iGEM 2025 — Team uclouvain

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Notebook

## Our Laboratory Logbook

Here you will find the detailed progress of our experiments carried out in the laboratory.
This document gathers our protocols, observations, and the evolution of our experimental project week by week.

You can view it below or download it using the button.

[Download the Notebook (PDF)](https://static.igem.wiki/teams/5897/pdf/notebook.pdf)
[Back to top](#top)

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{% block title %}Engineering{% endblock %}
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Our iterative engineering cycle — Design → Build → Test → Learn
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On this page

 [**Module 1**  - Division of labour & HMO production](#mod-hmo)
 [**Module 2**  - Controlled aggregation](#mod-agg)
 [**Module 3**  - Metabolic interdependence](#mod-meta)

Our project unfolds through three dynamic modules, each composed of key steps that build on one another.
At its core lies the engineering cycle (*Design, Build, Test, Learn*) which we have embedded as a driving
force behind every stage. Each iteration within every module has influenced the others, sparking innovation,
refining our approach, and shaping the project into its final form. Because learning never stops, and
improvement is always within reach.

![Engineering cycle illustration](https://static.igem.wiki/teams/5897/images/prot-ingen.webp)

## Division of labour & HMO production

As a proof of concept, we began by concentrating on one HMO family, the fucosylated HMOs. To access the fucose building blocks, the first step would be to depolymerize fucoidan into fucose monomers using a surface-displayed fucanase, thereby avoiding the need to transport large polysaccharides across the cell membrane. In parallel, a second subpopulation would be engineered to express both a fucose permease and a fucosyltransferase, a combination expected to enable the first steps of HMO biosynthesis, with room for future optimization.

Module 1

Iteration 1
▶

#### Design

The first step of HMO production consists in converting fucoidan into fucose monomers. Therefore, we designed a genetic construction to try to express a fucanase to be displayed on the cell surface using an appropriate signal sequence. We incorporated an antibiotic resistance cassette in the construct for selection, which we flanked with *loxP* sites to excise it later from the strain.

#### Build

We ordered the DNA sequence and introduced it naturally into *S. thermophilus*. We selected the bacteria that had incorporated the fragment by plating them on antibiotic containing medium. We then re-isolated a clone by replating, amplified the region where the insertion had occurred by PCR and confirmed the construct by Sanger sequencing.

#### Test

We used the L-fucose assay kit to detect the presence of fucose. Only one test was done. To detect fucose, we used the Tecan Spark spectrophotometer.

#### Learn

We didn’t detect any fucose. This led us to different hypotheses; First, It is possible that our fucanase is not displayed at the surface of bacteria. This could be due to a degradation by HtrA since we used a strain in which HtrA was still present. The second hypothesis could be that our fucanase is displayed at the surface but not active due to different conditions.

![L-fucose detection kit first attempt](https://static.igem.wiki/teams/5897/images/lab/fuc1-2.avif)

Highlight of the control curves for the L-fucose detection kit first attempt. In blue, the kit reaction with given fucose, in green, culture medium with 12ng/mL of fucoidan, in red, growth medium only

![L-fucose detection kit first attempt](https://static.igem.wiki/teams/5897/images/lab/fuc2-2.avif)

Highlight of the fucoidan concentrations for the L-fucose kit first attempt. We can observe a more intense absorbance at 340nm depending on the fucoidan concentration

The only primary result coming from these graphs is the fact that fucoidan absorb at 340nm, probably because this polymer traps ions from the extraction from *Undaria pinnatifida*.
This absorption should not impact our usage of the kit since we do not go over the detection spectrum of our machine (abs = 2) and the information we need is the slope of the curves. For the next graph, the representation of the slope (derivative) should be preferred.

## Controlled Aggregation

Controlled aggregation is expected to enhance the efficiency of HMOs production. By physically bringing the subpopulations closer together at the onset of production, we aim to enhance the diffusion of intermediates between them. However, simply forcing cells to aggregate can stress or even harm them. Therefore we wanted to develop a controllable cell–cell adhesion system, allowing us to promote or fine-tune aggregation only when and where it benefits production.

To achieve this, cells display either a nanobody or an α-rep targeting distinct epitopes of GFP, creating a “molecular glue” that promotes close cell–cell contact.

Module 2

Iteration 1
▶

#### Design

We began working with a strain already available in the lab, in which the three loci responsible for exopolysaccharide synthesis had been deleted. We expected that removing these surface structures would help reduce steric hindrance at the cell surface. However, we observed that this strain displayed a reduced growth rate compared to the wild type (WT). We decided to address this issue before moving forward with the strain.

#### Build

We performed 10 rounds of random evolution by continuously subculturing the same population for 10 days.

#### Test

After this adaptive evolution process, we sent the final strain for whole-genome sequencing.

#### Learn

The evolved strain showed improved growth, but sequencing revealed a duplication of one exopolysaccharide locus, suggesting that exopolysaccharides were still being produced. Since these structures appear to play an important role for the cells, we chose not to continue working with this strain.

Iteration 2
▶

#### Design

We needed to construct two distinguishable strains, in which we wanted to split the metabolic pathway later. To do so, we wanted to express cytosolic free mScarlet and mTurquoise in distinct LMD-9 cells. We incorporated an antibiotic resistance cassette in the construct for selection, which we flanked with loxP sites to excise it later from the strain.

#### Build

We ordered the DNA sequences and introduced it via natural transformation into S. thermophilus. We selected the bacteria that had incorporated the fragment by plating them on antibiotic containing medium. We then re-isolated a clone by replating, amplified the region where the insertion had occurred by PCR and confirm the construction by Sanger sequencing.

#### Test

Visualization of both fluorescent strains under an epifluorescence microscope.

#### Learn

These strains are easily distinguishable under an epifluorescence microscope, we can now incorporate the nanobody and the α-rep in these fluorescent strains.

![LMD9 transformed with mTurquoise](https://static.igem.wiki/teams/5897/images/lab/mturquoise.webp)

LMD9 transformed with mTurquoise

![LMD9 transformed with mScarlet ](https://static.igem.wiki/teams/5897/images/lab/mscarlet.webp)

LMD9 transformed with mScarlet

Iteration 3
▶

#### Design

We designed two genetic constructions to express the nanobody and the α-rep and display them at the surface of the cell using an appropriate signal sequence. We incorporated an antibiotic resistance cassette in the construct for selection, which we flanked with loxP sites to excise it later from the strain.

We also needed purified GFP to serve as a molecular glue between the partners. Fortunately, we already had a GFP-overexpressing strain in the lab. This GFP carried a C-terminal 6His tag, which facilitated its purification via an Ni-NTA column.

#### Build

We ordered the DNA sequences and introduced it via natural transformation into both mScarlet and mTurquoise producing strains. We selected the bacteria that had incorporated the fragment by plating them on antibiotic containing medium. We then re-isolated a clone by replating, amplified the region where the insertion had occurred by PCR and confirmed the construction by Sanger sequencing.

In parallel, we purified the GFP on a column of nickel and measured its concentration.

#### Test

We performed a visual aggregation test, which consists of looking at our cells under epifluorescence microscopy after incubating our two subpopulations for 30 minutes in the presence or absence of GFP. We chose to work with 10 µM of GFP as a first trial, since we didn’t know what to expect.

#### Learn

There was far too much GFP, and we observed an intense GFP background. In these conditions, it is very unlikely that our cells will aggregate, since they probably bound to different GFP molecules. We cannot draw conclusions about the efficiency of our system, but we learned that we will have to use lower GFP concentrations (nM concentrations).

![Aggregation trial with 10 µM of GFP](https://static.igem.wiki/teams/5897/images/lab/first-results.webp)

Aggregation trial with 10 µM of GFP

![Aggregation trial with 10 µM of GFP](https://static.igem.wiki/teams/5897/images/lab/first-results3.webp)

Aggregation trial with 10 µM of GFP zoom

Iteration 4
▶

#### Design

We wanted to test the functioning of our aggregation module, therefore we had to think about a suitable GFP concentration. We chose to test aggregation at 10 or 100 nM of GFP.

#### Build

Preparation of cocultures with or without GFP.

#### Test

Same visual aggregation test that in the iteration 2.

#### Learn

This primary data drew a first evidence of our aggregation. Statistics has been made to reinforce the microscopy images (see [measurments](https://2025.igem.wiki/uclouvain/measurement) ). Nevertheless, we lack proper controls and repetitions to be sure about our aggregation system. So, we planned to go back into microscopy, take more snaps of representative statistical analysis and same for control using mScarlet alpharep cocultured with mTurquoise alpharep (same with nano-body).

![Aggregation trial without GFP.](https://static.igem.wiki/teams/5897/images/lab/no-gfp.webp)

Aggregation trial without GFP

![Aggregation trial with 10 nM GFP.](https://static.igem.wiki/teams/5897/images/lab/10-gfp.webp)

Aggregation trial with 10 nM GFP

![Aggregation trial with 100 nM GFP.](https://static.igem.wiki/teams/5897/images/lab/100-gfp.webp)

Aggregation trial with 100 nM GFP.

## Metabolic interdependence

We needed a strategy to maintain a stable equilibrium between our bacterial subpopulations. Without some form of regulation, one strain could easily outgrow the other, jeopardizing the efficiency of the entire system.

Metabolic interdependency could help maintain this balance between subpopulations.
We imagined using one *S. thermophilus* strain engineered to metabolize lactose into glucose and galactose,
and another that can’t metabolize lactose but only galactose.
So, the goal would be to engineer a Gal+ LacZ− strain.
This mutual reliance would foster a stable, self-regulating consortium.

Module 3

Iteration 1
▶

#### Design

It was described in the literature that some strains of *S. thermophilus* can metabolize galactose due to spontaneous mutations. Therefore, we wanted to try to select and isolate those mutants.

#### Build

/

#### Test

We plated LMD-9 on M17gal to select spontaneous mutants.

#### Learn

We obtained spontaneous mutants, that we will use for this module!

![Aggregation trial with 10 µM of GFP](https://static.igem.wiki/teams/5897/images/lab/m17gallmd9.webp)

LMD9 on M17 galactose

![Aggregation trial with 10 µM of GFP](https://static.igem.wiki/teams/5897/images/lab/m17galhsiss4.webp)

HSISS4 on M17 galactose

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Detailed experimental design, materials, methods, and protocols used throughout our project
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On this page

[**Introduction**](#intro)
[**Materials**](#materials)
[**Methods**](#methods)
[**Protocols**](#protocols)
[**Primer List**](#primers)
[**References**](#references)

## 1. Introduction

With syntcoLAB, our goal is to provide a novel platform to produce HMOs. As a reminder, HMOs are Human Milk Oligosaccharides. Since their production in the industry, through several processes, is very difficult, we thought about using consortia of lactic acid bacteria. This is a big project that we had to divide into different parts.

First, the controlled aggregation of our bacteria (*Streptococcus thermophilus*), then the metabolic interdependence of our different strains and lastly the labour division and production of HMOs.

This year, our research mainly focused on the aggregation of our bacteria, while we also explored the other related aspects.

## 2. Materials

### 2.1 Strains

Bacterial strains and oligonucleotides used in this project are listed are in the table 1 and ??? (list of primers), respectively.

| Strain | Description | Origin |
| --- | --- | --- |
| **LMD-9** | WT *S. thermophilus* | Soumillion lab collection |
| **LMD-9 *mScarlet*** | LMD9 tRNAthr::P32-hlpA-mScarlet-spec | This study |
| **LMD-9 *mTurquoise*** | LMD9 tRNAthr::P32-hlpA-mTurquoise-spec | This study |
| **LMD-9 *mNeon*** | LMD9 tRNAthr::P32-hlpA-mNeon-spec | This study |
| **LMD-9 *fucanase1*** | LMD9 tRNAser::P32-PrtS signal peptide-Fucanase 168A-HtrA linker-PrtS CWSS-Cat | This study |
| **LMD-9 gal** | Random evolution on M17gal plates | This study |
| **LMD-9 *mScarlet nanobody*** | LMD9 tRNAthr::P32-hlpA-mScarlet-spec +   LMD9 PrtS::PrtS signal peptide-Nanobody LaG16-HtrA linker-PrtS CWSS-P32::Cat | This study |
| **LMD-9 *mTurquoise nanobody*** | LMD9 tRNAthr::P32-hlpA-mTurquoise-spec +   LMD9 PrtS::PrtS signal peptide-Nanobody LaG16-HtrA linker-PrtS CWSS-P32::Cat | This study |
| **LMD-9 *mScarlet alpha-rep*** | LMD9 tRNAthr::P32-hlpA-mScarlet-spec +   LMD9 PrtS::PrtS signal peptide-alpha-rep BGFP-HtrA linker-PrtS CWSS-P32::Cat | This study |
| **LMD-9 *mTurquoise alpha-rep*** | LMD9 tRNAthr::P32-hlpA-mTurquoise-spec +   LMD9 PrtS::PrtS signal peptide-alpha-rep BGFP-HtrA linker-PrtS CWSS-P32::Cat | This study |
| **LMD-9 ΔHtrA** | LMD9 HtrA::P32-cat | This study |
| **ExoPS-** | LMD9 Δeps Δrgp hasAB::P32-Cat | Soumillon lab collection |
| **TOP10** | WT *Escherichia coli* TOP10 | Soumillion lab collection |

*Table 1. List of strains used in our project.*

### 2.2 Media & Culture Conditions

*S. thermophilus* LMD9 and derivatives were grown at 37°C without shaking in M17 (Difco Laboratories, Detroit, MI) or in chemically deﬁned medium (CDM) (Letort, 2001) supplemented with 1% (wt/vol) glucose (M17G and CDMG, respectively). The composition of CDM is in the following table. We added spectinomycin (200 mg/mL) or chloramphenicol (5 mg/mL) when required. Solid plates inoculated with streptococcal cells were incubated anaerobically (BBL GasPak systems; Becton, Dickinson, Franklin Lakes, NJ) at 37°C.

![Composition of CDM](https://static.igem.wiki/teams/5897/images/lab/cdm.webp)

Table 2. Composition of CDM, from Letort et al, 2001.

### 2.3 Chemicals

Below is the list of chemicals used in our experiments, with their source and application.

#### Ri03

We used the peptide **Ri03** to inhibit exopolysaccharide production,
since they could interfere with our surface display system.
This peptide was purchased from ThermoFisher under the name
*5-(4-Chlorophenyl)-2-furoic acid*(CAS 41019-45-8).

#### sXIP

To induce natural competence, we used the pheromone **sXIP**(synthetic XIP); (LPYFAGCL) (purity >95%) supplied by Peptide 2.0 (Chantilly, VA) and resuspended ﬁrst in dimethylformamide (DMF) and diluted in water to reach a low DMF concentration (ﬁnal concentration of 0.02%).

#### PBS

For epifluorescence microscopy, we used **PBS** and **PBS-agar**
(PBS supplemented with 1% v/v agar) as resuspension medium and for microscopy pads.
PBS composition: 137 mM NaCl, 2.7 mM KCl, 1.8 mM KH2PO4,
10 mM Na2HPO4·H2O.

#### Fucoidan

We used fucoidan as fucose source. We used:
[Fucoidan = 95 9072-19-9](https://www.sigmaaldrich.com/BE/en/product/sigma/f8315)

Product Name : Product Number : Batch Number : Source Batch : CAS Number : MDL Number : Quality Release Date : Test Fucoidan from Undaria pinnatifida ≥95% F8315-500MG 0000467431 0000452853 9072-19-9 MFCD00131109 02 Jun 2025

## 3. Methods

### 3.1 Construct amplification

During our work a lot of polymerase chain reaction (PCR) had to be done to amplify constructions our to replace some genes by others. All PCR were performed on reaction volumes of 25 µl using the Q5 2x Master Mix (New England Biolabs, USA) or the GoTaq polymerase (see PCR protocols). From the 25 µl of PCR products, 5µl were mixed with 7µl of purple dye and loaded on a 0.8% agarose gel in 1x TAE buffer for a 25 min run at 125V after which the gel was placed in a BET bath for 15-20 minutes before the reveal.

The 20 µl of PCR products left were used for the DNA purification using either the Monarch Kit (New England Biolabs, USA) or the PEG/ethanol protocol. The purified DNA was then measured with the NanoDrop spectrophotometer and used for the natural transformation.

### 3.2 Natural transformation

*Streptococcus thermophilus* brings a huge advantage in terms of synthetic biology as it is a natural competent bacterium. This characteristic allows us to be able to work with linear and not circular DNA. This process of natural transformation was used to integrate our constructions in our bacteria. For this part the general protocol was followed. Shortly, cells were cultivated overnight in CDMg at 37°C. Then, they were transferred into milk for a little over an hour before adding the peptide allowing the competence (sXIP) and the DNA of interest. This step is followed by 3 hours of incubation at 37°C before plating the transformed bacteria at different dilutions on gM17 agar plates. This step is followed by the restreaking and cultivation in gM17 of different clones. Each big step of the transformation is followed by an overnight incubation at 37°C in the typical protocol. In our case, since the constructions are quite big after the restreaking step we had to wait for 48 hours minimum to see isolated colonies.

### 3.3 Controlled aggregation module

#### 3.3.1 HtrA deletion, Alpha-rep, Nanobody & Fluorophore amplification

Two very important proteins found at the surface of *Streptococcus thermophilus* are HtrA and PrtS. The first thing we wanted to verify was the co-lethal character of the HtrA deletion in an ExoPS- strain as we needed to be able to display our proteins on the surface of the bacteria. To do so, we used the overlap PCR which allowed us to get rid of HtrA by replacing it with a resistance cassette.

Before performing HtrA deletion, we first needed to get rid of the resistance cassette (*ΔhasAB::P32-Cat*) present in ExoPS- in order to use it for deleting HtrA and have a cleaned strain to work with. This cassette is flanked by lox sequences (lox 66 upstream and lox 71 downstream) on which we performed a loxing by transforming ExoPS- with a pGost plasmid encoding for a Cre recombinase specific to our both lox sequences, this pGhostCre is part of our lab collection. The performed loxing was failed with over 500 colonies screened. We checked the plasmid integrity using Bsph1 and Xba1 restriction enzymes. Bsph1 is predicted to cut at two sites in the plasmid, one being in the Cre recombinase gene. The agarose gel confirming this digestion showed a sole band, indicating that one of the two restriction sites was not as expected. To face this problem, we removed the resistance cassette using natural transformation by designing only homology arms, creating a recombination removing the P32-Cat. This resistance-less ExoPS- had an impaired growth by comparing it to LMD9. We so performed random evolution by cultivating the strain 10 consecutive days. The last evolved strain was sent to whole genome sequencing. From this sequencing, we saw that the eps and *hasAB* loci had reverted and that the *rgp* one was duplicated in the genome, indicating a critic role. To mimic ExoPS-, we ordered 5-(4-Chlorophenyl)-2-furoic acid, known inhibitor to *rgp* activity.

Alpha-rep and Nanobody are two of the three proteins that allow the controlled aggregation. They will each recognize a specific part of the GFP. The fluorophores are very important as our goal is to control the aggregation of bacteria that are genetically different.

The amplification of these three constructions was performed using the classical PCR protocol and specific primers (see table of primers).

#### 3.3.2 Controlled aggregation assays

Controlled aggregation was the focus of our team. For this part, we used our fluorescent LMD9 strains that we transformed with Alpha-rep and Nanobody constructions. The first step was to cultivate them in CDMg at 37°C overnight. The following day, we made sure that each strain was at the same optical density (0.05) to avoid any imbalance. After this step, we did some co-culture of strains with different colour and different proteins (e.g. mScarlet/Alpha-rep and mTurquoise/Nanobody). These co-cultures were incubated for 3 hours before the washing step with PBS. After that, we added 10 µM of GFP in the co-culture and let it at room temperature for 1h30. The GFP was first produced in E.coli thanks to a plasmid. We transformed *E.coli* with the plasmid containing the GFP via electroporation. After we induced the overexpression with arabinose. Finally, we purified it using the Protein purification protocol. To visualize our results, we used the Epifluorescence microscopy.

With the first try, the GFP concentration was too high, leading to cluster of GFP and a signal that was everywhere. Then, we tried this aggregation with 10 and 100 nM of GFP. For this try, we also changed the incubation time. We went from 3 hours at room temperature to 2 hours of incubation with GFP at 37°C. At this point, we saw a difference between the different conditions. The more GFP we added, the more aggregate our samples were. But we didn’t see any GFP and other results from our lab suggests that the bacterial envelop could be a problem for the surface display.

Therefore, in the future we will have to test the growth of bacteria along with Ri03 (a compound inhibiting the production of exopolysaccharides).  Before using epifluorescence microscopy, we are going to test the sedimentation of our sample. The more aggregation we have, the quicker the sedimentation will be. For this test, we will just re-suspend our bacteria and use the time laps method to see whether the sedimentation happened quicker in some sample. This test allows us to determine the conditions for an optimal sedimentation and therefore an optimal aggregation. It is a pre-test before using the epifluorescence microscopy with too many conditions.

### 3.4 Metabolic interdependency module

#### 3.4.1 LMD9 on M17 supplemented with galactose

As a part of our project is the metabolic interdependence, we thought about using auxotrophy which has the advantage
of avoiding any antibiotics or other compounds that could be harmful for babies.
To maintain balanced co-cultures with approximately 50% of each strain,
we plan to exploit metabolic interdependence as a stabilizing strategy.
The long-term goal is to engineer a **Gal⁺ LacZ⁻ strain** that depends on its partner for growth.

To do so, we plated *LMD9* on M17 supplemented with galactose and after 72 hours we obtained a strain
that was able to grow on a medium with galactose as a carbon source.

### 3.5 Division of labour module

#### 3.5.1 Fucanase amplification & transformation

Fucanase is an enzyme allowing the break of fucoidan (Fucose polymers) into fucose monomers.
The fucose is very important in our case as it is a component of **2’FL (fucosyllactose)**,
a simple human milk oligosaccharide. Using fucoidan and not fucose directly has an economic interest
as fucose is more expensive than fucoidan.

The amplification of the fucanase was done using the typical PCR protocol
and the transformation was done using the natural transformation protocol.

The next step will be to use the **L-fucose assay kit** from Megazyme.
This assay is based on the absorbance of our sample.

## 4. Protocols

### 4.1 Transformation in *S.thermophilus*

#### Day -1

* Place the strain in culture in **1.3 mL of CDM (Chemically Defined Medium)** with **1% glucose**.

#### Day 0

1. Place **50 µL** of culture in **1 mL** of Campina semi-skimmed milk.
2. Incubate for **1 h 15 min at 37 °C**, then add **2 µL XIP** (ComR activating hormone) from the 500 µM stock and mix gently (do not vortex → invert the tubes several times).
3. Take **400 µL** in an Eppendorf tube and add **6 µL DNA of interest**.
4. Incubate for **3 h at 37 °C**.
5. Dilute from 10² to 10⁷ in milk (15 µL in 135 µL milk) on a sterile 96-well plate.
   Plate **100 µL** on M17-Glu + selection marker plate and use **3 µL** for the multi-channel dilution control.
6. Incubate plates at **37 °C in an anaerobic jar**.

#### Day 1

1. Pick **4 colonies** and streak them (2 colonies per plate).
2. Incubate plates at **37 °C in an anaerobic jar**.

#### Day 2

* Place each clone in culture in M17.

#### Day 3

* Prepare glycerol stocks: **800 µL culture + 800 µL sterile glycerol**.

### 4.2 DNA Extraction

Minimum **500 µL** is required for DNA extraction.
Therefore, from a **1300 µL liquid culture**, use **800 µL** for glycerol storage
and the rest for DNA extraction.

1. Centrifuge for **1 min at 13,000 g** → recover pellet, discard supernatant.
2. Add **400 µL TRIS-EDTA (TE 1x)** to resuspend.
3. Centrifuge for **1 min at 13,000 g** → recover pellet, discard supernatant.
4. Resuspend in **400 µL TE 1x** and homogenize.
5. Transfer the **400 µL** to a screw-cap tube + add **100 µL beads (~0.18 mm)** (cut and dip tips).
6. Fastprep at **6.5** for **1 min**.
7. Centrifuge at **16,000 g for 10 min**, then place on ice.
8. Collect **350 µL of supernatant**.
9. Store at **-20 °C** or proceed to PCR.

### 4.3 Polymerase Chain Reaction (Q5 High-Fidelity DNA Polymerase)

PCR (Polymerase Chain Reaction) is a technique used to amplify a template DNA by cycling
through denaturation, annealing, and extension phases with a thermostable DNA polymerase.

#### Reaction Mix

| Component | Volume |
| --- | --- |
| Template DNA | 1 µL |
| Forward Primer (10 µM) | 1.25 µL |
| Reverse Primer (10 µM) | 1.25 µL |
| Q5 High-Fidelity DNA Polymerase | 0.25 µL |
| Q5 Reaction Buffer (5X) | 5 µL |
| dNTP Solution Mix (10 mM) | 0.5 µL |
| Nuclease-free water | to 25 µL |

#### PCR Cycle

| Step | Temperature | Time | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 °C | 30 s | - |
| Denaturation | 98 °C | 10 s | 30× |
| Annealing | 50–72 °C | 30 s |
| Extension | 72 °C | 30 s/kb |
| Final Extension | 72 °C | 2 min | - |
| Hold | 16 °C | ∞ | - |

### 4.4 Polymerase Chain Reaction (GoTaq Polymerase)

#### Reaction Mix

| Component | Volume |
| --- | --- |
| Template DNA | 1 µL |
| MgCl₂ | 2.5 µL |
| Forward Primer (10 µM) | 1.25 µL |
| Reverse Primer (10 µM) | 1.25 µL |
| GoTaq Polymerase | 0.125 µL |
| Buffer (5X) | 5 µL |
| dNTP Solution Mix (10 mM) | 0.5 µL |
| Nuclease-free water | to 25 µL |

#### PCR Cycle

| Step | Temperature | Time | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 95 °C | 30 s | - |
| Denaturation | 95 °C | 15 s | 30× |
| Annealing | 45–68 °C | 15 s |
| Extension | 72 °C | 30 s/kb |
| Final Extension | 72 °C | 2 min | - |
| Hold | 16 °C | ∞ | - |

⚠️ The annealing temperature and the extension time may change depending respectively
on the primers and the length of the target DNA.
In our work, only these two parameters were adjusted.

### 4.5 PEG/Ethanol Purification

Polyethylene glycol (PEG) selectively precipitates high-molecular-weight DNA while leaving
small fragments (primers, dimers) in solution.

#### Reagents

* **PEG solution**: PEG 8000 20% + NaCl 2.5 M

  *Note:* PEG takes more than 20 minutes to dissolve when preparing the initial solution.
* **Cold 70–80% ethanol**
* **Sterile ultrapure water**

#### Preparation of PEG solution (for 50 mL)

1. Weigh **10.0 g PEG 8000** (MW 6000–8000 ideal).
2. Add **7.3 g NaCl**.
3. Add **qH₂O to 45 mL**.
4. Stir and let PEG dissolve. If necessary, place in incubator or water bath at 37 °C and stir regularly.
5. Once dissolved, adjust to **50 mL final volume** with qH₂O.

#### Protocol

1. Add **0.6–1 volume PEG solution** to PCR product (v/v).
2. Mix by inversion (5–10 times) or gently pipette up & down. Incubate **15 min at room temperature**.

   *Note: longer incubation (up to 2 h) can improve yield; avoid mixing during incubation.*
3. Centrifuge **15 min at 12,000–14,000 rpm** (RT or 4 °C).
4. Carefully discard supernatant without disturbing the pellet.
5. Wash pellet with **5–10 volumes cold 70% ethanol**.
6. Centrifuge **5 min at 13,000 rpm** (optionally at 4 °C).
7. Repeat steps 5–6 if higher DNA purity is required.
8. Remove ethanol and dry pellet **5–10 min at room temperature**.

   *Note: using a SpeedVac may improve DNA quality. Ensure no ethanol remains before next step.*
9. Resuspend pellet in **20–50 µL ultrapure water**.

### 4.6 Epifluorescence Microscopy & Co-culture

#### Day -1

* Prepare PBS solution: **137 mM NaCl**, **2.7 mM KCl**,
  **1.8 mM KH₂PO₄**, **10 mM Na₂HPO₄·H₂O**.
* Start overnight cultures at **37 °C** in **1.3 mL CDMg**.

#### Day 0

1. Measure optical density and dilute cultures in fresh CDMg to reach **OD = 0.05**.
2. Incubate samples at **37 °C for 4 h**.
3. Centrifuge cells and wash **2× with 500 µL PBS**. Resuspend pellet in CDMg.
4. Add **GFP** (final concentration ≤ 5 µM) and incubate at **37 °C for 2–3 h**.
5. Centrifuge cells and wash **2× with 500 µL PBS**. Resuspend pellet in **50 µL PBS**.
6. Prepare slides with PBS solution supplemented with **1.5% agarose**
   and deposit **3 µL of cells**.

### 4.7 Overexpression and Purification of Proteins

#### Day -1

1. Prepare **4 cultures of 50 mL LB medium** with *E. coli* from the cryostock.
2. Incubate cultures at **37 °C overnight**.
3. Pre-heat **2 × 1 L LB medium** at **37 °C overnight**.

#### Day 0

1. Check OD with a spectrophotometer (**900 µL LB + 100 µL culture**).
2. Dilute culture to reach **final OD = 0.08** in the **1 L LB medium**.
3. Incubate at **37 °C** until OD reaches **0.4–0.6** (~2 h).
4. Add **20 mL arabinose (50×)** to induce protein production (GFP in our case).
5. Incubate at **30 °C for 4 h**.
6. Keep **5 mL** for a miniprep and centrifuge the rest at **3900 g, 15 min**.
7. Discard supernatant and resuspend pellet in **wash buffer** (100 mM Tris + 150 mM NaCl).
8. Centrifuge **10 min at 5000 g** and discard supernatant.
9. Store pellet at **-80 °C**.

#### Day 1

1. Resuspend pellet in **20 mL wash buffer**.
2. Apply solution on a **Ni-NTA column** (for His-tagged proteins).
3. Wash with **20 mM imidazole solution** to remove non-specific binding.

   *Note:* Use Bradford reagent (**15 µL flow-through + 135 µL Bradford reagent**) to monitor washing. Continue until blue color disappears.
4. Elute protein of interest using **300 mM imidazole solution**.

### 4.8 DNA Purification using Monarch Kit (New England Biolabs)

1. Add **5 volumes Monarch Buffer BZ** (e.g., 100 µL) to **1 volume sample** (e.g., 20 µL).
   Mix well by pipetting or flicking the tube. *Do not vortex.*
2. Insert **Monarch Spin Column S1A** into the collection tube and load the sample.
   Spin **1 min**, then discard flow-through.
3. Re-insert column into the collection tube. Wash by adding **200 µL Monarch Buffer WZ** and spin **1 min**.
   *Discarding flow-through is optional.*
4. Repeat wash (step 3).
5. Transfer column to a clean **1.5 mL microfuge tube**.
6. Add **5–20 µL Monarch Buffer EY** to the center of the matrix to elute DNA.
   Wait **1 min**, then spin **1 min**.

### 4.9 Plasmid Purification — Miniprep (SMARTPURE Kit)

1. Add **1–1.5 mL overnight bacterial culture** to a 1.5 mL microcentrifuge tube.
2. Centrifuge **30 s at 10,000 × g**. Discard supernatant.
3. Repeat steps 1–2 to collect more cells.
4. Resuspend pellet completely in **250 µL Resuspension Buffer**.
5. Add **250 µL Lysis Buffer** and gently invert tube 4–6 times.
   *Do not exceed 5 min. Do not vortex to avoid genomic DNA shearing.
   Close Lysis Buffer bottle immediately after use to avoid acidification.*
6. Add **350 µL Neutralization Buffer** and mix by inverting 4–6 times until cloudy and homogeneous.
7. Centrifuge **10 min at >14,000 × g** until a compact white pellet forms.
8. Place a **SmartPure column** in a collection tube, apply supernatant, and centrifuge **30–60 s at 6,000 × g**. Discard flow-through.
9. Add **650 µL Wash Buffer** to the column. Centrifuge **30–60 s at 12,000 × g**. Discard flow-through.
10. Repeat wash (step 9).
11. Centrifuge an additional **1 min at 12,000 × g** to remove residual liquid.
12. Transfer column to a sterile **1.5 mL microcentrifuge tube**.
13. Add **50 µL Elution Buffer, ddH₂O or TE Buffer** to the column. Let stand **1 min at RT**.
14. Centrifuge **1 min at 12,000 × g** to recover purified plasmid DNA.

The purified plasmid DNA can be used directly or stored at **-20 °C** for long-term storage.

### 4.10 SDS-PAGE

1. Add **5 µL bromophenol blue** to **15 µL proteins**.
2. Incubate mixture at **98 °C for 10 min**.
3. Load sample on a **4–20% polyacrylamide gel**.
4. Run electrophoresis at **120 V for 1 h 30**.
5. Stain gel in ~**20 mL Coomassie Brilliant Blue** for at least **20 min**.

### 4.11 Plasmid Extraction

#### Day 1

1. Work under **laminar flow hood / flame sterilization**.
2. Inoculate strain of interest in **10 mL appropriate medium**
   (M17G for *L. lactis*, LB for *E. coli*) supplemented with the relevant antibiotic
   (encoded by the plasmid resistance cassette).
3. Incubate overnight at **30 °C without shaking**.

#### Day 2

1. Work under **laminar flow hood / flame sterilization**.
2. Aliquot culture into **4 × 2 mL microtubes** and centrifuge
   **2 min at 13,000 rpm, RT**. Discard supernatant.
   *Remove remaining liquid with pipette if necessary.*
3. **Outside hood/flame:** Prepare lysozyme + S1 solution (2 mg/mL) by adding
   **25 µL lysozyme (−20 °C, 20 mg/mL)** to **250 µL S1 buffer**.
4. Resuspend pellets:
   * Pellet 1 → 275 µL S1 + lysozyme
   * Pellet 2 → resuspend in suspension from Pellet 1
   * Pellet 3 → 275 µL S1 + lysozyme
   * Pellet 4 → resuspend in suspension from Pellet 3➝ Results in 2 tubes (Pellet 2 & Pellet 4).
5. Incubate **30 min at 37 °C water bath**, mixing gently every 10 min.
6. Add **500 µL S2 buffer** and mix gently (invert 6–7×).
7. Pre-cool centrifuge to **4 °C**. Incubate samples **5 min at 4 °C**.
8. Add **375 µL S3 buffer**, mix gently (invert 6–7×). Two phases should appear.
   Incubate **5 min at 4 °C**.
9. Centrifuge **10 min at 13,000 rpm, 4 °C**. Collect supernatant into 2 mL screw-cap tube.

#### Phenol–Chloroform Extraction

1. **Under chemical fume hood:** Add **1 volume phenol–chloroform–isoamyl alcohol** (~900 µL).
   Close tube tightly, seal with parafilm, vortex **20 s**.
2. Centrifuge **7 min at 13,000 rpm, RT**.
3. Recover upper aqueous phase into clean 2 mL screw-cap tube.
   *Repeat transfer twice (3× total), ending in a clean Eppendorf tube.*

#### Ethanol Precipitation

1. **Outside chemical hood:** Add **500 µL 100% ethanol (−20 °C)**, vortex 5 s, incubate **5 min at −80 °C**.
2. Centrifuge **7 min at 13,000 rpm, 4 °C**. Carefully discard supernatant.
3. Add **700 µL 70% ethanol (−20 °C)**, mix gently.
4. Centrifuge **7 min at 13,000 rpm, 4 °C**. Discard supernatant.
5. Dry pellet in **SpeedVac 30 min at 60 °C**.
6. Resuspend pellet in **20 µL sterile H₂O**.
7. Measure DNA concentration with Nanodrop, label tube, store at **−20 °C**.
8. *Repeat the procedure 2 more times (3 extractions total).*

### 4.12 Loxage Protocol

#### Day –1

1. Inoculate strains to be loxed in **1.3 mL CDM medium + appropriate antibiotic**.
2. Incubate overnight at **37 °C**.

#### Day 0

1. Transfer **50 µL culture** into **1 mL semi-skimmed Campina milk**. Incubate **1 h 15 at 37 °C**.
2. Add **10.5 µL XIP carrying the Cre plasmid**. Mix gently by inversion (do not vortex).
3. Incubate **3 h at 37 °C**.
4. Plate cultures on **Erythromycin (Ery) agar**. Incubate **24 h at 30 °C**.

#### Day 1

1. Select at least **8 isolated colonies**.
2. Resuspend each colony in **1 mL M17G broth (no antibiotic)**.
3. Incubate overnight at **37 °C**.

#### Day 2

1. Prepare serial dilutions and plate on **M17G agar without antibiotic (10⁻⁶ dilution)**.
2. Incubate overnight at **37 °C**.

#### Day 3

1. Pick **10 colonies** from each isolate.
2. Streak each colony onto 3 plates:
   * Without antibiotic
   * With Erythromycin (Cre plasmid marker)
   * With Chloramphenicol (loxed cassette marker)
3. Incubate overnight at **37 °C**.

#### Day 4

1. Compare plates.
2. Inoculate liquid M17G medium with strains that show **no resistance to either antibiotic**.
3. Incubate overnight at **37 °C**.

#### Day 5

1. Add glycerol and store strains at **−80 °C**.
2. Perform PCR to confirm cassette loss.

### 4.13 L-Fucose Assay Kit

### 5. List of Primers

#### Table 1. Primers

| ID | Primer | Sequence (5’→3’) |
| --- | --- | --- |
| 844 | AR MD UpPrtS fwd | tcttttcagcgatttcc |
| 849 | AR MD DownPrtS rev | ctgtacaccatttctgatc |
| 853 | AR\_MD\_M039bls | ttgaagaaataactgaattttcag |
| 1028 | SA.Up\_tRNAser | cgttgtaacgtcactaacaatggaac |
| 1024 | SA.Dw\_tRNAser | gagctgcgtaagttcagagagacc |
| 1621 | CV\_tRNAthr\_DW | tgcaccctagaggagtc |
| 1690 | CV\_FP\_tRNAthr\_1\_UP | cgcattagaaaATTACTGTCCTCGGGATATG |
| 1691 | CV\_FP\_tRNAthr\_1\_DW | GACAGTAATtttctaatgcgggtgaag |
| 1692 | CV\_FP\_tRNAthr\_2\_UP | GTAACGTGAaaagttagccggcttagc |
| 1693 | CV\_FP\_tRNAthr\_2\_DW | ggctaactttTCACGTTACTAAAGGG |
| 1715 | CV\_tRNAthr\_long\_UP | CGGCCGTAACTATAACGGTC |
| 1716 | CV\_tRNAthr\_long\_DW | CCGTCCTCTTCAGCCTCTTG |
| 1723 | CV\_1692\_bis | GTAACGTGAaaagttagccggcttagctcag |
| 1736 | CV\_htra\_F | ctctagttgcgtttttcttttg |
| 1737 | CV\_htra::CAT\_1F | gaggactaaacTAAGGAAGATAAATCCCATAAG |
| 1738 | CV\_htra::CAT\_1R | CTTCCTTAgtttagtcctccttcttg |
| 1739 | CV\_htra::CAT\_2F | GTAACGTGAAttattgacaaatctgtc |
| 1740 | CV\_htra::CAT\_2R | gtcaataaTTCACGTTACTAAAGGGAATGTAG |
| 1741 | CV\_htra\_R | ctccatctttattcttgtcc |
| 1743 | MB – STER0849 DOWN rev | gtatctctcaagtatcttttgaaataagaatg |
| CV1 | CV\_DhtrA\_R | GTAACGTGAaaagttagccggcttagc |
| j1 | Cat to Ery UP R | GAGAATATTTTATATTTTTGTTCATTTGATATGCCTCCTAAATTTTTATC |
| j2 | Cat to Ery Up F | GATAAAAATTTAGGAGGCATATCAAATGAACAAAAATATAAAATATTCTC |
| j3 | Cat to Ery Dw R | CAGTCGGCATTATCTCATATTATTTCCTCCCGTTAAATAATAG |
| j4 | Cat to Ery Dw F | CTATTATTTAACGGGAGGAAATAATATGAGATAATGCCGACTGTAC |

#### Table 2. Primer Combinations

| Primers | Target / Product |
| --- | --- |
| 1715 + 1691 | Up fragment Fluorescent constructions (1542 bp) |
| 1690 + 1693 | Fluorophores amplification (NeonGreen, mScarlet, mTurquoise) (2396 bp) |
| 1692 + 1716 | Down fragment Fluorescent constructions (1088 bp), failed: replaced by 1723 |
| 1723 + 1716 | Down fragment Fluorescent constructions (1088 bp) |
| 1715 + 1716 | Overlap PCR for fluorescent cassette and homology fragments (4987 bp) |
| 1736 + 1738 | Up fragment for HtrA deletion (1393 bp), failed: replaced by CV1 |
| 1736 + CV1 | Up fragment for HtrA deletion (1395 bp) |
| 1737 + 1740 | CAT cassette (Chloramphenicol resistance) for HtrA deletion (1326 bp) |
| 1739 + 1741 | Down fragment for HtrA deletion (770 bp) |
| 1736 + 1741 | HtrA deletion |
| 983 + 1833 | Nanobody (3663 bp) and Alpha-rep (3744 bp) amplification |
| 1020 + 1024 | Fucanase amplification (4883 bp) |
| 983 + i1 | Up fragment for resistance cassette swapping (CAT → Ery) (1375 bp) |
| i2 + i3 | Erythromycin resistance gene (782 bp) |
| i4 + 1833 | Down fragment for resistance cassette swapping (CAT → Ery) (1767 bp) |
| 983 + 1833 | Overlap PCR for resistance cassette swapping (α-rep: 3831 bp, Nanobody: 3750 bp) |
| 844 + 849 | PrtS locus amplification |

## References

1. Zhang Y, Li X, Wang Z, et al. “Structural investigation of Fun168A unraveling the recognition mechanism of endo-1,3-fucanase towards sulfated fucan”. International Journal of Biological Macromolecules. 2024; Volume:132622. doi: [10.1016/j.ijbiomac.2024.132622](https://doi.org/10.1016/j.ijbiomac.2024.132622)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% set hero\_image = "https://static.igem.wiki/teams/5897/images/lab/100-gfp.webp" %}
{% block lead %}
What we have managed to achieve
{% endblock %}
{% block page\_content %}

On this page

[**Controlled aggregation**](#agg)
[— Fluorescent bacteria](#agg-fluo)
[— Aggregation assay](#agg-assay)
[**Metabolic interdependence**](#meta)
[— LMD9 on galactose](#meta-lmd9)
[**Division of labour**](#division)
[**Perspectives**](#perspectives)

## Controlled aggregation

### 1. Fluorescent bacteria

To distinguish genetically different strains during controlled aggregation assays, we generated fluorescently
tagged variants. While GFP is used as the adhesion “glue,” we constructed additional strains expressing either
**mTurquoise (blue)** or **mScarlet (red)** fluorescent proteins. This allows us to track strain identity and monitor
mixed populations in aggregation experiments.

![Schematic of fluorescence constructs](https://static.igem.wiki/teams/5897/images/lab/constru-mturquoise.webp)

**Figure 1.** Fluorescence construction (mTurquoise was also replaced by mScarlet and mNeonGreen).

![Fluorescent strain 1](https://static.igem.wiki/teams/5897/images/lab/mturquoise.webp)

![Fluorescent strain 2](https://static.igem.wiki/teams/5897/images/lab/mscarlet.webp)

**Figure 2.** LMD9 transformed with mTurquoise (left) and mScarlet (right).

### 2. Aggregation assay

To harness metabolic interdependence between genetically distinct strains, it is essential to control their
spatial organization. By minimizing the distance between complementary strains, we can ensure efficient metabolite
exchange and functional cooperation. For this purpose, we use an αRep and a nanobody recognizing GFP as adhesion
modules, enabling controlled aggregation. In addition, strains were engineered to express distinct fluorescent
markers: mTurquoise (blue) or mScarlet (red), which allow us to distinguish between different populations and
confirm their spatial proximity.

The αRep and nanobody constructs were first integrated via natural transformation in LMD9 (see natural transformation protocol). To know if our construct was integrated in the genome we used antibiotic resistance. Our construct was composed of the gene coding the protein of interest but also the gene conferring the antibiotic resistance (here to chloramphenicol).

![Integration of the constructions verification](https://static.igem.wiki/teams/5897/images/lab/boite-constru.webp)

**Figure 3.** Colonies after overnight growth on M17g-chloramphenicol (20 µg/mL). In the center: serial dilutions 10by 10, from up (100) to bottom (10-7) of each transformation performed, which are insertion of an alpharep, nanobody, fucanase in a WT LMD-9 background, from left to right. On the left: controls. A-rep, Nano, Fuca are LMD-9 which received sXIP to induce natural competence but not their respective DNA fragment. Medium is only the medium (milk) in which we performed the transformation.

At this point, we knew that our constructs were well integrated in the genome of the bacteria.
What we also wanted to verify was the spot of integration but also their length.
To do so, we did a DNA extraction after all the transformation steps.
From this DNA extraction, we did a polymerase chain reaction with primers that anneal themselves
on the specific region that we wanted to verify.

![PCR verification 1](https://static.igem.wiki/teams/5897/images/lab/gel-nano-et-alpha.webp)

**Figure 4.** Agarose gel as a proof that the constructions were inserted at the right spot in the genome and that they were completely inserted. The left band being our Nanobody construction and the right band being our Alpha-rep construction. Both band are at 5000bp.

After that, we performed a visual aggregation test, which consists of looking at our cells under epifluorescence microscopy after incubating our two subpopulations for 30 minutes in the presence or absence of GFP. The results were quite convincing. We observed aggregation between red and blue strains, but we also observed aggregation between strains that had the same colour. With an increase of GFP concentration, we observed more aggregation. We also need to be critical about our results because we couldn’t see any ring of GFP (which is the phenotype that we were expecting).

The reason why we didn’t see the GFP could also be the fact that CFP and GFP signals overlap. Thus, it might be that with the epifluorescence microscopy, the resolution isn’t high enough to distinguish CFP and GFP. For this issue, we will probably change the signal that we use from EGFP to YFP. This will allow us to potentially avoid any overlap between signals.

Due to this, we are not sure that our constructs are displayed at the surface. Thus, further controls and tests (e.g. Ri03, aggregation test with LMD9 where HtrA is deleted) will have to be done to confirm that the increase of aggregation is due to our tripartite system.

![Aggregation trial without GFP.](https://static.igem.wiki/teams/5897/images/lab/no-gfp.webp)

**Figure 5.** Epifluorescence microscopy of, in red, LMD9 nano-body and, in cyan, LMD9 α-rep. This sample does not contain any GFP.

![Aggregation trial with 10 nM GFP.](https://static.igem.wiki/teams/5897/images/lab/10-gfp.webp)

**Figure 6.** Epifluorescence microscopy of, in red, LMD9 nano-body and, in cyan, LMD9 α-rep. This sample contains 10 nM of GFP.

![Aggregation trial with 100 nM GFP.](https://static.igem.wiki/teams/5897/images/lab/100-gfp.webp)

**Figure 7.** Epifluorescence microscopy of, in red, LMD9 nano-body and, in cyan, LMD9 α-rep. This sample contains 100 nM of GFP.

After converting our microscopy images to a map and extract the coordinates of every cell, we looked after the closest cell to every cell and extracted the distance separating them. We obtained data on 12977 cells in total. By grouping our data by GFP concentration in different samples, we obtained a significant decrease in the mean distance between neighboring cells upon the increase of GFP concentration.

![Mean distance between neighboring cells grouped by GFP concentration](https://static.igem.wiki/teams/5897/images/lab/stat1-2.avif)

**Figure 8.** Mean distance between neighboring cells from 11 samples, grouped by the GFP concentration used during co-culture.

**\*\*** = p ≤ 0.01;
**\*\*\*\*** = p ≤ 0.0001;
*ns* = not significant between 0 and 10 nM of GFP.

These results indicate that upon GFP concentration increase, we observe a decrease in mean distance between neighboring cells that we can assimilate to our construction. But this does not ensure that our assumed aggregation is heterologous as expected. To further confirm this aggregation, we plot the same dataset by the identity of the origin cell (color) and the neighbor cell. This led to this plot:

![Mean distance between neighboring cells depending on the pair](https://static.igem.wiki/teams/5897/images/lab/stat2-2.avif)

**Figure 9.** Mean distance between neighboring cells depending on the pair.
“From” (columns) indicates the color of the reference cell, and “to” the color of the neighboring cell.

*n* = 12 977;
*ns* = no significant groups.

Even if this barplot shows you equivalent means, we are quite interested in its analysis. Ideally, we would want to see significant decrease in the mean distance in heterologous couples (mScarlet to mTurquoise and mTurquoise to mScarlet) or this is not the case here. The fact is that our statistics are biased by our own chassis. Indeed, “strepto” in Streptococcus thermophilus means chain. And like a chain, *S. thermophilus* cells are making a small thread of 3 to 4 cells (as observed in our microscopy results), genetically the same, implying that the neighboring cell of a cell inside a chain is, except correct aggregation, the neighbor of a cell of the same color. These data are so biased towards homogenous couples (mScarlet to mScarlet and mTurquoise to mTurquoise). Taking that into account, this non-significative barpot seems more hyping for our project.

## Division of labour

For the division of labour, the first part was to transform our strain with the fucanase construct. This transformation was successful (see figure 3). After that, we verified the insertion site and the length of our construct.

![PCR verification 2](https://static.igem.wiki/teams/5897/images/lab/gel-fuc.webp)

**Figure 10.** Agarose gel as a proof that the fucanase construction was inserted at the right spot with the right length. The fourth band at the right end of the gel is at 5000 bp which is the awaited length.

After that, we tested whether the fucanase was functional, i.e. whether it could degrade fucoidans into fucose. Unfortunately, this test did not yield conclusive results (see [Engineering](https://2025.igem.wiki/uclouvain/engineering#mod-hmo) ).

## Metabolic interdependence

### 1. Production of an LMD9 strain that can grow on galactose

To maintain balanced co-cultures with approximately 50% of each strain, we plan to exploit metabolic
interdependence as a stabilizing strategy. The long-term goal is to engineer a Gal⁺ LacZ⁻ strain that depends
on its partner for growth. At present, our construct is still Gal⁺ LacZ⁺, and future work will focus on eliminating
LacZ to enforce the desired interdependence. This approach should ensure stable coexistence of the strains and
allow robust cooperative behaviour in mixed cultures.

By plating LMD9 on M17 supplemented with galactose, we saw a colony that grew after 72 hours. We used
*Streptococcus salivarius* as a first control since this bacterium is able to grow on galactose.

![LMD9 on M17 galactose](https://static.igem.wiki/teams/5897/images/lab/m17gallmd9.webp)

![HSISS4 on M17 galactose](https://static.igem.wiki/teams/5897/images/lab/m17galhsiss4.webp)

**Figure 11.** LMD9 on M17 galactose (left) and HSISS4 on M17 galactose (right).

In the future, we will perform a PCR targeting the PrtS locus as a second control, since PrtS is quite specific to *Streptococcus thermophilus*. As a third control, could be a PCR and a sequencing of the gal locus of our single colony that we will compare to the gal locus of wild-type LMD9.

A fourth control, we will plate a fluorescent LMD9 strain; if colonies appear, we will confirm their fluorescence using an epifluorescence microscope.

## Perspectives

In the future, for the division of labour using the fucanase and for the controlled aggregation using the Alpha-rep and nanobody, we are planning to use an inhibitor of the production of exopolysaccharides. This compound is Ri03 also known as 5-(4-Chlorophenyl)-2-furoic acid. It inhibits dTDP-L-rhamnose formation. This compound will allow us to have strains with less exopolysaccharides and having less of those is potentially required for our proteins to be well displayed. Before using this compound on our strain transformed with our constructs, we will have to determine the bacterial growth in presence of Ri03. This test will allow us to determine the right concentration and conditions to have the optimal effect on our strains. For the next steps, it would be interesting to use a confocal microscope to obtain higher-quality images and thus better visualize the GFP.
We would also like to perform a sedimentation assay to gain another insight into cell aggregation. This test is carried out using a spectrophotometer — the faster the sample sediments, the more aggregated it is.
Finally, for the metabolic interdependence module, we plan to delete the β-galactosidase gene in the gal⁺ strain so that it becomes lacZ⁻.

{% endblock %}