iGEM 2020 — Team Paris_Bettencourt

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# Team:Paris Bettencourt/Notebook

[![Paris Bettencourt logo](https://static.igem.org/mediawiki/2020/4/44/T--Paris_Bettencourt--TeamLogo.png)](https://2020.igem.org/Team%3AParis_Bettencourt)

* [Overview![Dropdown Down Arrow](https://static.igem.org/mediawiki/2020/7/7c/T--Paris_Bettencourt--DropDownArrow.png)](https://2020.igem.org/Team%3AParis_Bettencourt)
* Project
  ![Dropdown Down Arrow](https://static.igem.org/mediawiki/2020/7/7c/T--Paris_Bettencourt--DropDownArrow.png)
* Public Engagement![Dropdown Down Arrow](https://static.igem.org/mediawiki/2020/7/7c/T--Paris_Bettencourt--DropDownArrow.png)
* [Parts](https://2020.igem.org/Team%3AParis_Bettencourt/Parts)

* [Lab Notebook](https://2020.igem.org/Team%3AParis_Bettencourt/Notebook)
* [Team![Dropdown Down Arrow](https://static.igem.org/mediawiki/2020/7/7c/T--Paris_Bettencourt--DropDownArrow.png)](https://2020.igem.org/Team%3AParis_Bettencourt/Team)
* [Judging Form](https://2020.igem.org/Team%3AParis_Bettencourt/Judging)

* [Skin Microbiome](https://2020.igem.org/Team%3AParis_Bettencourt/SkinMicrobiome)
* [Description](https://2020.igem.org/Team%3AParis_Bettencourt/Description)
* [Implementation](https://2020.igem.org/Team%3AParis_Bettencourt/Implementation)

* [Quaranskin](https://2020.igem.org/Team%3AParis_Bettencourt/Human_Practices)
* [EpiFlex](https://2020.igem.org/Team%3AParis_Bettencourt/Engineering)
* [EpiGrow](https://2020.igem.org/Team%3AParis_Bettencourt/Contribution)
* [EpiGlow](https://2020.igem.org/Team%3AParis_Bettencourt/Proof_Of_Concept)

* [Human Practices* [Science Communication](https://2020.igem.org/Team%3AParis_Bettencourt/Education)](https://2020.igem.org/Team%3AParis_Bettencourt/Human_Practices)

* [Members & Mentors](https://2020.igem.org/Team%3AParis_Bettencourt/Team)
* [Attributions](https://2020.igem.org/Team%3AParis_Bettencourt/Attributions)
* [Collaborations](https://2020.igem.org/Team%3AParis_Bettencourt/Collaborations)
* [Diversity & Inclusion](https://2020.igem.org/Team%3AParis_Bettencourt/Inclusion)

![Lab Notebook](https://static.igem.org/mediawiki/2020/9/9e/T--Paris_Bettencourt--LabNotebook.jpg)

### Lab Notebook

[![Lab Notebook](https://static.igem.org/mediawiki/2020/f/fd/T--Paris_Bettencourt--lab_notebook.png "Click to view Lab Notebook")](https://static.igem.org/mediawiki/2020/9/9e/T--Paris_Bettencourt--nb.pdf)

![Paris Bettencourt logo](https://static.igem.org/mediawiki/2020/4/44/T--Paris_Bettencourt--TeamLogo.png)

Centre de Recherches Interdisciplinaires (CRI)
 8 rue Charles V
 75004 Paris, France
![](https://static.igem.org/mediawiki/2020/6/60/T--Paris_Bettencourt--Email.png) paris-bettencourt-2020@cri-paris.org

[![](https://static.igem.org/mediawiki/2020/b/bf/T--Paris_Bettencourt--Twitter.png)](https://twitter.com/iGem_Paris)
[![](https://static.igem.org/mediawiki/2020/d/d7/T--Paris_Bettencourt--Facebook.png)](https://www.facebook.com/ParisiGEM2020)
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[![](https://static.igem.org/mediawiki/2020/c/cc/T--Paris_Bettencourt--CRI.png)](https://www.cri-paris.org/en)

[![](https://static.igem.org/mediawiki/2020/d/da/T--Paris_Bettencourt--Fondation_Bettencourt.png)](https://www.fondationbs.org/)

[![](https://static.igem.org/mediawiki/2020/2/2a/T--Paris_Bettencourt--iGEM.png)](https://igem.org/Main_Page)

© 2020 iGEM Paris Bettencourt | [CRI](https://www.cri-paris.org/en)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Paris Bettencourt/Engineering

[![Paris Bettencourt logo](https://static.igem.org/mediawiki/2020/4/44/T--Paris_Bettencourt--TeamLogo.png)](https://2020.igem.org/Team%3AParis_Bettencourt)

* [Overview![Dropdown Down Arrow](https://static.igem.org/mediawiki/2020/7/7c/T--Paris_Bettencourt--DropDownArrow.png)](https://2020.igem.org/Team%3AParis_Bettencourt)
* Project
  ![Dropdown Down Arrow](https://static.igem.org/mediawiki/2020/7/7c/T--Paris_Bettencourt--DropDownArrow.png)
* Public Engagement![Dropdown Down Arrow](https://static.igem.org/mediawiki/2020/7/7c/T--Paris_Bettencourt--DropDownArrow.png)
* [Parts](https://2020.igem.org/Team%3AParis_Bettencourt/Parts)

* [Lab Notebook](https://2020.igem.org/Team%3AParis_Bettencourt/Notebook)
* [Team![Dropdown Down Arrow](https://static.igem.org/mediawiki/2020/7/7c/T--Paris_Bettencourt--DropDownArrow.png)](https://2020.igem.org/Team%3AParis_Bettencourt/Team)
* [Judging Form](https://2020.igem.org/Team%3AParis_Bettencourt/Judging)

* [Skin Microbiome](https://2020.igem.org/Team%3AParis_Bettencourt/SkinMicrobiome)
* [Description](https://2020.igem.org/Team%3AParis_Bettencourt/Description)
* [Implementation](https://2020.igem.org/Team%3AParis_Bettencourt/Implementation)

* [Quaranskin](https://2020.igem.org/Team%3AParis_Bettencourt/Human_Practices)
* [EpiFlex](https://2020.igem.org/Team%3AParis_Bettencourt/Engineering)
* [EpiGrow](https://2020.igem.org/Team%3AParis_Bettencourt/Contribution)
* [EpiGlow](https://2020.igem.org/Team%3AParis_Bettencourt/Proof_Of_Concept)

* [Human Practices* [Science Communication](https://2020.igem.org/Team%3AParis_Bettencourt/Education)](https://2020.igem.org/Team%3AParis_Bettencourt/Human_Practices)

* [Members & Mentors](https://2020.igem.org/Team%3AParis_Bettencourt/Team)
* [Attributions](https://2020.igem.org/Team%3AParis_Bettencourt/Attributions)
* [Collaborations](https://2020.igem.org/Team%3AParis_Bettencourt/Collaborations)
* [Diversity & Inclusion](https://2020.igem.org/Team%3AParis_Bettencourt/Inclusion)

![Quaranskin](https://static.igem.org/mediawiki/2020/e/e2/T--Paris_Bettencourt--EpiFlex.png)

### EpiFlex, a MoClo Toolkit for *S. epidermidis*

##### The little history of EpiFlex genesis...

After investigating the skin microbiome composition with the Quaranskin study, the iGEM Paris
Bettencourt team aspired to engineer the microbiome. The final outlook being, the ability to control
populations dynamics of the skin microbiome in order to maintain its equilibrium and avoid dysbiosis
induced pathologies.This is where we envision the role of synthetic biology in the probiotic arena.

To pursue this, we focused on a commensal bacteria of the skin microbiome to use as a chassis for
synthetic biology: *Staphylococcus epidermidis*. On this quest we discovered that this
bacterium has few tools available for efficient expression of recombinant DNA and genetic
engineering. This generated the idea to develop a MoClo toolkit for *S.epidermidis*, the
EpiFlex toolkit. Here we hope to establish a robust and useful set of genetic parts for standardized
rational design of genetic circuits within this cool microbe, for the staphylococcus and synthetic
biology community alike.

##### The concept of the MoClo toolkit A MoClo kit is a modular cloning concept based on Golden Gate assembly. EpiFlex is a Moclo toolikit developed specifically for parts that function in *S.epidermidis* The aim of this toolkit is to allow the cloning of transcriptional units (TU) in a direct and rapid fashion. It also allows efficient construction of many different TUs or even genetic circuits by assembling various combinations of parts or TUs which have standard fusion sites. The concept of developing a kit is to furnish a set of parts, such as a promoter, RBS, CDS, or terminator, all stored in plasmids.A backbone receiving the differents parts of TUs to allows scientists to assemble their own genes choosing the parts they want like pieces of a puzzle. The plasmid containing individual parts is called a level 0 plasmid (p0). The plasmids containing different parts assembled creating a TU are called level 1 plasmids (p1), and finally the level 2 plasmids (p2) are plasmids containing several TUs to create a genetic circuit or metabolic pathway (Fig.1).

![Figure 1](https://static.igem.org/mediawiki/2020/1/1e/T--Paris_Bettencourt--EpiFlex_Fig1.png)

**Figure 1. Concept of MoClo :**  All the parts individually stored in p0
are assembled in a TU stored in a P1 using Golden Gate assembly. In the same way, several TUs of
different p1 can be assembled in a p2 (inset). Blue tint squares and green tint squares are
respectively level 1 fusion sites allowing assembly the p1 and level 2 fusion sites for assembly in
the p2.

![Figure 2](https://static.igem.org/mediawiki/2020/f/fb/T--Paris_Bettencourt--EpiFlex_Fig2.png)

**Figure 2. Design of the backbones and parts :**  **A.** Design of the
backbone of the plasmid of level 0 (p0), level 1 (p1), level 2 (p2). Grey tint squares are level 0
fusion sites (FSp0), blue tint squares are level 1 fusion sites (FSp1) and green tint squares are
level 2 fusion sites. Pink tint and blue antibiotic resistance genes are respectively E. coli and S.
epidermis ones **B.** Design of a part to be assembled in the p0 backbone using Golden Gate
assembly. Here is an exemple of a part with the fusion sites FSp1\_A and FSp1\_B, so the part is
designed to be in the 1st position in the p1 TU. These fusion sites of p1 (in blue) has to be
modified in function of the position of the part in the TU.

##### Plasmids and Parts Design The p0, p1 and p2 plasmids contain ampicillin, kanamycin and chloramphenicol resistance genes respectively (Fig.2) for selection in *E. coli*. Each part is designed by flanking the ends with level 1 fusion sites (depending on its relative position inside the TU), and BsaI restriction sites. This allows the assembly of multiple parts in the p1 backbone. These sequences (Part + level 1 fusion site + BsaI recognition site) are also surrounded on both sides by p0\_fusion sites and BbsI restriction sites allowing to insert the part into the p0 backbone. The p0 backbone has a GFP dropout cassette in between the the pairs of p0 fusion sites and BbsI restriction sites allowing us to verify the insertion of the part. The removal of the GFP cassette is a visual indicator of successful transformants, as the lack of a green suggests successful interruption of the cassette. In the same way, the p1 backbones are designed with a GFP cassette in between pairs of A/H fusion sites and BbsI restriction site needed to link to the first and the last part of the TU. It also has a level 2 fusion site and BbsI restriction site allowing the assembly of different TUs in the p2 backbone following the same principle as the TU assembly.

We chose a fluorescent reporter as a cloning selection marker to avoid the extra cost of reagents
needed in traditional screening methods such as blue-white screening which requires X-gal.

An important feature of our p1 and p2 backbones is the fact that they're *E. coli* -> *S.
epidermidis* shuttle vectors. This means that they contain origins of replication for both
*E. coli* and *S. epidermidis* as well antibiotic resistance selection for each microbe.
This is important for a two step cloning workflow as *E. coli* is an easy host for cloning and
plasmid amplification before seeing the performance of the cassette in S. epidermidis. The
difficulty of cloning into *S. epidermidis* is further illustrated in project EpiGlow page [here.](https://2020.igem.org/Team%3AParis_Bettencourt/Proof_Of_Concept)

![Figure 3](https://static.igem.org/mediawiki/2020/7/74/T--Paris_Bettencourt--EpiFlex_Fig3.png)

**Figure 3. Scheme of EpiFlex part design map :**  Each DNA part can be
designed with terminal fusion sites that correspond to a particular position within a TU, with a
total of 7 possible positions as the kit currently stands.

Table 1 shows the nomenclature of the parts and the backbones in the MoClo toolkit for now. Each
part is
added to the toolkit after being designed to be EpiFlex compatible, should follow the same
nomencalture.

For now one p1 has been successfully assembled using the EpiFlex toolkit, with a TU coding for the
mCherry protein. This plasmid has been characterized and this is part of the EpiGlow project which
is a
proof of concept of EpiFlex.You'll find more information about this project on the EpiGlow page [here.](https://2020.igem.org/Team%3AParis_Bettencourt/Proof_Of_Concept)

![Figure 4](https://static.igem.org/mediawiki/2020/b/bf/T--Paris_Bettencourt--EpiFlex_Fig4.png)

**Figure 4. Nomenclature and plasmids assembled :** **A.** Nomenclature
of
the plasmids. pPBEF : plasmid Paris Bettencourt EpiFlex. FS : Fusion Site. **B.** As an example,
a
table of the plasmids designed and assembled to create a TU coding for mCherry used in the EpiGrow
Project.

![Paris Bettencourt logo](https://static.igem.org/mediawiki/2020/4/44/T--Paris_Bettencourt--TeamLogo.png)

Centre de Recherches Interdisciplinaires (CRI)
 8 rue Charles
V
 75004 Paris, France
![](https://static.igem.org/mediawiki/2020/6/60/T--Paris_Bettencourt--Email.png)paris-bettencourt-2020@cri-paris.org

[![](https://static.igem.org/mediawiki/2020/b/bf/T--Paris_Bettencourt--Twitter.png)](https://twitter.com/iGem_Paris)
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[![](https://static.igem.org/mediawiki/2020/2/2a/T--Paris_Bettencourt--iGEM.png)](https://igem.org/Main_Page)

© 2020 iGEM Paris Bettencourt | [CRI](https://www.cri-paris.org/en)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Paris Bettencourt/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Paris Bettencourt/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)