Difference between revisions of "Team:Paris Bettencourt/Poster"

 
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             <!--This is the first row of the poster, it contains the title section --->
 
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             <!-- Poster Title-->
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             <!-- Poster title -->
 
             <div class="row">
 
             <div class="row">
 
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                     <img src="https://static.igem.org/mediawiki/2020/f/fb/T--Example--Poster_Title.png">
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                     <img src="https://static.igem.org/mediawiki/2020/0/0f/T--Paris_Bettencourt--Poster_Title1.jpg">
  
 
                     <!--Info: Place the title and text for this section, it will appear when the user clicks on it since the first_text class is used above, this section will appear by default -->
 
                     <!--Info: Place the title and text for this section, it will appear when the user clicks on it since the first_text class is used above, this section will appear by default -->
 
                     <div class="info">
 
                     <div class="info">
 
                         <!--Write the title of the section -->
 
                         <!--Write the title of the section -->
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                         <div class="title">SynDerma - The skin microbiome in the spotlight: From Sampling to Engineering</div>
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                         <div class="title">SynDerma - The Skin Microbiome in the Spotlight: <br /> From Sampling to
 +
                            Engineering
 +
                        </div>
 
                         <!--Write the text explaining this section -->
 
                         <!--Write the text explaining this section -->
 
                         <div class="text">
 
                         <div class="text">
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                             <p>Presented by Team Paris Bettencourt 2020</p>
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                             <p>By Team Paris Bettencourt 2020</p>
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                             <br/>
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                             <br />
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                             <p><b>Amandine Maire<sup>1</sup>, Anu Susan Kurian<sup>1</sup>, Chetan Kumar Velumurugan<sup>1</sup>, Nicolas Levrier<sup>1</sup>, Nikola Zarevski<sup>1</sup>, Valerie March<sup>1</sup>, Xavier Olessa-Daragon<sup>1</sup>,</b></p>
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                             <p><b>Amandine Maire<sup>1</sup>, Anu Susan Kurian<sup>1</sup>, Chetan Kumar
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                             <br/>
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                                    Velumurugan<sup>1</sup>, Nicolas Levrier<sup>1</sup>, Nikola Zarevski<sup>1</sup>,
 +
                                    Valerie March<sup>1</sup>, Xavier Olessa-Daragon<sup>1</sup></b>, Ariel
 +
                                Lindner<sup>2</sup>, Jake Wintermute<sup>2</sup>, Radoslaw Ejsmont<sup>2</sup>,
 +
                                Alexis Casas<sup>2</sup>, Darshak Bhatt<sup>2</sup></p>
 +
                             <br />
 
                             <p><b><sup>1</sup>Student Team Member</b>, <sup>2</sup>Team Mentor</p>
 
                             <p><b><sup>1</sup>Student Team Member</b>, <sup>2</sup>Team Mentor</p>
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                             <br/>
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                             <br />
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                             <b>Abstract</b>
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                             <b class="heading">Abstract</b>
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                             <p></p>
+
                             <p>SynDerma envisions therapeutics being administered by engineered microbes
 +
                                integrated into the skin microbiome. First, to understand the influence on the skin
 +
                                microbiome of individual habits such as hygiene, social interaction and exercise, which
 +
                                are all affected by this current unprecedented context of COVID-19 pandemic, we
 +
                                developed a community science project called Quaranskin. In Quaranskin we developed an
 +
                                at-home sampling kit, protocol and survey, in which participants swab four body sites
 +
                                for metagenomic analysis. Further implementation of this study will enable us to
 +
                                correlate the diversity and composition of those microbiome-data, to behaviours noted in
 +
                                the surveys, to uncover any trends. In parallel, we chose the skin commensal microbe
 +
                                <i>Staphylococcus epidermidis</i> to be a chassis for our future vision of microbial
 +
                                therapeutics enabled by synthetic biology. In projects EpiFlex, EpiGlow, and EpiGrow, we
 +
                                built a MoClo kit, expressed fluorescent proteins as a proof of concept, and optimised
 +
                                growth conditions, respectively.
 +
                            </p>
 
                         </div>
 
                         </div>
 
                     </div>
 
                     </div>
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                 <div class="section full" id="objective">
 
                 <div class="section full" id="objective">
 
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                     <img src="https://static.igem.org/mediawiki/2020/9/90/T--Paris_Bettencourt--Poster_Objectives1.jpg">
  
 
                     <!--Info: Place the title and text for this section, it will appear when the user clicks on it-->
 
                     <!--Info: Place the title and text for this section, it will appear when the user clicks on it-->
 
                     <div class="info">
 
                     <div class="info">
 
                         <!--Write the title of the section -->
 
                         <!--Write the title of the section -->
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                         <div class="title" id="objective_title"> Objectives </div>
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                         <div class="title"> Objectives </div>
 
                         <!--Write the text explaining this section -->
 
                         <!--Write the text explaining this section -->
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                         <div class="text"> <b>Objectives 1.</b> Investigate the impact of behavioural factors on the
+
                         <div class="text">
−
                            skin microbiome and provide novel data base to explore its composition within the context of
+
                            <!-- <p><b>Objectives 1.</b></p> -->
−
                            a pandemic. <br> <br> <b>Objectives 2.</b> Develop tools to engineer S. epidermidis. <br>
+
                            <p><b class="heading">1. Sample and Sequence the Human Skin Microbiome</b></p>
−
                             Control populations dynamics of the skin microbiome in order to maintain its equilibrium and
+
                            <p>Our first objective, pursued as part of the Quaranskin project, is to study the impact of
−
                            avoid dysbiosis induced pathologies.
+
                                environmental and behavioral factors on the skin microbiome, and to provide a new
 +
                                database to study its composition in the context of reduced social and environmental
 +
                                interactions.
 +
                            </p>
 +
                            <br />
 +
                            <!-- <p><b>Objectives 2.</b></p> -->
 +
                            <p><b class="heading">2. Design tools to engineer the bacterium <i>S. epidermidis</i></b>
 +
                             </p>
 +
                            <p>Our second objective is to make <i>S. epidermidis</i> an efficient synthetic biology chassis
 +
                                that can be used to monitor the population dynamics of the skin microbiome. This in
 +
                                order to maintain its equilibrium and to avoid pathologies induced by dysbiosis.
 +
                            </p>
 
                         </div>
 
                         </div>
 
                     </div>
 
                     </div>
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                 <div class="section third" id="inspiration">
 
                 <div class="section third" id="inspiration">
 
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                     <img src="https://static.igem.org/mediawiki/2020/1/18/T--Paris_Bettencourt--Poster_Inspiration31.jpg">
  
 
                     <!--Info: Place the title and text for this section, it will appear when the user clicks on it-->
 
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                         <div class="title"> Inspiration </div>
 
                         <div class="title"> Inspiration </div>
 
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                         <!--Write the text explaining this section -->
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                         <div class="text"> What inspired your team? What motivated you to work on this particular
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                         <div class="text">
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                             project? </div>
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                            <p>In the context of COVID-19 pandemic, we were, as most of the other iGEM teams confined at
 +
                                home.</p>
 +
 
 +
                            <div class="subsection third">
 +
                                <img
 +
                                    src="https://static.igem.org/mediawiki/2020/e/e5/T--Paris_Bettencourt--Poster_Inspiration1.png">
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                            </div>
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                            <div class="subsection two_thirds">
 +
                                <br />
 +
                                <br />
 +
                                <p class="padding-left">Public health was one of our primary concerns during the lockdown. We were intrigued by the question of the impact that the lockdown could have on our health. This led us to examine the skin microbiome.
 +
 
 +
 
 +
                                </p>
 +
                            </div>
 +
 
 +
                            <div class="clear"></div>
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                            <br />
 +
 
 +
                            <div class="subsection two_thirds">
 +
                                <p class="padding-right">Through our experience and the advice gathered from health experts, we decided to study the human skin microbiome. The impact of sanitary measures on the skin microbiome, in particular frequent washing of hands and other surfaces of the body, which has proven to be crucial for populations with eczema symptoms, legitimated our approach. This discussion drew our interest for dysbiosis induced pathologies and possible treatments involving synthetic biology.
 +
 
 +
                                </p>
 +
                             </div>
 +
                            <div class="subsection third">
 +
                                <img
 +
                                    src="https://static.igem.org/mediawiki/2020/0/03/T--Paris_Bettencourt--Poster_Inspiration2.png">
 +
                            </div>
 +
 
 +
                            <div class="clear"></div>
 +
                            <br />
 +
 
 +
                            <div class="subsection third">
 +
                                <img
 +
                                    src="https://static.igem.org/mediawiki/2020/6/63/T--Paris_Bettencourt--Poster_Inspiration3.png">
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                            </div>
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                            <div class="subsection two_thirds">
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                                <br />
 +
                                <br />
 +
                                <p class="padding-left">While lockdown posed a threat to social interactions, we wanted  to connect people through science by putting Open and Citizen Science at the core of our 2020 iGEM project.
 +
 
 +
                                </p>
 +
                            </div>
 +
                        </div>
 
                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
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                 <div class="section third" id="challenges">
 
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                     <img src="https://static.igem.org/mediawiki/2020/7/7e/T--Paris_Bettencourt--Poster_Challenges32.jpg">
  
 
                     <!--Info: Place the title and text for this section, it will appear when the user clicks on it-->
 
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                         <!--Write the title of the section -->
 
                         <!--Write the title of the section -->
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                         <div class="title"> Problem </div>
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                         <div class="title"> Challenges </div>
 
                         <!--Write the text explaining this section -->
 
                         <!--Write the text explaining this section -->
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                         <div class="text"> What is the problem your team is working to solve? How does it affect the
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                         <div class="text">
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                             world? </div>
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                            <p><b class="heading">1. Study the Skin Microbiome</b></p>
 +
                            <p>Sampling the human skin microbiome involved reaching out to a large cohort of people in the European Union, given the constraints imposed for collecting human derived samples and shipping of sampling kits during the lockdown. Social distancing measures brought additional constraints that had to be dealt with to achieve our objectives. 
 +
 
 +
.</p>
 +
 
 +
                             <img
 +
                                src="https://static.igem.org/mediawiki/2020/8/89/T--Paris_Bettencourt--Poster_SkinMicrobiomeStudy.png">
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 +
                            <p><b class="heading">2. Engineer the Skin Microbiome</b></p>
 +
                            <ul>
 +
                               
 +
                                <li>
 +
The main difficulties we have encountered in processing <i>S. epidermidis</i> in synthetic biology are twofold:</li>
 +
                            </ul>
 +
                            <img
 +
                                src="https://static.igem.org/mediawiki/2020/1/1f/T--Paris_Bettencourt--Poster_SkinMicrobiomeEngineering.png">
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                        </div>
 
                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
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                 <div class="section third" id="responses">
 
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                         <!--Write the title of the section -->
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                         <div class="title">Idea </div>
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                         <div class="title"> Responses </div>
 
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                         <div class="text"> How are you going to solve the problem? Where did the idea come from? </div>
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                         <div class="text">
 +
                            <p><b class="heading">1. Quaranskin</b></p>
 +
                            <p>While under lockdown, we sought to build a project that could involve citizens in science while maintaining social distancing.
 +
                                The following solutions were developed accordingly.
 +
                            </p>
 +
                          <br />
 +
                            <ul>
 +
                                <li>Designing a skin microbiome cohort study involving participants from the European Union
 +
                                </li>
 +
                                <li>Setting up a self-sampling kit delivered by post, for participants to anonymously provide samples of their skin microbiome along with a questionnaire detailing their habits
 +
                                </li>
 +
                                <li>Building an open database of the skin microbiome to foster open science
 +
                                </li>
 +
                                <li>Obtain approval from the Committee for People’s Protection and the assistance of legal experts to ensure project safety and compliance
 +
                                </li>
 +
 
 +
                            </ul>
 +
                            <br />
 +
                            <p><b class="heading">2. EpiFlex, EpiGlow, EpiGrow</b></p>
 +
                            <p>These three projects aims to develop tools to make <i>S. epidermidis</i> a good chassis for
 +
                                Synthetic biology.
 +
                            </p>
 +
                            <ul>
 +
                                <li><b>EpiFlex</b> is a MoClo tool kit for <i>S. epidermidis</i> </li>
 +
                                <li><b>EpiGlow</b> is the proof of concept of EpiFlex that aims to optimize
 +
                                    transformation protocol <i>S. epidermidis</i></li>
 +
                                <li><b>EpiGrow</b> is the optimization of <i>S. epidermidis</i> growth</i> </li>
 +
                            </ul>
 +
                        </div>
 
                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
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 +
                        src="https://static.igem.org/mediawiki/2020/a/a9/T--Paris_Bettencourt--Poster_TheSkinMicrobiome.jpg">
  
 
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                         <div class="title"> Skin Microbiome</div>
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                         <div class="title">The Skin Microbiome</div>
 
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                         <div class="text"> Here you can explain the engineering process behind your project. </div>
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                         <div class="text">
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                            <p><b class="heading">What is the Skin Microbiome?</b></p>
 +
                            <p>The human skin microbiome is a rich ecosystem of microorganisms that occupy the human skin at the level of the hypodermis, dermis, and    epidermis.
 +
                            </p>
 +
                            <br />
 +
                            <p><b class="heading">What role does the skin microbiome play in skin health? </b></p>
 +
                            <p>The human skin microbiome is tightly linked to the health of its host. Microorganisms in the skin microbiome prevent colonization of the skin by pathogenic microorganisms, and help train the immune system to be better prepared to fight pathogens. The skin microbiome is also involved in various pathologies, like atopic dermatitis, where its overall diversity is reduced in favor of Staphylococcus aureus which is over represented.
 +
                            </p>
 +
                            <br />
 +
                            <p><b class="heading">Why study the Skin Microbiome composition?</b></p>
 +
                            <p>
 +
                            <ul>
 +
                                <li>Most of the previous studies which on the skin microbiome performed by sampling human volunteers, were based on North American subjects. From these results, current evidence suggests a greater microbial diversity to be a beneficial trait. However there is a need for greater diversity in the participant pools from which this data is derived. Thus, a study observing human skin microbiome among a European population would provide an interesting perspective on skin microbiome studies.
 +
                                </li>
 +
                                <li>In today's global environment a significant number of people minimize their interactions with others. This represents a good opportunity to observe the environmental impact on the skin microbiome in a context of simplified interactions.
 +
                                </li>
 +
                                <li>By taking samples from the skin microbiome during the COVID-19 pandemic, we will have preserved evidence of the influence of the social restrictions induced by the pandemic on the skin microbiome.
 +
                                </li>
 +
                            </ul>
 +
                            </p>
 +
                            <br />
 +
                            <img
 +
                                src="https://static.igem.org/mediawiki/2020/8/80/T--Paris_Bettencourt--Poster_SkinMicrobiomeIllustration.png">
 +
 
 +
                            <br/>
 +
 
 +
                            <p>References</p>
 +
                            <ol class="references">
 +
                                <li>“Your Body Is a Wonderland ... of Bacteria.” Science | AAAS, May 28, 2009. https://www.sciencemag.org/news/2009/05/your-body-wonderland-bacteria.
 +
                                </li>                             
 +
                                <li>Baviera, Giuseppe, Maria Chiara Leoni, Lucetta Capra, Francesca Cipriani, Giorgio Longo, Nunzia Maiello, Giampaolo Ricci, and Elena Galli. “Microbiota in Healthy Skin and in Atopic Eczema.” Review Article. BioMed Research International, July 13, 2014. https://doi.org/https://doi.org/10.1155/2014/436921.
 +
                                </li>                             
 +
                                <li>OpenStax. “5.1 Layers of the Skin.” In Anatomy and Physiology. OpenStax, 2013. https://opentextbc.ca/anatomyandphysiology/chapter/5-1-layers-of-the-skin/.
 +
                                </li>                               
 +
                                <li>Byrd, Allyson L., Yasmine Belkaid, and Julia A. Segre. “The Human Skin Microbiome.” Nature Reviews Microbiology 16, no. 3 (March 2018): 143–55. https://doi.org/10.1038/nrmicro.2017.157.
 +
                                </li>                               
 +
                                <li>Schommer, Nina N., and Richard L. Gallo. “Structure and Function of the Human Skin Microbiome.” Trends in Microbiology 21, no. 12 (December 2013): 660–68. https://doi.org/10.1016/j.tim.2013.10.001.
 +
                                </li>
 +
                                <li>Alexeyev, Oleg A. “Bacterial Landscape of Human Skin: Seeing the Forest for the Trees.” Experimental Dermatology 22, no. 7 (2013): 443–46. https://doi.org/10.1111/exd.12160.
 +
                                </li>
 +
                            </ol>
 +
                        </div>
 
                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
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                <!-- Quaranskin -->
 
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                         <div class="title"> Engineering</div>
+
                         <div class="title">Quaranskin</div>
 
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                         <div class="text"> Here you can explain the engineering process behind your project. </div>
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 +
                            <p>Quaranskin, a word derived by the combination of Quarantine and Skin, is a project that brings together all the components to collect and analyze the skin microbiome samples collected from participants across Europe. Within this study, we aimed to investigate the impact of behavioral factors such as hygiene, exercise, and social interaction, on the composition of the human skin microbiome.
 +
                            </p>
 +
             
 +
                            <img
 +
                                src="https://static.igem.org/mediawiki/2020/6/6e/T--Paris_Bettencourt--Poster_QuaranskinStudy1.png">
 +
                            <br />
 +
                            <br />
 +
                            <p><b class="heading">Study Pipeline</b></p>
 +
                            <br />
 +
                            <img
 +
                                src="https://static.igem.org/mediawiki/2020/1/11/T--Paris_Bettencourt--Poster_QuaranskinStudy2.png">
 +
                          <br />                           
 +
                          <p><b>Participants recruitment</b></p>
 +
                            <p>Emails are sent to European citizens and  iGEMers, focusing on people whose countries where subject to                            social restrictions due to the pandemic. The expected number of  participants is 70.
 +
                            </p>
 +
                            <p><b>Participants enrollment</b></p>
 +
                            <p>Each participant has to fill a participation form and sign a consent form to be part of the study. After signing up to participate in the study, they are asked to create an account on a platform called Open Humans. Open Humans is a community-based platform that enables personal data collections across data streams, giving individuals more personal data access and control of sharing authorizations, and enabling academic research as well as patient-led projects.
 +
                            </p>
 +
                            <p>Participants are sent a link allowing them to create an account on the Open Humans platform that will generate an eight digit unique ID for each of them. This unique ID helps preserve anonymity of the participants in the study.
 +
                            </p>
 +
                            <p><b>Participants action</b></p>
 +
                            <p>Once officially enrolled in the study, each participant is shipped a kit containing all the  tools needed to sample their skin microbiome from 4 body sites. In parallel, the participants are asked to answer an online questionnaire with questions covering 4 main topics: personal characteristic (age, sex, nationality, etc.), hygiene habits, level of confinement and potential skin disorders.
 +
                            </p>
 +
                            <p><b>Microbiome sequencing</b></p>
 +
                            <p>Samples are then sent to a sequencing company (Genewiz). Bacterial DNA extraction is performed from the samples, before amplification of the V3-V4 regions of the 16S RNA gene, and sequencing of  the amplicons.
 +
                            </p>
 +
                            <p><b>Statistical analysis</b></p>
 +
                            <p>The results from composition and diversity analyses will be correlated to the questionnaires, to unveil any trend between the microbiome composition and characteristics of the lifestyle or skin disorders.
 +
                            </p>
 +
                            <br />
 +
 
 +
                            <p><b class="heading">Data Analysis</b></p>
 +
                            <p><b>1. Correlations between microbiome diversity and indexes of hygiene, personal information, and level of  social restriction
 +
                                </b></p>
 +
                            <p>The microbiomes in the generated database are grouped by index values in each of these 3 categories. By setting two indices, we can study the impact of the third element on the diversity of the microbiome.
 +
                            </p>
 +
                            <p><b>2. Identify environmental factors influencing the proportions of  Staphylococci
 +
                                </b></p>
 +
                            <p>A proportion threshold is defined and all individuals with a staphylococcal population above this threshold are isolated. Finally, we look for parameters common to these individuals.
 +
                            </p>
 +
                            <p><b>3. Investigate eczematic people’s microbiome
 +
                                </b></p>
 +
                            <p>We want to compare a typical composition of eczematic microbiome, found in the literature, to data collected from our subjects.
 +
                            </p>
 +
                            <p><b>4.  Analyse data based on common symptoms
 +
                                </b></p>
 +
                            <p>When a significant number of people present the same symptom, independently of the environment and lifestyle, we want to make a synthesis of the microbiome composition related to this symptom.
 +
                            </p>
 +
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                         <div class="title"> Engineering</div>
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                         <div class="title">Implementation</div>
 
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 +
                            <p><b class="heading">Ethical considerations</b></p>
 +
                            <p>As Quaranskin is a collection of data extracted from human derived samples and contains personal information, our research depends on the legal procedures related to research involving human being. This leads to particular care to respect people’s privacy and data protection.
 +
                            </p>
 +
                            <p>In France, Research Involving Human Being (RIPH) has to be approved and framed by the Committees for the Protection of Persons (CPP).
 +
                            </p>
 +
                            <p>We have submitted to the ethics committee a complete file presenting our study in detail. This file consisted mainly of a research protocol plus an information leaflet for the participants. Even though we already recruited participants and set up all the logistic aspects of the study, we are still waiting for their feedback to collect and analyse the samples.
 +
                            </p>
 +
 
 +
                            <p><b class="heading">Contribution to the scientific community</b></p>
 +
                            <ol>
 +
                                <li>Increase knowledge on the skin microbiome, more specifically the environmental factors that influence it
 +
                                </li>
 +
                                <li>Create a skin microbiome open database in the special context of a pandemic with living restrictions
 +
                                </li>
 +
                                <li>Develop Science@Home by providing  reproducible pipeline and protocols which can be used in future studies
 +
                                </li>
 +
                                <li>Instruct participants about their skin microbiome by sending them back their skin microbiome profile after analysis
 +
                                </li>
 +
                            </ol>
 +
                        </div>
 
                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
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                         <div class="title"> Engineer </div>
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                         <div class="title"> Engineering </div>
 
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                         <div class="text"> <b>Objectives 1.</b> Investigate the impact of behavioural factors on the
+
                         <div class="text">
−
                             skin microbiome and provide novel data base to explore its composition within the context of
+
                         
−
                             a pandemic. <br> <br> <b>Objectives 2.</b> Develop tools to engineer S. epidermidis. <br>
+
                            <p><b class="heading">Purpose</b></p>
−
                             Control populations dynamics of the skin microbiome in order to maintain its equilibrium and
+
                             <p>We aim to sense and modulate population dynamics of the skin microbiome in order to help
−
                             avoid dysbiosis induced pathologies.
+
                                maintain its equilibrium and avoid dysbiosis induced pathologies. This is where we
 +
                                envision the role of synthetic biology in the probiotic arena.
 +
                             </p>
 +
                            <p>More precisely, we thought about engineering <i>S. epidermidis</i> in order to control
 +
                                overgrowth of <i>S. aureus</i> which can induce eczema.
 +
                            </p>
 +
                            <p><b class="heading"><i>S. epidermidis</i></b></p>
 +
                             <p><i>S. epidermidis</i> represents approximately 90% of the aerobic skin flora, and about
 +
                                5% of
 +
                                the overall skin microbiome species. It is a non-motile gram-positive coccus, and a
 +
                                facultative anaerobe. Its mainly mutualistic behavior makes it a very good candidate to
 +
                                become a vector for sensors or ways to modulate the skin microbiome.
 +
                             </p>
 +
                            <p><b class="heading">Research Focus</b></p>
 +
                            <p>Three essential stages that we want to optimize to help engineer <i>S. epidermidis</i>:
 +
                            </p>
 +
                            <p></p>
 +
                            <ol>
 +
                                <li>Grow</li>
 +
                                <li>Transform</li>
 +
                                <li>Clone</li>
 +
                            </ol>
 
                         </div>
 
                         </div>
 
                     </div>
 
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                     <img src="https://static.igem.org/mediawiki/2020/9/91/T--Paris_Bettencourt--Poster_EpiFlex71.jpg">
 
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                     <div class="info">
 
                     <div class="info">
 
                         <!--Write the title of the section -->
 
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                         <div class="title"> Section 1 </div>
+
                         <div class="title">EpiFlex</div>
 
                         <!--Write the text explaining this section -->
 
                         <!--Write the text explaining this section -->
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                         <div class="text"> Use this section to explain whatever you would like! Suggestions: Safety,
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                         <div class="text">
−
                             Human Practices, Measurement, etc. </div>
+
                            <p>The bacterium <i>Staphylococcus epidermidis</i> has only 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.
 +
                             </p>
 +
 
 +
                            <div class="subsection full logo-align">
 +
                                <img src="https://static.igem.org/mediawiki/2020/b/b0/T--Paris_Bettencourt--Poster_EpiFlexLogo.png"
 +
                                    class="logo">
 +
                            </div>
 +
                            <p>EpiFlex aims to make the <i>Staphylococcus epidermidis</i> bacterium a chassis for synthetic biology.
 +
                            </p>
 +
 
 +
                            <p><b class="heading">MoClo Toolkit</b></p>
 +
                            <p>MoClo is a modular cloning method based on Golden Gate assembly. EpiFlex is a Moclo
 +
                                toolkit developed with parts that function specifically in S.epidermidis.
 +
                            </p>
 +
                            <p><b class="heading">The MoClo Concept</b></p>
 +
                            <img
 +
                                src="https://static.igem.org/mediawiki/2020/6/6b/T--Paris_Bettencourt--Poster_MoCloConcept.png">
 +
 
 +
                            <p><b class="heading">Plasmid and Parts Design</b></p>
 +
                            <img
 +
                                src="https://static.igem.org/mediawiki/2020/9/9c/T--Paris_Bettencourt--Poster_PlasmidParts.png">
 +
                            <p>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.
 +
                            </p>
 +
                            <p>An important feature of our p1 and p2 backbones is the fact that they're <i>E. coli</i>
 +
                                -> <i>S.
 +
                                    epidermidis</i> shuttle vectors. This is important for a two step cloning workflow
 +
                                as <i>E.
 +
                                    coli</i> is an easy host for cloning and plasmid amplification before seeing the
 +
                                performance
 +
                                of the cassette in <i>S. epidermidis</i>.
 +
                            </p>
 +
                        </div>
 
                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
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                 <div class="section third" id="epiglow">
 
                 <div class="section third" id="epiglow">
 
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                     <img src="https://static.igem.org/mediawiki/2020/9/9b/T--Paris_Bettencourt--Poster_EpiGlow72.jpg">
  
 
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                     <div class="info">
 
                     <div class="info">
 
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                         <!--Write the title of the section -->
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                         <div class="title"> Section 2 </div>
+
                         <div class="title">EpiGlow</div>
 
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                         <!--Write the text explaining this section -->
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                         <div class="text"> Use this section to explain whatever you would like! Suggestions: Safety,
+
                         <div class="text">
−
                             Human Practices, Measurement, etc. </div>
+
                            <p>EpiGlow is the proof of concept of our EpiFlex toolkit. We chose to express mCherry
 +
                                in <i>S. epidermidis</i> to demonstrate that our parts are functional.
 +
                            </p>
 +
 
 +
                            <div class="subsection full logo-align">
 +
                                <img src="https://static.igem.org/mediawiki/2020/b/b6/T--Paris_Bettencourt--Poster_EpiGlowLogo.png"
 +
                                    class="logo">
 +
                            </div>
 +
 
 +
                            <p>The expression of mCherry, a fluorescent protein, allows the easy characterization of
 +
                                different regulatory sequences, such as promoter, RBS and terminator.
 +
                            </p>
 +
 
 +
                            <p><b class="heading">Cloning Pipeline</b></p>
 +
                            <ul>
 +
                                <li>To evade the type IV restriction barriers in <i>S. epidermidis</i>, we used a
 +
                                    dam-/dcm- strain of <i>E. coli</i>.</li>
 +
                                <li>We had to clone into an efficient cloning strain before growing our plasmid in the
 +
                                    dam-/dcm- <i>E. coli</i> since the latter is not very efficient for cloning.</li>
 +
                            </ul>
 +
                            <img
 +
                                src="https://static.igem.org/mediawiki/2020/6/60/T--Paris_Bettencourt--Poster_EpiGlowProtocol.png">
 +
 
 +
                            <p><b class="heading">Protocols</b></p>
 +
                            <ul>
 +
                                <li>We built a TU coding for mCherry. We used the following parts of the EpiFlex toolkit
 +
                                    :
 +
                                </li>
 +
                            </ul>
 +
 
 +
                            <img
 +
                                src="https://static.igem.org/mediawiki/2020/b/bd/T--Paris_Bettencourt--Poster_EpiGlowProtocol2.png">
 +
 
 +
                            <ul>
 +
                                <li>We explored and optimized electroporation protocols<sup>1</sup> by testing different voltages and a
 +
                                    newer heat shock/ electroporation combination<sup>2</sup>, to see which protocol would yield
 +
                                    more transformants.
 +
                                </li>
 +
                            </ul>
 +
 
 +
                            <p><b class="heading">Results</b></p>
 +
                            <ul>
 +
                                <li>Using our EpiFlex system, we were able to successfully build a construct that
 +
                                    expressed mCherry in <i>S. epidermidis</i>
 +
                                </li>
 +
                            </ul>
 +
 
 +
                            <img
 +
                                src="https://static.igem.org/mediawiki/2020/1/14/T--Paris_Bettencourt--Poster_EpiGolwResult1.png">
 +
 
 +
                            <ul>
 +
                                <li>In our optimization of the electroporation of <i>S. epidermidis</i>, we found that a
 +
                                    voltage of 2.5kV yielded the greatest transformation efficiency. We achieve around 3
 +
                                    - 7 transformants per plate using this protocol.
 +
                                </li>
 +
                             </ul>
 +
 
 +
                            <img
 +
                                src="https://static.igem.org/mediawiki/2020/3/3b/T--Paris_Bettencourt--Poster_EpiGolwResult2.png">
 +
 
 +
                            <br/>
 +
 
 +
                            <p>References</p>
 +
                            <ol class="references">
 +
                                <li>Lee, Jean YH, et al. "Mining the Methylome Reveals Extensive Diversity in
 +
                                    Staphylococcus epidermidis Restriction Modification."Mbio10.6 (2019).
 +
                                </li>
 +
                                <li>Chen, Y. Erin, et al. "Decoding commensal-host communication through genetic
 +
                                    engineering of <i>Staphylococcus epidermidis</i>."bioRxiv(2019): 664656.
 +
                                </li>
 +
                            </ol>
 +
                        </div>
 
                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
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                 <div class="section third" id="epigrow">
 
                 <div class="section third" id="epigrow">
 
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                     <img src="https://static.igem.org/mediawiki/2020/b/b0/T--Paris_Bettencourt--Poster_EpiGrow.jpg">
  
 
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                     <div class="info">
 
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                         <div class="title"> Section 3 </div>
+
                         <div class="title">EpiGrow</div>
 
                         <!--Write the text explaining this section -->
 
                         <!--Write the text explaining this section -->
−
                         <div class="text"> Use this section to explain whatever you would like! Suggestions: Safety,
+
                         <div class="text">
−
                             Human Practices, Measurement, etc. </div>
+
                            <p>To effectively implement our goal of developing <i>Staphylococcus epidermidis</i> as a
 +
                                chassis
 +
                                for synthetic biology of the skin microbiome, it was essential to explore the growth of
 +
                                our target organism.
 +
                            </p>
 +
 
 +
                            <img
 +
                                src="https://static.igem.org/mediawiki/2020/3/3e/T--Paris_Bettencourt--Poster_EpiGrowLogo.png">
 +
 
 +
                            <p>We used the <i>S. epidermidis</i> strain ATCC12228
 +
                            </p>
 +
 
 +
                            <p><b class="heading">Parameters Tested</b></p>
 +
                            <p><b>1. Temperature</b></p>
 +
                            <p>We chose to test the temperature range from 32°C-42°C. In fact, skin may reach
 +
                                temperatures as low as 33°C (hands, feet, nose), up to 40°C (severe conditions).
 +
                             </p>
 +
                            <p>At each temperature, the growth was measured in TSB for 15 hours via optical density measurements.
 +
                            </p>
 +
                            <p><b>2. Acidity</b></p>
 +
                            <p>The acid mantle of the skin is key as it neutralizes alkaline-based aggressors (such as
 +
                                harsh surfactants) and maintains the optimal acid environment in which skin’s natural
 +
                                flora can thrive. The skin’s pH generally fluctuates between 5.6 and 6.4 (armpit,
 +
                                genital area) depending on the region of the body it is covering.
 +
                            </p>
 +
                            <p>We tested in pH range from pH3 to pH10. The measurement has been done in the same way as for
 +
                                the Temperature tests.
 +
                            </p>
 +
                            <p><b>3. Salinity</b></p>
 +
                            <p>With respect to the skin, there can be variations in salinity caused mainly by sweat and
 +
                                sebaceous glands. The evaporation of water from the release of heat enables the salts to
 +
                                remain on the skin.
 +
                            </p>
 +
                            <p>We varied the salinity of the media from 0.5% to 5.5% of NaCl. Again, the measurements
 +
                                have been done over 15 hours of growth in TSB.
 +
                            </p>
 +
 
 +
                            <p><b class="heading">Results</b></p>
 +
                            <ol>
 +
                                <li>The growth of <i>S. epidermidis</i> is stunted at lower temperatures, and it grows best at
 +
                                    37°C which is the normal human body temperature.
 +
                                </li>
 +
                                <li><i>S. epidermidis</i> thrives better in the neutral and basic pH conditions.
 +
                                </li>
 +
                                <li><i>S. epidermidis</i> is halo tolerant and it grows best in media with about 0.5
 +
                                    percent of NaCl.
 +
                                </li>
 +
                            </ol>
 +
                            <br/>
 +
 
 +
                            <img
 +
                                src="https://static.igem.org/mediawiki/2020/b/b6/T--Paris_Bettencourt--Poster_EpiGrowGrowthRates.png">
 +
                            <br/>
 +
 
 +
                            <p>References</p>
 +
                            <ol class="references">
 +
                                <li>Bierman, William (1936-04-04). "The Temperature of the Skin Surface". Journal of the
 +
                                    American Medical Association. 106 (14): 1158. doi:10.1001/jama.1936.02770140020007.
 +
                                    ISSN 0002-9955
 +
                                </li>
 +
                                <li>Kanitakis, Jean (2002-07-02). "Anatomy, histology and immunohistochemistry of normal
 +
                                    human skin". European Journal of Dermatology. 12 (4): 390–9, quiz 400–1. ISSN
 +
                                    1167-1122. PMID 12095893
 +
                                </li>
 +
                                <li>Benedict, FG; Miles, WR; Johnson, A (June 1919). "The Temperature of the Human
 +
                                    Skin". Proceedings of the National Academy of Sciences of the United States of
 +
                                    America. 5 (6): 218–22. Bibcode:1919PNAS....5..218B. doi:10.1073/pnas.5.6.218. PMC
 +
                                    1091574. PMID 16576376
 +
                                </li>
 +
                                <li>Edmonds-Wilson S,Nurinova N, Zapka C et al. “Review of human hand microbiome
 +
                                    research”.Journal of Dermatological Science (2015), 3-12,80(1).
 +
                                    doi.org/10.1016/j.jdermsci.2015.07.006
 +
                                </li>
 +
                                <li>Marples, M. J. The Ecology of the Human Skin. Charles C Thomas Publisher.
 +
                                    Springfield, Ill. (1965) pgs103-154
 +
                                </li>
 +
                                <li>Aly, Raza. Clinical Skin Microbiology. Springfield, IL: Thomas Books, 1987. 11-35.
 +
                                </li>
 +
                                <li>Elias, Peter M., and Kenneth R. Feingold, eds. Skin Barrier. Danbury: Marcel Dekker
 +
                                    Incorporated, 2006
 +
                                </li>
 +
                            </ol>
 +
                        </div>
 
                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
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                         <div class="title"> Results</div>
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                         <div class="title"> Future Work - EpiFlex and EpiGlow</div>
 
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                         <div class="text"> What did your team achieve? What do you plan to work on moving forward?
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                            <p><b class="heading">In the short run
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                                </b></p>
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                            <ul>
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                                <li>Improve transformation efficiency from electroporation
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                                </li>
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                                <li>Build more constructs using the EpiFlex toolkit to characterize all the parts
 +
                                </li>
 +
                            </ul>
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                            <p><b class="heading">In the long run
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                                </b></p>
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                            <ul>
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                                <li>Using EpiFlex to build a genetic circuit aiming to control population dynamics in the
 +
                                    skin microbiome.
 +
                                </li>
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                            </ul>
 
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                         <div class="title">Future Work - EpiGrow</div>
 
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                            <p><b class="heading">EpiGrow</b></p>
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                            <ul>
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                                <li>We would like to investigate the difference of growth kinetics between <i>S. epidermidis</i>
 +
                                    grown in 2D and in 3D media.
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                                </li>
 +
                                <li>We decided to build an artificial skin model of the human palm to develop a method to
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                                    measure growth on 2D media while having comparable data with growth in 3D media.
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                                </li>
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                            </ul>
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                                helped broaden our knowledge on both human and scientific topics! We truly enjoyed our
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                                experience while attending the virtual meetings and discussing on various topics with
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                                iGEMers across the world.
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                            </p>
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                            <p><b class="heading">A Chit-Chat around the Skin Microbiome</b></p>
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                            <p>In early June, our team enjoyed hosting a ChitChat session to discuss on the subject of
 +
                                the Skin Microbiome. Several iGEM teams joined us in this session and provided us with
 +
                                the opportunity in sharing our experience on this topic with them.
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                            </p>
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                            <p><b class="heading">iGEMeetParis: A Parisian Virtual Meetup</b></p>
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                            <p>During the first weekend of September, we hosted our own virtual meetup, together with
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                                the other Parisian teams. It took two months of preparation to set up an amazing weekend
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                                lined up with workshops, hackathons, discussions, pitch presentations and other cool
 +
                                social events!
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                            </p>
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                            <p><b class="heading">Cité des Sciences</b></p>
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                            <p>We had the wonderful opportunity to display our project at the Cité des Sciences Annual
 +
                                Science Fair in Paris where we shared our scientific communication with the general
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                                public.
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                            <p>We would like to thank the following people for their support during the course of this
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                                project</p>
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                            <b>
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                                <p>Valérie Antonio</p>
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                                <p>Mad Price Ball</p>
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                                <p>Céline Couteau</p>
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                                <p>Paulina Ejsmont</p>
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                                <p>Stéphanie Leclerc-Mercier</p>
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                                <p>Valérie Lerouyer</p>
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                                <p>Hamid Mebrouki</p>
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                                <p>Piers Millett</p>
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                                <p>Ian Monk</p>
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                                <p>Julia Oh</p>
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                                <p>Alexandre Singier</p>
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                                <p>Jean-Christophe Thalabard</p>
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                                <p>Bastian Greshake Tzovaras</p>
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                            </b>
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                            <br />
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Latest revision as of 03:54, 11 November 2020

SynDerma - The Skin Microbiome in the Spotlight:
From Sampling to Engineering

By Team Paris Bettencourt 2020


Amandine Maire1, Anu Susan Kurian1, Chetan Kumar Velumurugan1, Nicolas Levrier1, Nikola Zarevski1, Valerie March1, Xavier Olessa-Daragon1, Ariel Lindner2, Jake Wintermute2, Radoslaw Ejsmont2, Alexis Casas2, Darshak Bhatt2


1Student Team Member, 2Team Mentor


Abstract

SynDerma envisions therapeutics being administered by engineered microbes integrated into the skin microbiome. First, to understand the influence on the skin microbiome of individual habits such as hygiene, social interaction and exercise, which are all affected by this current unprecedented context of COVID-19 pandemic, we developed a community science project called Quaranskin. In Quaranskin we developed an at-home sampling kit, protocol and survey, in which participants swab four body sites for metagenomic analysis. Further implementation of this study will enable us to correlate the diversity and composition of those microbiome-data, to behaviours noted in the surveys, to uncover any trends. In parallel, we chose the skin commensal microbe Staphylococcus epidermidis to be a chassis for our future vision of microbial therapeutics enabled by synthetic biology. In projects EpiFlex, EpiGlow, and EpiGrow, we built a MoClo kit, expressed fluorescent proteins as a proof of concept, and optimised growth conditions, respectively.

Objectives

1. Sample and Sequence the Human Skin Microbiome

Our first objective, pursued as part of the Quaranskin project, is to study the impact of environmental and behavioral factors on the skin microbiome, and to provide a new database to study its composition in the context of reduced social and environmental interactions.


2. Design tools to engineer the bacterium S. epidermidis

Our second objective is to make S. epidermidis an efficient synthetic biology chassis that can be used to monitor the population dynamics of the skin microbiome. This in order to maintain its equilibrium and to avoid pathologies induced by dysbiosis.

Inspiration

In the context of COVID-19 pandemic, we were, as most of the other iGEM teams confined at home.



Public health was one of our primary concerns during the lockdown. We were intrigued by the question of the impact that the lockdown could have on our health. This led us to examine the skin microbiome.


Through our experience and the advice gathered from health experts, we decided to study the human skin microbiome. The impact of sanitary measures on the skin microbiome, in particular frequent washing of hands and other surfaces of the body, which has proven to be crucial for populations with eczema symptoms, legitimated our approach. This discussion drew our interest for dysbiosis induced pathologies and possible treatments involving synthetic biology.




While lockdown posed a threat to social interactions, we wanted to connect people through science by putting Open and Citizen Science at the core of our 2020 iGEM project.

Challenges

1. Study the Skin Microbiome

Sampling the human skin microbiome involved reaching out to a large cohort of people in the European Union, given the constraints imposed for collecting human derived samples and shipping of sampling kits during the lockdown. Social distancing measures brought additional constraints that had to be dealt with to achieve our objectives. .

2. Engineer the Skin Microbiome

  • The main difficulties we have encountered in processing S. epidermidis in synthetic biology are twofold:
Responses

1. Quaranskin

While under lockdown, we sought to build a project that could involve citizens in science while maintaining social distancing. The following solutions were developed accordingly.


  • Designing a skin microbiome cohort study involving participants from the European Union
  • Setting up a self-sampling kit delivered by post, for participants to anonymously provide samples of their skin microbiome along with a questionnaire detailing their habits
  • Building an open database of the skin microbiome to foster open science
  • Obtain approval from the Committee for People’s Protection and the assistance of legal experts to ensure project safety and compliance

2. EpiFlex, EpiGlow, EpiGrow

These three projects aims to develop tools to make S. epidermidis a good chassis for Synthetic biology.

  • EpiFlex is a MoClo tool kit for S. epidermidis
  • EpiGlow is the proof of concept of EpiFlex that aims to optimize transformation protocol S. epidermidis
  • EpiGrow is the optimization of S. epidermidis growth
The Skin Microbiome

What is the Skin Microbiome?

The human skin microbiome is a rich ecosystem of microorganisms that occupy the human skin at the level of the hypodermis, dermis, and epidermis.


What role does the skin microbiome play in skin health?

The human skin microbiome is tightly linked to the health of its host. Microorganisms in the skin microbiome prevent colonization of the skin by pathogenic microorganisms, and help train the immune system to be better prepared to fight pathogens. The skin microbiome is also involved in various pathologies, like atopic dermatitis, where its overall diversity is reduced in favor of Staphylococcus aureus which is over represented.


Why study the Skin Microbiome composition?

  • Most of the previous studies which on the skin microbiome performed by sampling human volunteers, were based on North American subjects. From these results, current evidence suggests a greater microbial diversity to be a beneficial trait. However there is a need for greater diversity in the participant pools from which this data is derived. Thus, a study observing human skin microbiome among a European population would provide an interesting perspective on skin microbiome studies.
  • In today's global environment a significant number of people minimize their interactions with others. This represents a good opportunity to observe the environmental impact on the skin microbiome in a context of simplified interactions.
  • By taking samples from the skin microbiome during the COVID-19 pandemic, we will have preserved evidence of the influence of the social restrictions induced by the pandemic on the skin microbiome.



References

  1. “Your Body Is a Wonderland ... of Bacteria.” Science | AAAS, May 28, 2009. https://www.sciencemag.org/news/2009/05/your-body-wonderland-bacteria.
  2. Baviera, Giuseppe, Maria Chiara Leoni, Lucetta Capra, Francesca Cipriani, Giorgio Longo, Nunzia Maiello, Giampaolo Ricci, and Elena Galli. “Microbiota in Healthy Skin and in Atopic Eczema.” Review Article. BioMed Research International, July 13, 2014. https://doi.org/https://doi.org/10.1155/2014/436921.
  3. OpenStax. “5.1 Layers of the Skin.” In Anatomy and Physiology. OpenStax, 2013. https://opentextbc.ca/anatomyandphysiology/chapter/5-1-layers-of-the-skin/.
  4. Byrd, Allyson L., Yasmine Belkaid, and Julia A. Segre. “The Human Skin Microbiome.” Nature Reviews Microbiology 16, no. 3 (March 2018): 143–55. https://doi.org/10.1038/nrmicro.2017.157.
  5. Schommer, Nina N., and Richard L. Gallo. “Structure and Function of the Human Skin Microbiome.” Trends in Microbiology 21, no. 12 (December 2013): 660–68. https://doi.org/10.1016/j.tim.2013.10.001.
  6. Alexeyev, Oleg A. “Bacterial Landscape of Human Skin: Seeing the Forest for the Trees.” Experimental Dermatology 22, no. 7 (2013): 443–46. https://doi.org/10.1111/exd.12160.
Quaranskin

Quaranskin, a word derived by the combination of Quarantine and Skin, is a project that brings together all the components to collect and analyze the skin microbiome samples collected from participants across Europe. Within this study, we aimed to investigate the impact of behavioral factors such as hygiene, exercise, and social interaction, on the composition of the human skin microbiome.



Study Pipeline



Participants recruitment

Emails are sent to European citizens and iGEMers, focusing on people whose countries where subject to social restrictions due to the pandemic. The expected number of participants is 70.

Participants enrollment

Each participant has to fill a participation form and sign a consent form to be part of the study. After signing up to participate in the study, they are asked to create an account on a platform called Open Humans. Open Humans is a community-based platform that enables personal data collections across data streams, giving individuals more personal data access and control of sharing authorizations, and enabling academic research as well as patient-led projects.

Participants are sent a link allowing them to create an account on the Open Humans platform that will generate an eight digit unique ID for each of them. This unique ID helps preserve anonymity of the participants in the study.

Participants action

Once officially enrolled in the study, each participant is shipped a kit containing all the tools needed to sample their skin microbiome from 4 body sites. In parallel, the participants are asked to answer an online questionnaire with questions covering 4 main topics: personal characteristic (age, sex, nationality, etc.), hygiene habits, level of confinement and potential skin disorders.

Microbiome sequencing

Samples are then sent to a sequencing company (Genewiz). Bacterial DNA extraction is performed from the samples, before amplification of the V3-V4 regions of the 16S RNA gene, and sequencing of the amplicons.

Statistical analysis

The results from composition and diversity analyses will be correlated to the questionnaires, to unveil any trend between the microbiome composition and characteristics of the lifestyle or skin disorders.


Data Analysis

1. Correlations between microbiome diversity and indexes of hygiene, personal information, and level of social restriction

The microbiomes in the generated database are grouped by index values in each of these 3 categories. By setting two indices, we can study the impact of the third element on the diversity of the microbiome.

2. Identify environmental factors influencing the proportions of Staphylococci

A proportion threshold is defined and all individuals with a staphylococcal population above this threshold are isolated. Finally, we look for parameters common to these individuals.

3. Investigate eczematic people’s microbiome

We want to compare a typical composition of eczematic microbiome, found in the literature, to data collected from our subjects.

4. Analyse data based on common symptoms

When a significant number of people present the same symptom, independently of the environment and lifestyle, we want to make a synthesis of the microbiome composition related to this symptom.

Implementation

Ethical considerations

As Quaranskin is a collection of data extracted from human derived samples and contains personal information, our research depends on the legal procedures related to research involving human being. This leads to particular care to respect people’s privacy and data protection.

In France, Research Involving Human Being (RIPH) has to be approved and framed by the Committees for the Protection of Persons (CPP).

We have submitted to the ethics committee a complete file presenting our study in detail. This file consisted mainly of a research protocol plus an information leaflet for the participants. Even though we already recruited participants and set up all the logistic aspects of the study, we are still waiting for their feedback to collect and analyse the samples.

Contribution to the scientific community

  1. Increase knowledge on the skin microbiome, more specifically the environmental factors that influence it
  2. Create a skin microbiome open database in the special context of a pandemic with living restrictions
  3. Develop Science@Home by providing reproducible pipeline and protocols which can be used in future studies
  4. Instruct participants about their skin microbiome by sending them back their skin microbiome profile after analysis
Engineering

Purpose

We aim to sense and modulate population dynamics of the skin microbiome in order to help maintain its equilibrium and avoid dysbiosis induced pathologies. This is where we envision the role of synthetic biology in the probiotic arena.

More precisely, we thought about engineering S. epidermidis in order to control overgrowth of S. aureus which can induce eczema.

S. epidermidis

S. epidermidis represents approximately 90% of the aerobic skin flora, and about 5% of the overall skin microbiome species. It is a non-motile gram-positive coccus, and a facultative anaerobe. Its mainly mutualistic behavior makes it a very good candidate to become a vector for sensors or ways to modulate the skin microbiome.

Research Focus

Three essential stages that we want to optimize to help engineer S. epidermidis:

  1. Grow
  2. Transform
  3. Clone
EpiFlex

The bacterium Staphylococcus epidermidis has only 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.

EpiFlex aims to make the Staphylococcus epidermidis bacterium a chassis for synthetic biology.

MoClo Toolkit

MoClo is a modular cloning method based on Golden Gate assembly. EpiFlex is a Moclo toolkit developed with parts that function specifically in S.epidermidis.

The MoClo Concept

Plasmid and Parts Design

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 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.

EpiGlow

EpiGlow is the proof of concept of our EpiFlex toolkit. We chose to express mCherry in S. epidermidis to demonstrate that our parts are functional.

The expression of mCherry, a fluorescent protein, allows the easy characterization of different regulatory sequences, such as promoter, RBS and terminator.

Cloning Pipeline

  • To evade the type IV restriction barriers in S. epidermidis, we used a dam-/dcm- strain of E. coli.
  • We had to clone into an efficient cloning strain before growing our plasmid in the dam-/dcm- E. coli since the latter is not very efficient for cloning.

Protocols

  • We built a TU coding for mCherry. We used the following parts of the EpiFlex toolkit :
  • We explored and optimized electroporation protocols1 by testing different voltages and a newer heat shock/ electroporation combination2, to see which protocol would yield more transformants.

Results

  • Using our EpiFlex system, we were able to successfully build a construct that expressed mCherry in S. epidermidis
  • In our optimization of the electroporation of S. epidermidis, we found that a voltage of 2.5kV yielded the greatest transformation efficiency. We achieve around 3 - 7 transformants per plate using this protocol.

References

  1. Lee, Jean YH, et al. "Mining the Methylome Reveals Extensive Diversity in Staphylococcus epidermidis Restriction Modification."Mbio10.6 (2019).
  2. Chen, Y. Erin, et al. "Decoding commensal-host communication through genetic engineering of Staphylococcus epidermidis."bioRxiv(2019): 664656.
EpiGrow

To effectively implement our goal of developing Staphylococcus epidermidis as a chassis for synthetic biology of the skin microbiome, it was essential to explore the growth of our target organism.

We used the S. epidermidis strain ATCC12228

Parameters Tested

1. Temperature

We chose to test the temperature range from 32°C-42°C. In fact, skin may reach temperatures as low as 33°C (hands, feet, nose), up to 40°C (severe conditions).

At each temperature, the growth was measured in TSB for 15 hours via optical density measurements.

2. Acidity

The acid mantle of the skin is key as it neutralizes alkaline-based aggressors (such as harsh surfactants) and maintains the optimal acid environment in which skin’s natural flora can thrive. The skin’s pH generally fluctuates between 5.6 and 6.4 (armpit, genital area) depending on the region of the body it is covering.

We tested in pH range from pH3 to pH10. The measurement has been done in the same way as for the Temperature tests.

3. Salinity

With respect to the skin, there can be variations in salinity caused mainly by sweat and sebaceous glands. The evaporation of water from the release of heat enables the salts to remain on the skin.

We varied the salinity of the media from 0.5% to 5.5% of NaCl. Again, the measurements have been done over 15 hours of growth in TSB.

Results

  1. The growth of S. epidermidis is stunted at lower temperatures, and it grows best at 37°C which is the normal human body temperature.
  2. S. epidermidis thrives better in the neutral and basic pH conditions.
  3. S. epidermidis is halo tolerant and it grows best in media with about 0.5 percent of NaCl.


References

  1. Bierman, William (1936-04-04). "The Temperature of the Skin Surface". Journal of the American Medical Association. 106 (14): 1158. doi:10.1001/jama.1936.02770140020007. ISSN 0002-9955
  2. Kanitakis, Jean (2002-07-02). "Anatomy, histology and immunohistochemistry of normal human skin". European Journal of Dermatology. 12 (4): 390–9, quiz 400–1. ISSN 1167-1122. PMID 12095893
  3. Benedict, FG; Miles, WR; Johnson, A (June 1919). "The Temperature of the Human Skin". Proceedings of the National Academy of Sciences of the United States of America. 5 (6): 218–22. Bibcode:1919PNAS....5..218B. doi:10.1073/pnas.5.6.218. PMC 1091574. PMID 16576376
  4. Edmonds-Wilson S,Nurinova N, Zapka C et al. “Review of human hand microbiome research”.Journal of Dermatological Science (2015), 3-12,80(1). doi.org/10.1016/j.jdermsci.2015.07.006
  5. Marples, M. J. The Ecology of the Human Skin. Charles C Thomas Publisher. Springfield, Ill. (1965) pgs103-154
  6. Aly, Raza. Clinical Skin Microbiology. Springfield, IL: Thomas Books, 1987. 11-35.
  7. Elias, Peter M., and Kenneth R. Feingold, eds. Skin Barrier. Danbury: Marcel Dekker Incorporated, 2006
Future Work - EpiFlex and EpiGlow

In the short run

  • Improve transformation efficiency from electroporation
  • Build more constructs using the EpiFlex toolkit to characterize all the parts

In the long run

  • Using EpiFlex to build a genetic circuit aiming to control population dynamics in the skin microbiome.
Future Work - EpiGrow

EpiGrow

  • We would like to investigate the difference of growth kinetics between S. epidermidis grown in 2D and in 3D media.
  • We decided to build an artificial skin model of the human palm to develop a method to measure growth on 2D media while having comparable data with growth in 3D media.
Our iGEM Experience

iGEM 2020 was a unique opportunity to meet other aspiring and motivated researchers that helped broaden our knowledge on both human and scientific topics! We truly enjoyed our experience while attending the virtual meetings and discussing on various topics with iGEMers across the world.

A Chit-Chat around the Skin Microbiome

In early June, our team enjoyed hosting a ChitChat session to discuss on the subject of the Skin Microbiome. Several iGEM teams joined us in this session and provided us with the opportunity in sharing our experience on this topic with them.

iGEMeetParis: A Parisian Virtual Meetup

During the first weekend of September, we hosted our own virtual meetup, together with the other Parisian teams. It took two months of preparation to set up an amazing weekend lined up with workshops, hackathons, discussions, pitch presentations and other cool social events!

Cité des Sciences

We had the wonderful opportunity to display our project at the Cité des Sciences Annual Science Fair in Paris where we shared our scientific communication with the general public.

Acknowledgements

We would like to thank the following people for their support during the course of this project

Valérie Antonio

Mad Price Ball

Céline Couteau

Paulina Ejsmont

Stéphanie Leclerc-Mercier

Valérie Lerouyer

Hamid Mebrouki

Piers Millett

Ian Monk

Julia Oh

Alexandre Singier

Jean-Christophe Thalabard

Bastian Greshake Tzovaras

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