iGEM 2024 — Team univlyon1-insalyon

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

# Notebook

*Explore our calendar by hovering over each colored day to see what we have done!*

Legend

: The Team

: Sciences

: WETLAB

: Wiki

: Communication

: Presentation video

: Financement

: Human Practices

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{% endblock %}

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

# Engineering

![](https://static.igem.wiki/teams/5143/engineering/cycle-eng.png)

**BIO Snare’s goal** was to produce functionalized cellulose to make it adaptable for
specifically attracting and trapping insects through the use of different colors and a bioglue. We were inspired by
an article [(Gilbert. *et al.*, 2021)](https://www.nature.com/articles/s41563-020-00857-5) and decided to use a coculture of the bacterium *K. rhaeticus*, which
overproduces cellulose, along with a genetically modified yeast, *S. cerevisiae*. The initial construction of
our project is **BIO** **Snare**, a plasmid containing the genes encoding a **yellow chromoprotein** and a
**bioglue**.

 To build our trap, we followed three main steps: the creation of the **BioBrick**, the
**transformation** of *S. cerevisiae* and finally the production of the functionalized **cellulose**. You
can find the corresponding enineering cycles by clicking on the step you want.

*Click on the buttons to discover the engineering cycle associated.*

---

[BioBrick](#BioBrick)

[Transformation](#Transformation)

[Cellulose](#Cellulose)

**1. Construction of the final composite Biobrick**

![Image](https://static.igem.wiki/teams/5143/engineering/design1.png)

This biobrick contains:

*Click on each one of them to learn more about what this biobrick
contains.*

* **Constitutive GAP promoter**

Drives the continuous expression of both YFP
and the bioglue.

* **fwYellow gene**

Encodes Yellow Fluorescent Protein (YFP), the
chromoprotein we selected. The sequence has been optimized for expression in *S. cerevisiae*.
The fwYellow gene is a BioBrick created by the [Uppsala iGEM team in 2013](https://parts.igem.org/Part%3ABBa_K1033910).

* **Bioglue genetic sequence**

Encodes a bioglue made from a fusion of two
natural proteins to significantly enhance adhesion properties: Masp1 from spider silk and Cp19k from
barnacles. These proteins have individual adhesion strengths of 22.2 mJ/m² and 2.2 mJ/m²,
respectively, but their fusion increases this to 39.9 mJ/m² [(Ye et al. (2023).](https://pubmed.ncbi.nlm.nih.gov/37776922/) The sequences were optimized for
expression in *S. cerevisiae*.

* **Cellulose Binding Domain (CBD)**

Each protein (both the colored and the
adhesive) contains a CBD to allow binding to cellulose. The CBD sequence was adapted from [Gilbert *et al.* (2021).](https://www.nature.com/articles/s41563-020-00857-5)

* **Alpha factor signal peptide**

Derived from the *MAT alpha* mating
system, this sequence acts as a signal peptide to facilitate protein secretion. Once secreted, the
signal peptide is cleaved, leaving only the mature protein.

* **P2A system**

Enables co-expression of both YFP and the bioglue protein
from a single mRNA transcript. This system is optimized for eukaryotic cells and allows simultaneous
synthesis of YFP and the adhesive protein in our context.

* **URA3 gene**

Selected as a marker for yeast transformation. This gene
allows the *URA-* yeast strain to synthesize uracil, enabling cells that have successfully
integrated the plasmid containing URA3 to grow in uracil-deficient medium.

* **Homologous sequences (URA5’ and URA3’)**

Facilitate plasmid integration
into the yeast chromosome via homologous recombination.

![Image](https://static.igem.wiki/teams/5143/engineering/build1.png)

1. We started by integrating the **URA3-URA3'** fragment into **Plasmid A**, which already
   contained the **URA5' fragment, GAP promoter, alpha factor, fwYellow, and CBD**. The result of
   this construction is **Plasmid C**.
   The alpha factor, fwYellow, and CBD genes had to be synthesized, as their sequence were optimized
   for *S. cerevisiae* and were not available in any strains we had access to. As a result, we
   chose to synthesize the entire plasmid. Initially, we aimed to amplify the URA3-URA3' fragment via
   PCR, but due to the presence of an additional BsaI restriction site in the organism we used for
   amplification, we decided to synthesize this fragment as well. We used the HiFi Assembly technique
   to seamlessly join the DNA fragments, ensuring the precise integration of the **URA3-URA3'
   fragment**.
2. The fragments containing the **P2A sequence**, the **alpha factor**, and the coding
   sequences of **Masp1, Cp19k, and CBD** were synthesized, the resulting plasmid is **plasmid
   B**.
3. Finally, biobricks from **plasmid C** and **plasmid B** were PCR amplified and fused using
   **HiFi assembly** resulting in **plasmid D**.

![Image](https://static.igem.wiki/teams/5143/engineering/test1.png)

The conformity of the plasmid was assessed by (i) performing **PCR** to amplify the fragment of
interest (ii) performing a **restriction map** to validate the assembly and finally (iii) by
**sequencing** to verify the conformity of the final plasmid.

![Figure 1](https://static.igem.wiki/teams/5143/team/fig-eng1.png)

![Figure 2](https://static.igem.wiki/teams/5143/engineering/eng-fig2.png)

![Image](https://static.igem.wiki/teams/5143/engineering/learn1.png)

During the process, we identified the presence of an **unexpected BsaI** site in plasmid A. In
hindsight, we realized that if we had carefully checked the sequence of synthesized Plasmid A earlier,
we could have identified the extra BsaI site **sooner and saved time**. Moving forward, we decided to
amplify the **linear form of our plasmid using PCR** instead of relying on restriction enzymes for
the next steps.

**2. Construction of the engineered *S. cerevisiae* strain**

![Image](https://static.igem.wiki/teams/5143/engineering/design1.png)

Once Plasmid D is assembled, it needs to be recombined into *S. cerevisiae* (BY4741). The
homologous regions **URA5'and URA3'** will facilitate the insertion of the plasmid into the
**yeast
chromosome**.

![Image](https://static.igem.wiki/teams/5143/engineering/build1.png)

We made *S. cerevisiae* competent using the **Lithium Acetate method**. We then transformed
the
yeast with the 6.3 kbp digestion product of **Plasmid D using XhoI**. Selection was carried out on
a
medium **lacking uracil** since the yeast strain is **URA-**. If the URA3 gene from the plasmid
is
successfully integrated into the genome, it will restore the yeast’s ability to synthesize uracil,
allowing growth on the selective medium.

![Image](https://static.igem.wiki/teams/5143/engineering/test1.png)

First, the correct chromosomal integration of our construct had to be checked. Then, the production
of
the proteins had to be assayed.
To verify the correct insertion of the plasmid into the yeast genome, we extracted genomic DNA from 8
clones and performed PCR amplification using the primers **iGEM1 and iGEM17**.

![](https://static.igem.wiki/teams/5143/engineering/eng-fig3.png)

Then we cultured the yeasts to detect the production of the engineered proteins. While we don’t have
a
way to directly verify the secretion of **Masp1-Cp19k-CBD**, we can check the secretion of
**YFP**. We anticipated that, according to our design, the fluorescence should be retrieved in the
soluble fraction of the cultures as the protein encompass a secreting signal peptide (**Alpha factor
signal peptide**).
We recorded the fluorescence of the wt strain and the engineered strain after growth in YPG medium and
collection of the pellet fraction as well as the supernatant fraction. However, no significant
fluorescence could be measured.

![Image](https://static.igem.wiki/teams/5143/engineering/learn1.png)

The correct insertion of the genetic construct into the yeast chromosome was demonstrated using
**colony PCR**, we can thus conclude that the **strain is conform**. However, the test of the
phenotype failed. Thus, we designed another test, more sensitive to detect the YFP.

![Image](https://static.igem.wiki/teams/5143/engineering/test1.png)

To detect the production of the **YFP** we decided to perform a **western blot** using
commercial
**anti-GFP antibodies**. These antibodies should recognize YFP, as the sequences of the two
proteins
only differ from a few amino acids and the 3D structure is the same. To test our hypothesis
(production
of YFP in the supernatant), we prepared **yeast crude extracts**, **soluble fraction** (culture
supernatant) and decided to **concentrate the soluble fraction** to enhance the signal,
anticipating
that, if produced, the YFP may be present in small amounts.

![](https://static.igem.wiki/teams/5143/engineering/eng-fig4.png)

![Image](https://static.igem.wiki/teams/5143/engineering/learn1.png)

We confirmed that our construct is functional: **YFP is synthesized and secreted** as expected. The
observed protein sizes match the predicted values, indicating that the **P2A system is functioning
correctly**. Additionally, since YFP is secreted, we can conclude that the **alpha factor is also
working** as intended, facilitating the secretion of the protein. In conclusion, both the **P2A
system and the alpha factor are effective** in our construct, **the chromoprotein is
produced**.
We can expect that the **bio-adhesive protein is produced** as well but didn’t have time to test
it.

**3. Production and conditioning of cellulose**

The matrix of the trap is **cellulose**. It is the most crucial component of our trap because, without
it,
it is **impossible** to immobilize the proteins of interest, such as the bioglue and the yellow
chromoprotein.

![Image](https://static.igem.wiki/teams/5143/engineering/design1.png)

1. The production of cellulose by *K. rhaeticus*. In the article [(Gilbert *et .al*, 2021)](https://www.nature.com/articles/s41563-020-00857-5) on which
   the
   co-culture system was based, the cellulose-producing microorganism is *Komagateibacter
   rhaeticus*. The objective is to verify that *K. rhaeticus* is indeed a cellulose-producing
   bacterium.
2. Once the cellulose producing medium is optimized, the conditions leading to a massive production
   had
   to be determined.
3. Finally, to package the cellulose it must be dried and sterilized

![Image](https://static.igem.wiki/teams/5143/engineering/build1.png)

1. *K. rhaeticus* was cultured in both YPG and HS media. YPG medium was used as described in the
   article, while HS medium was also employed, HS medium is optimal for cultivating this type of
   bacteria
   according to the manufacturer.
2. Cellulose production is a process that quickly depletes the co-culture medium. The cellulose
   patches
   produced from a co-culture are placed in fresh culture after 3 days to prevent production from being
   halted by medium depletion.
3. In the foundational article, cellulose is dried between two sheets of absorbent paper and two
   sheets
   of parafilm. This procedure was replicated in our experiments. To attempt to sterilize the cellulose
   patches, we used UV irradiation.

![Image](https://static.igem.wiki/teams/5143/engineering/test1.png)

Following the various cultures in different media, it was observed that both YPG and HS media
support
cellulose production.
![](https://static.igem.wiki/teams/5143/engineering/eng-fig5.png)

 Then, we aimed at proving that the material observed in YPG is cellulose. YPG
medium and YPG medium supplemented with 2% cellulase were inoculated with *K. rhaeticus*.
![](https://static.igem.wiki/teams/5143/engineering/eng-fig6.png)
The
nature of the material present in the medium is concluded from visual observation. In the medium
without
cellulase, cellulose flakes are observed, while no material is present in the medium with
cellulase.
To enhance the production, the cellulose patches produced from a co-culture are placed
in
fresh culture after 3 days to prevent production from being halted by medium depletion. However, the
cellulose is not thicker after several passages.
The cellulose was dried between two sheets of
absorbent paper and two sheets of parafilm.
Finally, the cellulose patches were placed in a UV box
for 30 minutes. Following this, the cellulose patches were digested and diluted. These solutions were
spread on solid YPG medium, and the plates were incubated for 2 days at 30°C. Regardless of the media
tested, the cellulose patches are not sterile.

![Image](https://static.igem.wiki/teams/5143/engineering/learn1.png)

The results obtained in the article were successfully reproduced, as *K.
rhaeticus* produced cellulose in YPG medium. *K. rhaeticus* also produced cellulose in HS
medium. The results for cultures in HS and YPG media were similar. Given that YPG medium was used in
the
article, it was selected for the remaining experiments. The observed material is indeed cellulose, as
the addition of cellulase to the medium results in no observable material. The cellulose is not
thicker
after several passages. A continuous culture system (fed-batch) should be considered after
establishing
the appropriate medium conditions. We succeeded in drying the cellulose between two sheets of paper.
Finally, the sterilization procedure we used was not effective enough. A different sterilization
technique for the cellulose patches needs to be developed. We are in a situation where the cellulose
patch is not functional; if adherent and colored proteins are fixed to the cellulose, sterilization
methods applied to the cellulose membrane alone should be tested, as these methods may not be
effective
on functional patches.

---

Participating in **iGEM** is not just about conducting science; it’s also a **social adventure**, where we come
together as a **team** of people who often start off not knowing each other. We’ve faced numerous
**challenges**, learning to collaborate with individuals from **diverse backgrounds** and working styles, and
successfully **overcoming obstacles** along the way. It’s **crucial** for our team to highlight the engineering
cycles that have shaped this part of **our journey**.

*Click on the buttons to know more about our team.*

[Project](#Project)

[Team](#Team)

**1. Project Development and Implementation in the Current Context**

**1st iteration: Project Theme**

![Image](https://static.igem.wiki/teams/5143/engineering/design1.png)

To participate in the iGEM competition, it is necessary to find a theme to work on. We had several
ideas, but one issue stood out to us more than the others: bed bugs.

![Image](https://static.igem.wiki/teams/5143/engineering/build1.png)

During the search for a topic, France was experiencing an infestation of public places such as
libraries, public transportation, and cinemas by bed bugs. This significant event prompted us to work on
the topic of pest insects. After discussing this idea with Erwan Gueguen, he advised us to read the
article that forms the foundation of our project.

![Image](https://static.igem.wiki/teams/5143/engineering/test1.png)

After a meeting aimed at bringing together the team and the PIs to discuss the theme of our project, the
idea of a pest insect trap produced by yeast/bacteria co-culture was chosen.

![Image](https://static.igem.wiki/teams/5143/engineering/learn1.png)

The theme of the project has been chosen.

**2nd iteration: Which Insects to Target with Our Prototype ?**

![Image](https://static.igem.wiki/teams/5143/engineering/design1.png)

After deciding to work on a 100% bio-based insect trap using a yeast/bacteria co-culture, it was
necessary to target pest insects. The aim of BIO Snare is to be modular, so we needed to select at least
one pest insect for the prototype.

![Image](https://static.igem.wiki/teams/5143/engineering/build1.png)

After discussing with Erwan Gueguen, he brought up the issue of cherry fruit flies in cherry orchards.

![Image](https://static.igem.wiki/teams/5143/engineering/test1.png)

The Lyon region has many cherry orchards, and sticky traps are commonly used in these fields; thus,
targeting the cherry fruit fly with our trap was accepted by the team.

![Image](https://static.igem.wiki/teams/5143/engineering/learn1.png)

The team decided to target the cherry fruit fly with the patch produced using the yeast/bacteria
co-culture.

**3rd iteration: In which village is the BIO Snare project situated?**

![Image](https://static.igem.wiki/teams/5143/engineering/design1.png)

During the course of the project, it was necessary to choose a village to participate in the iGEM
competition.

![Image](https://static.igem.wiki/teams/5143/engineering/build1.png)

We had numerous discussions among team members and with the PIs to decide which village to integrate.

![Image](https://static.igem.wiki/teams/5143/engineering/test1.png)

In light of the context in which our project is situated, we chose the "food and nutrition" village as
our first choice.

![Image](https://static.igem.wiki/teams/5143/engineering/learn1.png)

The team has chosen the village in which the BIO Snare project will be proposed.

**2. Team Organization**

**1st iteration: Team Formation**

![Image](https://static.igem.wiki/teams/5143/engineering/design1.png)

The team, composed of students from various fields (microbiology, engineering school, pharmaceuticals,
public health, oncology), found it very challenging to align schedules, work times, etc. Solutions
needed to be found to enhance communication among the different sectors of the team.

![Image](https://static.igem.wiki/teams/5143/engineering/build1.png)

A Discord server was established, allowing us to centralize communication among team members and PIs,
who were also on the server. Teams were formed in the areas of "sciences," "communication," "funding,"
and "human practices." This structure enabled us to focus everyone's efforts on specific tasks. To
ensure the smooth operation of these teams, team leaders were appointed, making it easier to relay
information to the team leader.

![Image](https://static.igem.wiki/teams/5143/engineering/test1.png)

All these efforts led to a better work dynamic, but inter-team communication issues arose.

**2nd iteration: Communication within the Team**

![Image](https://static.igem.wiki/teams/5143/engineering/design1.png)

In light of the inter-team communication issues, a solution needed to be found to improve
communication.

![Image](https://static.igem.wiki/teams/5143/engineering/build1.png)

Weekly meetings were implemented for a time, which established open communication to avoid tension and
misunderstandings.

![Image](https://static.igem.wiki/teams/5143/engineering/test1.png)

This more open communication allowed everyone to express themselves freely, helping to reduce tensions.

![Image](https://static.igem.wiki/teams/5143/engineering/learn1.png)

Communicating more openly, while being mindful of the language used and the character of one's
interlocutor, helped improve communication among team members and between the sectors.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

# Experiments

**BIO Snare’s goal** was to produce functionalized cellulose to make it adaptable for specifically trapping insects through the use of different **colors and bioglue**. We were inspired by an article and decided to use a **coculture** of the bacterium ***K. rhaeticus***, which overproduces **cellulose**, along with a genetically modified yeast, ***S. cerevisiae***.

Our main plasmid, plasmid D, has been constructed as following:

This biobrick contains:

*Click on each of the components to learn more about what this biobrick contains.*

* **Constitutive GAP promoter**

Drives the continuous expression of both YFP and the bioglue.

* **fwYellow gene**

Encodes Yellow Fluorescent Protein (YFP), the chromoprotein we selected. The sequence has been optimized for expression in *S. cerevisiae*. The fwYellow gene is a BioBrick created by the [Uppsala iGEM team in 2013](https://parts.igem.org/Part%3ABBa_K1033910).

* **Bioglue genetic sequence**

Encodes a bioglue made from a fusion of two natural proteins to significantly enhance adhesion properties: Masp1 from spider silk and Cp19k from barnacles. These proteins have individual adhesion strengths of 22.2 mJ/m² and 2.2 mJ/m², respectively, but their fusion increases this to 39.9 mJ/m² [(Ye *et al.* (2023).](https://pubmed.ncbi.nlm.nih.gov/37776922/) The sequences were optimized for expression in *S. cerevisiae*.

* **Cellulose Binding Domain (CBD)**

Each protein (both the colored and the adhesive) contains a CBD to allow binding to cellulose. The CBD sequence was adapted from [Gilbert et al. (2021).](https://www.nature.com/articles/s41563-020-00857-5)

* **Alpha factor signal peptide**

Derived from the *MAT alpha* mating system, this sequence acts as a signal peptide to facilitate protein secretion. Once secreted, the signal peptide is cleaved, leaving only the mature protein.

* **P2A system**

Enables co-expression of both YFP and the bioglue protein from a single mRNA transcript. This system is optimized for eukaryotic cells and allows simultaneous synthesis of YFP and the adhesive protein in our context.

* **URA3 gene**

Selected as a marker for yeast transformation. This gene allows the *URA-* yeast strain to synthesize uracil, enabling cells that have successfully integrated the plasmid containing URA3 to grow in uracil-deficient medium.

* **Homologous sequences (URA5’ and URA3’)**

Facilitate plasmid integration into the yeast chromosome via homologous recombination.

This plasmid was designed to make the **cellulose sticky and yellow-colored**, attracting insects. However, the ultimate goal of our project is to make the trap adaptable and specific to targeted insects, **avoiding any negative impact on biodiversity**. To achieve this, we aim to modify features like the **color or other attractants** in order to capture specific insects while minimizing effects on non-target species.

The steps we followed are outlined below. Diagrams for each construction and key stages are provided. Additionally, you will find a toolbox containing detailed protocols used for each step.

## Main steps of our project

*Click on the buttons to discover a diagram for each step of our project.*

## CREATING A YELLOW AND STICKY BIOBRICK

Here is a diagram to sum up the different steps to produce **the plasmid D**, the original BIO Snare plasmid coloring the bacterial cellulose in yellow and making it sticky.

![](https://static.igem.wiki/teams/5143/experiments/sche-ma-pour-experiments-2.png)

## INTEGRATION IN *S. cerevisiae*

Here is a diagram to sum up the different steps to transform S. cerevisiae with the plasmid D encoding for the yellow protein and the bioadhesive protein.
The insertion of the fragment in the yeast chromosome is possible thanks to the homologous sequences URA3’ and URA5’.

![](https://static.igem.wiki/teams/5143/experiments/sche-ma-pour-experiments.png)

## CO-CULTURE: PRODUCTION OF SNARE

Here is a diagram to sum up the different steps to do the coculture of our two organisms to produce the trap. *K. rhaeticus* naturally overproduces cellulose and *S. cerevisiae* that has been genetically modified to produce both a yellow and a bio-adhesive proteins.

![](https://static.igem.wiki/teams/5143/experiments/experiments3.png)

## Tool box

Here are detailed all protocols used in labs. They are separated in 3 main categories: microbiological methods with all media used for each microorganisms we used, genetic methods and molecular biology with all the protocols related to DNA and biochemical methods with protocols related to proteins.

*Click on the buttons to discover the protocols associated.*

## Microbiological methods

We have used different media for each organism alone, and others for their coculture.

*Click on the organism you want to study to discover the media to use.*

---

[*K. rhaeticus*](#Krhaeticus)

[*S. cerevisiae*](#Yeast)

[Coculture](#Coculture)

## ***K. rhaeticus* medium**

**For 1L of medium:**

|  |  |
| --- | --- |
| Glucose | 20 g |
| Tryptone | 5 g |
| Yeast extract | 5 g |
| Agar (for plates) | (25 g) |
| HCl 37% | ~ 200 µL |

## ***S. cerevisiae* media**

**Minimum medium:**

| Components | Quantities for 1L | Quantities for 0.25L |
| --- | --- | --- |
| YNE (without (NH4)2SO4) | 1.7 g | 0.425 g |
| Glucose | 20 g | 5 g |
| (NH4)2SO4 | 5 g | 1.25 g |
| Agar | 24 g | 6 g |
| Demineralised water | qsf 1L | qsf 250mL |

**Selective yeast medium -URA:**

Add sterilely each component to a minimum medium kept cooled down at 60°C.

| Solutions | Quantities for 1L | Quantities for 0.25L |
| --- | --- | --- |
| Minimum medium | 10mL | 2.5mL |
| 1% ADE | 1mL | 0.25mL |
| 1% TYR | 5mL | 1.25mL |

**YPD medium**

Composition:

| Component | Concentration (g/L) |
| --- | --- |
| Yeast extract | 10 |
| Bacto Peptone | 10 |
| Glucose | 20 |
| To make agar plates | 20 |

Protocol:

|  |
| --- |
| Dissolve all components in the required amount of demineralized water. |
| Sterilize for 30 min at 110°C. |
| Store at 4°C. |

## **Coculture media**

**YPD + 1% Cellulase**

Before the coculture, *K. rhaeticus* has to grow in a YPD+1%cellulase medium because the cellulase improves its growth. So, to an YPD medium, 0.5 g of cellulase have to be added. Then, the medium is filtrated.

**YPS medium**

To avoid the dominance of *K. rhaeticus* on *S. cerevisiae*, a YPS medium was used in the coculture. Indeed, *K. rhaeticus* can't metabolize sucrose and need *S. cerevisiae* to survive in YPS medium.

Composition:

| Component | Concentration (g/L) |
| --- | --- |
| Yeast extract | 10 |
| Bacto Peptone | 10 |
| Sucrose | 20 |
| To make agar plates | 20 |

Protocol:

|  |
| --- |
| Dissolve all components in the required amount of demineralized water. |
| Sterilize for 30 min at 110°C. |
| Store at 4°C. |

## Genetic methods and molecular biology

In this section, you can find all protocols related to DNA: **extraction**, **digestion**, **amplification**, analysis on **agarose** gel, **DNA purification**, **DNA cloning** and finally **transformation** in *E. coli* firstly and then in *S. cerevisiae*

*Click on the buttons to discover the protocols used in the lab.*

---

[Extraction](#Extraction)

[Digestion](#Digestion)

[PCR](#Amplification)

[Agarose](#Agarose)

[Purification](#Purification)

---

[Cloning](#Cloning)

[Transformation](#Transformation)

## **DNA Extraction**

**Quick and Dirty**

**Aim:** extract DNA from bacterial colonies for a PCR reaction to check the efficiency of the recombination.
For each clone:

|  |
| --- |
| Put 25 µL of NaOH 20 mM in a microtube. |
| With a sterile cone, take one colony and resuspend it in the tube. |
| Incubate 15 min at 95°C. |
| Vortex. |
| Spin 1 min. |
| For a PCR, take 1 µL of supernatant. |

**Plasmid Extraction**

**Aim:** extract plasmid from bacteria.
*Kit “Nucleospin Plasmids” (Macherey-Nagel).*

Prepare 2 heat blocks: 50°C and 70°C, 1 tube of 2 mL, 1 column, 1 tube of 1.5 mL per culture. Put aliquots of AW (500 µL per culture) at 50°C and of AE (60µL per culture) at 70°C.

|  |
| --- |
| Pick colonies. |
| Inoculate 5 mL of LB medium with a colony and incubate bacterial culture O/N at 37°C. |
| Transfer 2 mL in a micro tube, centrifuge 1 min at 13,000 rpm, discard supernatant. |
| Resuspend the pellet with 250 µL of A1, vortex. |
| Lyse cells by adding 250 µL of A2, incubate 3 min at RT. |
| Neutralize lysate by adding 300 µL of A3, spin 10 min at 13,000. |
| Load supernatant on the column. |
| Spin 1 min at 13,000 rpm. |
| Discard supernatant. |
| Add 500µL of Aw at 50°C. |
| Spin 1 min at 13,000 rpm. |
| Discard supernatant. |
| Add 600 µL of A4 to wash DNA. |
| Spin 1 min at 13,000 rpm. |
| Discard supernatant. |
| Dry the column by spinning 1 min at 13,000 rpm. |
| Put the column in a new micro-tube , add 30 µL of AE at 70°C and incubate 2 min at 70°C. |
| Pick colonies |
| Inoculate 5 mL of LB medium with a colony and incubate bacterial culture O/N at 37°C. |
| Transfer 2 mL in a micro tube, centrifuge 1 min at 13,000 rpm, discard supernatant. |
| Resuspend the pellet with 250 µL of A1, vortex. |
| Lyse cells by adding 250 µL of A2, incubate 3 min at RT. |
| Neutralize lysate by adding 300 µL of A3, spin 10 min at 13,000 rpm. |
| Load supernatant on the column. |
| Spin 1 min at 13,000 rpm. |
| Discard the supernatant. |
| Add 500 µL of Aw at 50°C. |
| Spin 1 min at 13,000 rpm. |
| Discard the supernatant. |
| Add 600 µL of A4 to wash DNA. |
| Elute DNA by spinning 1 min at 13,000 rpm. |
| Quantify DNA with Nanodrop. |

**Extraction of chromosomal DNA yeast**

**Aim:** extract DNA from yeast clones for a PCR reaction to check the efficiency of the transformation.

|  |
| --- |
| Add 25 mL of NaOH 20 mM in a 1.5 mL tube. |
| With a sterile cone, take one colony and resuspend it. |
| Incubate 15 min at 95°C. |
| Vortex and spin 1 min. |
| Use 1-2 µL/PCR. |

## **Digestion by restriction enzymes**

**Aim:** Cut a plasmid on targeted sites thanks to specific restriction enzymes. It enables either to check the integrity or to linearize a plasmid.
The specific restriction enzyme has to be chosen using the Snapgene software. Be careful to take the buffer corresponding to the specific enzyme.
The conditions of the reaction are as follow:

| 10X CutSmart Buffer | Restriction enzyme (HF) | DNA | Water |
| --- | --- | --- | --- |
| 5 µL | 1 µL | 200 ng | complete to 50 µL |
| Incubate at 37°C for 4h. |

## **DNA Amplification by PCR**

**PCR on bacterial and yeast colonies**

* Take a colony and put it in 20 µL of UP water and then streak it on the required medium to have a pure culture.
* Put the colonies at 95°C in 20 µL of UP water and centrifuge 1 min at 13,000 rpm.

**PCR PrimeSTAR max**

Mix:

|  |  |
| --- | --- |
| DNA matrix | 1 ng |
| Primer 1 (10 µM) | 1.2 µL |
| Primer 2 (10 µM) | 1.2 µL |
| PrimeSTAR max master mix (2X) | 20 µL |
| Pure water | qsf 40 µL |

30 cycles:

|  |  |
| --- | --- |
| 98°C | 10 s |
| 55°C | 10 s |
| 72°C | 5s/kb\* |

*\*In the case of gDNA, as the DNA is less available, a longer time is required.*

**PCR Dream Taq**

For colonies PCR, suspend each colony in 50 µL of pure water. Depending on the strain, place 5 to 10 min at 98°C.
Mix:

|  |  |
| --- | --- |
| Bacterial suspension | 6.3 µL |
| Primer 1 (10 µM) | 0.6 µL |
| Primer 2 (10 µM) | 1.2 µL |
| Primer 2 (10 µM) | 0.6 µL |
| DreamTaq Mastermix (2X) | 7.5 µL |

Cycles:

|  |  |
| --- | --- |
| 98°C | 30 s |
| 98°C | 10 s |
| Depending of the primers (NEB sites to know) | 30 s |
| 72°C | 25s/kb |
| 72°C | 5 min |

## **Agarose gel**

**Aim:** Separate DNA fragments by molecular sizes.
If you only want to **control sizes** of your fragments, perform an **EtBr** gel coloration. If you want to **purify** a specific DNA fragment, perform the gel coloration with **Gel Green**.

Firstly, choose the **required agarose gel percentage** according to the following table:

|  |  |
| --- | --- |
| Gel percent | DNA Size Range (bp) |
| 0.5% | 1000 - 30,000 |
| 0.8% | 800 - 12,000 |
| 1.0% | 500 - 10,000 |
| 1.5% | 200 - 3,000 |
| 2.0% | 50 - 2,000 |

Protocol:

* In an Erlenmeyer, add the required quantity of agarose in 0.5X TAE buffer.
* Heat in the microwave to melt agarose.
* When the liquid is transparent, take it out of the microwave and pour in a casting
  stand.

Coloration:

|  |  |
| --- | --- |
| EtBr gel | Gel green |
| Bath the gel into an EtBr solution after migration. | Add 2 µL of the Gel Green solution |

## **Purification**

**Aim:** Extracted DNA band from an agarose gel.

|  |
| --- |
| Excise band of interest on blue light, weight it. |
| Add Gel dissolving buffer NT1 (200 µL / 100 mg agarose). |
| Incubate at 50°C until the gel is dissolved. |
| Load the dissolved gel on a column. |
| Spin 1 min at 13,000 rpm, discard supernatant. |
| Wash the column with 700 µL of NT3, spin 1 min at 13,000 rpm, discard supernatant x2. |
| Dry the column by spinning 1 min at 13000. |
| Put the column on an micro tube. |
| Incubate 2 min at 70°C. |
| Add 30 µL of NE Buffer in the column, incubate 2 min at 70°C x2. |
| Spin 1 min at 13000 rpm. |
| Quantify DNA. |

## **Cloning**

**Hifi Cloning**

**Aim:** Assembling fragments by recombination thanks to their identical ends.
The conditions of the reaction are as followed:

|  |  |
| --- | --- |
|  | Volume |
| NEBuilder HiFi DNA Assembly Master Mix | 10µL |
| 2X |  |
| DNA fragments | Use NEBuilder Calculator (required length and concentration of each fragments) |
| H2O | Up to 20 µL |
| Incubate 15 min (up to 60 min) at 50°C |

**TEDA Cloning**

**Principle:** This technique enables building DNA assembly with overlapping ends thanks to T5 exonuclease.

Mix:

|  |  |
| --- | --- |
| Vector : Insert | Ratio 1:3 or 1:4 |
| TEDA reagents | 4 µL |
| Pure water | qsf 20 µL |

Control tube:

|  |  |
| --- | --- |
| Vector |  |
| TEDA reagents | 4 µL |
| Pure water | qsf 20 µL |

Reaction: incubation 40 min at 30°C.

Transformation: use 10 µL, [see protocol](#Transformation).

Reagents:

|  |  |
| --- | --- |
| TrisHCl 1 M pH 7.5 | 500 µL |
| MgCl2 1M | 50 µL |
| DTT | 50 µL |
| PEG8000 | 250 mg |
| T5 exonuclease | 1 µL |
| Water qsf 1 mL | 1 mL |

## **Transformation**

**Transformation in *E. coli***

**Aim:** Introducing a plasmid into a competent *E. coli*.

Preparation before starting:

* Put 1 heat block at 42°C + 1 Thermomixer micro tube eppendorf at 37°C.
* Preheat 900 µL of SOC per transformation (37°C).
* Preheat petri dishes at 37°C, beads on the lid. Let competent cells thaw on ice.

Protocol:

|  |
| --- |
| Add a maximum of 1 ng/µL of DNA Add a maximum of 1ng of DNA per µL of competent cells. |
| Mix by tapping on tube. |
| Incubate 45 min on ice. |
| Heat shock : incubate at 42°C for 45 sec. |
| Incubate 5 min on ice. |
| Add 900 µL of SOC preincubated at 37°C. |
| Centrifuge 1h at 37°C at 900 rpm. |
| Spread 100 µL on a Petri dish. |

**Transformation integrative in *S. cerevisiae***

**Aim:** Introduction of a DNA in *S. cerevisia* genome.

**Preparation of cells:**

The day before the transformation:
Preparing a preculture by inoculating a colony in 2 mL of YPD overnight at 30°C 120 rpm.

The day of the transformation:

Inoculate the preculture (2 mL) in 50 mL of YPD. Measure the OD600. When OD600 is between 1 and 2, it means the cells are in the exponential phase. Transformation can start.

**Protocol:**

Solutions:

* Lithium acetate 10X: 1M Lithium acetate pH 7.5 (adjust pH with diluted acetic acid).
* TE 10X: 100 mM Tris-HCl pH 7.5; 10 mM EDTA pH 8.0.
* 50% PEG: 50% polyethylene glycol 4000 (weight/volume).
* LiAcTE: 1vol 10X TE, 1vol 10X lithium acetate, 8 vol H2O.
* LiAcTE/PEG: 1vol 10X TE, 1vol 10X lithium acetate, 8 vol PEG.

Make the cells competent:

|  |
| --- |
| Transfer 50 mL of yeast preculture in a 50 mL tube. |
| Centrifuge 3 min at 3000 rpm. |
| Discard supernatant and add 10 mL of LiAcTE buffer. |
| Incubate 15 min at RT (room temperature). |
| Centrifuge 3 min at 3000 rpm. |
| Discard Supernatant. |
| Resuspend in 1 mL of LiAcTE buffer. |

Transformation:

|  |
| --- |
| In a 1.5 mL tube, add between 0.5-1 ng of DNA fragment. |
| Add 10 µL of ssDNA (salmon sperm DNA) at 10 mg/mL predenaturated 5 min at 95°C. Keep on the ice. |
| Add 100 µL of competent cells for each transformation. Mix by tapping.. |
| Incubate 5 min at RT. |
| Add 280 µL of solution LiAcTE/PEG. Mix by inverting.. |
| Incubate 1h at 30°C. |
| Add 43 µL of DMSO (10% final) and mix by inverting. |
| Heat shock : 42°C for 10 min. |
| Centrifuge at 5000 rpm for 2 min at RT. |
| Discard the supernatant. |
| Resuspend in 1 mL of YPD, incubate at 30°C O/N. |
| Centrifuge. |
| Resuspend in 200 µL of H2O |
| Spread 100 µL / petri dish (make 2 petri dish/transformation) |

## Biochemical methods

In this section, you can find all protocols related to proteins: **microplate reader** to analyze the fluorescence in the media, checking if YFP is produced, **PAGE** and **western blot**.

*Click on the buttons to discover the protocols used in the lab.*

[Microplate reader](#microplate)

[SDS PAGE](#PAGE)

[Western Blot](#western)

## **Microplate reader**

**Aim:** measuring the production of fluorochrome.

• From a culture, keep a sample. If you have several cultures to test, be careful to have the same concentration of cells, or you can take the absorbance at 600 nm.

• From the same culture, take 108 cells (knowing that for a DO600 = 1, there are 3x107 cell/mL).

• Spin 5 min at 5000 rpm.

• Keep 200 µL of supernatant, this is the second sample.

• Read with Tecan.

| Protein | Excitation 𝛌 | Emission 𝛌 |
| --- | --- | --- |
| GFP | 488 nm | 510 nm |
| YFP | 522 nm | 536 nm |
| mRuby | 558 nm | 605 nm |
| Venus | 515 nm | 528 nm |

## **SDS PAGE**

**Aim:** separating proteins according to their molecular mass.

**Preparation**
Buffers

* APS 10% (stored: -20°C ∞, 4°C 1 month):

|  |  |
| --- | --- |
| APS | 1 g |
| Water | 10 mL |
| Aliquot per 1 mL |

* Stacking gel preparation:

|  |  |
| --- | --- |
| Water | 23.7 mL |
| Tris 0.5M pH 6.8 | 2 mL |
| Acrylamide 30% | 4 mL |
| SDS 10% | 300 µL |
| APS 10% | 40 µL |
| Temed | 5 µL |

* Running buffer SDS-PAGE 10X:

|  |  |
| --- | --- |
| Tris | 30.3 g |
| SDS | 10 g |
| Glycine | 144.1 g |
| Water | qsf 1L |

*Start by assembling the plates and putting in water to check that the system is not leaking. Remove the water and wipe with whatmann paper.*

Resolving Gel preparation:

| Components | Volume for 2 gels 10% | Volumes for 2 gels 12.5% | Volumes for 2 gels 15% |
| --- | --- | --- | --- |
| Water | 3.89 mL | 3.06 mL | 2.22 mL |
| Tris 1.5M pH 8.8 | 2.64 mL | 2.64 mL | 2.64 mL |
| Acrylamide 30% | 3.33 mL | 4.16 mL | 5 mL |
| SDS 10% | 100 µL | 100 µL | 100 µL |
| APS 10% | 40 µL/ | 40 µL | 40 µL |
| Temed | 5 µL | 5 µL | 5 µL |

Pour in the casting stand Put a layer of water on top to smooth out and avoid air bubbles.

**Sample preparation and migration**
Buffers

* 2X SDS-PAGE sample buffer, RT.

|  |  |
| --- | --- |
| Tris 0.5M pH 6.8 | 16 mL |
| SDS 10% | 16 mL |
| Glycerol 10% | 10 mL |
| Bromophenol blue 1% | 800 µL |
| Water | 7.2 mL |

* Cracking buffer (-20°C)

|  |  |
| --- | --- |
| Water | 4.5 mL |
| 2X SDS-PAGE Sample buffer | 5 mL |
| ß mercaptoéthanol | 500 µL |

Protocol
**Sample preparation:**
*Crude extract:*

1. Pick colonies
2. Inoculate
3. Incubate bacterial culture, O/N, 30°C for *S. cerevisiae*
4. Inoculate in 5 mL of YPD the required volume to have OD = 0.2. Incubate until OD = 1, to be in the exponential phase.
5. Take 108 cells, spin 5 min 5,000 rpm.
6. Keep the supernatant if you want to precipitate secreted proteins. Wash the pellet with 1 mL of cold water and transfer in a tube, spin.
7. Discard supernatant with pipetman, resuspend pellet with 100 µL NaOH 0.2M.
8. Incubate 10 min at RT.
9. Spin 3 min at 7830 rpm, discard supernatant.
10. Add 50 µL of cracking buffer 1X and mix by pipetting.
11. Incubate 5 min at 95°C.
12. Spin 2 min at 13,000 rpm.
13. Store at -20°C or put 18 µL in SDS Page Gel

*Supernatant:*

1. Mix 300 µL of supernatant with 900 µL of acetone.
2. Incubate 15 min at 4°C.
3. Spin 10 min at 12000 rpm at 4°C.
4. Discard supernatant.
5. Short spin 5/10 seconds to dry the pellet.
6. Let the pellet dry O/N, cover it with alumnium.
7. The next day, add 50 µL of SDS 0.5%.
8. Take 30 µL of this sample and add 10 µL of cracking buffer 4X.
9. Incubate 5 min at 95°C.
10. Spin 2 min at 13,000 rpm.
11. Store at -20°C or put 18 µL in SDS Page.

## **Western Blot**

**Transfer**
Buffers:

* Transfer buffer 10X

| Tris | 3.1 g |
| --- | --- |
| Glycine | 144 g |
| Water | qsf 1L |

* Transfer buffer 1X (4°C)

| Transfer buffer 10X | 100 mL |
| --- | --- |
| Ethanol 100% | 200 mL |
| SDS 10% | 10 mL |
| Water | 690 mL |

Protocol
20 min before the end of the SDS-PAGE migration, soak the whatmannspaper in 1X transfer buffer.
*Activate membrane:*

* PVDF membrane 3 min in ethanol, then soak it in transfer buffer.
* Soak the gel in transfer buffer.

*Transfer*
Order of the sandwich: (-) whatmann, gel, membrane, whatmann (+)
Rake between each layer to remove bubbles, remove excess pad with paper towel.
For one gel: 20 V, 100 mA, for two gels: 20V, 200 mA, 20 min.

**Antibodies binding**
Buffers:

* TBS (4°C)

| Tris-HCl 1M, pH8 | 20 mL |
| --- | --- |
| NaCl 5M | 27 mL |
| Water | qsf 1L |

* Blocking solution (to be prepared the same day)

| TBS | 50 mL |
| --- | --- |
| Skim milk | 2.5 g |

* TBST (4°C)

| TBS | 50 mL |
| --- | --- |
| Skim milk | 2.5 g |
| Tween 20 | 1 mL |
| Water | qsf 1L |

*Protocol*

|  |
| --- |
| Aspecific sites Blocking: put PVDF membrane in blocking solution at least 1h at RT. |
| Incubate with ab I O/N at 4°C. |
| Wash 3 times in TBST 15 min at RT. |
| Incubate with ab II 2h at RT. |
| Wash 4 times in TBST 15 min at RT. |
| Put in TBS. |
| Reveal: PVDL membrane in substrate HRP 1-2 min at RT, Streatchable film plastic, chemidoc.. |

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

# Results

## Cellulose production and functionalized cellulose

Here is a diagram to sum up our results:

![](https://static.igem.wiki/teams/5143/results/results-sche-ma-global.png)

*If you want to know more about our results, click on the buttons below.*

## Production of cellulose by *K. rhaeticus*

Following the receipt of the **cellulose-producing** bacterial strain *K. rhaeticus*, we conducted **cellulose production tests** in various containers using YPS, YPG, and HS media. The **highest cellulose yield** was observed in **YPG** medium, as indicated in the referenced article [(Gilbert et al., 2021)](https://www.nature.com/articles/s41563-020-00857-5). According to the article, *K. rhaeticus* does not grow in **YPS** since it cannot metabolize **sucrose**. These trials **confirmed** the capacity of the *K. rhaeticus* strain to produce cellulose (Figure 1). This strain was **selected** for subsequent experiments.

![Cellulose patches obtained following YPG inoculation by <em>K. rhaeticus</em>](https://static.igem.wiki/teams/5143/results/results-figure1.png)

Figure 1: Cellulose patches obtained following YPG inoculation by *K. rhaeticus* in 24-well plates.

## Co-culture between *K. rhaeticus* and wild-type *S. cerevisiae*

### Co-culture protocol:

---

We chose to replicate the protocols from the article **Living materials with programmable functionalities grown from engineered microbial co-cultures** between the selected strain of *K. rhaeticus* and the wild-type (WT) *S. cerevisiae* BY4741 strain. **Various containers** were tested, including 60 mm Petri dishes, 90 mm Petri dishes, 24-well plates, and rectangular 4-well plates. **Different media** were also tested in accordance with the article's experiments, specifically YPS and YPG media. According to the article, **YPS** is the **preferred** medium for producing the patches because the bacterium requires an enzyme produced by the yeast to metabolize sucrose. Therefore, in **YPS**, the bacterium is **less likely** to outcompete the yeast, which is **essential** for its growth.

The YPS and YPG media were inoculated with *K. rhaeticus* and *S. cerevisiae* BY4741 according to the article's protocol: a mixture of **1/50** of YPS or YPG from a *K. rhaeticus* culture at an **OD of 2.5**, combined with **1/100** of YPS or YPG from a *S. cerevisiae* culture at an **OD of 0.01**. These mixtures were incubated for **three days at 30°C**, as recommended in the article.

Cellulose **patches** were obtained under **all co-culture conditions** (across all containers and media). However, the cellulose patches were **more compact** and **resilient** when produced in **YPG** medium (Figure 2). This result aligns with the observations from the article, as the bacterium cannot metabolize sucrose independently and relies on the yeast for growth in **YPS**, resulting in **slower development** and cellulose production. Conversely, the bacterium does not require the yeast for growth in **YPG**, leading to **faster growth** and **cellulose production** compared to YPS.

![Cellulose patches obtained following YPG and YPS inoculation by <em>K. rhaeticus</em> and <em>S. cerevisiae</em>](https://static.igem.wiki/teams/5143/results/results-figure2.png)

Figure 2: Cellulose patches obtained following YPG and YPS inoculation by *K. rhaeticus* and *S. cerevisiae* in 24-well plates.

### Passaging

---

Subsequent **passages** of the co-cultures were conducted according to the article's recommendations to determine whether it was possible to produce **functionalized cellulose** from the **same** co-cultures over several weeks. Given that the project's objective is to produce functionalized cellulose patches, it would be **advantageous** to generate an **initial** co-culture and then **renew** only the medium every three days without having to restart the cultures for new patches. After three days of co-cultivation, **fresh** YPS and YPG media were inoculated with a volume of co-culture corresponding to **1/100** of the final medium volume. These passages were incubated for **three days at 30°C**.

One of the **goals** of these passage tests was to verify that the **quality** of the patches did not decline over successive passages, allowing for extended use of the patches. A total of **three passages** were performed following the aforementioned protocol.

During the first passage, the **texture** of the cellulose patches was **similar** to that of the initial co-culture. However, from the second passage onward, the texture of the patches produced in **YPG** changed noticeably. Specifically, the patches **lost rigidity, opacity, and firmness**, becoming **soft** and **translucent**, with **very little** material remaining (Figure 3).

In contrast, the quality of the patches in **YPS** did not change significantly across passages, resulting in cellulose that **remained soft and thin**, making it difficult to handle.

![](https://static.igem.wiki/teams/5143/results/results-figure3.png)

Figure 3: Soft and translucent cellulose patches produced after second passage.

In conclusion, the **quality** of cellulose patches is **superior** when the co-culture is performed in **YPG**, but this is only observed during the **initial co-culture**. A possible explanation is that the culture was not sufficiently **homogenized** before being introduced into the fresh medium.

## Co-culture of *K. rhaeticus* and recombinant *S. cerevisiae*

The goal of this project is to produce functionalized cellulose patches through the co-culture of ***K. rhaeticus*** and recombinant ***S. cerevisiae*** expressing YFP and bioglue proteins. After generating and validating the genetic constructs for YFP and bioglue in the genome of ***S. cerevisiae***, two clones have been selected (**ScpD1** and **ScpD7**) we proceeded to test co-culture with ***K. rhaeticus***. The aim was to confirm that cellulose quality would not be compromised by co-culturing with recombinant yeast. Cellulose patches were successfully produced (Figure 7), displaying similar texture and appearance to those obtained from wild-type yeast co-culture (Figure 2). In conclusion, cellulose patches retain their quality regardless of whether ***K. rhaeticus*** is co-cultured with wild-type or recombinant yeast. However, the cellulose produced was not yellow, and further analyses are required to assess the adhesive properties of the cellulose.

![Cellulose patches from <em>K. rhaeticus</em> and recombinant <em>S. cerevisiae</em> co-culture](https://static.igem.wiki/teams/5143/results/results-figure7.png)

Figure 7: Cellulose patches from the coculture between *K. rhaeticus* and ScpD1 and 7 (Clone 1 and 7 of *S. cerevisiae* transformed with plasmid D).

## Drying of cellulose

To **preserve** the cellulose patches produced from the co-culture, various **drying** methods were tested. Initially, **absorbent paper** was used to wrap the cellulose. However, this method made it **difficult** to detach the patches from the paper. Subsequently, following the article's recommendations, we employed the **"sandwich" method**. In this approach, the cellulose was encased in **parafilm** and then surrounded by absorbent paper. When the absorbent paper was changed after 24 hours, the cellulose was **dry** after **48 hours**. However, detaching the cellulose without deforming the parafilm proved challenging, often resulting in the tearing of the patch.

![Sandwich method for drying cellulose](https://static.igem.wiki/teams/5143/results/results-figure4.jpg)

Figure 4: Sandwich method for drying the cellulose: encapsing cellulose with grease-proof paper and absorbent paper.

Ultimately, the **most effective** and **straightforward** technique for recovering the patches was the **sandwich** method (Figure 4), which involved encasing the cellulose with **grease-proof paper**, as described in the article, along with **absorbent paper**.

Additionally, the drying of the cellulose was conducted to **weigh** the patches, allowing us to determine the **weight variations** associated with the **different** co-culture **media** tested.

Figure 5: Table summarizing the weights of cellulose patches based on the media used and the passage performed.

| Conditions | Average weight of the patches when the co-culture was performed in YPS medium (mg) | Average weight of the patches when the co-culture was performed in YPG medium (mg) |
| --- | --- | --- |
| First co-culture | 0.7 | 3.14 |
| Third co-culture | 1.5 | 0 |

## Sterilization of cellulose

The production of a **functionalized cellulose** patch through the co-culture of *K. rhaeticus* and recombinant *S. cerevisiae* involves the integration of **genetically modified yeast** within the **patch**. Given the necessity to **avoid** the use of cellulose containing these genetically modified microorganisms, **UV sterilization** of the produced cellulose was tested. It is **crucial** to identify a **sterilization method** that does **not degrade** the recombinant proteins, ensuring their continued **functionality**.

To **quantify** the yeast present in the cellulose patches, the patches were **washed** with phosphate-buffered saline (PBS) and subsequently **immersed** in a **2% cellulase** PBS solution to degrade the cellulose. This mixture was left at room temperature until complete **degradation** of the patches occurred (24 to 48 hours). The yeast present in the digested patch solution was then **enumerated** using a **Malassez cell**. However, this counting method does not distinguish between **dead yeast**, resulting from sterilization, and **viable yeast**.

To further assess **yeast viability**, the digested patch solutions were **centrifuged**, and the pellet was diluted before being **plated** on YPG medium and incubated at 30°C. The **count** of **yeast colonies** obtained represents the cells that **survived UV sterilization**.

Preliminary results indicate that **UV sterilization** effectively **reduces** the number of **yeast cells** within the cellulose patches but does **not eliminate** them entirely. Future investigations should also focus on evaluating the **functionality** of the **recombinant yeast proteins** post-sterilization.

Figure 6: Table summarizing the enumeration data (using a Malassez cell or CFU technique after cellulase digestion) for cellulose patches treated with UV for 30 or 45 minutes.

| Condition | Without UV sterilisation | With UV sterilisation for 30 minutes  (45 minutes for the last two lines). |
| --- | --- | --- |
| Number of cells/mL for a cellulose patch | | |
| YPS | 9.58 x 105 | 7.08 x 105 |
| YPG | 2.54 x 106 | 1.42 x 106 |
| YPS | 9.58 x 105 | 7.08 x 105 |
| YPG | 2.54 x 106 | 1.42 x 106 |
| YPS | 3.67 x 106 | 8.33 x 105 |
| YPG | 2.50 x 105 | 7.50 x 105 |
| YPS | 9.50 x 106 | 2.12 x 106 |
| YPG | 6.75 x 106 | 2.37 x 106 |
| CFU/mL for a cellulose patch | | |
| YPS | 3.80 x 107 | 4.0 x 106 |
| YPG | 1.70 x 106 | 8.30 x 106 |
| YPS | 3.90 x 107 | 5.20 x 106 |
| YPG | 2.20 x 107 | 9.0 x 106 |

### Perspectives for sterilization

The ultimate objective of our product is not to release genetically modified yeast into the environment, but rather to provide **sterile**, **functionalized cellulose patches**. To achieve this, various **sterilization methods** have to be tested to ensure the elimination of any genetically modified organisms (GMOs) without compromising the functionality of the proteins integrated within the patch.

Current efforts focus on **UV sterilization**, which has shown promising results, but other approaches, such as **gamma irradiation**, could also be explored to sterilize larger volumes of cellulose patches. Gamma irradiation, for instance, is commonly used in medical and cosmetic industries for its ability to treat entire palettes of products in a single process.

Moreover, a recent study ([Chong *et al.*, 2024](https://www.nature.com/articles/s41589-024-01602-8#citeas)) found a novel method to kill GMO ***S. cerevisiae***: the **kill-switch method**. This method is composed of an **ER destabilizing domain** (ERdd) fused to an essential gene (spc110) in the chromosome of ***S. cerevisiae***. ERdd protein is **stabilized with estradiol** in the cytosol of the cell and allows the survival of the yeast. If no estradiol is added to the extracellular medium, then ERdd protein fused to Spc110 protein will be **destabilized** and so will be destroyed by the proteasome of the cell, causing the **cell death**. The **kill-switch** method could be a promising device to produce a **sterile functionalized cellulose**. Further investigations into this and other methods are necessary to optimize the sterilization of cellulose patches for broader industrial applications.

## Plasmid D BioBrick design, cloning, and expression in *S. cerevisiae*

## Designing a Biobrick for cellulose functionalization: BioGlue and chromoprotein selection

### BioGlue and chromoprotein ideas

---

To impart **adhesive** and **visual properties** to cellulose, we employed a biological system capable of producing **proteins** that **bind** to and **functionalize** cellulose. The selected system is *Saccharomyces cerevisiae*, a yeast widely used for **heterologous protein production**. Although several experts suggested using alternative yeasts, such as *Pichia pastoris*, we adhered to the protocol described in a reference article [(Gilbert et al., 2021)](https://www.nature.com/articles/s41563-020-00857-5), as it provided a **well-characterized** and **functional** co-culture system.

The primary objective of this study is to enable *S. cerevisiae* to co-produce an **adhesive** protein (**bioglue**) and a **colored** protein, both designed to **bind** cellulose, rendering it both visually appealing and **adhesive**. We first identified **adhesive proteins** in nature, selecting **MaSp1** (spider silk protein) and **Cp19k** (barnacle protein), which, when fused, exhibit **enhanced adhesive strength**, making them ideal for our bioglue (Ye et al., 2023).

After selecting the **bioglue**, we searched for **colored** proteins, or **chromoproteins**. Based on findings from the iGEM Uppsala 2014 team, we identified **fwYellow** ([BBa\_K1033910](https://parts.igem.org/Part%3ABBa_K1033910)), a yellow **chromoprotein** suitable for **attracting pest insects**. At this stage, we had identified the proteins needed to **functionalize** cellulose. The next step involved **optimizing** *S. cerevisiae* to produce these proteins and ensuring their binding to cellulose.

### Protein secretion and binding

---

To achieve this, we utilized a **cellulose-binding domain (CBD)** system described in a reference article [(Gilbert et al., 2021)](https://www.nature.com/articles/s41563-020-00857-5). This domain **binds** directly to **cellulose** and, when fused to a protein of interest, **facilitates** its **attachment** to the cellulose surface.

Finally, we investigated **protein secretion strategies** in *S. cerevisiae*, selecting the well-documented **alpha-factor signal sequence**, which enables the secretion of fused proteins (O’Riordan et al., 2024). This ensures that both **adhesive** and **colored** proteins are secreted into the surrounding medium, promoting their **interaction** with cellulose.

Once all target sequences were identified, they were fused using the **SnapGene** software as follows:

![Figure 8](https://static.igem.wiki/teams/5143/results/results-figure8.png)

Figure 8 : [BBA\_K5143022](https://parts.igem.org/Part%3ABBA_K5143022)

![Figure 9](https://static.igem.wiki/teams/5143/results/results-figure9.png)

Figure 9 :[BBA\_K5143023](https://parts.igem.org/Part%3ABBA_K5143023)

### Final BioBrick construction

---

We then identified a **viral peptide** called **P2A**, which enables the generation of **polycistronic RNAs** in **eukaryotic** organisms (Mukherjee, M. & Wang, 2023, Liu et al., 2017). This approach was particularly suited to our project, as it allows the expression of **two constructs** under a **single promoter**. Consequently, we **fused** the two constructs using the P2A system and incorporated a **strong constitutive yeast promoter**, the **GAP** promoter:

![Figure 10](https://static.igem.wiki/teams/5143/results/results-figure10.png)

Figure 10: [BBA\_K5143024](https://parts.igem.org/Part%3ABBA_K5143024)

## Cloning of the BioBrick in Plasmid D and transformation into *S. cerevisiae*

Once our construct was finalized, the goal was to **integrate** it into *S. cerevisiae* to enable the expression of our **two recombinant proteins**. To achieve this, we added 500 bp **homologous regions** flanking the construct, targeting the **URA3** gene locus. Additionally, we included the URA3 gene itself to facilitate the **selection** of transformants:

![Figure 11](https://static.igem.wiki/teams/5143/results/results-figure11.png)

Figure 11: SnapGene map of plasmid D ; [BBA\_K5143025](https://parts.igem.org/Part%3ABBA_K5143025)

### Transformation of plasmid D in *S. cerevisiae*

---

However, achieving the final plasmid, referred to as **plasmid D**, required several construction steps, which are summarized on the [Engineering](https://2024.igem.wiki/univlyon1-insalyon/engineering) page. Once the plasmid was **assembled**, it was **linearized** and **transformed** into *S. cerevisiae*, resulting in **successful transformants**.

![Figure 12](https://static.igem.wiki/teams/5143/results/results-figure12.png)

Figure 12: Colonies on URA- medium after plating the transformation of *S. cerevisiae* BY4741 strain with the linearized plasmid D.

To **confirm** the correct **integration** of the fragment into the yeast genome, eight transformants were selected, and **colony PCR** was performed:

![Figure 13](https://static.igem.wiki/teams/5143/results/results-figure13.png)

Figure 13: Colony PCR amplifying the MaSp1-Cp19k-CBD genes from the plasmid D integrated into the *S. cerevisiae* genome. The expected fragment size is 1633 bp.

For the eight selected clones, a band of the **expected size** was observed, confirming the **integration** of the fragment into the *S. cerevisiae* genome. This result demonstrates the **efficacy** of plasmid D in integrating into the *S. cerevisiae* genome and its capability to **insert heterologous genes**.

## Proteins secretion by *S. cerevisiae*

While the genes have been **successfully integrated** into the genome, it remains **uncertain** whether they are being **expressed** and yet to produce a protein. To clarify this, further analyses are necessary. Consequently, **positive PCR clones** were isolated on selective plates, and additional assessments were conducted to validate the various mutant yeast strains.

### SDS-PAGE

---

For each clone, it was necessary to determine whether the proteins of interest were **produced** and subsequently **secreted** by the yeast. To achieve this, first a **SDS-PAGE** was conducted for two clones confirmed by PCR (**ScpD1** and **ScpD7**) under three different conditions: crude extract, supernatant, and precipitated supernatant.

![Figure 14](https://static.igem.wiki/teams/5143/results/results-figure14.png)

Figure 14: Detection of proteins in CE = Crude Extract, S = Supernatant, CS = Concentrated Supernatant. WT: *S. cerevisiae* BY4741. ScpD1 and ScpD7 : clone 1 and clone 7 of *S. cerevisiae* transformed with plasmid D.

**No bands** were detected in the **supernatant** or **concentrated supernatant** from cultures of clones 1 and 7 of *S. cerevisiae* transformed with plasmid D. Similarly, no bands were observed in the WT condition. Additionally, **no apparent differences** in protein bands were seen between **WT** and **recombinant yeast** for protein extraction from the cell pellets. In conclusion, **no proteins** were **secreted** in sufficient quantities to be detected by SDS-PAGE. A **western blot** should therefore be performed to **specifically** verify the **production** and **secretion** of YFP and bioglue.

### Protein detection in supernatant via Western-Blot

---

Thus, a **Western blot** was conducted on the clones ScpD1 and ScpD7 under the same three different conditions as the SDS-PAGE: crude extract, supernatant, and precipitated supernatant. In each Western blot analysis, the presence of **YFP**, the product of the fwYellow gene, was detected using **anti-GFP antibodies** present in the plasmid D integrated into the *S. cerevisiae* genome.

![Figure 15](https://static.igem.wiki/teams/5143/results/results-figure15.png)

Figure 15: Detection of fluorescent proteins. CE = Crude Extract, S = Supernatant and CS = Concentrated Supernatant. A: Positive control: Purified GFP. GFP ctrl: *S. cerevisiae* GFP-producer. WT: *S. cerevisiae* BY4741 B: ScpD1 and ScpD7: clone 1 and clone 7 of *S. cerevisiae* transformed with plasmid D. Expected sizes: alphafactor-YFP-CBD 49.2 kDa, alphafactor-GFP-CBD 48.1 kDa, GFP 27 kDa.

The expected size for the **alpha-factor-YFP-CBD** fusion protein present in the plasmid D integrated into *S. cerevisiae* is **49.2 kDa**. A band of this size was **observed** for clones 1 and 7 (YFP1 and YFP7) in the **precipitated supernatant**. This result suggests that the fusion protein is indeed **expressed and secreted** by the yeast. Furthermore, it indicates that the **P2A** system is **functioning** correctly, allowing for the **translation** of two recombinant proteins from the same messenger RNA.

However, numerous **non-specific bands** were observed in the precipitated supernatants, necessitating the performance of additional **replicates** of this experiment to confirm these results.

The objective of the fwYellow gene was to produce a **yellow-colored protein** that would be visible to the **naked eye** in order to color our trap. This protein appears to be **expressed and secreted** by the yeast, as previously observed in the Western blot analysis. However, **no yellow** coloration was detected, either **visually** on the yeast colonies or through **fluorescence** (data not shown). Thus, the product of the fwYellow gene is **not functional** when expressed in yeast. To address this issue, it will be necessary to identify **other** functional yellow chromoproteins in yeast and clone them as **replacements** for fwYellow.

## Perspectives

To confirm the results observed in the Western blot using anti-GFP antibodies, this manipulation needs to be **repeated**.
In this study, we were **unable** to determine the **expression, production, and secretion** of the **α-factor–BioGlue–CBD fusion protein** because we did not have the necessary antibodies available.
We could utilize **anti-CBD antibodies** to detect our two proteins of interest: α-factor–YFP–CBD and α-factor–BioGlue–CBD. This would allow us to **confirm** the presence of each protein in the **supernatant** and further **validate** the **P2A** and **α-factor** systems.
To impart colored characteristics to cellulose, it will be necessary to identify **new functional chromoproteins** in yeast.
To address the issue of **low protein production**, one possible solution would be to use a **stronger promoter** to drive higher levels of gene expression. Replacing the current promoter with a well-characterized, high-strength promoter, such as the **PGK1** or **TEF1** promoters, could **enhance** the transcription of the fusion protein. This would likely **increase** protein **production**, making it easier to detect and evaluate its functionality in yeast. To confirm the **effectiveness** of our BioGlue, **adhesion tests** on cellulose should be conducted using a **dynamometer**.

References

[[1]](https://www.nature.com/articles/s41563-020-00857-5) Gilbert, C. et al. Living materials with programmable functionalities grown from engineered microbial co-cultures. Nat Mater 20, 691–700 (2021).

[[2]](https://pubmed.ncbi.nlm.nih.gov/37776922/) Ye, L. et al. A bioinspired synthetic fused protein adhesive from barnacle cement and spider dragline for potential biomedical materials. Int J Biol Macromol 253, 127125 (2023).

[[3]](https://2013.igem.org/Team%3AUppsala/chromoproteins) Team:Uppsala/chromoproteins - 2013.igem.org. https://2013.igem.org/Team:Uppsala/chromoproteins.

[[4]](https://pubmed.ncbi.nlm.nih.gov/38483353/) O’Riordan, N. M., Jurić, V., O’Neill, S. K., Roche, A. P. & Young, P. W. A Yeast Modular Cloning (MoClo) Toolkit Expansion for Optimization of Heterologous Protein Secretion and Surface Display in Saccharomyces cerevisiae. ACS Synth. Biol. 13, 1246–1258 (2024).

[[5]](https://www.nature.com/articles/s41598-017-02460-2) Liu, Z. et al. Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci Rep 7, 2193 (2017).

[[6]](https://pubmed.ncbi.nlm.nih.gov/36168285/) Mukherjee, M. & Wang, Z. Q. A well-characterized polycistronic-like gene expression system in yeast. Biotechnology and Bioengineering 120, 260–271 (2023).

[[7]](https://www.nature.com/articles/s41589-024-01602-8#citeas)Chong, G. Reeling in microorganisms. Nat Chem Biol 20, 393 (2024).

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