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

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                             </p>
 
                             </p>
  
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                             <img src="https://static.igem.org/mediawiki/2020/3/3e/T--Paris_Bettencourt--Poster_EpiGrowLogo.png">
+
                             <img
 +
                                src="https://static.igem.org/mediawiki/2020/3/3e/T--Paris_Bettencourt--Poster_EpiGrowLogo.png">
  
 
                             <p>We used the S. epidermidis strain ATCC12228
 
                             <p>We used the S. epidermidis strain ATCC12228
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                             <p>For each temperature, the growth was measured in TSB for 15 h via optical density.
 
                             <p>For each temperature, the growth was measured in TSB for 15 h via optical density.
 
                             </p>
 
                             </p>
−
                             <img src="https://static.igem.org/mediawiki/2020/1/12/T--Paris_Bettencourt--Poster_Temperature.png">
+
                             <img
 +
                                src="https://static.igem.org/mediawiki/2020/1/12/T--Paris_Bettencourt--Poster_Temperature.png">
 
                             <p><b>2. Acidity</b></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>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>
−
                             <p>We tested pH range from pH3 to pH10. The measurement has been done in the same way as for the Temperature tests.
+
                             <p>We tested pH range from pH3 to pH10. The measurement has been done in the same way as for
 +
                                the Temperature tests.
 
                             </p>
 
                             </p>
 
                             <img src="https://static.igem.org/mediawiki/2020/2/23/T--Paris_Bettencourt--Poster_Salinity.png">
 
                             <img src="https://static.igem.org/mediawiki/2020/2/23/T--Paris_Bettencourt--Poster_Salinity.png">
 
                             <p><b>3. Salinity</b></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 present on the skin.
+
                             <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 present on the skin.
 
                             </p>
 
                             </p>
−
                             <p>We made vary the salinity of the media from 0.5% to 5.5% of NaCl. Again, the measurements have been done over 15h of growth in TSB.
+
                             <p>We made vary the salinity of the media from 0.5% to 5.5% of NaCl. Again, the measurements
 +
                                have been done over 15h of growth in TSB.
 
                             </p>
 
                             </p>
 
                             <img src="https://static.igem.org/mediawiki/2020/1/11/T--Paris_Bettencourt--Poster_Acidity.png">
 
                             <img src="https://static.igem.org/mediawiki/2020/1/11/T--Paris_Bettencourt--Poster_Acidity.png">
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                             <p><b class="heading">Results</b></p>
 
                             <p><b class="heading">Results</b></p>
 
                             <ol>
 
                             <ol>
−
                                 <li>The growth of S. epidermidis is stunted at lower temperatures, and it grows best at 37°C which is the normal human body temperature.
+
                                 <li>The growth of S. epidermidis is stunted at lower temperatures, and it grows best at
 +
                                    37°C which is the normal human body temperature.
 
                                 </li>
 
                                 </li>
 
                                 <li><i>S. epidermidis</i> thrive better in the neutral and basic pH conditions.
 
                                 <li><i>S. epidermidis</i> thrive better in the neutral and basic pH conditions.
 
                                 </li>
 
                                 </li>
−
                                 <li><i>S. epidermidis</i> is halo tolerant and it grows best in media with about 0.5 percent of NaCl.
+
                                 <li><i>S. epidermidis</i> is halo tolerant and it grows best in media with about 0.5
 +
                                    percent of NaCl.
 
                                 </li>
 
                                 </li>
 
                             </ol>
 
                             </ol>
−
                             <img src="https://static.igem.org/mediawiki/2020/b/b6/T--Paris_Bettencourt--Poster_EpiGrowGrowthRates.png">
+
                             <img
 +
                                src="https://static.igem.org/mediawiki/2020/b/b6/T--Paris_Bettencourt--Poster_EpiGrowGrowthRates.png">
  
 +
                            <p>References</p>
 +
                            <ol>
 +
                                <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>

Revision as of 01:35, 11 November 2020

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

Presented 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 perturbaility of the skin microbiome by 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.



As this situation was unprecedented, we were wondering if the lockdown could have an impact on human health and more specifically on the composition of our skin microbiome.


We met some dermatologists as Stéphanie Leclerc-Mercier. She pointed out the fact that the new sanitary measures and hygiene habits imposed by the pandemic were a real issue for people with eczema symptoms since they cannot wash their hands as often as healthy people. This discussion drew our interest for dysbiosis induced pathologies and ways to treat them using synthetic biology.




Our team personal values led us to create a project who enters in the framework of Open and Citizen Science. Especially during the lockdown which forced us to be physically isolated of each other, we wanted to find a way to connect people through science.

Challenges

1. Study the Skin Microbiome

Human skin microbiome sampling is a challenge in itself since it implies reaching out to a large cohort and collecting human being derived samples. In the context of a pandemic other constraints are added because of social distancing measures.

2. Engineer the Skin Microbiome

  • Staphylococcus epidermidis is not used in synthetic biology and no tools are currently available to engineer it
  • Thw two main challenges in using S. epidermidis in synthetic biology are:
Responses

1. Quaranskin

Quaranskin is a study involving the collection of skin microbiome samples during the COVID-19 pandemic and observing the impact of environmental factors on the skin microbiome.

  • We took ethical considerations very seriously by submitting our project to the French national ethics committee in order to obtain their approval before starting the study
  • In order to include citizen participation, we designed a smapling kit containing all the material required for sampling the skin microbiome and set up mailing system to ship and collect back the samples from participants
  • We are building an Open Database of skin microbiome during the COVID-19 pandemic

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 vast and very large ecosystem of microorganisms that occupy the human skin at the level of the hypodermis, dermis, and epidermis. It is very numerous and very diverse, a real petri dish hidden in plain sight.


How the Skin Microbiome plays a role in the skin health?

The human skin microbiome is very tightly linked to the health of its host in numerous ways. First of all the microorganisms of the skin microbiome prevent the colonization of the skin by pathological microorganisms, and help to train the immune system to make it more prepared to face pathogens. It also directly involved in various pathologies, like atopic dermatitis, where its overall diversity is reduced and Staphylococcus aureus is over represented.


Why studying the composition of the Skin Microbiome?

  • Most of the previous studies which lead to understand the skin microbiome by sampling human volunteers were based on North American subjects. Form 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 give new data increasing our knowledge of the skin microbiome.
  • Besides the current global environment wherein a significant number of people are minimizing their interactions with other people is a good opportunity to observe environmental impact on skin microbiome in a more simplified model.
  • Also, 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.


Quaranskin

Quaranskin, a combination of quarantine and skin, is a project based on the collection and analysis of skin microbiome samples, collected from participants across Europe.

We aim to understand if there are correlations between behavioral characteristics that involve activity, hygiene and human interaction, and the diversity and composition of skin microbes at four body sites.


Study Pipeline


Participants recruitment

Emails are sent to all the members of our institution, the CRI, and all the european iGEMers, by focusing on people form countries where confinement or social distanciation are set up. The total number of participants expected is 70.

Participants enrollment

To be involved in the study each participants has to fill in a participation form and link to it a signed and dated consent form.

A link is sent to them allowing them to create a account on the OpenHumans platform in order to furnish them an ID code which we use to keep anonymity

Participants action

Once they are officially enroll in the study, we send to the participants by mail a kit containing all tools needed to sample microbiome from 4 body sites and to send them back to us, and by email a link to answer an online questionnaire asking questions covering 4 main topics : The intrinsic characteristic (age, sexe, nationality...), the hygiene habits, the level of confinement and the skin disorder appeared since February 2020.

Microbiome sequencing

Once we receive the microbiome samples we send them directly to an external company, Genewiz. They’ll extract bacterial DNA from the samples, amplify the V3-V4 regions of the 16S RNA gene, and then sequence the amplicons.

Statistical analysis

After having analyzed the composition and diversity of each microbiome we’ll link these results to the answer to the questionnaire to finally find some correlations between some microbiome compositions and some characteristics of the lifestyle or some skin disorder


Data Analysis

1. Diversity analysis by index of hygiene, personal information, level of restriction

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

2. Environmental factors which influence proportion of Staphylococcus

We define a proportion threshold, then we identify all the people who present a population of Staphylococcus beyond this threshold. We finally look for parameters common to these individuals,

3. Researching existing microbiome composition among our data set

We want to compare a typical composition of eczematic microbiome found in literature, with our data

4. Analysis of 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 typical composition of the microbiome for this symptom.

Implementation

Ethical consideration

As Quaranskin is a collection of data extracted from human derived samples (skin microbiome) and personal information (answer to the questionnaire), it’s important to learn about the ethical rules that govern research involving human being in Europe in order to respect the protection of the participants in our study.

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 and all the information documents for the study participants. Even though we already recruit participant and set up all the logistic aspect of the study, we are still waiting for their approval to collect and analyse samples.

Contribution to the scientific community

  1. Increase the knowledge we have about the microbiome and more precisely about the external factors that can impact it.
  2. Create an open database of skin microbiome from people in the context of a pandemic.
  3. Develop Science@Home by providing pipeline and protocols useful for future microbiome studies based on citizen science
  4. Instruct participants about their microbiome by sending them back their microbiome profile after analysis.
Engineering

Biological engineering allowed us to develop solutions for our objectives.

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 bacterium a chassis for synthetic biology.

MoClo Toolkit

The 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 choose 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 optimised 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 optimisation 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).

For each temperature, the growth was measured in TSB for 15 h via optical density.

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 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 present on the skin.

We made vary the salinity of the media from 0.5% to 5.5% of NaCl. Again, the measurements have been done over 15h 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 thrive 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 EpiGrow)

In the short run

  • Improve transformation efficiency form 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 dynamic in the skin microbiome.
Future Work (EpiGrow)

EpiGrow

  • We would like to investigate the difference of growth kinetic between S. epidermidis grown in 2D and in 3D media.
  • To do so, we decided to build an artificial skin model and 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

Acknowledgements

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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