general-rid.png)

**Figure 2:** Schematic representation of the Rubber-binding domain system.

In literature, we found a protein domain (Als3) that has been shown to bind
to rubber.[3](#section-ref) As the rubber domain is too big to be directly
fused to *csgA* and exported, we used a different approach. We fused a SpyTag to
*csgA* and from a second plasmid hosted in BL21 *E. coli* cells we expressed the
Rubber-binding protein fused to the SpyCatcher. These cells are lysed and
the supernatant is then added to the Curli expressing cell, in this case our
working strain PB\_002.

Hence, to make this system work, we used two different plasmids: one plasmid
contains the genes for the *csg* domain fused to the Spytag, and the second plasmid
contains the genes for the Rubber-binding protein fused to the SpyCatcher.

The plasmid pFM\_tag that contains the gene for SpyTag domain was kindly given to us by Roberto Avendaño Vega.
The plasmid that contains the genes for the Rubber-binding protein and SpyCatcher was engineered by our team.

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/results/rubber-double-rb-lc.png)

**Figure 3:** Schematic representation of the starting plasmid PN2\_013 (on the left) that
we used as a template for our engineered plasmid pFM\_RB. The main objective was to substitute
the *lacZ* gene with the *als3*, that codes for the Rubber-binding protein

## Build

We started the cloning process (see section [Experiments, Cloning protocol](https://2023.igem.wiki/unilausanne/experiments))
from the plasmid PN2\_013 that we have used as our backbone. We have also ordered
from IDT the sequence of the gene *als3* that codes for the Rubber-binding protein. The first step
was the amplification of the backbone (fragments size: 3198 bp). To do that we designed a pair of
primers that would amplify almost all the sequence of PN2\_013 except for the *lacZ* gene, that encodes for a
beta-galactosidase, that in our project is not of any interest. With a PCR we have also amplified the *als3* gene (fragments size: 1323 bp).

We continued the building process with the Gibson Assembly which joined the two previously amplified
fragments to create our engineered plasmid pFM\_RB.

The cloning process was concluded with the transformation of the plasmid in *E. coli* strain NEB5α and
plating on the medium LB + Spec50.

To check the correct assembly of the plasmid, we performed a Colony PCR. We verified the junction between the gene encode for the SpyCatcher and *als3*.
For each candidate we obtained a single band and the length of the fragment that we expected (609 kb) (see Figure 4).

![](https://static.igem.wiki/teams/4685/wiki/engineering/colonypcr-pfm-rb.png)

**Figure 4:** Result of the Colony PCR on plasmid pFM\_RB on two different candidate colonies from the Gibson Assembly.
We have obtained the band of the expected length (609 kb).

This result was confirmed also by the sequencing (see Figure 5).

![](https://static.igem.wiki/teams/4685/wiki/engineering/sequencing-rbd.png)

**Figure 5:** Alignment of the sequencing result and the map of the plasmid pFM\_RB in the region between genes *SpyCatcher* and *als3*.

After confirming the successful building we have transformed the plasmid pFM\_RB in the working strain *E. coli* BL21.

## Test

![](https://static.igem.wiki/teams/4685/wiki/engineering/test-rbd.png)

**Figure 6:** Schematic representation of the SpyTag/SpyCatcher system, after transforming the plasmids pFM\_RB
and pFM\_tag in the respective working strain. The strain BL21 that was transformed with the plasmid pFM\_RB
(coding for the Rubber-binding domain system) will be lysed and as a consequence the Rubber-binding proteins are
going to interact with the SpyTag protein present on the Curli fibers produced by the strain PB\_002 transformed
with the plasmid pFM\_tag.

Once we knew that we had obtained the correct plasmid, the next step was to test whether
the Rubber-binding protein can be correctly expressed and whether the SpyTag/SpyCatch interaction was working correctly.

As we said in the section before, the Rubber-binding system is composed of two different plasmids that have been
transformed into two different working strains. In the strain BL21 we inserted the cloned plasmid called pFM\_RB
and in our working strain PB\_002 we inserted the plasmid pFM\_tag.

As the Rubber-binding domain does not have an export tag, the cell is not capable of exporting it.
So it is necessary to lyse the BL21 cells. Once the cells are lysed, the Rubber-binding proteins can
interact with the Spytag present on the Curli fibers produced by our working strain PB\_002 (strain lacking the *csg* operon).

To test the SpyTag-SpyCatcher interaction, we followed the protocol for Bacterial Lysate SDS-PAGE Gel
(see section [Experiments](https://2023.igem.wiki/unilausanne/experiments)). Briefly the idea of this protocol was to lyse both
strains BL21 and PB\_002 and then create three different solutions: The first solution contains BL21 cell lysate
and the Rubber-binding domain-SpyCatcher protein. The second solution contains PB\_002 cell lysate and the Curli
fibers modified with the SpyTag. In the last one we would mix the two lysates.

After preparing the solutions we run the proteins on an SDS-PAGE gel. The three overexpressed protein
complexes differ in their molecular weight so we would expect to see three different bands that would
stand out from the background of the cellular proteins.

First, we decided to run the gel on a crude extract hoping for the fact
that the proteins will be enough overexpressed to detect the bands. Unfortunately, we could not identify
clear bands at the expected sizes (Figure 7).

![](https://static.igem.wiki/teams/4685/wiki/engineering/sds-rbd.png)

**Figure 7:** SDS-PAGE gel of the three different solutions containing the crude extract after the
lysing process of the strain BL21 (Rubber-binding domain system) and PB\_002 (Spytage system).
From left to right: CsgA\_Spytag, SpyCatcher-Rubber-binding protein, CsgA\_Spytag-SpyCatcher-Rubber-binding protein.

## Learn

Since the proteins were not enough overexpressed to be seen in the crude extract,
we decided to redo the process by adding a step of purification that would allow us to isolate our desired proteins.

## Second cycle: Test

To improve the result quality of the precedent SDS-PAGE, we decided to add to the protocol Bacterial Lysate SDS-PAGE Gel a
purification step, more precisely a His-tag purification with metal beads (see section [Experiments, SDS-PSGE protocol](https://2023.igem.wiki/unilausanne/experiments)).
At the base of this procedure is the interaction between metal ions and the imidazole ring of histidine.
[1](#section-ref)

Indeed, our Rubber-domain also contains a His-tag. This purification process enriches the Rubber-binding domain
(SpyCatcher protein + Rubber-binding protein) and the Rubber-binding domain bound to CsgA through the SpyTag protein
from the crude lysate. This process would allow us to hopefully see more clean bands, as the cellular proteins should
not bind to the beads. It is important to underline that this method would not work for the isolation of the CsgA-Spytag
as it does not contain a His-tag, so we used it as a negative control.

![](https://static.igem.wiki/teams/4685/wiki/engineering/sds-rbd2.png)

**Figure 8:** Picture of the SDS-PAGE of three different solutions containing the purified His-tag purification
solution after the lysing process of the strain BL21 (Rubber-binding domain system) and PB\_002 (CsgA-Spytag).
From left to right: CsgA\_Spytag, SpyCatcher-Rubber-binding protein (RBD), CsgA\_Spytag-SpyCatcher-Rubber-binding protein (RBD).

From the gel, we still cannot identify clear bands for the proteins we are looking for.
We think this result is due to an error made during the His-tag purification. In fact,
even after the purification process we still obtained several bands corresponding to cell fragments from the lysis process.

## Second cycle: Learn

Due to the lack of time it was not possible to redo the process with the His-tag purification.
From the results that we have obtained until now it is possible to conclude that we were able to
correctly assemble the plasmid that contains the Rubber-binding domain (Colony PCR and sequencing).
If we would have more time, we would repeat the purification to see if we can see SpyTag and SpyCatcher
interacting. Afterwards we would test if this biofilm can successfully capture rubber from tyres.

This is a new composite part that we added to the registry:

[BBa\_K685000](https://parts.igem.org/Part%3ABBa_K4685000)

# Functionalization of the Curli fibers with Plastic-binding peptides

---

## Overview

![](https://static.igem.wiki/teams/4685/wiki/engineering/peptide-cycle.png)

**Figure 9:** Engineering cycle. We began by designing our primers then building the
constructs through PCR and Gibson Assembly, we tested our method using an electrophoresis
gel and then learned why it did not work and started again.

In order to improve the plastic binding capacity of the biofilm, we wanted to add Plastic-binding peptides to the Curli fibers.

## Design

We identified Plastic-binding peptides from the literature.[2](#section-ref) Our aim was to fuse them to the *csgA* gene via a linker (Figure 10).

For more information check the [Design](https://2023.igem.wiki/unilausanne/design)

![](https://static.igem.wiki/teams/4685/wiki/engineering/plasmid-with-peptides.png)

**Figure 10:** Design of the plasmid pC3\_peptide after adding the linker and the Plastic-binding peptide.

To clone the DNA sequence coding for the Plastic-binding peptides into our plasmid we first decided
to design a short forward primer (PR\_010) and a long reverse primer (PR\_peptide) containing the linker,
the peptide and the overhang with the reverse primer (see Figure 11). Furthermore, we divided our plasmid into two
fragments as it was too long for one PCR (~8500 bp).

![](https://static.igem.wiki/teams/4685/wiki/engineering/peptide-primer1.png)

**Figure 11:** Primer design to include a linker and peptide into the original plasmid.
PR\_peptide contains the linker and the peptide and together with PR\_010 they will amplify the backbone.
As amplifying 8500 bp is too long we used PR\_004 and PR\_003. PR\_003 and PR\_peptide amplify one half of
the backbone and PR\_004 and PR\_010 amplify the other half of the backbone and add the peptide and linker.

## Build

After having designed our primers, we started the building part. We performed two PCRs.
The first PCR consisted of adding the peptides and amplifying one half of the plasmid from *csgG* to *csgA*
(using PR\_003 and PR\_peptide). The second PCR amplified the second half of the plasmid from *csgC* to *csgG*.
As a template we used the plasmid pC3.

## Test

To test if the two PCRs yielded fragments of the sizes we expected we performed a gel electrophoresis.

![peptide failed gel](https://static.igem.wiki/teams/4685/wiki/engineering/peptide-failed-gel.png)

**Figure 12:** Result of the gel electrophoresis of our two fragments with different clones.
The numbers (1, 2, 1\* and 2\*) represent the backbone and the letters represent the fragment containing the linker and the peptide.
The different letters represent the different peptides binding to a specific plastic type.
The numbers and letters with stars represent the fragments where we used pC3 (Curli V3) as the backbone and the others are different clones.

We expected a size of ~2200 bp for fragment 1 and a size of ~5300 bp for fragment 2.
As you can see in Figure 12, we did not get the results that we expected. For the backbone,
we got two bands and none of them were of the right size. For the fragments, some of them just
did not get amplified and some got multiple bands from which one had the right length.

## Learn

The PCRs carried out did not yield the expected fragments. Our hypothesis for those results is the following:
As the primer PR\_pepitde is really long, it has a high chance of forming homodimer, this would explain the
smallest bands we got on our gel.

## Second cycle: Design

We decided to change our design. We first created a fragment (“template”) containing the linker and the specific peptide.
To amplify this part, we designed two primers (PR\_041 and PR\_peptide\_R) and designed more primers to amplify pC3 as backbone (PR\_042 and PR\_043). The power of this technique compared to our former one is that no primer has an excessive length thus limiting homodimer formation. The primer designed to amplify pC3, the backbone (PR\_042 and PR\_043) contain a region of overhang with the primers used to amplify the template (PR\_041 and PR\_peptide\_R) in order to close the plasmid during the Gibson Assembly.

![Second set of primers with peptides](https://static.igem.wiki/teams/4685/wiki/engineering/primer-peptide2.png)

**Figure 13:** This primer design includes a template containing the linker and the specific peptide.
For this template to be amplified we used a forward primer (PR\_041) which is the same for every peptide
as it does not amplify the peptide region and a reverse primer (PR\_peptide\_R) which is peptide specific.
To amplify pC3, the backbone we use reverse primer PR\_042 and forward primer PR\_043.

## Second cycle: build

We did a 1st PCR to amplify the template and a second to amplify the backbone.
After having amplified the sequence of interest and the backbone we carried out a Gibson Assembly using pC3.

## Second cycle: Test

To test if the template containing the linker and the peptide had been correctly amplified we ran a
PCR using primers PR\_041 and PR\_peptide\_R. We were expecting a size of 149 bp which is what we got.

![gel of the site of the peptides](https://static.igem.wiki/teams/4685/wiki/engineering/peptide-gel.png)

**Figure 14:** Result of the gel electrophoresis used to determine if the template was amplified correctly.
The size of the template is 149 bp, which is what we obtained. p1-p14 stands for 14 different peptides.

By PCR we also amplified the plasmid pC3 the backbone. After having carried out the Gibson Assembly we
transformed the plasmids in the cloning strain NEB5α.

To check that our sequence contained the specific peptide and the linker we did a Colony PCR using primers
RAV\_14 and RAV\_16 and ran an electrophoresis. We expected a size of 1036 bp. We got a fragment of the corresponding size.

![peptide into pC3 gel](https://static.igem.wiki/teams/4685/wiki/engineering/peptide-into-pc3-gel.png)

**Figure 15:** Results of the gel electrophoresis carried out after Colony PCR.
We expected a plasmid size of 1036 bp and we got a fragment of the corresponding size.
Numbers indicate the peptide and letters are the different colonies from the same plate.

Unfortunately, the sequencing did not show any traces of the linker nor the peptide.
We suspected that the primers we used amplified the *csg* operon present in NEB5α cells and that we
sequenced the genome region. Therefore, we isolated the cloned plasmids and sent the plasmids for sequencing.
The results confirmed that the peptide and the linker were not present.

## Second cycle: Learn

We hypothesize that the colonies that grow after transformation contain the original pC3 that we used as template during PCR.
As the modification is only 149 bp it is impossible to differentiate in our gel the two plasmids. To prevent this from happening again,
we should digest our PCRs with Dpn1, an enzyme that digests methylated DNA. The original plasmid in NEB5α
is methylated and would be digested, leaving only the amplified non-methylated DNA. Thus, we are sure that all
transformants come from the Gibson Assembly.

In summary, we designed and made progress in building a library of plastic-binding peptides. We uploaded the peptides as parts ([BBa\_K685003](http://parts.igem.org/Part%3ABBa_K4685003),
[BBa\_K685004](http://parts.igem.org/Part%3ABBa_K4685004), [BBa\_K685005](http://parts.igem.org/Part%3ABBa_K4685005),
[BBa\_K685007](http://parts.igem.org/Part%3ABBa_K4685007), [BBa\_K685008](http://parts.igem.org/Part%3ABBa_K4685008),
[BBa\_K685010](http://parts.igem.org/Part%3ABBa_K4685010), [BBa\_K685014](http://parts.igem.org/Part%3ABBa_K4685014),
[BBa\_K685015](http://parts.igem.org/Part%3ABBa_K4685015), [BBa\_K685020](http://parts.igem.org/Part%3ABBa_K4685020),
[BBa\_K685022](http://parts.igem.org/Part%3ABBa_K4685022), [BBa\_K685023](http://parts.igem.org/Part%3ABBa_K4685023))
and their fusions to csgA as new composite part ( [BBa\_K685034](https://parts.igem.org/Part%3ABBa_K4685034), [BBa\_K685035](https://parts.igem.org/Part%3ABBa_K4685035)
[BBa\_K685036](https://parts.igem.org/Part%3ABBa_K4685036), [BBa\_K685033](https://parts.igem.org/Part%3ABBa_K4685033)
[BBa\_K685038](https://parts.igem.org/Part%3ABBa_K4685038), [BBa\_K685040](https://parts.igem.org/Part%3ABBa_K4685040),
[BBa\_K685042](https://parts.igem.org/Part%3ABBa_K4685042), [BBa\_K685037](https://parts.igem.org/Part%3ABBa_K4685037),
[BBa\_K685039](https://parts.igem.org/Part%3ABBa_K4685039), [BBa\_K685041](https://parts.igem.org/Part%3ABBa_K4685041),
[BBa\_K685043](https://parts.igem.org/Part%3ABBa_K4685043) ).
Once built, we would characterize them using the protocol we established to measure plastic capturing [Measurement page](https://2023.igem.wiki/unilausanne/measurement)

## References

1. [His-Tag | Definition & Data [WWW Document], n.d. . Cube Biotech. (accessed 10.6.23).](https://cube-biotech.com/knowledge/protein-purification/his-tag/)
2. [Woo, H., Kang, S. H., Kwon, Y., Choi, Y., Kim, J., Ha, D. H., Tanaka, M., Okochi, M., Kim, J. S., Kim, H. K., & Choi, J. (2022). Sensitive and specific capture of polystyrene and polypropylene microplastics using engineered peptide biosensors. RSC Advances, 12(13), 7680–7688.](https://doi.org/10.1039/D1RA08701K)
3. [Zhao, X., Daniels, K.J., Oh, S.-H., Green, C.B., Yeater, K.M., Soll, D.R., Hoyer, L.L., 2006. Candida albicans Als3p is required for wild-type biofilm formation on silicone elastomer surfaces. Microbiology 152, 2287–2299.](https://doi.org/10.1099/mic.0.28959-0)

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{% block title %}Experiments{% endblock %}
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https://static.igem.wiki/teams/4685/wiki/experiments/national-cancer-institute-jet-de3essi-unsplash.jpg
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[Cloning protocols](#section-cloning)
[Bacterial lysate SDS-PAGE protocols](#section-sds)
[Microplastics protocols](#section-micro)
[Biofilm protocols](#section-biofilm)
[Gel extraction protocols](#section-gel)
[Primers](#section-primers)
[Plasmids](#section-plasmids)
[Strains](#section-strains)
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# Our Protocols

We decided to divide the work in the laboratory into four clearly defined protocols. This division allows us to efficiently manage the different stages of our project. Each protocol represents a key step in our research process and is designed to address specific aspects of our project.
Below you will find each protocol explained in detail, where we outline the procedures and objectives associated with each one.

* ## Cloning protocols

* ## Bacterial lysate SDS-PAGE protocols

* ## Mircroplastics protocols

* ## Biofilm protocols

* ## Gel extraction protocol

# Primers

---

| Primer name | Sequence | Description |
| --- | --- | --- |
| PR\_001 | gcacgaagtatcattgttatccgctcacaagtcaattgttatccgctcacaattagccctgcaaagtaaactgg | PCR primer and promoter for pFM\_1300 amplification |
| PR\_002 | aattgtgagcggataacaattgacttgtgagcggataacaatgatacttcgtgcTAAATGTACGACCAGGTCCAGG | PCR primer with promoter sequence (lower case) for pFM\_1300 amplification |
| PR\_003 | CGATCAGATTGCCGTGAACC | Forward PCR primer for split pFM\_1300 amplification |
| PR\_004 | GGTACTCACATTGACGACGC | Reverse PCR primer for split pFM\_1300 amplification |
| PR\_005 | GTACTGATGAGCGGTCGCGTTGT | Primer without linker and peptide |
| PR\_006 | TCCCGCTGCAATCTTTAACG | Primer for sequencing the region between primers PR\_003 and PR\_004 |
| PR\_007 | ATGTCATTCTGCCGTTCTGC | Primer for sequencing the region between primers PR\_003 and PR\_004 |
| PR\_008 | cgctgttgagatccagttcg | Primer for sequencing the region between primers PR\_001 and PR\_002 |
| PR\_009 | AAAGTTTTGAGCCTCCCTGC | Primer for sequencing the region between primers PR\_001 and PR\_002 |
| PR\_010 | TACATCATTTGTATTACAGAAACAGGGCGCAAG | Primer with overhang |
| PR\_011 | CTCAATCGATCTGACCCAACG | Primer for sequencing the region between primer with plastics peptide |
| PR\_012 | TTGCTTCGTCTGACTTTGCC | Primer for sequencing the region between primer with plastics peptide |
| PR\_013 | TACTGATTGGGTGAACTACGATACTTCCAGAATAACGAGAAATACGCTACTCTTttgacggctagctcagtcctaggtacagtgctagcAATGTACGACCAGGTCCAGG | Primer to add constitutive promoter ([BBa\_J23100](http://parts.igem.org/Part%3ABBa_J23100)) for csgB/A/C + random sequence for overhang |
| PR\_014 | gctagcattatacctaggactgagctagctgtcaaagagtcactaagggctaactaact | Primer to add constitutive promoter (overhang) associate with PR\_015 |
| PR\_015 | ttgacagctagctcagtcctaggtataatgctagcAGATAACCTCAGGCGATAAAGC | Primer to add constitutive promoter ([BBa\_J23119](http://parts.igem.org/Part%3ABBa_J23119)) for csgD/E/F |
| PR\_016 | GGAAGTATCGTAGTTCACCCAATCAGTActgcaaagtaaactggatggc | Primer to add constitutive promoter ([BBa\_J23100](http://parts.igem.org/Part%3ABBa_J23100)) associate with PR\_015 + overhang of 20-30 bp of PR\_013 |
| PR\_017 | CCGGAGAATACGATTGCTGC | Primer for negative control amplification to delete csgA |
| PR\_018 | GCAGCAATCGTATTCTCCGGTGTATTACAGAAACAGGGCGC | Primer for negative control amplification to delete csgA |
| PR\_019 | aagggaataagggcgacacg | Primer for sequencing the region between PR\_016 and PR\_013 |
| PR\_020 | aagggaataagggcgacacg | Primer for sequencing the region between PR\_016 and PR\_013 |
| PR\_021 | TTGCTTCGTCTGACTTTGCC | Primer for sequencing region between PR\_017 and PR\_018 |
| PR\_022 | GCATATATTGATCAGGCGGGC | Primer for sequencing the region between PR\_017 and PR\_018 |
| PR\_023 | GTTACCCGTATAGTCACTTTCCC | Primer for sequencing the region between PR\_015 and PR\_014 |
| PR\_024 | TTATCACAACAATGGCCGCC | Primer for sequencing the region between PR\_015 and PR\_014 |
| PR\_025 | GAGAACTGCTGCCTGGTAGTAGATAGGTTGTTATTGAGTAAGAAGGTAtctggtaaggttgggaagcc | Primer that replace PR\_001 (primer + random sequence at the end) for the amplification of pFM\_1300 |
| PR\_026 | TACCTTCTTACTCAATAACAACCTATCTACTACCAGGCAGCAGTTCTCaattgtgagcggataacaattgacttgtgagcggataacaatgatacttcgtgcAATGTACGACCAGGTCCAGG | Primer that replace the PR\_002 (primer + promoter + random sequence at the end) for the amplification of pFM\_1300 |
| PR\_027 | TTCTTCTGACCTGTAACGAATAaattgtgagcggataacaattgacttgtgagcggataacaatgatacttcgtgcTAAATGTACGACCAGGTCCAGG | Primer that replace PR\_002 for the amplification of pFM\_1300 |
| PR\_028 | gcacgaagtatcattgttatccgctcacaagtcaattgttatccgctcacaattTATTCGTTACAGGTCAGAAGAAagccctgcaaagtaaactgg | Primer that replace PR\_001 for the amplification of pFM\_1300 |
| PR\_029 | ATGCTGCAGCAATACACCCT | Primer for the amplification of *ALS3* gene (rubber domain gene) |
| PR\_030 | GATCAGCAGAAGGGTGTATTGCTGCAGCATaccgctaccaccgctaccag | Primer for the amplification of the backbone for the rubber domain plasmid |
| PR\_031 | TGCTCAATCGTGTAGAAATCTCTTGGATCCttaCTGAACGATTACTGTATCGATACTATCTGT | Primer for the amplification of *ALS3* gene (rubber domain gene) |
| PR\_032 | GGATCCAAGAGATTTCTACACGATTGAGC | Primer for the amplification of the backbone for the rubber domain plasmid |
| PR\_033 | TAAAGCCCATCCCGACTACC | Primers for sequencing of the region between primers PR\_031 and PR\_032 |
| PR\_034 | TGTTATCGCTACCCTGTCCC | Primers for sequencing the region between primers PR\_031 and PR\_032 |
| PR\_035 | CAGGGTAAACGTGTCGCC | Primers for sequencing the region between primers PR\_029 and PR\_030 |
| PR\_036 | catcatcatcacagcagcgg | Primer for sequencing the region between primers PR\_029 and PR\_030 |
| PR\_037 | TGCTAACCTTATTCAGCGAA | Primer for sequence all the fragment (SpyCatcher + *Als3*) with PR\_036 |
| PR\_038 | TCACCGCAGGAACGAATACG | Primer for sequence all the fragment (SpyCatcher + *Als3*) with PR\_034 |
| PR\_041 | TTTGGTAACAACGCGACCGCTCATCAGTACggcagcggcggcag | Primer to amplify the template for the plasmid with the peptide |
| PR\_042 | CATCATTTGTATTACAGAAACAGGG | Primer to amplify the backbone for the plasmid with the peptide |
| PR\_043 | AGCGGTCGCGTTGTT | Primer to amplify the backbone for the plasmid with the peptide |
| PR\_PE1t | TCTGTAATACAAATGATGTAttacataacaaaagcagcccgctgaccgcggcgctgccggccgctgccgccgctgccGTACTGATGAGCGGTCGCGTTGT | Primer to amplify the fragment with the peptide PE1 (assay 1) |
| template\_PE1 | ggcagcggcggcagcggccggcagcgccgcggtcagcgggctgcttttgttatgTAA | Primer to amplify the template for PE1 (assay 2) |
| PR\_PE1\_R | GCGCCCTGTTTCTGTAATACAAATGATGTATTAcataacaaaagcagcccgctg | Primer to amplify the template for PE1 (assay 2) |
| PR\_PE2t | TCTGTAATACAAATGATGTAttacgcccagccgctggttttatgtttccacggcggcaggccgctgccgccgctgccGTACTGATGAGCGGTCGCGTTGT | Primer to amplify the fragment with the peptide PE2 (assay 1) |
| template\_PE2 | ggcagcggcggcagcggcctgccgccgtggaaacataaaaccagcggctgggcgTAA | Primer to amplify the template for PE2 (assay 2) |
| PR\_PE2\_R | GGCTTGCGCCCTGTTTCTGTAATACAAATGATGTATTAcgcccagccgctggt | Primer to amplify the template for PE2 (assay 2) |
| PR\_PE3t | TCTGTAATACAAATGATGTAttacggcagcagatagctatcgcgcagccaccacggcaggccgctgccgccgctgccGTACTGATGAGCGGTCGCGTTGT | Primer to amplify the fragment with the peptide PE3 (assay 1) |
| template\_PE3 | ggcagcggcggcagcggcctgccgtggtggctgcgcgatagctatctgctgccgTAA | Primer to amplify the template for PE3 (assay 2) |
| PR\_PE3\_R | CTTGCGCCCTGTTTCTGTAATACAAATGATGTATTAcggcagcagatagctatcgc | Primer to amplify the template for PE3 (assay 2) |
| PR\_PE4t | TCTGTAATACAAATGATGTAttacggccacggcagcggcggatgtttccaccacggccagccgctgccgccgctgccGTACTGATGAGCGGTCGCGTTGT | Primer to amplify the fragment with the peptide PE4 (assay 1) |
| template\_PE4 | ggcagcggcggcagcggctggccgtggtggaaacatccgccgctgccgtggccgtaa | Primer to amplify the template for PE4 (assay 2) |
| PR\_PE4\_R | TTGCGCCCTGTTTCTGTAATACAAATGATGTAttacggccacggcagc | Primer to amplify the template for PE4 (assay 2) |
| PR\_PE6t | TCTGTAATACAAATGATGTAttacatgcgcagaatcggatgggttttccacgccggccagccgctgccgccgctgccGTACTGATGAGCGGTCGCGTTGT | Primer to amplify the fragment of the peptide PE6 (assay 1) |
| template\_PE6 | ggcagcggcggcagcggctggccggcgtggaaaacccatccgattctgcgcatgtaa | Primer to amplify the template for PE6 (assay 2) |
| PR\_PE6\_R | GCGCCCTGTTTCTGTAATACAAATGATGTAttacatgcgcagaatcggatgg | Primer to amplify the template for PE6 (assay 2) |
| template\_PS1 | ggcagcggcggcagcggccgcgcgtttattgcgagccgccgcattaaacgcccgTAA | Primer to amplify the tamplate for PS1 (assay 2) |
| PR\_PS1\_R | CGCCCTGTTTCTGTAATACAAATGATGTATTAcgggcgtttaatgcggc | Primer to amplify the template for PS1 (assay 2) |
| PR\_PS2t | TCTGTAATACAAATGATGTAttacgggcggcgaatgcggcggctcgcaataaacgcgcggccgctgccgccgctgccGTACTGATGAGCGGTCGCGTTGT | Primer to amplify the fragment with the peptide PS2 (assay 1) |
| template\_PS2 | ggcagcggcggcagcggccgcgcgtttattgcgagccgccgcattcgccgcccgtaa | Primer to amplify the template for PS2 (assay 2) |
| PR\_PS2\_R | CGCCCTGTTTCTGTAATACAAATGATGTAttacgggcggcgaatgc | Primer to amplify the template (assay 2) |
| PR\_PS3t | TCTGTAATACAAATGATGTATTAcagtttcagcagttttttcagcagtttcagcagttttttcaggccgctgccgccgctgccGTACTGATGAGCGGTCGCGTTGT | Primer to amplify the fragment with the peptide PS3 (assay 1) |
| template\_PS3 | ggcagcggcggcagcggcctgaaaaaactgctgaaactgctgaaaaaactgctgaaactgTAA | Primer to amplify the template for PS3 (assay 2) |
| PR\_PS3\_R | CGCCCTGTTTCTGTAATACAAATGATGTATTAcagtttcagcagttttttcagcagt | Primer to amplify the template for PS3 (assay 2) |
| PR\_PS3\_R | CGCCCTGTTTCTGTAATACAAATGATGTATTAcagtttcagcagttttttcagcagt | Primer to amplify the template for PS3 (assay 2) |
| PR\_PS4t | TCTGTAATACAAATGATGTATTAgcgaattttgctctgcatggtcgggctcgcggtgctggtgccgctgccgccgctgccGTACTGATGAGCGGTCGCGTTGT | Primer to amplify the fragment with the peptide PS4 (assay 1) |
| template\_PS4 | ggcagcggcggcagcggcaccagcaccgcgagcccgaccatgcagagcaaaattcgcTAA | Primer to amplify the template for PS4 (assay 2) |
| PR\_PS4\_R | CGCCCTGTTTCTGTAATACAAATGATGTATTAgcgaattttgctctgcatggtc | Primer to amplify the template for PS4 (assay 2) |
| PR\_PS5t | TCTGTAATACAAATGATGTATTAgcgcagggtcggaaagttcgcgcgtttcagctgcgcgcgaaacgcgcggcgatagctgcccgcgcgcgggccgctgccgccgctgccGTACTGATGAGCGGTCGCGTTGT | Primer to amplify the fragment with the peptide PS5 (assay 1) |
| template\_PS5 | ggcagcggcggcagcggcccgcgcgcgggcagctatcgccgcgcgtttcgcgcgcagctgaaacgcgcgaactttccgaccctgcgcTAA | Primer to amplify the template for PS5 (assay 2) |
| PR\_PS5\_R | CGCCCTGTTTCTGTAATACAAATGATGTATTAgcgcagggtcggaaagt | Primer to amplify the template for PS5 (assay 2) |
| PR\_PP1t | TCTGTAATACAAATGATGTAttaaccagcgatattaaaagccgcagcccgcatcatcgcgccgctgccgccgctgccGTACTGATGAGCGGTCGCGTTGT | Primer to amplify the fragment with the peptide PP1 (assay 1) |
| template\_PP1 | ggcagcggcggcagcggcgcgatgatgcgggctgcggcttttaatatcgctggttaa | Primer to amplify the template for PP1 (assay 2) |
| PR\_PP1\_R | CGCCCTGTTTCTGTAATACAAATGATGTAttaaccagcgatattaaaagccgca | Primer to amplify the template for PP1 (assay 2) |
| PR\_PP3t | TCTGTAATACAAATGATGTAttacgccaggctgcccggcggcaggccgctgccgccgctgccGTACTGATGAGCGGTCGCGTTGT | Primer to amplify the fragment with the PP3 (assay 1) |
| template\_PP3 | ggcagcggcggcagcggcctgccgccgggcagcctggcgtaa | Primer to amplify the template for PP3 (assay 2) |
| PR\_PP3\_R | GCGCCCTGTTTCTGTAATACAAATGATGTAttacgccaggctgcccg | Primer to amplify the template for PP3 (assay 2) |
| PR\_PP5t | TCTGTAATACAAATGATGTAttaggtcgcgccatgcgggctcatggtgctcagaatgctgccgctgccgccgctgccGTACTGATGAGCGGTCGCGTTGT | Primer to amplify the fragment with the peptide PP5 (assay 1) |
| template\_PP5 | ggcagcggcggcagcggcagcattctgagcaccatgagcccgcatggcgcgacctaa | Primer to amplify the template for PP5 (assay 2) |
| PR\_PP5\_R | CGCCCTGTTTCTGTAATACAAATGATGTAttaggtcgcgccatgcgg | Primer to amplify the template for the PP5 (assay 2) |
| PR\_PP6t | TCTGTAATACAAATGATGTAttacagcgccggcgcggtgctatggctatatttcatgctgccgctgccgccgctgccGTACTGATGAGCGGTCGCGTTGT | Primer to amplify the fragment with peptide PP6 (assay 1) |
| template\_PP6 | ggcagcggcggcagcggcagcatgaaatatagccatagcaccgcgccggcgctgtaa | Primer to amplify the template for PP6 (assay 2) |
| PR\_PE6\_R | CGCCCTGTTTCTGTAATACAAATGATGTAttacagcgccggcgc | Primer to amplify the template for PP6 (assay 2) |

# Plasmids

---

| Plasmid name | Characteristics | Reference or source |
| --- | --- | --- |
| pFM\_1300 | Plasmid we used to clone our engineered plasmid (pFM\_indu\_ctrl, pFM\_pos\_Ctrl and pFM\_neg\_Ctrl) | Given to us by Roberto Avendaño Vega, Schaerli Lab |
| pC3 | Used as working plasmid for biofilm production | Given to us by Roberto Avendaño Vega, Schaerli Lab |
| pFM\_indu\_ctrl | IPTG inducible plasmid, with *csg* operon and ampicillin resistance | Engineered by us during this project |
| pFM\_pos\_Ctrl | Constitutive plasmid, with *csg* operon and ampicillin resistance | Engineered by us during this project |
| pFM\_neg\_Ctrl | Constitutive plasmid, without *csgA* and ampicillin resistance | Engineered by us during this project |
| PN2\_013 | Plasmid used as a template for our engineered plasmid pFM\_RB | Given to us by Roberto Avendaño Vega, Schaerli Lab |
| pFM\_RB | Plasmid that encode for the SpyCatcher protein, due to the presence of the als3 gene | Given to us by Roberto Avendaño Vega, Schaerli Lab |
| pFM\_tag | Plasmid that encode for the SpyTag protein | Given to us by Roberto Avendaño Vega, Schaerli Lab |
| pKD13 | Plasmid for gene deletion contained the FRT regions flanking a kanamycin resistance cassette that was then inserted into the genome in place of our operons | Given to us by Schaerli Lab |
| pDK46 | Plasmid for gene deletion contained the red recombinase genes that allowed recombination between our resistance cassette and the *csg* operon | Given to us by Schaerli Lab |
| pCP20 | Plasmid for gene deletion encoding for the flippase enzyme that removed our resistance cassette via recombineering of the FRT regions | Given to us by Schaerli Lab |

# Strains

---

| Name of the Strains | Characteristics | Reference or source |
| --- | --- | --- |
| *E. coli* NEB5α | Used for cloning | Schearli Lab |
| *E. coli* [PB\_002](http://parts.igem.org/Part%3ABBa_K4685044) | Used to test our plasmid, strain without *csg* operons | Created during this project |
| *E. coli* MG1655 | Used for the gene deletion project | Schearli Lab |
| *E. coli* M037 | Used as a control strain | Schearli Lab |

{% endblock %}

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Results
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[Gene delection](#section-gene)
[Capturing microplastic](#section-microplastics)
[Functionalization of the Curli fibers with Plastic-binding peptides](#section-curli)
[Functionalization of the Curli fibers with a Rubber-binding domain](#section-rubber)
[Plasmid engineering](#section-plasmid)
[Constitutive Biofilm Production and Negative control](#section-control)
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# Achievements

---

- We successfully deleted the *csg* operons from the genome of MG1655.
- We successfully overproduced Curli fibers.
- We successfully established a protocol to quantify captured microplastics.
- We successfully captured microplastics.
- We successfully cloned the Rubber-binding domain.
- We designed the addition of Plastic-binding peptides and started cloning them.

# Abstract

---

The aim of our project was to create an engineered biofilm, based on overproducing Curli fibers, to be able to capture microplastics.
In this section, we summarized our results and the problems we encountered along the way of our project.
It was not always a linear path, as we encountered several problems, but we always created new solutions and new ways to solve these problems.

# Gene deletion

---

The first step in realizing our goal was to create a working strain. Our working strain had to possess a deletion of the *csg* operons, the operons that allows the production of biofilms, so that we could test the functioning of our plasmid.
We decided to perform the *csg* operons deletion on *E. coli* strain MG1655.
We chose the technique of red lambda deletion because this method allows precise insertion or deletion of a sequence inside the *E. coli* genome.
We used three principal plasmids for performing lambda-red gene deletion. The first plasmid, pKD13, contained the FRT regions flanking a kanamycin resistance cassette that was then inserted into the genome in place of our operons. The second plasmid, pDK46, contained the red recombinase genes that allowed recombination between our resistance cassette and the *csg* operon. The red recombinases need to be induced by the addition of L-arabinose.
The third plasmid, pCP20, encoding for the flippase enzyme that removed our resistance cassette via recombineering of the FRT regions (see section [Engineering](https://2023.igem.wiki/unilausanne/engineering)).
The first step of this recombination process was to amplify the fragment of the pKD13 plasmid containing the kanamycin resistance cassette and the two FRT sites using PCR. By adding them to the primers, we incorporated the two homologous regions during the PCR amplification. We then checked the size of our fragment on a gel electrophoresis. We expected a size of 1330 bp and indeed, we obtained a fragment of this size.

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/results-1-gel.png)

**Figure 1:** Electrophoresis gel of the fragment amplified by pKD13, that contains the kanamycin cassette, the FRT sites and the homology region

We then transformed the pKD46 plasmid into our MG1655 strain, and selected the cells containing pKD46 by the ampicillin resistance.
We then proceeded with recombination by transforming the fragment containing the kanamycin resistance cassette into strain MG1655 with the pKD46 plasmid. We induced the expression of the Red genes with L-arabinose and allowed the recombination to occur.
To verify that the recombination had taken place correctly, we first performed colony PCR on four colonies, amplifying the region of the genome that contained the *csg* operons to check whether the resistance cassette had been inserted. We could see on the electrophoresis gel (Figure 2) that the fragment size corresponded to what we expected and that the resistance cassette had therefore been inserted.

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/results-2-gel.png)

**Figure 2:** Gel that verifies the insertion of the resistance cassette into the gene.

To be sure, we still decided to send the DNA fragment for sequencing. Sequencing (Figure 3) showed us that both the kanamycin resistance cassette and the two FRT regions had been inserted.

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/sequencing-1.png)

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/sequencing-2.png)

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/sequencing-3.png)

**Figure 3:** Aligning on Benchling of the sequencing result for the insertion of the selection marker (kanamycin cassette) and the two FRT sites, in the genome.

Once we were sure that we had the correct insertion of the kanamycin resistance cassette, we started the procedure for its removal from the genome, so that we would have the strain without a *csg* operon and without kanamycin resistance that would prevent us from using plasmids with kanamycin resistance in the future.
To do this, we inserted the pCP20 plasmid, coding for a flippase, into the strain containing the kanamycin resistance.
To check the success of this step, we streaked single colonies on one plate containing kanamycin and one LB only.
This allowed us to check which colonies were growing on kanamycin and which were not, so we could discard those that still had kanamycin resistance.
We isolated a strain that had the *csg* operons deleted and the kanamycin resistance cassette was removed. We named this strain PB\_002.
Since we successfully performed the deletion of *csg* operons, we decided to test the phenotype of MG1655 onto plates with Congo red (CR) and Coomassie blue (CB) to see if it produced biofilm.
We used strain M037 (a strain from the lab of our PI also containing a *csg* deletion) as a negative control. We expected that the colonies producing Curli fibers will appear red and dry, while those not producing Curli will appear white or light pink and moist (Figure 4).
The colony of our deletion strain was pink. This visual test allowed us to conclude that the gene deletion had worked correctly, and therefore we were able to create our own strain with the *csg* operons deleted that did not produce biofilm.

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/results-1-cell.png)

**Figure 4:** Colonies grown on agar plates containing in LB Congo red and Coomassie blue (to improve contrast).
A) A colony of strain M037
 B) Colony of our deletion strain MG1655 *Δcsg*. Both strains did not uptake the Congo red.

### Conclusion

In conclusion, we succeeded in deleting the *csg* operons using the red lambda recombination method, thus obtaining our working strain.
We confirmed this deletion by Sanger sequencing and phenotype analysis on the ability of our *csg* knock-out strain to form biofilm.
The creation of our working strain was essential for the continuation of our project, in particular to proceed with the testing of our plasmids.

## **Capturing microplastics**

### Curli fiber overexpression

Here we describe how we overproduced Curli fibers and then could show that we can use the resulting biofilm to capture microplastics. This part was carried out while we were constructing our deletion strain and therefore used a strain from the lab of our PI (M037), also containing a deletion of the *csg* operons.
We transformed it with pC3 - a plasmid overproducing the Curli genes (see section [Design](https://2023.igem.wiki/unilausanne/design)). We tested the phenotype of M037 and M037 pC3 onto plates with Congo Red and Coomassie Blue to see if it produced biofilm.
The use of plates with Congo red (CR 40 µg/ml) allowed us to visualize the expression of Curli fibers.
In the plates we add Coomassie blue (CB 1 µg/ml) to enhance the contrast of the Congo red staining when looking at the colonies with the light microscope.
We expected M037, does not produce the Curli fiber, due to the deletion of *csg* operons in the genome; and M037 pC3 does produce the Curli fiber, thanks to the plasmid pC3 constitutively expressing the *csg* operons.
We followed the Biofilm protocol (see section [Experiments, Biofilm protocols](https://2023.igem.wiki/unilausanne/experiments#section-biofilm)). We prepared and inoculated two different types of plates: LB CR CB for M037 strain and LB CR CB + Amp for M037 pC3 strain. After 24 hours of incubation, we expected that the colonies producing Curli fibers will appear red and dry, while those not producing Curli will appear white or light pink and moist.
The results we obtained show us that M037 strain, with the *csg* operons deletion cannot produce Curli fibers, as we can see the colony is pink (Figure 5, A). While M037 pC3 strain shows an overexpression of Curli fibers production, as we can see the colony is red (Figure 5, B).

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/results/results-2-cell.png)

**Figure 5**
A: M037 in LB Congo red Coomassie blue, colony not producing biofilm, colony is pink;
 B: M037 pC3 in LB CR CB Amp, colony is red, overexpression of Curli fibers production.

Pictures taken with a light microscope.

### Microplastics capturing capacity

Having a strain that produced biofilm, we wanted to begin to understand how we could study the interaction between biofilm and microplastics and how we could quantify how much microplastic could be captured.
As a first test to quantify the mass of captured microplastics, we tried to reproduce an experiment found in an article: [“Quantification of dry weight mass of microplastics”](https://www.sciencedirect.com/science/article/pii/S1385894720332071) . We expected the microplastics to end up as pellets, we dried and we weighed it. But this did not allow us to determine how much microplastic had been captured by the biofilm. We therefore decided to try another way.
The next idea was to cultivate the biofilm with microplastics in a multi-well plate. Hoping that, after 24 hours, we could see aggregates of microplastics at the bottom of the well. But also with this technique, we could not get satisfactory results.

Finally, by reading articles, we managed to find a [method](https://2023.igem.wiki/unilausanne/measurement) for quantifying the captured microplastics and also the biomass produced by the strains. The idea was to use a vacuum pump system and filters with pores of the size that would allow us to determine what we were interested in: the biomass produced in one case and the microplastics captured in the other.
The idea of the first test is to use the vacuum system with a filter with a certain pore size that allows us to retain the microplastics and the biofilm that has been produced. The cells that have not produced biofilm will pass through the filter, allowing us to quantify the biofilm mass produced.
The idea of the second test is almost the same, only in this case we initially want to have the microplastics captured by the biofilm as pellets, while the microplastic remaining free is present in the supernatant. We then lyse the cells so that only the captured microplastics remain attached to the filter. This allows us to quantify the ability of the biofilm to capture microplastics.
We prepared two different samples, (M037 and M037 pC3) for the two tests: the “Biomass quantification of biofilm” and the “Quantification of microplastics captured by biofilm”. For each sample, we made three replicates.
The first test allows us to quantify the biomass of biofilm produce by the two strains: M037, does not produce the Curli fiber, due to the deletion of *csg* operons in the genome; and M037 pC3 does produce the Curli fiber, thanks to the plasmid pC3 constitutively expressing the *csg* operons (Figure 6). The results prove the overexpression of Curli fibers produced by the pC3 plasmid.

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/result-1-stat.png)

**Figure 6:**  barplot of the quantification of biomass produced by M037 strain, with a deletion of the operon *csg* in the genome and M037 pC3 strain, which contains the plasmid pC3 constitutively expressing the *csg* operons (Welch two samples t-test: p-value=0.026). This significant result shows us the overexpression of Curli fibers produced by the pC3 plasmid.

The second test allows us to quantify the microplastics captured by biofilm produced by the strains.
We used two types of microplastics for the quantification: PE (polyethylene) and PP (polypropylene). Figure 7 shows us that the overexpression of Curli fiber allows us to successfully capture more microplastics.

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/results/results-2-stat.png)

**Figure 7:** The first two barplots show the ability of the strain overexpressing Curli fibers (M037 pC3) to capture PP microplastics compared to the strain with the *csg* operon deletion (M037), not producing Curli fiber (Welch two samples t-test: p-value=0.003). The next two barplots show the ability of the M037 pC3 strain to catch PE microplastic compared to the M037 strain (Welch two samples t-test: p-value=0.02).
We obtained significant results for the two types of microplastics. That shows us that the overexpression of Curli fiber allows us to capture more microplastics.

# Functionalization of the Curli fibers with Plastic-binding peptides

---

In order to further improve the plastic binding capacity of the biofilm, we wanted to add Plastic-binding peptides to the *csgA*.
To read all the details about our different attempts of doing this, check the page [Engineering success](https://2023.igem.wiki/unilausanne/engineering) . Here we summarize the most promising attempt.
We identified Plastic-binding peptides from the literature. Our aim was to fuse them to *csgA* via a linker. We PCR-amplified fragments containing the linker and the specific peptides. We were expecting a size of 149 bp, which is what we obtained (see Figure 8).

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/results/results-1-electro.png)

**Figure 8:** Result of the gel electrophoresis used to determine if the template was amplified correctly. The size of the template is 149 bp, which is what we obtained. p1-p14 stands for 14 different peptides.

By PCR we also amplified the plasmid pC3. Next, we tried to assemble the two fragments using Gibson Assembly. After transformation, we checked by Colony PCR and obtained fragments that correspond to the expected size (1036 bp) (Figure 9).

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/results/results-2-electro.png)

**Figure 9:**  Results of the gel electrophoresis carried out after Colony PCR the Gibson Assembly. We expected a plasmid size of 1036bp and we got a fragment of the corresponding size, which is what we got. Numbers indicate the different peptides and letters are the different colonies coming from the same plate.

We then sent these PCRs for sequencing.

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/results/results-2-new-seq.png)

**Figure 10:** Alignment of sequences where the linker and peptide should be. Example of the peptide PP3. On the top is our plasmid map and at the bottom is the actual sequence. The red represents the missing part on our plasmid.

Unfortunately, the linker and specific peptides were not present in the plasmid (see Figure 10). We suspected that the primers we used amplified the *csgA* present in NEB5α cells and that we sequenced the genome region. Therefore, we isolated the cloned plasmids and sent the plasmids plasmid for sequencing. The results confirmed that the peptide and the linker were not present.

# Conclusions and outlook

---

We hypothesize that the colonies that grow after transformation contain the original pC3 that we used as template during PCR. As the modification is only 149 bp it is impossible to differentiate in our gel the two plasmids.
To prevent this from happening again, we should digest our PCRs with Dpn1, an enzyme that digests methylated DNA. The original plasmid in NEB5α is methylated and would be digested, leaving only the amplified non-methylated DNA. Thus, we are sure that all transformants come from the Gibson Assembly .
After inserting the peptide sequences successfully into the plasmid we would then next test whether their catching to the specific microplastic capacity is greater than with the Curli fibers alone.

# Functionalization of the Curli fibers with a Rubber-binding domain

---

In parallel to design the plastic specific peptides we decided to extend our project and find a way to also catch the rubber particles from car tyres. In literature, we found a protein domain (Als3) that has been shown to bind to rubber. [2](#Ref)
As the rubber domain is too big to be directly fused to CsgA and exported, we used a different approach: we used a system composed of two parts, that would interact via the SpyTag/SpyCatcher system.

So we created a new composite part, in which the Rubber-binding protein is fused to the SpyCatcher and this would interact with the second part by forming a covalent bond to the SpyTag domain fused to *csgA*.
To achieve this, we used two different plasmids: one plasmid containing the *csg* genes including *csgA* fused to the SpyTag, and the second plasmid containing the genes for the Rubber-binding protein fused to the SpyCatcher (Figure 11).

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/pfm-tag-new.png)

**Figure 11:** Schematic representation of the plasmids used for the Rubber-binding functionalization.
A) plasmid pFM\_Tag coding for the *csg* genes, including *csgA* fused to SpyTag.

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/pfm-rb-new.png)

**Figure 11:** Schematic representation of the plasmids used for the Rubber-binding functionalization.
B) plasmid pFM\_RB coding for the Rubber-binding domain with the SpyCatcher

The plasmid pFM\_tag is identical to the plasmid pC3, with the only difference that the *csgA* gene fused to the SpyTag sequence. This plasmid was kindly provided by one of our assistants (Roberto Avendaño Vega).
The plasmid that contains the genes for the Rubber-binding protein fused to the SpyCatcher was engineered with success by our team. (see section [Engineering - Rubber-binding domain](https://2023.igem.wiki/unilausanne/engineering#section-rbd))

Once we obtained the correct plasmid for the synthesis of the Rubber-binding domain, that is specifically constructed for high-level expression of recombinant proteins. Our working strain PB\_002 (strain lacking the *csg* operon) will instead express the Curli fibers fused to the SpyTag.

#### Binding between the two parts

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/rbd-and-spytag.png)

**Figure 12:** Schematic representation of the SpyTag/SpyCatcher system, after transforming the plasmids pFM\_RB and pFM\_tag in the respective working strain.
The strain BL21 that was transformed with the plasmid pFM\_RB (coding for the Rubber-binding domain system) will be lysed and as a consequence the Rubber-binding proteins are going to be able to interact with the SpyTag protein present on the Curli fibers produced by the strain PB\_002 transformed with the plasmid pFM\_tag.

To test if the interaction SpyTag/SpyCatcher worked correctly we lysed the cells and run a SDS-PAGE (see section [Engineering](https://2023.igem.wiki/unilausanne/engineering)). The three overexpressed protein complexes (CsgA-SpyTag, Rubber-binding domain-SpyCatcher and the complex of the two) differ in their molecular weight so we would expect to see three different bands that would stand out from the background of the cellular proteins.
We decided to run the first SDS-PAGE directly on the crude cell lysates, hoping that the proteins would be overexpressed enough to see them clearly in the gel. Unfortunately, we see in Figure 13 it was not the case. There are bands in the expected regions, but we can be sure that they correspond to the proteins that we want to identify.

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/results/sds-page-1.png)

**Figure 13:** Picture of the SDS-PAGE result of the crude extract of three different samples: CsgA-Spytag (1), SpyCatcher-Rubber-binding domain (2) , CsgA-SpyTag + SpyCatcher-Rubber-binding domain (3)

Faced with these results, we decided to redo the process, but adding the purification step with the His-tag method. As the Rubber-binding domain contains a His-tag, this purification process would enrich the Rubber-binding domain and the Rubber-binding domain linked to the CsgA through the SpyTag-SpyCatcher interaction for the mixture of the two strains. This process would allow us to hopefully have more clean bands.

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/results/sds-2.png)

**Figure 14:**  Picture of the SDS-PAGE of three different solutions containing the purified His-tag purification solution after the lysing process of the strain BL21 (Rubber-binding domain system) and PB\_002 (CsgA-SpyTag). 1) CsgA-SpyTag, 2) SpyCatcher-Rubber-binding protein (RBD) 3) CsgA-SpyTag/SpyCatcher-Rubber-binding protein (RBD).

From the gel, we still cannot identify clear bands for the proteins we are looking for. We think this result is due to an error made during the His-tag purification.

### Conclusions and Outlook

From the results that we have obtained until now it is possible to conclude that we were able to correctly assemble the plasmid that contains the Rubber-binding domain fused to SpyCatcher (as shown by Colony PCR and sequencing). Future experiments would repeat the His-tag purification.
Next, we would test the effectiveness of this mechanism within a solution with rubber particles. In fact, it would be necessary to adapt the microplastic quantification test with the vacuum flask to the rubber particles and the two-plasmid system. We would then compare the quantity of rubber particles captured by the wild type Curli fibers and the modified Curli fibers with the Rubber-binding system.

# Plasmid engineering

---

### IPTG-inducible plasmid for Curli fibers

During our project, we also wanted to develop a plasmid that would allow us to control the overexpression of Curli fibers (the main protein component of the biofilm matrix) using Isopropyl β-d-1-thiogalactopyranoside (IPTG). After obtaining the plasmid, we wanted to transform it into our MG1655 *Δcsg* strain so that the expression of biofilm will be conditional only in the presence of IPTG induction.
We decided to create the plasmid by modifying an already existing plasmid, called pFM\_1300.[1](#Ref) This plasmid encodes the *csg* operons (containing *csgABC* and *csgDEF* genes), with promoters that respond to light and IPTG, respectively. There is also the *lacI* sequence, necessary for IPTG induction. We wanted to replace the light-inducible promoter with an IPTG-inducible promoter.

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/results/pfm-pos-ctrl.png)

**Figure 15:**  Schematic representation of construction of pFM\_indu\_Ctrl, by replacing the light inducible promoter by the Plac IPTG inducible promoter.

To build the plasmid, we used the cloning protocol (see section [Experiments](https://2023.igem.wiki/unilausanne/experiments)). The template for the construction of pFM\_indu\_Ctrl was pFM\_1300. The first step of the process was to do two PCRs on pFM\_1300 to obtain two fragments. The first one would contain the Plac promoter followed by the gene *cgsABC* and the gene for the ampicillin resistance (fragments size: 3884 bp). The second fragment instead would contain the genes *cgsEFG* (fragment size: 4384 bp)
To confirm the correct amplification of the two fragments necessary for assembling pFM\_indu\_Ctrl, we run an electrophoresis (See Figure 16). We successfully obtained the correct fragments since their size corresponds to the expected sizes.

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/results/gel-plasmid-engineering.jpeg)

**Figure 16:**  Result of gel electrophoresis of the PCR on the plasmid pFM\_1300 to amplify the fragments needed for the assembling of pFM\_indu\_Ctrl.

After obtaining the correct fragments, we performed a Gibson Assembly to create the pFM\_indu\_Ctrl plasmid. Then, we transformed the Gibson Assembly mix by electroporation into *E. coli* NEB5ɑ and plated the cells to obtain individual transformants. As the last step, we did a Colony PCR to select for a transformant with correctly assembled plasmid. Unfortunately with the Colony PCR, we did not obtain the results that we were hoping for. For the first junction (region after the *csgG* gene) we have obtained the expected length, but for the second junction (region that contains the Plac) we have obtained two bands instead of one (see Figure 17).

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/results/gel-2-engigneering.png)

**Figure 17:** Result of gel electrophoresis of the Colony PCR on two plasmid pMF\_indu\_Ctrl taken from two different colonies and plasmid pMF\_1300 from one Colony PCR using the sequencing primers.
From left to right: The first three bands correspond to the first junction that we have tested on pMF\_indu\_Ctrl candidate n°1, pMF\_indu\_Ctrl candidate n°1 and pFM\_1300. The last three bands correspond to the second junction that we have tested on candidate n°1, pMF\_indu\_Ctrl candidate n°1 and pFM\_1300. For the first junction we have obtained the correct length, while for the second junction we have obtained two bands instead of one.

We hypothesized that the problem was caused by the primers. There was a possibility that the primers that were responsible to amplify the junction in the pLac region, were capable of attaching to multiple sites on the plasmid or even to the genome of *E. coli* strain NEB5ɑ.
We decided to redesign a new couple of sequencing primers. We also decided that to identify the correct plasmid, we would extract the plasmid from the colonies with a Miniprep instead of doing a Colony PCR and then directly send for Sanger sequencing with the new primers. In this way, we could avoid possible attachments of the primers to the *E. coli* genome.

This time, we received a positive result from one of the candidates that we sent for sequencing (see Figure 18), indicating that we successfully inserted the Plac promoter.

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/results/pfm-indu-ctrl-sequencing.png)

**Figure 18:** Aligning on Benchling the Sanger sequencing result for the pFM\_indu\_Ctrl sequencing result and the map of pFM\_indu\_Ctrl.

We also sent our plasmid for full plasmid sequencing to be completely sure that our plasmid is correct and contains all the necessary genes.
Unfortunately, from these sequencing results it seemed that the genes *csgB*, *csgA* and *lacI* were missing.
Our engineered plasmid pFM\_indu\_Ctrl seemed to be incapable of producing biofilm since the genes coding for the basic unit of Curli fibers are missing.

While we were waiting for the sequencing results, we decided to start testing for biofilm formation. We wanted to know whether the plasmid was IPTG inducible and whether it could produce biofilm. We used 'Qualitative characterisation of biofilm using Congo red' protocol (put link for experiments). The use of plates with Congo red (CR 40 µg/ml) allowed us to visualize the Curli fibers, a characteristic of biofilm formation. In the plates we add Coomassie blue (CB 1 µg/ml) to enhance the contrast of the Congo red staining when looking at the colonies at the light microscope. For this experiment, we included the deletion strain PB\_001 (MG1655 *Δcsg*::kan strain), and strain PB\_001 with the pFM\_indu\_Ctrl plasmid. After 24 hours, we expected that the colonies producing Curli fibers will appear red and dry, while those not producing Curli will appear pink and moist.
The results we obtained showed us that the pFM\_indu\_Ctrl plasmid enabled biofilm production.

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/results/biofilm-final.png)

**Figure 19:** left: PB\_001 in LB CR CB, not producing biofilm;
 right: PB\_001 pFM\_indu\_Ctrl in LB CR CB IPTG, producing biofilm.

### Conclusions

In this section we have described the construction of the plasmid pFM\_indu\_Ctrl. From the results it seems that the cells when transformed with our plasmid are capable of producing biofilm. Unfortunately the result of full plasmid sequencing of pFM\_indu\_Ctrl does not support this statement, since it has shown a lack of the fundamental genes for the biofilm formation.
After lengthy discussions with assistants, we came to the conclusion that we were probably not working with a single plasmid all this time, but with a mix of plasmids of different sizes and composition. Probably during the Gibson Assembly, several plasmid versions were assembled and transformed into the same colony. This hypothesis might explain the fact that despite the sequencing result, the cells are able to form the biofilm.
Faced with this, we decided not to invest more time into getting a clean version of pFM\_indu\_Ctrl, but continue with plasmid pC3 instead. This did not stop us from continuing our project and finding a way to demonstrate that the biofilm is capable of capturing microplastics and to make it specific for capturing certain types of plastics through the addition of specific peptides.
The next phases of the project were based exclusively on the pC3 plasmid.

# Constitutive Biofilm Production and Negative control

---

One of our objectives at the beginning of the project was the designing of two other plasmids: pFM\_pos\_Ctrl and pFM\_neg\_Ctrl (Figure 20).
The pFM\_pos\_Ctrl would allow the production of Curli fibers constitutively, while pFM\_neg\_Ctrl would lack one of the necessary genes for the Curli production.
For both plasmids we used as a template pFM\_1300.
To build the pFM\_pos\_Ctrl we have decided to replace the Plac promoter with two different constitutive promoters, which sequence was taken from the iGEM database (Bba\_23100 for *csgBAC* and Bba\_23119 for *csgDEF*)

![](https://static.igem.wiki/teams/4685/wiki/results/results/results/results/negative-positive.png)

**Figure 20:** Schematic representation of plasmid pFM\_pos\_Ctrl (A) and of plasmid pFM\_neg\_Ctrl (B)

Instead for the construction of the pFM\_neg\_Ctrl we have not only replaced the pLac promoters with constitutive ones, but we have also deleted the gene *csgA*, so it will not produce the Curli fibers.
To build the two plasmids we have followed the Cloning protocol (see section [Experiments](https://2023.igem.wiki/unilausanne/experiments#section-cloning)). Unfortunately, we were not able to clone these two plasmids. We faced several challenges as documented in our notebook. One of the problem was that the primers we used, also bound to the *csg* operon of our cloning strain NEB5α. A potential solution would be to carry out the cloning in our working strain with the *csg* operon deleted. However, as these plasmids were not absolutely essential to continue our project, we decided to not invest more time in creating them and instead focused on demonstrating the plastic capturing capability of our biofilm.

## References

1. [Moser, F., Tham, E., González, L.M., Lu, T.K., Voigt, C.A., 2019. Light-Controlled, High-Resolution Patterning of Living Engineered Bacteria Onto Textiles, Ceramics, and Plastic. Advanced Functional Materials 29, 1901788.](https://doi.org/10.1002/adfm.201901788)
2. [Zhao, X., Daniels, K.J., Oh, S.-H., Green, C.B., Yeater, K.M., Soll, D.R., Hoyer, L.L., 2006. *Candida albicans* Als3p is required for wild-type biofilm formation on silicone elastomer surfaces. Microbiology 152, 2287–2299.](https://doi.org/10.1099/mic.0.28959-0)

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