http://2014.igem.org/wiki/index.php?title=Special:Contributions/FannyPineau&feed=atom&limit=50&target=FannyPineau&year=&month=2014.igem.org - User contributions [en]2024-03-29T06:31:37ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:Toulouse/ethicsTeam:Toulouse/ethics2014-10-17T22:10:23Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<script type='text/javascript' src='http://ajax.googleapis.com/ajax/libs/jquery/1.9.0/jquery.min.js'></script><br />
<br />
<script type='text/javascript'> $(function(){ <br />
$(window).scroll(function () {<br />
if ($(this).scrollTop() > 250) {<br />
$('#column-left').addClass("fixNavigation"); <br />
} else { <br />
$('#column-left').removeClass("fixNavigation");<br />
} <br />
}); <br />
}); <br />
</script><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.title4{color:#5a6060; font-family:'Open Sans'; font-weight:700; font-size:15px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align:justify;}<br />
<br />
.citation{color:#5a6060; font-family:'Open Sans'; font-size:18px; margin:0 0 50px 0; line-height:24px; font-style: italic; text-align: center;}<br />
<br />
p.legend{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: center;}<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/d/d0/Template-igem2014-slide1.jpg') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
<br />
.Title{<br />
text-shadow: 1px 3px 3px rgba(70, 156, 133, 0.75);<br />
color: #2CC23E;<br />
font-size: 50px;<br />
text-align: center;<br />
}<br />
<br />
.Sub_title{<br />
color: #2CC23E;<br />
font-size: 30px;<br />
}<br />
<br />
.Article {<br />
text-align: justify;<br />
text-indent:15px;<br />
}<br />
<br />
a.Link {<br />
font-size:10pt;<br />
color :#A4A4A4; <br />
text-decoration:none;<br />
}<br />
<br />
a.Link:hover{<br />
text-decoration:underline;<br />
}<br />
/*Overview*/<br />
.CropImg{<br />
position : absolute;<br />
width: 700px;<br />
left :50%;<br />
margin-left:-350px;<br />
height: auto; <br />
overflow: hidden;<br />
display :block;<br />
<br />
}<br />
<br />
table {<br />
display: table;<br />
border-collapse: separate;<br />
border-spacing: 2px;<br />
border-color: gray;<br />
}<br />
<br />
table[Attributes Style] {<br />
border-top-width: 1px;<br />
border-right-width: 1px;<br />
border-bottom-width: 1px;<br />
border-left-width: 1px;<br />
width: 100%;<br />
height: 20%;<br />
}<br />
<br />
#column-left{<br />
float:left;<br />
width: 190px;<br />
float: left;<br />
padding: 15px 10px 15px 15px;<br />
border: 1px solid #ccc;<br />
border-radius: 5px;<br />
background-color: #eee;<br />
}<br />
<br />
.fixNavigation{ <br />
z-index: 9999;<br />
position: fixed;<br />
top:15px;<br />
width:240px;<br />
}<br />
<br />
ul.menuleft {<br />
padding-left:10px;<br />
}<br />
<br />
<br />
ul.menuleft li {<br />
padding-bottom: 10px;<br />
}<br />
<br />
ul.menuleft li a{<br />
color:#5a6060;<br />
font-family:'Open Sans';<br />
font-size:14px;<br />
margin:0 0 0 0;<br />
line-height:24px;<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed; margin-top:30px;"><br />
<div style="margin:0 auto; width:960px;"><br />
<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Human practice&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Ethics</p> <br />
</div> <br />
</div><br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<div id="column-left"><br />
<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1">Protection of the beauty</a></li><br />
<li><a href="#select2">Human intervention in the nature</a></li><br />
<li><a href="#select3">SubtiTree</a></li><br />
</ul><br />
</div><br />
<br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<!--CITATION--><br />
<p class="citation"><br />
"Ethics is as important as laws."<br />
</p><br />
<br />
<p class="texte">The ethical<br />
questioning turned out to be one of the major starting points of our project.<br />
Acting on an established environment and modifying it is by no mean trivial and<br />
our combined technical and philosophical points of view. The actual purpose of our project also leads us to undertake an<br />
ethical questioning about the role of the scientist regarding “useless” things<br />
such as the trees lining along the Canal du Midi. </p><br />
<br />
<p class="title1" id="select1">Protection of the beauty</p><br />
<p class="title2">Is it the scientists’ role to protect beauty?<br />
</p><br />
<br />
<p class="texte"> Beauty is a<br />
feeling of satisfaction and is selfless. It is more a feeling than the property<br />
of a thing, this is not a notion we can clearly understand. Indeed, we can find<br />
something beautiful even when we don’t know the purpose of the object...</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2014/f/fa/Fontaine_Duchamp.jpg" width="400px"><br />
<p class="legend">Figure 1: Fontaine (Marcel Duchamp). Yes this is also art...</p></center><br />
<br />
<p class="texte">There is always<br />
a distinction between natural beauty and artistic beauty according to Hegel,<br />
the famous philosopher. The artistic beauty is born from our mind and our<br />
spirit: it is an element of signification of the work of art whereas the<br />
natural beauty of the object is external. In a way, the Canal du Midi combines<br />
both types of beauty: a natural one regarding the Nature, the centenary plane<br />
trees but also an artistic one since the Canal was built by the human hands.<br />
Usually, science judges beauty as a superficial feature not deserving to<br />
undertake any kind of scientific efforts to maintain it. The traditional role<br />
of science is to solve global issues and to elaborate complex strategies in<br />
order to find useful solutions for everyone’s life. Once made this observation,<br />
one may wonder why synthetic biology would be used only to protect the useless<br />
beauty of a local heritage such as the trees lining the Canal du Midi. <br />
</p><br />
<br />
<p class="texte"><br />
This crucial<br />
interrogation leads us to consider science and synthetic biology from a<br />
different point of view. <b>What if the role of scientists was also to make<br />
people rediscovering the beauty of Nature? What if the bases of new scientific<br />
challenges resulted from a more local scale? </b>Science does not have to be it has so much to gain opening itself to these<br />
challenges. First scientifically, as research is never<br />
useless and as we never know the impact and the scope of our results.<br />
Then socially, as we could measure the deep interest raised by our<br />
project within the population and the media. Adopting a new vision of synthetic<br />
biology, we will probably make people change their mind about this innovative<br />
discipline. <br><br />
The traditional cold objectivity of science distances itself from the society.<br />
However, scientists are also being capable of feeling the beauty, sensitive to<br />
the charm of landscapes and <b>able to understand the usefulness of<br />
"useless" trees</b>…<br><br />
The design of a strategy to protect useless beauty may seem senseless but we<br />
believe that it is also the scientist’s duty. We have to remember that thinking<br />
is what distinguish <i>Homo sapiens</i> from other species on earth and this<br />
"thinking" feature allows us to understand the world and be conscious<br />
of our human Nature (Descartes: <i>Cogito ergo sum</i>). The art is an object<br />
of philosophical thought. Consciousness raises humans above all others living<br />
creatures. Thus, it is necessary to respect and protect art. And thus, it<br />
becomes essential to preserve the beauty of the Canal du Midi. <br />
</p><br />
<br />
<br />
<p class="title1" id="select2">Human<br />
intervention on Nature</p><br />
<p class="texte">Our main<br />
question is to understand the complicated relationship between man and Nature.<br />
Does mankind have the proper right to change the Nature? Is modified Nature<br />
considered as artificial?</p><br />
<p class="title2"> Mankind & Nature</p><br />
<br />
<p class="texte"> Nature deserves<br />
to be respected and loved. Mankind has always been linked to Nature as its<br />
survival depends on what comes out of the ground, the trees, the oceans… The<br />
Nature is a source of wealth for mankind. It ensures survival and development<br />
by giving men the wood, the rocks, the soil to build shelters. Being in contact<br />
with Nature can allow men to feel strong emotion, as describe by poets like<br />
Hugo and Lamartine.</p><br />
<br />
<br />
<p class="texte">Since the birth<br />
of humanity, man himself understood the importance of studying and mastering<br />
Nature to develop the civilization. Still today the most advanced technologies<br />
often try to mimic natural phenomena. With the development of civilization, men<br />
modified their environment, changing it for their own comfort depending on<br />
their own desire. With the increase of human activity, the natural environment<br />
is modified profundly. With industrialization, the<br />
natural environment suffered from waste discharges, oil slicks, intensive<br />
fishing (and many others...) but also the introduction of devastating species<br />
such as the pathogen,<i> Ceratocystis<br />
platani</i>. However, despite these negative aspects, men<br />
are capable of favorable actions to help the environment and fix their<br />
mistakes. The current trend is to limit the impact of human interventions on<br />
Nature, and hopefully this trend is not transient and will not vanish. A new<br />
desire is born, a wish to protect Nature and wilderness. Humanity can adhere to<br />
this position: human take advantage of the<br />
environment and the environment takes advantage of the reasoned human<br />
interventions. There is an adaptation of mankind to Nature. Moreover, humans<br />
can have empathy: people are capable of understanding emotions and cognitive<br />
states of other organisms. To respond to these feelings, humans have<br />
technological tools allowing them to fight against enemies. This is the case<br />
with our project: fighting <i>Ceratocystis<br />
platani</i>. <br />
</p><br />
<br />
<p class="texte">In conclusion,<br />
by destroying and hammering the Nature, we jeopardize our lives. We need<br />
Nature, we come from Nature and we depend on Nature for survival, food,<br />
discoveries and civilisation. Respecting, loving and<br />
preserving the beauty of it is also a question of<br />
survival.</p><br />
<br />
<p class="title2">Nature and artifice<br />
</p><br />
<br />
<p class="texte">Talking about<br />
the Nature refers to the whole world with an exception: all the transformations<br />
made by mankind. Nature exists regardless of men and his interventions whereas<br />
artificial is everything that exists because to humans.<br />
</p><br />
<br />
<p class="texte">However,<br />
pretending that natural and artificial are opposite does not seem to be true.<br />
Man cannot create without the various elements provided by Nature, he then justs transform Nature. Thus we may wonder if there is a<br />
true difference between natural and artificial. The border between these two<br />
notions is not as obvious as it seems. The landscapes are shaped by the hand of<br />
man, animals are domesticated, and now bacteria are considered as cell<br />
factories. A natural reserve is artificially preserved as a result of human<br />
actions. Is there still something natural since the birth of mankind? Actually,<br />
the artifice is a slight modification of Nature and couldn’t exist by itself.<br />
The distinction between natural and artificial seems sterile and we clearly<br />
understand that these notions are inextricably linked and need each other to<br />
exist. </p><br />
<br />
<p class="texte">In conclusion,<br />
isn't it our duty to use our unique position in the history of life and our human<br />
approach to try to replace the evolutive processes?</p><br />
<br />
<br />
<p class="title2">Back to our project</p><br />
<br />
<br />
<p class="texte">These<br />
inextricable links are obviously the basis of our project. We aim to<br />
artificially preserve a natural heritage shaped by Pierre Paul Riquet hundreds years ago.Fighting a<br />
naturally occurring form of life that threatens it maybe just an imitation of<br />
the natural evolution process. What is considered today as ‘non-natural’<br />
may be one day regarded differently. To the extent that everything is done not<br />
to unbalance the ecosystem, our intervention can be judged rightful, even more<br />
than the use of chemicals.</p><br />
<br />
<br />
<p class="title1" id="select3">SubtiTree</p><br />
<br />
<p class="title2"> Potential strategies discussed<br />
<br> (See more details in the <a href="https://2014.igem.org/Team:Toulouse/Project/Spreading">Spreading</a> dedicated page)<br />
</p><br />
<br />
<p class="texte">To be sure that<br />
SubtiTree will not survive and spread in the<br />
environment, many strategies were discussed to improve our bacterium: <br />
<br />
<br>- Avoid the survival in the natural environment (outside the tree) thanks to a proline auxotrophy system <br />
<br>- Prevent the sporulation of <i>B. subtilis</i> to make it annual <br />
<br>- Avoid gene transfers between SubtiTree and a wild<br />
type bacterium thanks to a toxin-antitoxin system <br />
<br>- Use an integrative plasmid to improve the genetic stability<br />
</p><br />
<br />
<br />
<br />
<p class="title2">Public perception<br />
</p><br />
<br />
<p class="texte"><I><CENTER>Political and public adhesion</I></CENTER></p><br />
<br />
<p class="texte">Due to our<br />
strong implication in preserving this magnificent work of art, our project<br />
interested several governmental services. Indeed some municipalities and<br />
regional councils supported our local engagement. Beyond that, our project<br />
interests the highest level of the “Canal du Midi” administration: the national<br />
navigation authority (VNF) and the Ministry of agriculture. Both of them funded<br />
this project. They are now looking for the continuation of the project after<br />
the iGEM competition. This is clearly a sign that we targeted the right<br />
question. </p><br />
<br />
<p class="texte">This project<br />
also received the attention of the public through several articles in<br />
newspapers, television, radio and internet. First we had just a local coverage,<br />
but days after days there were more and more media interested in SubtiTree. This mediatic coverage<br />
allowed us to contact concerned citizens who participated to the development of<br />
this project. This interaction with the public allowed us to explain and<br />
promote public knowledge of synthetic biology. </p><br />
<br />
<br />
<p class="texte"><I><CENTER>Safety principle</I></CENTER></p><br />
<br />
<p class="texte">One single tree<br />
infected by Canker, and all the trees located in an area of a couple of hundred<br />
meters around are included in the prophylactic cut. We acted to preserve the<br />
surrounding trees. The modification of the endophytic<br />
microbial fauna generated by the introduction of the engineered bacterium has<br />
to be compared to the introduction of chemicals. They contain chlorine atom and<br />
aromatic hydrocarbon, so their remediation is complicated and they represent a<br />
source of pollution. By shortening the lifespan to one season and minimizing<br />
the risks of spreading, we plan a safe and environmental-friendly way to fight<br />
Canker. <br />
<br />
<br />
<p class="title2">Feasability<br />
</p><br />
<br />
<p class="texte">We wonder about<br />
the feasibility of tree’s treatment. As we used endophytic<br />
bacteria, we can count on the natural growth of SubtiTree<br />
inside the sap. So we can inject few bacteria to be sure to have enough<br />
bacteria to protect the tree. Some researchers (Xianling<br />
Ji<sup>1</sup> et al) already injected <i>Bacillus subtilis</i> in plants and<br />
observed an increase of bacteria concentration to a maximum of 10<sup>5</sup><br />
bacteria/mL</p><br />
<br />
<p class="texte">As we aim to<br />
inject a small quantity of bacteria, this treatment remains cheaper than the<br />
injection of several liters of chemical fungicides. In addition, this injection<br />
prevents the preventive tree cutting, which is very expensive. Cutting one tree<br />
cost around € 3000. The administration in charge of the protection of the<br />
“Canal du Midi” already plans to spend 220 million euros to cut and replant all<br />
trees along the Canal. Besides the important cost of cutting trees, it will<br />
destroy one of the symbols of south-western France. </p><br />
<br />
<p class="texte">We know that SubtiTree could be improved in many ways, but in the <br />
iGEM’s circumstances we could not have the time to go<br />
deeper. First, we can improve the fixation module. Using chitin as fixation<br />
anchor is simple but not enough specific to fix just one fungus type. That’s<br />
why we first think to fix SubtiTree to one protein<br />
included in the <i>Ceratocystis<br />
platani</i>’s<br />
membrane: CP. The bacterial prototype designed this summer can be optimized to<br />
trigger the fungicides production when the binding is completed, and to be more<br />
specific changing the peptides produced.</p><br />
<br />
<p class="title1">References</p><br />
<br />
<li class="tree"><p class="texte"> Xianling Ji, Guobing Lu, Yingping Gai, Chengchao Zheng & Zhimei Mu.<b> Biological control against bacterialwilt and colonization of<br />
mulberry byan endophyticBacillus subtilis strain </b>. FEMS Microbiol Ecol. 65 (2008) 565–573. </p></li><br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/ethicsTeam:Toulouse/ethics2014-10-17T17:15:00Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<script type='text/javascript' src='http://ajax.googleapis.com/ajax/libs/jquery/1.9.0/jquery.min.js'></script><br />
<br />
<script type='text/javascript'> $(function(){ <br />
$(window).scroll(function () {<br />
if ($(this).scrollTop() > 250) {<br />
$('#column-left').addClass("fixNavigation"); <br />
} else { <br />
$('#column-left').removeClass("fixNavigation");<br />
} <br />
}); <br />
}); <br />
</script><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.title4{color:#5a6060; font-family:'Open Sans'; font-weight:700; font-size:15px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align:justify;}<br />
<br />
.citation{color:#5a6060; font-family:'Open Sans'; font-size:18px; margin:0 0 50px 0; line-height:24px; font-style: italic; text-align: center;}<br />
<br />
p.legend{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: center;}<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/d/d0/Template-igem2014-slide1.jpg') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
<br />
.Title{<br />
text-shadow: 1px 3px 3px rgba(70, 156, 133, 0.75);<br />
color: #2CC23E;<br />
font-size: 50px;<br />
text-align: center;<br />
}<br />
<br />
.Sub_title{<br />
color: #2CC23E;<br />
font-size: 30px;<br />
}<br />
<br />
.Article {<br />
text-align: justify;<br />
text-indent:15px;<br />
}<br />
<br />
a.Link {<br />
font-size:10pt;<br />
color :#A4A4A4; <br />
text-decoration:none;<br />
}<br />
<br />
a.Link:hover{<br />
text-decoration:underline;<br />
}<br />
/*Overview*/<br />
.CropImg{<br />
position : absolute;<br />
width: 700px;<br />
left :50%;<br />
margin-left:-350px;<br />
height: auto; <br />
overflow: hidden;<br />
display :block;<br />
<br />
}<br />
<br />
table {<br />
display: table;<br />
border-collapse: separate;<br />
border-spacing: 2px;<br />
border-color: gray;<br />
}<br />
<br />
table[Attributes Style] {<br />
border-top-width: 1px;<br />
border-right-width: 1px;<br />
border-bottom-width: 1px;<br />
border-left-width: 1px;<br />
width: 100%;<br />
height: 20%;<br />
}<br />
<br />
#column-left{<br />
float:left;<br />
width: 190px;<br />
float: left;<br />
padding: 15px 10px 15px 15px;<br />
border: 1px solid #ccc;<br />
border-radius: 5px;<br />
background-color: #eee;<br />
}<br />
<br />
.fixNavigation{ <br />
z-index: 9999;<br />
position: fixed;<br />
top:15px;<br />
width:240px;<br />
}<br />
<br />
ul.menuleft {<br />
padding-left:10px;<br />
}<br />
<br />
<br />
ul.menuleft li {<br />
padding-bottom: 10px;<br />
}<br />
<br />
ul.menuleft li a{<br />
color:#5a6060;<br />
font-family:'Open Sans';<br />
font-size:14px;<br />
margin:0 0 0 0;<br />
line-height:24px;<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed; margin-top:30px;"><br />
<div style="margin:0 auto; width:960px;"><br />
<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Human practice&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Ethics</p> <br />
</div> <br />
</div><br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<div id="column-left"><br />
<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1">Protection of the beauty</a></li><br />
<li><a href="#select2">Human intervention in the nature</a></li><br />
<li><a href="#select3">SubtiTree</a></li><br />
</ul><br />
</div><br />
<br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<!--CITATION--><br />
<p class="citation"><br />
"Ethics is as important as laws."<br />
</p><br />
<br />
<p class="texte">The ethical questioning turned out to be one of the major starting points of our project. Acting on an established environment and modifying it is no mean feat and our thinking combines technical and philosophical points of view. The actual purpose of our project also leads us to undertake an ethical questioning about the role of the scientist regarding “useless” things such as the trees lining the Canal du Midi. </p><br />
<br />
<p class="title1" id="select1">Protection of the beauty</p><br />
<p class="title2">Is it the scientists’ role to protect beauty?<br />
</p><br />
<br />
<p class="texte"> Beauty is a feeling of satisfaction and is selfless. It is more a feeling than the property of a thing, this is not a notion we can clearly understand. Indeed, we can find something beautiful even when we don’t know the purpose of the object...</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2014/f/fa/Fontaine_Duchamp.jpg" width="400px"><br />
<p class="legend">Figure 1: Fontaine (Marcel Duchamp). Yes this is also art...</p></center><br />
<br />
<p class="texte">There is always a distinction between natural beauty and artistic beauty according to Hegel, the famous philosopher. The artistic beauty is born from our mind and our spirit: it is an element of signification of the work of art whereas the natural beauty of the object is external. In a way, the Canal du Midi combines both types of beauty: a natural one regarding the nature, the centenary plane trees but also an artistic one since the Canal was built by the human hands.<br />
Usually, science judges beauty as a superficial feature not deserving to undertake any kind of scientific efforts to maintain it. The traditional role of science is to solve global issues and to elaborate complex strategies in order to find useful solutions for everyone’s life. <br />
Once made this observation, one may wonder why synthetic biology would be used only to protect the useless beauty of a local heritage such as the trees lining the Canal du Midi. <br />
</p><br />
<br />
<p class="texte"><br />
This crucial interrogation leads us to consider science and synthetic biology in another way. <B> What if the role of scientists was also to make people rediscovering the beauty of nature? What if the bases of new scientific challenges resulted from a more local scale? </B> Science does not have to be elitist, it has so much to gain opening itself to these challenges. First scientifically, as research is never useless and as we never know the impact and the scope of our results. Then socially, as we could measure the deep interest raised by our project within the population and the media. Adopting a new vision of synthetic biology, we probably make people change their mind about this innovative discipline. <br><br />
The traditional cold objectivity of science distances itself from the society. However, scientists are also being capable of feeling the beauty, sensitive to the charm of landscapes and <B>able to understand the usefulness of "useless" trees</B>…<br><br><br />
The design of a strategy to protect useless beauty may seem senseless but we believe that it is also the scientist’s duty. We have to remember that the thought is what distinguish <i>Homo sapiens</i> from other species on earth and this thought make us able to understand the world and be conscious of our being (Descartes: <i>Cogito ergo sum</i>). The art is an object of philosophical thought. Consciousness raises humans above all others living creatures. Thus, it is necessary to respect and protect art. And thus, it becomes essential to preserve the beauty of this site. <br />
</p><br />
<br />
<br />
<p class="title1" id="select2">Human intervention in the nature</p><br />
<p class="texte">Our main questioning aim to understand the complicated relationship between man and nature. Does the mankind have the proper right to change the nature? Is modified nature considered as artificial?</p><br />
<p class="title2"> Mankind & Nature</p><br />
<br />
<p class="texte"> Nature is known as a creation of God. Human is linked to the nature and for that reason the nature deserves to be respected and loved. Mankind has always been linked to Nature as its survival depends on what comes out of the ground, the trees, the oceans… The nature is a source of wealth for the humankind. It ensures survival and development by giving men the wood, the rocks, the soil to build shelters. Being in contact with nature can allow men to feel strong emotion, as describe by poets like Hugo and Lamartine.</p><br />
<br />
<br />
<p class="texte">Since the birth of humanity, man himself understood well the importance of studying and mastering Nature to develop the civilization. Still today the most advanced technologies often try to mimic natural phenomena. With the development of the civilizations, men modified their environment, changing it for their own comfort depending on their own desire. By increasing their cities and acitvities, humans modify the natural environment. With the industrialization of the societies, the natural environment has suffered from human activities such as waste discharges, oil slicks, intensive fishing but also the introduction of devastating species such as the pathogen,<I> Ceratocystis platani</I>. However, despite these negative aspects, men are capable of favorable actions to help the environment and fix their mistakes. The current trend is to limit the impact of human interventions on the nature, and hopefully this trend is not transient and will not vanish. A new desire is born, a wish to protect the nature and the wildness. Man fits with his position: he takes advantage of the environment and the environment takes advantage of the reasoned human interventions. There is an adaptation of the mankind toward the nature. Moreover, humans have the capacity of empathy: people are able to understand the emotions and cognitive states of other organisms and to identify to them. To respond to these feelings, humans have technological tools allowing them to fight against enemies such as <I>Ceratocystis platani.</I><br />
</p><br />
<br />
<p class="texte">In conclusion, by destroying and hammering the nature, we jeopardize our lives. We need the nature, we come from the nature and we depend on nature for our survival, our food, our discoveries and our civilisation. Respecting, loving and preserving the environment is a question of survival.</p><br />
<br />
<p class="title2">Nature and artifice<br />
</p><br />
<br />
<p class="texte">Talking about the nature refers to the whole world with an exception: all the transformations made by mankind. Thus, the nature consists in the real without all the artificial elements created by humans. The nature is existing regardless of men and his interventions whereas artificial is everything that exists thanks to humans.<br />
</p><br />
<br />
<p class="texte">However, pretending that natural and artificial are opposite does not seem to be true. Man cannot create without elements provided by the nature, he is just transforming the nature, changing the shape. Thus we may wonder if there is a true difference between natural and artificial. The border between these two notions is not as obvious as it seems. The landscapes are shaped by the hand of man, animals are domesticated, and now bacteria are considered as cell factories. A natural reserve is artificially preserved as the result of human actions. Is there still something natural since the birth of humankind? Actually, the artifice is a slight modification of Nature and couldn’t exist by itself. The distinction between natural and artificial seems sterile and we clearly understand that these notions are inextricably linked and need each other to exist. </p><br />
<br />
<p class="texte">In conclusion, isn't our duty to use our unique position in the history of life and our human approach to try to replace the evolutive processes?</p><br />
<br />
<br />
<p class="title2">Back to our project</p><br />
<br />
<br />
<p class="texte">These inextricable links are obviously the basis of our project. We aim to artificially preserve a natural heritage shaped by Pierre Paul Riquet hundreds years ago. The modification of a naturally occurring form of life to strengthen it is maybe just the imitation of the natural evolution process. What is considered today as ‘non-natural’ may be one day regarded differently. To the extent that everything is done not to unbalance the ecosystem, our intervention can be judged rightful, even more than the use of chemicals.</p><br />
<br />
<br />
<p class="title1" id="select3">SubtiTree</p><br />
<br />
<p class="title2"> Potential strategies discussed<br />
<br> (See more details in the <a href="https://2014.igem.org/Team:Toulouse/Project/Spreading">Spreading</a> dedicated page)<br />
</p><br />
<br />
<p class="texte">To be sure that SubtiTree will not survive and spread in the environment, many strategies were discussed to improve our bacterium: <br />
<br />
<br>- Avoid the survival in the environment thanks to a proline auxotrophy system<br />
<br>- Prevent the sporulation of <I> B. subtilis</I> to make it annual <br />
<br>- Avoid gene transfers between SubtiTree and a wild type bacterium thanks to a toxin-antitoxin system <br />
<br>- Use integrative plasmid to improve the genetic stability<br />
</p><br />
<br />
<br />
<br />
<p class="title2">Public perception<br />
</p><br />
<br />
<p class="texte"><I><CENTER>Political and public adhesion</I></CENTER></p><br />
<br />
<p class="texte">Because of the magnitude of our project,its application interested several civil services. Indeed some municipalities and regional councils supported our local engagement. Beyond that, our project interests the highest level of the “Canal du Midi” administration: the national navigation authority and the Ministry of agriculture funded this project. They are still looking for the best way to further this work after the iGEM competition. </p><br />
<br />
<p class="texte">This project also received the attention of the public through several articles in newspapers, television, radio and internet. First we had just a local coverage, but days after days there were more and more media intereseted in SubtiTree. This mediatic coverage allowed us to contact concerned citizens who participated to the development of this project. This interaction with the public allowed us to explain and promote public knowledge of synthetic biology. </p><br />
<br />
<br />
<p class="texte"><I><CENTER>Safety principle</I></CENTER></p><br />
<br />
<p class="texte">One single tree infected by Canker, and all the trees located in an area of a couple of hundred meters around are included in the prophylactic cut. We acted to preserve the surrounding trees. The modification of the endophytic microbial fauna generated by the introduction of the engineered bacterium has to be compared to the introduction of chemicals. They contain chlorine atom and aromatic hydrocarbon, so their remediation is complicated and they represent a source of pollution. By shortening the lifespan to one season and minimizing the risks of spreading, we plan a safe and environmental-friendly way to fight Canker. <br />
<br />
<br />
<p class="title2">Feasability<br />
</p><br />
<br />
<p class="texte">We wonder about the feasibility of tree’s treatment. As we used endophytic bacteria, we can count on the natural growth of SubtiTree inside the sap. So we can inject few bacteria to be sure to have enough bacteria to protect the tree. Some researchers (Xianling Ji1 et al) already injected <i>Bacillus subtilis</i> in plants and observe an increase of bacteria concentration to a maximum of 10^5 bacteria/mL/p><br />
<br />
<p class="texte">As we aim to inject a poor quantity of bacteria, this treatment remains cheaper than the injection of several litter of fungicides. In addition, this injection prevents the preventive tree cutting, which is very expensive. Cutting one tree cost around €3000. The administration in charge of the protection of the “Canal du Midi” already plans to spend 220 million euros to cut and replant all trees along the Canal. Besides the important cost of cutting trees, it will destroy one of the symbols of south-western France. </p><br />
<br />
<p class="texte">We know that SubtiTree could be improved in many ways, but in the iGEM’s circumstances we could not have the time to go deeper. First, we can improve the fixation module. Using chitin as fixation anchor is simple but not enough specific to fix just one fungus type. That’s why we first think to fix SubtiTree to one protein included in the <I>Ceratocystis platani</I>’s membrane: CP. The bacterial prototype designed this summer can be optimized to trigger the fungicides production when the binding is completed, and to be more specific changing the peptides produced.</p><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/ethicsTeam:Toulouse/ethics2014-10-17T17:00:21Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<script type='text/javascript' src='http://ajax.googleapis.com/ajax/libs/jquery/1.9.0/jquery.min.js'></script><br />
<br />
<script type='text/javascript'> $(function(){ <br />
$(window).scroll(function () {<br />
if ($(this).scrollTop() > 250) {<br />
$('#column-left').addClass("fixNavigation"); <br />
} else { <br />
$('#column-left').removeClass("fixNavigation");<br />
} <br />
}); <br />
}); <br />
</script><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.title4{color:#5a6060; font-family:'Open Sans'; font-weight:700; font-size:15px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align:justify;}<br />
<br />
.citation{color:#5a6060; font-family:'Open Sans'; font-size:18px; margin:0 0 50px 0; line-height:24px; font-style: italic; text-align: center;}<br />
<br />
p.legend{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: center;}<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/d/d0/Template-igem2014-slide1.jpg') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
<br />
.Title{<br />
text-shadow: 1px 3px 3px rgba(70, 156, 133, 0.75);<br />
color: #2CC23E;<br />
font-size: 50px;<br />
text-align: center;<br />
}<br />
<br />
.Sub_title{<br />
color: #2CC23E;<br />
font-size: 30px;<br />
}<br />
<br />
.Article {<br />
text-align: justify;<br />
text-indent:15px;<br />
}<br />
<br />
a.Link {<br />
font-size:10pt;<br />
color :#A4A4A4; <br />
text-decoration:none;<br />
}<br />
<br />
a.Link:hover{<br />
text-decoration:underline;<br />
}<br />
/*Overview*/<br />
.CropImg{<br />
position : absolute;<br />
width: 700px;<br />
left :50%;<br />
margin-left:-350px;<br />
height: auto; <br />
overflow: hidden;<br />
display :block;<br />
<br />
}<br />
<br />
table {<br />
display: table;<br />
border-collapse: separate;<br />
border-spacing: 2px;<br />
border-color: gray;<br />
}<br />
<br />
table[Attributes Style] {<br />
border-top-width: 1px;<br />
border-right-width: 1px;<br />
border-bottom-width: 1px;<br />
border-left-width: 1px;<br />
width: 100%;<br />
height: 20%;<br />
}<br />
<br />
#column-left{<br />
float:left;<br />
width: 190px;<br />
float: left;<br />
padding: 15px 10px 15px 15px;<br />
border: 1px solid #ccc;<br />
border-radius: 5px;<br />
background-color: #eee;<br />
}<br />
<br />
.fixNavigation{ <br />
z-index: 9999;<br />
position: fixed;<br />
top:15px;<br />
width:240px;<br />
}<br />
<br />
ul.menuleft {<br />
padding-left:10px;<br />
}<br />
<br />
<br />
ul.menuleft li {<br />
padding-bottom: 10px;<br />
}<br />
<br />
ul.menuleft li a{<br />
color:#5a6060;<br />
font-family:'Open Sans';<br />
font-size:14px;<br />
margin:0 0 0 0;<br />
line-height:24px;<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed; margin-top:30px;"><br />
<div style="margin:0 auto; width:960px;"><br />
<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Human practice&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Ethics</p> <br />
</div> <br />
</div><br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<div id="column-left"><br />
<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1">Protection of the beauty</a></li><br />
<li><a href="#select2">Human intervention in the nature</a></li><br />
<li><a href="#select3">SubtiTree</a></li><br />
</ul><br />
</div><br />
<br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<!--CITATION--><br />
<p class="citation"><br />
"Ethics is as important as laws."<br />
</p><br />
<br />
<p class="texte">The ethical questioning turned out to be one of the major starting points of our project. Acting on an established environment and modifying it is no mean feat and our thinking combines technical and philosophical points of view. The actual purpose of our project also leads us to undertake an ethical questioning about the role of the scientist regarding “useless” things such as the trees lining the Canal du Midi. </p><br />
<br />
<p class="title1" id="select1">Protection of the beauty</p><br />
<p class="title2">Is it the scientists’ role to protect beauty?<br />
</p><br />
<br />
<p class="texte"> Beauty is a feeling of satisfaction and is selfless. It is more a feeling than the property of a thing, this is not a notion we can clearly understand. Indeed, we can find something beautiful even when we don’t know the purpose of the object...</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2014/f/fa/Fontaine_Duchamp.jpg" width="400px"><br />
<p class="legend">Figure 1: Fontaine (Marcel Duchamp). Yes this is also art...</p></center><br />
<br />
<p class="texte">There is always a distinction between natural beauty and artistic beauty according to Hegel, the famous philosopher. The artistic beauty is born from our mind and our spirit: it is an element of signification of the work of art whereas the natural beauty of the object is external. In a way, the Canal du Midi combines both types of beauty: a natural one regarding the nature, the centenary plane trees but also an artistic one since the Canal was built by the human hands.<br />
Usually, science judges beauty as a superficial feature not deserving to undertake any kind of scientific efforts to maintain it. The traditional role of science is to solve global issues and to elaborate complex strategies in order to find useful solutions for everyone’s life. <br />
Once made this observation, one may wonder why synthetic biology would be used only to protect the useless beauty of a local heritage such as the trees lining the Canal du Midi. <br />
</p><br />
<br />
<p class="texte"><br />
This crucial interrogation leads us to consider science and synthetic biology in another way. <B> What if the role of scientists was also to make people rediscovering the beauty of nature? What if the bases of new scientific challenges resulted from a more local scale? </B> Science does not have to be elitist, it has so much to gain opening itself to these challenges. First scientifically, as research is never useless and as we never know the impact and the scope of our results. Then socially, as we could measure the deep interest raised by our project within the population and the media. Adopting a new vision of synthetic biology, we probably make people change their mind about this innovative discipline. <br><br />
The traditional cold objectivity of science distances itself from the society. However, scientists are also being capable of feeling the beauty, sensitive to the charm of landscapes and <B>able to understand the usefulness of "useless" trees</B>…<br><br><br />
The design of a strategy to protect useless beauty may seem senseless but we believe that it is also the scientist’s duty. We have to remember that the thought is what distinguish <i>Homo sapiens</i> from other species on earth and this thought make us able to understand the world and be conscious of our being (Descartes: <i>Cogito ergo sum</i>). The art is an object of philosophical thought. Consciousness raises humans above all others living creatures. Thus, it is necessary to respect and protect art. And thus, it becomes essential to preserve the beauty of this site. <br />
</p><br />
<br />
<br />
<p class="title1" id="select2">Human intervention in the nature</p><br />
<p class="texte">Our main questioning aim to understand the complicated relationship between man and nature. Does the mankind have the proper right to change the nature? Is modified nature considered as artificial?</p><br />
<p class="title2"> Mankind & Nature</p><br />
<br />
<p class="texte"> Nature is known as a creation of God. Human is linked to the nature and for that reason the nature deserves to be respected and loved. Mankind has always been linked to Nature as its survival depends on what comes out of the ground, the trees, the oceans… The nature is a source of wealth for the humankind. It ensures survival and development by giving men the wood, the rocks, the soil to build shelters. Being in contact with nature can allow men to feel strong emotion, as describe by poets like Hugo and Lamartine.</p><br />
<br />
<br />
<p class="texte">Since the birth of humanity, man himself understood well the importance of studying and mastering Nature to develop the civilization. Still today the most advanced technologies often try to mimic natural phenomena. With the development of the civilizations, men modified their environment, changing it for their own comfort depending on their own desire. By increasing their cities and acitvities, humans modify the natural environment. With the industrialization of the societies, the natural environment has suffered from human activities such as waste discharges, oil slicks, intensive fishing but also the introduction of devastating species such as the pathogen,<I> Ceratocystis platani</I>. However, despite these negative aspects, men are capable of favorable actions to help the environment and fix their mistakes. The current trend is to limit the impact of human interventions on the nature, and hopefully this trend is not transient and will not vanish. A new desire is born, a wish to protect the nature and the wildness. Man fits with his position: he takes advantage of the environment and the environment takes advantage of the reasoned human interventions. There is an adaptation of the mankind toward the nature. Moreover, humans have the capacity of empathy: people are able to understand the emotions and cognitive states of other organisms and to identify to them. To respond to these feelings, humans have technological tools allowing them to fight against enemies such as <I>Ceratocystis platani.</I><br />
</p><br />
<br />
<p class="texte">In conclusion, by destroying and hammering the nature, we jeopardize our lives. We need the nature, we come from the nature and we depend on nature for our survival, our food, our discoveries and our civilisation. Respecting, loving and preserving the environment is a question of survival.</p><br />
<br />
<p class="title2">Nature and artifice<br />
</p><br />
<br />
<p class="texte">Talking about the nature refers to the whole world with an exception: all the transformations made by mankind. Thus, the nature consists in the real without all the artificial elements created by humans. The nature is existing regardless of men and his interventions whereas artificial is everything that exists thanks to humans.<br />
</p><br />
<br />
<p class="texte">However, pretending that natural and artificial are opposite does not seem to be true. Man cannot create without elements provided by the nature, he is just transforming the nature, changing the shape. Thus we may wonder if there is a true difference between natural and artificial. The border between these two notions is not as obvious as it seems. The landscapes are shaped by the hand of man, animals are domesticated, and now bacteria are considered as cell factories. A natural reserve is artificially preserved as the result of human actions. Is there still something natural since the birth of humankind? Actually, the artifice is a slight modification of Nature and couldn’t exist by itself. The distinction between natural and artificial seems sterile and we clearly understand that these notions are inextricably linked and need each other to exist. </p><br />
<br />
<p class="texte">In conclusion,isn't it our duty to use our unique position in the history of life and our human approach to try to replace the evolutive processes?</p><br />
<br />
<br />
<p class="title2">Back to our project</p><br />
<br />
<br />
<p class="texte">These inextricable links are obviously the basis of our project. We aim to artificially preserve a natural heritage shaped by Pierre Paul Riquet hundreds years ago. The modification of a naturally occurring form of life to strengthen it is maybe just the imitation of the natural evolution process. What is considered today as ‘non-natural’ may be one day regarded differently. To the extent that everything is done not to unbalance the ecosystem, our intervention can be judged rightful, even more than the use of chemicals.</p><br />
<br />
<br />
<p class="title1" id="select3">SubtiTree</p><br />
<br />
<p class="title2"> Potential strategies discussed<br />
<br> (See more details in the <a href="https://2014.igem.org/Team:Toulouse/Project/Spreading">Spreading</a> dedicated page)<br />
</p><br />
<br />
<p class="texte">To be sure that SubtiTree will not survive and spread in the environment, many strategies were discussed to improve our bacterium: <br />
<br />
<br>- Avoid the survival in the environment thanks to a proline auxotrophy system<br />
<br>- Prevent the sporulation of <I> B. subtilis</I> to make it annual <br />
<br>- Avoid gene transfers between SubtiTree and a wild type bacterium thanks to a toxin-antitoxin system <br />
<br>- Use integrative plasmid to improve the genetic stability<br />
</p><br />
<br />
<br />
<br />
<p class="title2">Public perception<br />
</p><br />
<br />
<p class="texte"><I><CENTER>Political and public adhesion</I></CENTER></p><br />
<br />
<p class="texte">Because of the magnitude of our project,its application interested several civil services. Indeed some municipalities and regional councils supported our local engagement. Beyond that, our project interests the highest level of the “Canal du Midi” administration: the national navigation authority and the Ministry of agriculture funded this project. They are still looking for the best way to further this work after the iGEM competition. </p><br />
<br />
<p class="texte">This project also received the attention of the public through several articles in newspapers, television, radio and internet. First we had just a local coverage, but days after days there were more and more media intereseted in SubtiTree. This mediatic coverage allowed us to contact concerned citizens who participated to the development of this project. This interaction with the public allowed us to explain and promote public knowledge of synthetic biology. </p><br />
<br />
<br />
<p class="texte"><I><CENTER>Safety principle</I></CENTER></p><br />
<br />
<p class="texte">One single tree infected by Canker, and all the trees located in an area of a couple of hundred meters around are included in the prophylactic cut. We acted to preserve the surrounding trees. The modification of the endophytic microbial fauna generated by the introduction of the engineered bacterium has to be compared to the introduction of chemicals. They contain chlorine atom and aromatic hydrocarbon, so their remediation is complicated and they represent a source of pollution. By shortening the lifespan to one season and minimizing the risks of spreading, we plan a safe and environmental-friendly way to fight Canker. <br />
<br />
<br />
<p class="title2">Feasability<br />
</p><br />
<br />
<p class="texte">We wonder about the feasibility of tree’s treatment. As we used endophytic bacteria, we can count on the natural growth of SubtiTree inside the sap. So we can inject few bacteria to be sure to have enough bacteria to protect the tree. Some researchers (Xianling Ji1 et al) already injected <i>Bacillus subtilis</i> in plants and observe an increase of bacteria concentration to a maximum of 10^5 bacteria/mL/p><br />
<br />
<p class="texte">As we aim to inject a poor quantity of bacteria, this treatment remains cheaper than the injection of several litter of fungicides. In addition, this injection prevents the preventive tree cutting, which is very expensive. Cutting one tree cost around €3000. The administration in charge of the protection of the “Canal du Midi” already plans to spend 220 million euros to cut and replant all trees along the Canal. Besides the important cost of cutting trees, it will destroy one of the symbols of south-western France. </p><br />
<br />
<p class="texte">We know that SubtiTree could be improved in many ways, but in the iGEM’s circumstances we could not have the time to go deeper. First, we can improve the fixation module. Using chitin as fixation anchor is simple but not enough specific to fix just one fungus type. That’s why we first think to fix SubtiTree to one protein included in the <I>Ceratocystis platani</I>’s membrane: CP. The bacterial prototype designed this summer can be optimized to trigger the fungicides production when the binding is completed, and to be more specific changing the peptides produced.</p><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/ethicsTeam:Toulouse/ethics2014-10-17T16:55:14Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<script type='text/javascript' src='http://ajax.googleapis.com/ajax/libs/jquery/1.9.0/jquery.min.js'></script><br />
<br />
<script type='text/javascript'> $(function(){ <br />
$(window).scroll(function () {<br />
if ($(this).scrollTop() > 250) {<br />
$('#column-left').addClass("fixNavigation"); <br />
} else { <br />
$('#column-left').removeClass("fixNavigation");<br />
} <br />
}); <br />
}); <br />
</script><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.title4{color:#5a6060; font-family:'Open Sans'; font-weight:700; font-size:15px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align:justify;}<br />
<br />
.citation{color:#5a6060; font-family:'Open Sans'; font-size:18px; margin:0 0 50px 0; line-height:24px; font-style: italic; text-align: center;}<br />
<br />
p.legend{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: center;}<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/d/d0/Template-igem2014-slide1.jpg') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
<br />
.Title{<br />
text-shadow: 1px 3px 3px rgba(70, 156, 133, 0.75);<br />
color: #2CC23E;<br />
font-size: 50px;<br />
text-align: center;<br />
}<br />
<br />
.Sub_title{<br />
color: #2CC23E;<br />
font-size: 30px;<br />
}<br />
<br />
.Article {<br />
text-align: justify;<br />
text-indent:15px;<br />
}<br />
<br />
a.Link {<br />
font-size:10pt;<br />
color :#A4A4A4; <br />
text-decoration:none;<br />
}<br />
<br />
a.Link:hover{<br />
text-decoration:underline;<br />
}<br />
/*Overview*/<br />
.CropImg{<br />
position : absolute;<br />
width: 700px;<br />
left :50%;<br />
margin-left:-350px;<br />
height: auto; <br />
overflow: hidden;<br />
display :block;<br />
<br />
}<br />
<br />
table {<br />
display: table;<br />
border-collapse: separate;<br />
border-spacing: 2px;<br />
border-color: gray;<br />
}<br />
<br />
table[Attributes Style] {<br />
border-top-width: 1px;<br />
border-right-width: 1px;<br />
border-bottom-width: 1px;<br />
border-left-width: 1px;<br />
width: 100%;<br />
height: 20%;<br />
}<br />
<br />
#column-left{<br />
float:left;<br />
width: 190px;<br />
float: left;<br />
padding: 15px 10px 15px 15px;<br />
border: 1px solid #ccc;<br />
border-radius: 5px;<br />
background-color: #eee;<br />
}<br />
<br />
.fixNavigation{ <br />
z-index: 9999;<br />
position: fixed;<br />
top:15px;<br />
width:240px;<br />
}<br />
<br />
ul.menuleft {<br />
padding-left:10px;<br />
}<br />
<br />
<br />
ul.menuleft li {<br />
padding-bottom: 10px;<br />
}<br />
<br />
ul.menuleft li a{<br />
color:#5a6060;<br />
font-family:'Open Sans';<br />
font-size:14px;<br />
margin:0 0 0 0;<br />
line-height:24px;<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed; margin-top:30px;"><br />
<div style="margin:0 auto; width:960px;"><br />
<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Human practice&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Ethics</p> <br />
</div> <br />
</div><br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<div id="column-left"><br />
<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1">Protection of the beauty</a></li><br />
<li><a href="#select2">Human intervention in the nature</a></li><br />
<li><a href="#select3">SubtiTree</a></li><br />
</ul><br />
</div><br />
<br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<!--CITATION--><br />
<p class="citation"><br />
"Ethics is as important as laws."<br />
</p><br />
<br />
<p class="texte">The ethical questioning turned out to be one of the major starting points of our project. Acting on an established environment and modifying it is no mean feat and our thinking combines technical and philosophical points of view. The actual purpose of our project also leads us to undertake an ethical questioning about the role of the scientist regarding “useless” things such as the trees lining the Canal du Midi. </p><br />
<br />
<p class="title1" id="select1">Protection of the beauty</p><br />
<p class="title2">Is it the scientists’ role to protect beauty?<br />
</p><br />
<br />
<p class="texte"> Beauty is a feeling of satisfaction and is selfless. It is more a feeling than the property of a thing, this is not a notion we can clearly understand. Indeed, we can find something beautiful even when we don’t know the purpose of the object...</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2014/f/fa/Fontaine_Duchamp.jpg" width="400px"><br />
<p class="legend">Figure 1: Fontaine (Marcel Duchamp). Yes this is also art...</p></center><br />
<br />
<p class="texte">There is always a distinction between natural beauty and artistic beauty according to Hegel, the famous philosopher. The artistic beauty is born from our mind and our spirit: it is an element of signification of the work of art whereas the natural beauty of the object is external. In a way, the Canal du Midi combines both types of beauty: a natural one regarding the nature, the centenary plane trees but also an artistic one since the Canal was built by the human hands.<br />
Usually, science judges beauty as a superficial feature not deserving to undertake any kind of scientific efforts to maintain it. The traditional role of science is to solve global issues and to elaborate complex strategies in order to find useful solutions for everyone’s life. <br />
Once made this observation, one may wonder why synthetic biology would be used only to protect the useless beauty of a local heritage such as the trees lining the Canal du Midi. <br />
</p><br />
<br />
<p class="texte"><br />
This crucial interrogation leads us to consider science and synthetic biology in another way. <B> What if the role of scientists was also to make people rediscovering the beauty of nature? What if the bases of new scientific challenges resulted from a more local scale? </B> Science does not have to be elitist, it has so much to gain opening itself to these challenges. First scientifically, as research is never useless and as we never know the impact and the scope of our results. Then socially, as we could measure the deep interest raised by our project within the population and the media. Adopting a new vision of synthetic biology, we probably make people change their mind about this innovative discipline. <br><br />
The traditional cold objectivity of science distances itself from the society. However, scientists are also being capable of feeling the beauty, sensitive to the charm of landscapes and <B>able to understand the usefulness of "useless" trees</B>…<br><br><br />
The design of a strategy to protect useless beauty may seem senseless but we believe that it is also the scientist’s duty. We have to remember that the thought is what distinguish <i>Homo sapiens</i> from other species on earth and this thought make us able to understand the world and be conscious of our being (Descartes: <i>Cogito ergo sum</i>). The art is an object of philosophical thought. Consciousness raises humans above all others living creatures. Thus, it is necessary to respect and protect art. And thus, it becomes essential to preserve the beauty of this site. <br />
</p><br />
<br />
<br />
<p class="title1" id="select2">Human intervention in the nature</p><br />
<p class="texte">Our main questioning aim to understand the complicated relationship between man and nature. Does the mankind have the proper right to operate in nature? Is modified nature considered as artificial?</p><br />
<p class="title2"> Mankind & Nature</p><br />
<br />
<p class="texte"> Nature is known as a creation of God. Human is linked to the nature and for that reason the nature deserves to be respected and loved. Mankind has always been linked to Nature as its survival depends on what comes out of the ground, the trees, the oceans… The nature is a source of wealth for the humankind. It ensures survival and development by giving men the wood, the rocks, the soil to build shelters. Being in contact with nature can allow men to feel strong emotion, as describe by poets like Hugo and Lamartine.</p><br />
<br />
<br />
<p class="texte">Since the birth of humanity, man himself understood well the importance of studying and mastering Nature to develop the civilization. Still today the most advanced technologies often try to mimic natural phenomena. With the development of the civilizations, men modified their environment, changing it for their own comfort depending on their own desire. By increasing their cities and acitvities, humans modify the natural environment. With the industrialization of the societies, the natural environment has suffered from human activities such as waste discharges, oil slicks, intensive fishing but also the introduction of devastating species such as the pathogen,<I> Ceratocystis platani</I>. However, despite these negative aspects, men are capable of favorable actions to help the environment and fix their mistakes. The current trend is to limit the impact of human interventions on the nature, and hopefully this trend is not transient and will not vanish. A new desire is born, a wish to protect the nature and the wildness. Man fits with his position: he takes advantage of the environment and the environment takes advantage of the reasoned human interventions. There is an adaptation of the mankind toward the nature. Moreover, humans have the capacity of empathy: people are able to understand the emotions and cognitive states of other organisms and to identify to them. To respond to these feelings, humans have technological tools allowing them to fight against enemies such as <I>Ceratocystis platani.</I><br />
</p><br />
<br />
<p class="texte">In conclusion, by destroying and hammering the nature, we jeopardize our lives. We need the nature, we come from the nature and we depend on nature for our survival, our food, our discoveries and our civilisation. Respecting, loving and preserving the environment is a question of survival.</p><br />
<br />
<p class="title2">Nature and artifice<br />
</p><br />
<br />
<p class="texte">Talking about the nature refers to the whole world with an exception: all the transformations made by mankind. Thus, the nature consists in the real without all the artificial elements created by humans. The nature is existing regardless of men and his interventions whereas artificial is everything that exists thanks to humans.<br />
</p><br />
<br />
<p class="texte">However, pretending that natural and artificial are opposite does not seem to be true. Man cannot create without elements provided by the nature, he is just transforming the nature, changing the shape. Thus we may wonder if there is a true difference between natural and artificial. The border between these two notions is not as obvious as it seems. The landscapes are shaped by the hand of man, animals are domesticated, and now bacteria are considered as cell factories. A natural reserve is artificially preserved as the result of human actions. Is there still something natural since the birth of humankind? Actually, the artifice is a slight modification of Nature and couldn’t exist by itself. The distinction between natural and artificial seems sterile and we clearly understand that these notions are inextricably linked and need each other to exist. </p><br />
<br />
<p class="texte">In conclusion,isn't it our duty to use our unique position in the history of life and our human approach to try to replace the evolutive processes?</p><br />
<br />
<br />
<p class="title2">Back to our project</p><br />
<br />
<br />
<p class="texte">These inextricable links are obviously the basis of our project. We aim to artificially preserve a natural heritage shaped by Pierre Paul Riquet hundreds years ago. The modification of a naturally occurring form of life to strengthen it is maybe just the imitation of the natural evolution process. What is considered today as ‘non-natural’ may be one day regarded differently. To the extent that everything is done not to unbalance the ecosystem, our intervention can be judged rightful, even more than the use of chemicals.</p><br />
<br />
<br />
<p class="title1" id="select3">SubtiTree</p><br />
<br />
<p class="title2"> Potential strategies discussed<br />
<br> (See more details in the <a href="https://2014.igem.org/Team:Toulouse/Project/Spreading">Spreading</a> dedicated page)<br />
</p><br />
<br />
<p class="texte">To be sure that SubtiTree will not survive and spread in the environment, many strategies were discussed to improve our bacterium: <br />
<br />
<br>- Avoid the survival in the environment thanks to a proline auxotrophy system<br />
<br>- Prevent the sporulation of <I> B. subtilis</I> to make it annual <br />
<br>- Avoid gene transfers between SubtiTree and a wild type bacterium thanks to a toxin-antitoxin system <br />
<br>- Use integrative plasmid to improve the genetic stability<br />
</p><br />
<br />
<br />
<br />
<p class="title2">Public perception<br />
</p><br />
<br />
<p class="texte"><I><CENTER>Political and public adhesion</I></CENTER></p><br />
<br />
<p class="texte">Because of the magnitude of our project,its application interested several civil services. Indeed some municipalities and regional councils supported our local engagement. Beyond that, our project interests the highest level of the “Canal du Midi” administration: the national navigation authority and the Ministry of agriculture funded this project. They are still looking for the best way to further this work after the iGEM competition. </p><br />
<br />
<p class="texte">This project also received the attention of the public through several articles in newspapers, television, radio and internet. First we had just a local coverage, but days after days there were more and more media intereseted in SubtiTree. This mediatic coverage allowed us to contact concerned citizens who participated to the development of this project. This interaction with the public allowed us to explain and promote public knowledge of synthetic biology. </p><br />
<br />
<br />
<p class="texte"><I><CENTER>Safety principle</I></CENTER></p><br />
<br />
<p class="texte">One single tree infected by Canker, and all the trees located in an area of a couple of hundred meters around are included in the prophylactic cut. We acted to preserve the surrounding trees. The modification of the endophytic microbial fauna generated by the introduction of the engineered bacterium has to be compared to the introduction of chemicals. They contain chlorine atom and aromatic hydrocarbon, so their remediation is complicated and they represent a source of pollution. By shortening the lifespan to one season and minimizing the risks of spreading, we plan a safe and environmental-friendly way to fight Canker. <br />
<br />
<br />
<p class="title2">Feasability<br />
</p><br />
<br />
<p class="texte">We wonder about the feasibility of tree’s treatment. As we used endophytic bacteria, we can count on the natural growth of SubtiTree inside the sap. So we can inject few bacteria to be sure to have enough bacteria to protect the tree. Some researchers (Xianling Ji1 et al) already injected <i>Bacillus subtilis</i> in plants and observe an increase of bacteria concentration to a maximum of 10^5 bacteria/mL/p><br />
<br />
<p class="texte">As we aim to inject a poor quantity of bacteria, this treatment remains cheaper than the injection of several litter of fungicides. In addition, this injection prevents the preventive tree cutting, which is very expensive. Cutting one tree cost around €3000. The administration in charge of the protection of the “Canal du Midi” already plans to spend 220 million euros to cut and replant all trees along the Canal. Besides the important cost of cutting trees, it will destroy one of the symbols of south-western France. </p><br />
<br />
<p class="texte">We know that SubtiTree could be improved in many ways, but in the iGEM’s circumstances we could not have the time to go deeper. First, we can improve the fixation module. Using chitin as fixation anchor is simple but not enough specific to fix just one fungus type. That’s why we first think to fix SubtiTree to one protein included in the <I>Ceratocystis platani</I>’s membrane: CP. The bacterial prototype designed this summer can be optimized to trigger the fungicides production when the binding is completed, and to be more specific changing the peptides produced.</p><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/ethicsTeam:Toulouse/ethics2014-10-17T16:47:24Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<script type='text/javascript' src='http://ajax.googleapis.com/ajax/libs/jquery/1.9.0/jquery.min.js'></script><br />
<br />
<script type='text/javascript'> $(function(){ <br />
$(window).scroll(function () {<br />
if ($(this).scrollTop() > 250) {<br />
$('#column-left').addClass("fixNavigation"); <br />
} else { <br />
$('#column-left').removeClass("fixNavigation");<br />
} <br />
}); <br />
}); <br />
</script><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.title4{color:#5a6060; font-family:'Open Sans'; font-weight:700; font-size:15px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align:justify;}<br />
<br />
.citation{color:#5a6060; font-family:'Open Sans'; font-size:18px; margin:0 0 50px 0; line-height:24px; font-style: italic; text-align: center;}<br />
<br />
p.legend{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: center;}<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/d/d0/Template-igem2014-slide1.jpg') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
<br />
.Title{<br />
text-shadow: 1px 3px 3px rgba(70, 156, 133, 0.75);<br />
color: #2CC23E;<br />
font-size: 50px;<br />
text-align: center;<br />
}<br />
<br />
.Sub_title{<br />
color: #2CC23E;<br />
font-size: 30px;<br />
}<br />
<br />
.Article {<br />
text-align: justify;<br />
text-indent:15px;<br />
}<br />
<br />
a.Link {<br />
font-size:10pt;<br />
color :#A4A4A4; <br />
text-decoration:none;<br />
}<br />
<br />
a.Link:hover{<br />
text-decoration:underline;<br />
}<br />
/*Overview*/<br />
.CropImg{<br />
position : absolute;<br />
width: 700px;<br />
left :50%;<br />
margin-left:-350px;<br />
height: auto; <br />
overflow: hidden;<br />
display :block;<br />
<br />
}<br />
<br />
table {<br />
display: table;<br />
border-collapse: separate;<br />
border-spacing: 2px;<br />
border-color: gray;<br />
}<br />
<br />
table[Attributes Style] {<br />
border-top-width: 1px;<br />
border-right-width: 1px;<br />
border-bottom-width: 1px;<br />
border-left-width: 1px;<br />
width: 100%;<br />
height: 20%;<br />
}<br />
<br />
#column-left{<br />
float:left;<br />
width: 190px;<br />
float: left;<br />
padding: 15px 10px 15px 15px;<br />
border: 1px solid #ccc;<br />
border-radius: 5px;<br />
background-color: #eee;<br />
}<br />
<br />
.fixNavigation{ <br />
z-index: 9999;<br />
position: fixed;<br />
top:15px;<br />
width:240px;<br />
}<br />
<br />
ul.menuleft {<br />
padding-left:10px;<br />
}<br />
<br />
<br />
ul.menuleft li {<br />
padding-bottom: 10px;<br />
}<br />
<br />
ul.menuleft li a{<br />
color:#5a6060;<br />
font-family:'Open Sans';<br />
font-size:14px;<br />
margin:0 0 0 0;<br />
line-height:24px;<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed; margin-top:30px;"><br />
<div style="margin:0 auto; width:960px;"><br />
<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Human practice&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Ethics</p> <br />
</div> <br />
</div><br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<div id="column-left"><br />
<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3><br />
<ul class="menuleft"><br />
<li style="margin-top:25px;"><a href="#select1">Protection of the beauty</a></li><br />
<li><a href="#select2">Human intervention in the nature</a></li><br />
<li><a href="#select3">SubtiTree</a></li><br />
</ul><br />
</div><br />
<br />
<div class="column-right" style="width:75%; float:right;"><br />
<br />
<!--CITATION--><br />
<p class="citation"><br />
"Ethics is as important as laws."<br />
</p><br />
<br />
<p class="texte">The ethical questioning turned out to be one of the major starting points of our project. Acting on an established environment and modifying it is no mean feat and our thinking combines technical and philosophical points of view. The actual purpose of our project also leads us to undertake an ethical questioning about the role of the scientist regarding “useless” things such as the trees lining the Canal du Midi. </p><br />
<br />
<p class="title1" id="select1">Protection of the beauty</p><br />
<p class="title2">Is it the scientists’ role to protect beauty?<br />
</p><br />
<br />
<p class="texte"> Beauty is a feeling of satisfaction and is selfless. It is more a feeling than the property of a thing, this is not a notion we can clearly understand. Indeed, we can find something beautiful even when we don’t know the purpose of the object...</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2014/f/fa/Fontaine_Duchamp.jpg" width="400px"><br />
<p class="legend">Figure 1: Fontaine (Marcel Duchamp). Yes this is also art...</p></center><br />
<br />
<p class="texte">There is always a distinction between natural beauty and artistic beauty according to Hegel, the famous philosopher. The artistic beauty is born from our mind and our spirit: it is an element of signification of the work of art whereas the natural beauty of the object is external. In a way, the Canal du Midi combines both types of beauty: a natural one regarding the nature, the centenary plane trees but also an artistic one since the Canal was built by the human hands.<br />
Usually, science judges beauty as a superficial feature not deserving to undertake any kind of scientific efforts to maintain it. The traditional role of science is to solve global issues and to elaborate complex strategies in order to find useful solutions for everyone’s life. <br />
Once made this observation, one may wonder why synthetic biology would be used only to protect the useless beauty of a local heritage such as the trees lining the Canal du Midi. <br />
</p><br />
<br />
<p class="texte"><br />
This crucial interrogation leads us to consider science and synthetic biology in another way. <B> What if the role of scientists was also to make people rediscovering the beauty of nature? What if the bases of new scientific challenges resulted from a more local scale? </B> Science does not have to be elitist, it has so much to gain opening itself to these challenges. First scientifically, as research is never useless and as we never know the impact and the scope of our results. Then socially, as we could measure the deep interest raised by our project within the population and the media. Adopting a new vision of synthetic biology, we probably make people change their mind about this innovative discipline. <br><br />
The traditional cold objectivity of science distances itself from the society. However, scientists are also beings capable of feeling the beauty, sensitive to the charm of landscapes and <B>able to understand the usefulness of "useless" trees</B>…<br><br><br />
The design of a strategy to protect useless beauty may seem senseless but we believe that it is also the scientist’s duty. We have to remember that the thought is what distinguish <i>Homo sapiens</i> from other species on earth and this thought make us able to understand the world and be conscious of our being (Descartes: <i>Cogito ergo sum</i>). The art is an object of philosophical thought. Consciousness raises humans above all others living creatures. Thus, it is necessary to respect and protect art. And thus, it becomes essential to preserve the beauty of this site. <br />
</p><br />
<br />
<br />
<p class="title1" id="select2">Human intervention in the nature</p><br />
<p class="texte">Our main questioning aim to understand the complicated relationship between man and nature. Does the mankind have the proper right to operate in nature? Is modified nature considered as artificial?</p><br />
<p class="title2"> Mankind & Nature</p><br />
<br />
<p class="texte"> Nature is known as a creation of God. Human is linked to the nature and for that reason the nature deserves to be respected and loved. Mankind has always been linked to Nature as its survival depends on what comes out of the ground, the trees, the oceans… The nature is a source of wealth for the humankind. It ensures survival and development by giving men the wood, the rocks, the soil to build shelters. Being in contact with nature can allow men to feel strong emotion, as describe by poets like Hugo and Lamartine.</p><br />
<br />
<br />
<p class="texte">Since the birth of humanity, man himself understood well the importance of studying and mastering Nature to develop the civilization. Still today the most advanced technologies often try to mimic natural phenomena. With the development of the civilizations, men modified their environment, changing it for their own comfort depending on their own desire. By increasing their cities and acitvities, humans modify the natural environment. With the industrialization of the societies, the natural environment has suffered from human activities such as waste discharges, oil slicks, intensive fishing but also the introduction of devastating species such as the pathogen,<I> Ceratocystis platani</I>. However, despite these negative aspects, men are capable of favorable actions to help the environment and fix their mistakes. The current trend is to limit the impact of human interventions on the nature, and hopefully this trend is not transient and will not vanish. A new desire is born, a wish to protect the nature and the wildness. Man fits with his position: he takes advantage of the environment and the environment takes advantage of the reasoned human interventions. There is an adaptation of the mankind toward the nature. Moreover, humans have the capacity of empathy: people are able to understand the emotions and cognitive states of other organisms and to identify to them. To respond to these feelings, humans have technological tools allowing them to fight against enemies such as <I>Ceratocystis platani.</I><br />
</p><br />
<br />
<p class="texte">In conclusion, by destroying and hammering the nature, we jeopardize our lives. We need the nature, we come from the nature and we depend on nature for our survival, our food, our discoveries and our civilisation. Respecting, loving and preserving the environment is a question of survival.</p><br />
<br />
<p class="title2">Nature and artifice<br />
</p><br />
<br />
<p class="texte">Talking about the nature refers to the whole world with an exception: all the transformations made by mankind. Thus, the nature consists in the real without all the artificial elements created by humans. The nature is existing regardless of men and his interventions whereas artificial is everything that exists thanks to humans.<br />
</p><br />
<br />
<p class="texte">However, pretending that natural and artificial are opposite does not seem to be true. Man cannot create without elements provided by the nature, he is just transforming the nature, changing the shape. Thus we may wonder if there is a true difference between natural and artificial. The border between these two notions is not as obvious as it seems. The landscapes are shaped by the hand of man, animals are domesticated, and now bacteria are considered as cell factories. A natural reserve is artificially preserved as the result of human actions. Is there still something natural since the birth of humankind? Actually, the artifice is a slight modification of Nature and couldn’t exist by itself. The distinction between natural and artificial seems sterile and we clearly understand that these notions are inextricably linked and need each other to exist. </p><br />
<br />
<p class="texte">In conclusion,isn't it our duty to use our unique position in the history of life and our human approach to try to replace the evolutive processes?</p><br />
<br />
<br />
<p class="title2">Back to our project</p><br />
<br />
<br />
<p class="texte">These inextricable links are obviously the basis of our project. We aim to artificially preserve a natural heritage shaped by Pierre Paul Riquet hundreds years ago. The modification of a naturally occurring form of life to strengthen it is maybe just the imitation of the natural evolution process. What is considered today as ‘non-natural’ may be one day regarded differently. To the extent that everything is done not to unbalance the ecosystem, our intervention can be judged rightful, even more than the use of chemicals.</p><br />
<br />
<br />
<p class="title1" id="select3">SubtiTree</p><br />
<br />
<p class="title2"> Potential strategies discussed<br />
<br> (See more details in the <a href="https://2014.igem.org/Team:Toulouse/Project/Spreading">Spreading</a> dedicated page)<br />
</p><br />
<br />
<p class="texte">To be sure that SubtiTree will not survive and spread in the environment, many strategies were discussed to improve our bacterium: <br />
<br />
<br>- Avoid the survival in the environment thanks to a proline auxotrophy system<br />
<br>- Prevent the sporulation of <I> B. subtilis</I> to make it annual <br />
<br>- Avoid gene transfers between SubtiTree and a wild type bacterium thanks to a toxin-antitoxin system <br />
<br>- Use integrative plasmid to improve the genetic stability<br />
</p><br />
<br />
<br />
<br />
<p class="title2">Public perception<br />
</p><br />
<br />
<p class="texte"><I><CENTER>Political and public adhesion</I></CENTER></p><br />
<br />
<p class="texte">Because of the magnitude of our project,its application interested several civil services. Indeed some municipalities and regional councils supported our local engagement. Beyond that, our project interests the highest level of the “Canal du Midi” administration: the national navigation authority and the Ministry of agriculture funded this project. They are still looking for the best way to further this work after the iGEM competition. </p><br />
<br />
<p class="texte">This project also received the attention of the public through several articles in newspapers, television, radio and internet. First we had just a local coverage, but days after days there were more and more media intereseted in SubtiTree. This mediatic coverage allowed us to contact concerned citizens who participated to the development of this project. This interaction with the public allowed us to explain and promote public knowledge of synthetic biology. </p><br />
<br />
<br />
<p class="texte"><I><CENTER>Safety principle</I></CENTER></p><br />
<br />
<p class="texte">One single tree infected by Canker, and all the trees located in an area of a couple of hundred meters around are included in the prophylactic cut. We acted to preserve the surrounding trees. The modification of the endophytic microbial fauna generated by the introduction of the engineered bacterium has to be compared to the introduction of chemicals. They contain chlorine atom and aromatic hydrocarbon, so their remediation is complicated and they represent a source of pollution. By shortening the lifespan to one season and minimizing the risks of spreading, we plan a safe and environmental-friendly way to fight Canker. <br />
<br />
<br />
<p class="title2">Feasability<br />
</p><br />
<br />
<p class="texte">We wonder about the feasibility of tree’s treatment. As we used endophytic bacteria, we can count on the natural growth of SubtiTree inside the sap. So we can inject few bacteria to be sure to have enough bacteria to protect the tree. Some researchers (Xianling Ji1 et al) already injected <i>Bacillus subtilis</i> in plants and observe an increase of bacteria concentration to a maximum of 10^5 bacteria/mL/p><br />
<br />
<p class="texte">As we aim to inject a poor quantity of bacteria, this treatment remains cheaper than the injection of several litter of fungicides. In addition, this injection prevents the preventive tree cutting, which is very expensive. Cutting one tree cost around €3000. The administration in charge of the protection of the “Canal du Midi” already plans to spend 220 million euros to cut and replant all trees along the Canal. Besides the important cost of cutting trees, it will destroy one of the symbols of south-western France. </p><br />
<br />
<p class="texte">We know that SubtiTree could be improved in many ways, but in the iGEM’s circumstances we could not have the time to go deeper. First, we can improve the fixation module. Using chitin as fixation anchor is simple but not enough specific to fix just one fungus type. That’s why we first think to fix SubtiTree to one protein included in the <I>Ceratocystis platani</I>’s membrane: CP. The bacterial prototype designed this summer can be optimized to trigger the fungicides production when the binding is completed, and to be more specific changing the peptides produced.</p><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/CommunicationTeam:Toulouse/Communication2014-10-13T22:17:48Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify;}<br />
<br />
.nomsd{color:#5a6060; font-family:'Open Sans'; font-size:14px; font-weight:bold; margin:0 30px 50px 30px; line-height:24px; text-align: center;}<br />
<br />
.table{align="center"; text-align: center;<br />
}<br />
.tr,td{text-align: center; vertical-align="middle"}<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/d/d0/Template-igem2014-slide1.jpg') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
<br />
.Title{<br />
text-shadow: 1px 3px 3px rgba(70, 156, 133, 0.75);<br />
color: #2CC23E;<br />
font-size: 50px;<br />
text-align: center;<br />
}<br />
<br />
.Sub_title{<br />
color: #2CC23E;<br />
font-size: 30px;<br />
}<br />
<br />
.Article {<br />
text-align: justify;<br />
text-indent:15px;<br />
}<br />
<br />
a.Link {<br />
font-size:10pt;<br />
color :#A4A4A4; <br />
text-decoration:none;<br />
}<br />
<br />
a.Link:hover{<br />
text-decoration:underline;<br />
}<br />
/*Overview*/<br />
.CropImg{<br />
position : absolute;<br />
width: 700px;<br />
left :50%;<br />
margin-left:-350px;<br />
height: auto; <br />
overflow: hidden;<br />
display :block;<br />
<br />
}<br />
.Img {<br />
float:left;<br />
}<br />
<br />
#virginiabadge{<br />
background: url("https://static.igem.org/mediawiki/2014/6/6a/Virginia_Badge2.png");<br />
background-size:250px;<br />
width: 250px;<br />
height: 185px;<br />
background-repeat:no-repeat;<br />
}<br />
<br />
#virginiabadge:hover{<br />
background: url("https://static.igem.org/mediawiki/2014/2/2b/File-Virginia-Images-Badge1.png");<br />
background-repeat:no-repeat;<br />
width: 250px;<br />
height: 185px;<br />
background-size:250px;<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed; margin-top:30px;"><br />
<div style="margin:0 auto; width:960px;"><br />
<p style="color:#696969; padding-top:20px; font-size:16px; float:left;"> Human practice&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Communication</p> <br />
</div><br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<br />
<!--Press--><br />
<p class="title1">Press</p><br />
<p class="texte">As our project was very regional, a lot of different regional newspapers were interested into it.<br><br />
After the first articles, other newspapers have begun to be interested into our work. It helped us to have a better visibility.<br><br />
We have listed all the articles :<br><br />
<center><table width="60%"><br />
<tr><td><p class="texte"><br />
- <a href="http://www.20minutes.fr/toulouse/1416215-20140708-guerisseurs-platanes">20Minutes</a><br><br />
- <a href="http://www.metronews.fr/toulouse/ils-etudient-une-bacterie-pour-soigner-les-platanes-du-canal-du-midi/mngj!FOMnxoEasL2s/">Metronews</a><br><br />
- <a href="http://www.ladepeche.fr/article/2014/07/15/1918689-un-espoir-de-traitement-pour-les-platanes-du-canal.html">LaDépêche</a><br><br />
- La Voix du Midi<br><br />
- <a href="http://labiotech.fr/les-equipes-francaise-lassaut-digem-2014-competition-mondiale-biologie-synthetique/">LaBiotech</a><br><br />
- <a href="http://www.humanite.fr/un-bourgeon-despoir-pour-les-platanes-du-canal-du-midi-548812">L'Humanité</a><br><br />
- <a href="http://www.developpement-durable-networkvisio.com/n31-france/article-toulouse.html?id=10765">Développement durable</a><br><br />
- Midi Libre<br><br />
- <a href="http://www.lindependant.fr/2014/08/15/vnf-aide-la-recherche-pour-sauver-les-platanes,1918464.php">L'Indépendant</a><br><br />
- <a href="http://www.digischool.fr/initiatives/etudiants-veulent-sauver-platanes-canal-du-midi-23990.php">digiSchool</a><br><br />
- La Voix du Midi Lauragais<br><br />
- <a href="http://www.lurioaddl.com/Pages/CanalMidi.aspx">LURIO Addl</a><br><br />
- <a hrf="http://www.fluvialnet.com/murmures-pop-actualites-des-etudiants-toulousains-inventent-une-methode-de-lutte-contre-le-chancre-colore/9576">Fluvial</a><br><br />
- <a hrf="http://www.toulouseblog.fr/actualite-27679-etudiants-toulousains-participent-a-competition-internationale-a-boston.html">Toulouse Blog</a><br><br />
- <a hrf="http://etudiant.lefigaro.fr/flash/flash-actu/detail/article/des-etudiants-toulousains-au-mit-pour-sauver-les-platanes-du-canal-du-midi-8976/">Le Figaro étudiants</a><br></p><br />
</td><br />
<td><img width="200px"; src="https://static.igem.org/mediawiki/2014/1/11/Articles.png"></td></tr><br />
<tr><td><p class="texte"><br />
The Television was also interested in our project : the regional channel <a href="http://france3-regions.francetvinfo.fr/midi-pyrenees/2014/07/09/des-etudiants-toulousains-developpent-une-bacterie-pour-sauver-les-platanes-514659.html">France 3 Midi-Pyrénées</a><br />
came to our lab for an interview.</p></td><br />
<td><img width="250px"; src="https://static.igem.org/mediawiki/2014/3/37/France_3_interview.png"></td></tr><br />
<tr><td><img width="140px"; src="https://static.igem.org/mediawiki/2014/0/0d/France_inter_interview.png"></td><br />
<td><p class="texte">To finish, the radio France Inter interviewed us in our lab.</td></tr><br />
</p></table></center><br><br />
<br />
<!--Interventions--><br />
<p class="title1">Interventions<br></p><br />
<p class="texte">We have participated to a lot of interventions to talk about synthetic biology and our project :</p><br><br />
<br><center><table width="50%"><br />
<br />
<tr><td><p class="texte">- The <b>Agora</b> at the University Paul-Sabatier in Toulouse (22<sup>nd</sup> April 2014). It is a monthly conference set up by the association<br />
<a href="http://www.arborisscientiae.fr/">Arboris Scientiae</a></p></td><br />
<td><img style="width:180px"; src="https://static.igem.org/mediawiki/2014/7/7d/22042014-Agora-PierreFlorie1.jpg"><br>Pierre and Florie during the Agora.</td></tr><br />
<br />
<tr><td><img width="100px"; src="https://static.igem.org/mediawiki/2014/4/4e/Exposciences_Toulouse_2014.jpg"><br>Florie and Fanny during the Exposcience.</td><br />
<td valign="middle"><p class="texte">- The Journées de l’Ecole Doctorale Biologie, Santé, Biotechnologies (17th April 2014). This day was dedicated to the exhibition of doctor’s results with posters. <br />
We came with a poster explaining what is the iGEM competition.</td></tr><br />
<br />
<tr><td><p class="texte">- The <b>Exposcience</b> in Toulouse (5<sup>th</sup> june 2014). <br />
We held a stand during the whole day. <br />
We met a few people and explained our project. <br />
We brought lab stuff and a poster describing the iGEM competition.</p></td><br />
<td valign="middle"><img width="150px"; src="https://static.igem.org/mediawiki/2014/0/00/Exposciences_1.jpg"><br>Manon and Camille during the Exposcience</td></tr><br />
<br />
<tr><td valign="middle"><img width="200px"src="https://static.igem.org/mediawiki/2014/e/e5/Colloque_BioSynSys.jpg"><br>Pierre and Mathieu during the Biosynsys conference.</td><br />
<td valign="middle"><p class="texte">- The <a href="http://biosynsys2014.sciencesconf.org/"><b>Biosynsys conference</b></a> (2<sup>nd</sup> july 2014)<br />
where we made an oral presentation behind a panel of scientists specialized in synthetic biology.</p></td></tr><br />
<br />
<br />
<tr><td><p class="texte">- The <b>Novela</b> in Toulouse (4<sup>th</sup> and 11<sup>th</sup> october 2014) where we made an oral presentation about synthetic biology and human health.</p></td><br />
<td valign="middle"><img width="200px" src="https://static.igem.org/mediawiki/2014/9/95/Novela.png"><br>Camille and Manon during the Novela.</td></tr><br />
</table></center><br />
</p><br />
<br />
<!--Other iGEM teams--><br />
<p class="title1">Interactions with other iGEM teams<br></p><br />
<p class="texte"><br />
- Filling in the iGEM Virginia online survey to gauge acceptance and understanding of synthetic biology by the general public (11<sup>st</sup> July).</p><br><br />
<div id="virginiabadge"><br />
<a href=<br />
"https://2014.igem.org/Team:Virginia/HumanPractices"><img height="180"<br />
src="https://static.igem.org/mediawiki/2014/b/b0/Virginia_Transparent.png"<br />
width="250"></a><br />
</div><br><br />
<p class="texte">- Skype with iGEM Columbia (31<sup>st</sup> July) : they explained their contest "Low Budget". Finally, we did not have the time to take part of this contest.<br><br />
- Skype with iGEM York (29<sup>th</sup> August) : we answered questions about women in science</p><br><br />
<br />
<br />
<!--Newsletter--><br />
<p class="title1">Newsletters</p><br />
<p class="texte">We set up a monthly Newsletter to inform our sponsors and other people interested in our project. You can find these here (french version):<br> <br />
- <a href="https://static.igem.org/mediawiki/2014/a/ad/Newsletter_iGEM_n%C2%B01_-_juillet_2014.jpg">Newsletter n°1</a><br><br />
- <a href="https://static.igem.org/mediawiki/2014/3/32/Newsletter_iGEM_n%C2%B02_-_ao%C3%BBt_2014.pdf">Newsletter n°2</a><br><br />
- <a href="https://static.igem.org/mediawiki/2014/8/8b/Newsletter_iGEM_n%C2%B03_-_septembre_2014.pdf">Newsletter n°3</a><br><br />
- <a href="https://static.igem.org/mediawiki/2014/e/e1/Newsletter_iGEM_n%C2%B04_-_octobre_2014.pdf">Newsletter n°4</a><br><br />
</p><br />
<br />
<!--Communication tools--><br />
<p class="title1">Communication tools</p><br />
<p class="texte">Here you can find all our communication tools: <br><br />
- <a href="https://2014.igem.org/File:IGEM_plaquette.jpg">iGEM Toulouse presentation flyer (French version)</a><br><br />
- <a href="https://static.igem.org/mediawiki/2014/e/e2/Plaquette_SubtiTree.pdf">SubtiTree presentation flyer (French version)</a><br><br />
- <a href="https://static.igem.org/mediawiki/2014/d/de/Poster_igem_VF.jpg">iGEM Toulouse presentation poster (French version)</a><br><br />
- <a href="https://static.igem.org/mediawiki/2014/e/ed/IGEM_Toulouse_2014_logo.pdf">iGEM Toulouse logo</a><br><br />
- <a href="">iGEM Toulouse Giant Jamboree poster (Coming soon)</a><br><br />
- <a href="">iGEM Toulouse Giant Jamboree presentation (Coming soon)</a><br><br />
</p><br />
<br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Result/partsTeam:Toulouse/Result/parts2014-10-13T22:09:26Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#5a6060; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.title4{color:#5a6060; font-family:'Open Sans'; font-weight:700; font-size:15px; margin:0 0 20px 0; border:none;}<br />
<br />
.title5{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:14px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify; }<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify; }<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/9/95/Toulouse_Home_2.JPG') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
table {<br />
border-width:1px; <br />
border-style:solid; <br />
border-color:black;<br />
width:100%;<br />
}<br />
<br />
td {<br />
text-align:center;<br />
vertical-align:middle;<br />
border-width:1px; <br />
border-style:solid; <br />
border-color:black;<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Parts</h2><br />
<p>What did we send to the Registry?</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Parts</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<br />
<br />
<br />
<p class="texte">As the chassis <i>B. subtilis</i> is not as used as <i>E. coli</i>, <br />
it was sometimes hard for us to get or find desired parts.<br><br />
However, in collaboration with other iGEM teams and with a synthesis compagny, we managed to obtain or make the following parts:<br><br />
</p><br />
<br />
<p class="title1">Submitted parts</p><br />
<br />
<p class="texte"><br />
We have deposed 16 new BioBrick parts to the Registry. <br />
All of them were cloned into the standard plasmid pSB1C3, tested and sequenced.<br><br />
<!--à vérifier et confirmer--><br />
</p><br />
<br />
<br />
<p class="title2">Chemotaxis</p><br />
<br />
<!--K1364000 : RBS + Chimio + terminator--><br />
<br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a>:N-acetylglucosamine based chemotaxis for <i>Bacillus subtilis</i><br></p><br />
<p class="texte">This part is designed to enable a N-acetylglucosamine <br />
based chemotaxis in <i>Bacillus subtilis</i>.<br />
<p class="title4">Design</p><br />
<p class="texte">The encoded protein is a chimera of two proteins:<br><br />
- the methyl accepting Chemotaxis protein (McpA from <i>Bacillus subtilis</i> which is required for taxis towards glucose.<br><br />
- the N-acetylglucosamine regulated methyl-accepting chemotaxis protein from <i>Vibrio cholerae</i> (VCD).<br><br />
<br><br />
The chimeric protein contains the intracellular domains of McpA from <i>Bacillus subtilis</i> to enable the transduction of the signal: <br />
these domains correspond to the amino acids 1-37 and 282-610. <br />
The extracellular domain of VCD is inserted between the intracellular regions of McpA <br />
to sense N-acetylglucosamine and corresponds to the amino acids 31-310.<br></p><br />
<img src="https://static.igem.org/mediawiki/2014/a/a2/BBa_K1364000.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translation unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a><br></p><br />
<!--à compléter--><br />
</p><br />
<br />
<br><br />
<br />
<!--K1364004 : Promotor Pveg + Chimio--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364004">BBa_K1364004</a>: P<sub>veg</sub> + N-acetylatedglucosamine based chemotaxis for <i>B. subtilis</i> <br />
<br><br />
<p class="texte">This part is designed to enable a N-acetylglucosamine based chemotaxis in <i>Bacillus subtilis</i>.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This expression cassette is designed for the expression of an antifungal peptide, <br />
D4E1 and for its secretion in <i>Bacillus subtilis</i>.<br />
It is composed of the strong, constitutive promotor of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
strong RBS for <i>B. subtilis</i> (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of D4E1<br />
and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>)</p><br />
<img src="https://static.igem.org/mediawiki/2014/3/35/BBa_K1364004.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a><br><br />
</p><br />
<br />
<br><br />
<br />
<p class="title2">Binding</p><br />
<br />
<!--K1364001 : RBS + Binding + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364005">BBa_K1364005</a>: P<sub>veg</sub> + Chitin Binding Cell Wall protein</p><br />
<p class="texte">This part is designed to enable the binding of <i>Bacillus subtilis</i> to the fungi wall made of chitin.<p/><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Cell Wall Binding (CWB) sequence <br />
and a Chitin Binding Domain (CBD) linked with a 6 amino acids linker.<br><br />
<br><br />
The CWB domain has been extracted from LytC (<a href="http://parts.igem.org/Part:BBa_K316030">BBa_K316030</a>) of the iGEM 2010 Imperial College team.<br><br />
The CBD is composed of the Domain 4 of the GbpA protein of <i>Vibrio cholerae</i>,<br />
a protein reported to mediate bacterial attachment to chitin. <br />
It has been shown that the Domain 4 was essential to bind chitin.</p><br />
<img src="https://static.igem.org/mediawiki/2014/f/f5/BBa_K1364005.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested with chitin beeds. (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Binding module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br><br />
</p><br />
<br />
<br><br />
<br />
<br />
<p class="title2">Fungicides</p><br />
<p class="title3">GAFP-1</p><br />
<br />
<br><br />
<br />
<!--K1364002 : RBS + GAFP-1--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a>: RBS - Antifungal GAFP-1</p><br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>) and<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/5/5f/BBa_K1364002.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br></p><br />
<br><br />
<br />
<!--K1364007 : RBS + GAFP-1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364007">BBa_K1364007</a>: RBS + Antifungal GAFP-1 + Double terminator</p><br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator <a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>.<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/42/BBa_K1364007.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite part</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br></p><br />
<br><br />
<br />
<!--K13640008 : Pveg + RBS + GAFP-1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364008">BBa_K1364008</a>: P<sub>veg</sub> - strong RBS - Antifungal GAFP-1 - Double terminator</p><br />
<p class="texte">This expression cassette is designed for the expression and secretion of the <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin. GAFP-1 is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the strong, constitutive promoter of <i>Bacillus subtilis</i> P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/6/67/BBa_K1364008.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br><br />
</p><br />
<br />
<br><br />
<br />
<p class="title3">D4E1</p><br />
<br />
<br><br />
<br />
<!--K1364003 : RBS + D4E1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a>:RBS - Antifungal D4E1 - Double terminator</p><br />
<p class="texte">D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of D4E1 and <br />
a Double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
This part was optimized for the expression and its secretion in <i>Bacillus subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/b/bb/BBa_K1364003.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
<br />
<br />
<br><br />
<br />
<!--K1364009 : Pveg + RBS + D4E1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364009">BBa_K1364009</a>:P<sub>veg</sub> - RBS - Antifungal D4E1 - Double Terminator</p><br />
<p class="texte"><br />
D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the strong, constitutive promotor of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
a strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of D4E1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br />
This part was optimized for the expression and its secretion in <i>Bacillus subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/47/BBa_K1364009.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
<br><br />
<br />
<p class="title3">EcAMP-1</p><br />
<br />
<!--K1364010 : Pveg RBS + EcAMP --><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364010">BBa_K1364010</a>: P<sub>veg</sub> - SpoVG - Antifungal EcAMP-1</p><br />
<p class="texte"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>B. subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">K733013</a>) and<br />
the open reading frame of EcAMP-1.<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/5/5f/BBa_K1364010.png"><br />
<p class="title4">Type</p><br />
<p class="texte"> Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte"><!--à compléter : demander à emeline--></p><br />
<br />
<br><br />
<br />
<!--K1364011 : Pveg RBS + EcAMP + Double term--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364011">BBa_K1364011</a>: P<sub>veg</sub> - SpoVG - Antifungal EcAMP-1 - Double terminator</p><br />
<p class="texte"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>B. subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">K733013</a>),<br />
the open reading frame of EcAMP-1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/7/74/BBa_K1364011.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte"><!--à compléter : demander à emeline--></p><br />
<br />
<br><br />
<br />
<p class="title3">Fungicide operons</p><br />
<br />
<!--K1364013 : Pveg + RBS + GAFP + RBS + D4E1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364013">BBa_K1364013</a>: P<sub>veg</sub> - SpoVG - Antifungal EcAMP-1 - Double terminator</p><br />
<p class="texte"><br />
<p class="title4">Design</p><br />
<p class="texte">This part is designed for the co-expression of two different peptides <br />
with anti-fungal activities : D4E1 and GAFP-1. <br />
It is composed of the strong, constitutive promotor of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>) <br />
and the translation unit with GAFP-1 and D4E1 (BBa_K1364012).</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/BBa_K1364013.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
See <a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a> and <a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a>.</p><br />
<br><br />
<p class="title2">Basic tools</p><br />
<br />
<!--K1364015--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364015">BBa_K1364015</a>: P<sub>veg</sub> + RFP</p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--K1364016--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364016">BBa_K1364016</a>: P<sub>lepA</sub> + RFP</p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--K1364017--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364017">BBa_K1364017</a>: P<sub>lepA</sub> + RBS SpoVG</p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--K1364019--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364019">BBa_K1364019</a>: P<sub>veg</sub> + RBS + Antifungal EcAMP + Double terminator</p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--K1364021--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364021">BBa_K1364021</a>: Integrative plasmid for <i>Bacillus subtilis</i></p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--SOUS-TITRE--><br />
<p class="title1">Used parts</p><br />
<br />
<br><br />
<br />
<!--Table--><br />
<p class="texte"><center><br />
<table border="1" width="100%" height="20%"><br />
<!--1ere ligne--><br />
<tr><td><p class="texte"><b>BioBrick</b></p></td><br />
<td><p class="texte"><b>Description</b></p></td><br />
<td><p class="texte"><b>Origine</b></p></td><br />
<td><p class="texte"><b>Module(s)</b></p></td></tr><br />
<!--2eme ligne--><br />
<tr><td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K733013">K733013</a></p></td><br />
<td><p class="texte">P<sub>veg</sub> (<a href="http://parts.igem.org/Part:BBa_K316001">K316001</a>) + RBS-SpoVG (<a href="http://parts.igem.org/Part:BBa_K143021">K143021</a>) : Constitutive promotor P<sub>veg</sub> and strong RBS for <i>B. subtilis</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 7H</p></td><br />
<td><p class="texte">Fungicides (EcAMP)</p></td><br />
</tr><br />
<!--3eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823003">K823003</a></p></td><br />
<td><p class="texte">P<sub>veg</sub> : strong, constitutive promotor of <i>B. subtilis</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 20G</p></td><br />
<td><p class="texte">Fungicides (D4E1 GAFP-1)<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--4eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823002">K823002</a></p></td><br />
<td><p class="texte">P<sub>lepA</sub> : constitutive promotor of <i>B. subtilis</i>.<br />
<br>It is the promotor of the lepA gene of <i>B. subtilis</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 20E</p></td><br />
<td><p class="texte">Fungicides (D4E1)<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--5eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K606061">K606061</a></p></td><br />
<td><p class="texte">SpoVG RBS for <i>B. subtilis</i><br />
<br>Strong ribosome binding site from subtilis. Works fine in <i>E. coli</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 15A</p></td><br />
<td><p class="texte">Optional</p></td><br />
</tr><br />
<!--6eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K316027">K316027</a></p></td><br />
<td><p class="texte"><i>B. subtilis</i> transformation vector with LacI, targets amyE locus.</p></td><br />
<td><p class="texte">2014 Kit plate 1, 2N</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--7eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_B0015">B0015</a></p></td><br />
<td><p class="texte">Double terminator (<a href="http://parts.igem.org/Part:BBa_B0010">BBa_B0010</a> + <a href="http://parts.igem.org/Part:BBa_B0012">BBa_B0012</a>)</p></td><br />
<td><p class="texte">2014 Kit plate 3, 3F</p></td><br />
<td><p class="texte">Fungicides (GAFP-1, EcAMP-1)</p></td><br />
</tr><br />
<!--8eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780000">K780000</a></p></td><br />
<td><p class="texte">Terminator for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
<td></td><br />
</tr><br />
<!--9eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780001">K780001</a></p></td><br />
<td><p class="texte">Strong RBS for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
<td></td><br />
</tr><br />
<!--10eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780002">K780002</a></p></td><br />
<td><p class="texte">Strong RBS for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
<td></td><br />
</tr><br />
<!--11eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780003">K780003</a></p></td><br />
<td><p class="texte">Strong constitutive promotor for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--12eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K1162001">K1162001</a></p></td><br />
<td><p class="texte">EcAMP-1</p></td><br />
<td><p class="texte">Sent by the Utah State iGEM team</p></td><br />
<td><p class="texte">Fungicides (EcAMP-1)</p></td><br />
</tr><br />
<!--13eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823023">K823023</a></p></td><br />
<td><p class="texte">Empty backbone vector pSB<sub>BS</sub>1C for integration into <i>B. subtilis</i><br />
<br>It integrates in the amyE locus</p></td><br />
<td><p class="texte">Sent by the Munich iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td></tr><br />
<!--14eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823022">K823022</a></p></td><br />
<td><p class="texte">Empty backbone vector pSB<sub>BS</sub>4S for integration into<i>B. subtilis</i><br />
<br>It integrates in the thrC locus</p></td><br />
<td><p class="texte">Sent by the Munich iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--15eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823021">K823021</a></p></td><br />
<td><p class="texte">Reporter vector pSB<sub>BS</sub>1C-lacZ for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Munich iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td></tr><br />
</table></center><br />
</p><br />
<br />
<br><br />
<br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Experimental results<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Result/achievement" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Achievement</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Result/partsTeam:Toulouse/Result/parts2014-10-13T22:04:47Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#5a6060; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.title4{color:#5a6060; font-family:'Open Sans'; font-weight:700; font-size:15px; margin:0 0 20px 0; border:none;}<br />
<br />
.title5{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:14px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify; }<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify; }<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/9/95/Toulouse_Home_2.JPG') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
table {<br />
border-width:1px; <br />
border-style:solid; <br />
border-color:black;<br />
width:100%;<br />
}<br />
<br />
td {<br />
text-align:center;<br />
vertical-align:middle;<br />
border-width:1px; <br />
border-style:solid; <br />
border-color:black;<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Parts</h2><br />
<p>What did we send to the Registry?</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Parts</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<br />
<br />
<br />
<p class="texte">As the chassis <i>B. subtilis</i> is not as used as <i>E. coli</i>, <br />
it was sometimes hard for us to get or find desired parts.<br><br />
However, in collaboration with other iGEM teams and with a synthesis compagny, we managed to obtain or make the following parts:<br><br />
</p><br />
<br />
<p class="title1">Submitted parts</p><br />
<br />
<p class="texte"><br />
We have deposed 16 new BioBrick parts to the Registry. <br />
All of them were cloned into the standard plasmid pSB1C3, tested and sequenced.<br><br />
<!--à vérifier et confirmer--><br />
</p><br />
<br />
<br />
<p class="title2">Chemotaxis</p><br />
<br />
<!--K1364000 : RBS + Chimio + terminator--><br />
<br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a>:N-acetylglucosamine based chemotaxis for <i>Bacillus subtilis</i><br></p><br />
<p class="texte">This part is designed to enable a N-acetylglucosamine <br />
based chemotaxis in <i>Bacillus subtilis</i>.<br />
<p class="title4">Design</p><br />
<p class="texte">The encoded protein is a chimera of two proteins:<br><br />
- the methyl accepting Chemotaxis protein (McpA from <i>Bacillus subtilis</i> which is required for taxis towards glucose.<br><br />
- the N-acetylglucosamine regulated methyl-accepting chemotaxis protein from <i>Vibrio cholerae</i> (VCD).<br><br />
<br><br />
The chimeric protein contains the intracellular domains of McpA from <i>Bacillus subtilis</i> to enable the transduction of the signal: <br />
these domains correspond to the amino acids 1-37 and 282-610. <br />
The extracellular domain of VCD is inserted between the intracellular regions of McpA <br />
to sense N-acetylglucosamine and corresponds to the amino acids 31-310.<br></p><br />
<img src="https://static.igem.org/mediawiki/2014/a/a2/BBa_K1364000.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translation unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a><br></p><br />
<!--à compléter--><br />
</p><br />
<br />
<br><br />
<br />
<!--K1364004 : Promotor Pveg + Chimio--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364004">BBa_K1364004</a>: P<sub>veg</sub> + N-acetylatedglucosamine based chemotaxis for <i>B. subtilis</i> <br />
<br><br />
<p class="texte">This part is designed to enable a N-acetylglucosamine based chemotaxis in <i>Bacillus subtilis</i>.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This expression cassette is designed for the expression of an antifungal peptide, <br />
D4E1 and for its secretion in <i>Bacillus subtilis</i>.<br />
It is composed of the strong, constitutive promotor of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
strong RBS for <i>B. subtilis</i> (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of D4E1<br />
and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>)</p><br />
<img src="https://static.igem.org/mediawiki/2014/3/35/BBa_K1364004.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a><br><br />
</p><br />
<br />
<br><br />
<br />
<p class="title2">Binding</p><br />
<br />
<!--K1364001 : RBS + Binding + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364005">BBa_K1364005</a>: P<sub>veg</sub> + Chitin Binding Cell Wall protein</p><br />
<p class="texte">This part is designed to enable the binding of <i>Bacillus subtilis</i> to the fungi wall made of chitin.<p/><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Cell Wall Binding (CWB) sequence <br />
and a Chitin Binding Domain (CBD) linked with a 6 amino acids linker.<br><br />
<br><br />
The CWB domain has been extracted from LytC (<a href="http://parts.igem.org/Part:BBa_K316030">BBa_K316030</a>) of the iGEM 2010 Imperial College team.<br><br />
The CBD is composed of the Domain 4 of the GbpA protein of <i>Vibrio cholerae</i>,<br />
a protein reported to mediate bacterial attachment to chitin. <br />
It has been shown that the Domain 4 was essential to bind chitin.</p><br />
<img src="https://static.igem.org/mediawiki/2014/f/f5/BBa_K1364005.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested with chitin beeds. (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Binding module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br><br />
</p><br />
<br />
<br><br />
<br />
<br />
<p class="title2">Fungicides</p><br />
<p class="title3">GAFP-1</p><br />
<br />
<br><br />
<br />
<!--K1364002 : RBS + GAFP-1--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a>: RBS - Antifungal GAFP-1</p><br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>) and<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/5/5f/BBa_K1364002.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br></p><br />
<br><br />
<br />
<!--K1364007 : RBS + GAFP-1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364007">BBa_K1364007</a>: RBS + Antifungal GAFP-1 + Double terminator</p><br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator <a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>.<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/42/BBa_K1364007.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite part</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br></p><br />
<br><br />
<br />
<!--K13640008 : Pveg + RBS + GAFP-1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364008">BBa_K1364008</a>: P<sub>veg</sub> - strong RBS - Antifungal GAFP-1 - Double terminator</p><br />
<p class="texte">This expression cassette is designed for the expression and secretion of the <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin. GAFP-1 is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the strong, constitutive promoter of <i>Bacillus subtilis</i> P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator <a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>.<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/6/67/BBa_K1364008.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br><br />
</p><br />
<br />
<br><br />
<br />
<p class="title3">D4E1</p><br />
<br />
<br><br />
<br />
<!--K1364003 : RBS + D4E1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a>:RBS - Antifungal D4E1 - Double terminator</p><br />
<p class="texte">D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>,<br />
the open reading frame of D4E1 and <br />
a Double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
This part was optimized for the expression and its secretion in <i>Bacillus subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/b/bb/BBa_K1364003.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
<br />
<br />
<br><br />
<br />
<!--K1364009 : Pveg + RBS + D4E1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364009">BBa_K1364009</a>:P<sub>veg</sub> - RBS - Antifungal D4E1 - Double Terminator</p><br />
<p class="texte"><br />
D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the strong, constitutive promotor of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
a strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of D4E1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br />
This part was optimized for the expression and its secretion in <i>Bacillus subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/47/BBa_K1364009.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
<br><br />
<br />
<p class="title3">EcAMP-1</p><br />
<br />
<!--K1364010 : Pveg RBS + EcAMP --><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364010">BBa_K1364010</a>: P<sub>veg</sub> - SpoVG - Antifungal EcAMP-1</p><br />
<p class="texte"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>B. subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">K733013</a>) and<br />
the open reading frame of EcAMP-1.<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/5/5f/BBa_K1364010.png"><br />
<p class="title4">Type</p><br />
<p class="texte"> Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte"><!--à compléter : demander à emeline--></p><br />
<br />
<br><br />
<br />
<!--K1364011 : Pveg RBS + EcAMP + Double term--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364011">BBa_K1364011</a>: P<sub>veg</sub> - SpoVG - Antifungal EcAMP-1 - Double terminator</p><br />
<p class="texte"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>B. subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">K733013</a>),<br />
the open reading frame of EcAMP-1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/7/74/BBa_K1364011.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte"><!--à compléter : demander à emeline--></p><br />
<br />
<br><br />
<br />
<p class="title3">Fungicide operons</p><br />
<br />
<!--K1364013 : Pveg + RBS + GAFP + RBS + D4E1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364013">BBa_K1364013</a>: P<sub>veg</sub> - SpoVG - Antifungal EcAMP-1 - Double terminator</p><br />
<p class="texte"><br />
<p class="title4">Design</p><br />
<p class="texte">This part is designed for the co-expression of two different peptides <br />
with anti-fungal activities : D4E1 and GAFP-1. <br />
It is composed of the strong, constitutive promotor of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>) <br />
and the translation unit with GAFP-1 and D4E1 (BBa_K1364012).</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/BBa_K1364013.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
See <a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a> and <a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a>.</p><br />
<br><br />
<p class="title2">Basic tools</p><br />
<br />
<!--K1364015--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364015">BBa_K1364015</a>: P<sub>veg</sub> + RFP</p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--K1364016--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364016">BBa_K1364016</a>: P<sub>lepA</sub> + RFP</p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--K1364017--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364017">BBa_K1364017</a>: P<sub>lepA</sub> + RBS SpoVG</p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--K1364019--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364019">BBa_K1364019</a>: P<sub>veg</sub> + RBS + Antifungal EcAMP + Double terminator</p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--K1364021--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364021">BBa_K1364021</a>: Integrative plasmid for <i>Bacillus subtilis</i></p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--SOUS-TITRE--><br />
<p class="title1">Used parts</p><br />
<br />
<br><br />
<br />
<!--Table--><br />
<p class="texte"><center><br />
<table border="1" width="100%" height="20%"><br />
<!--1ere ligne--><br />
<tr><td><p class="texte"><b>BioBrick</b></p></td><br />
<td><p class="texte"><b>Description</b></p></td><br />
<td><p class="texte"><b>Origine</b></p></td><br />
<td><p class="texte"><b>Module(s)</b></p></td></tr><br />
<!--2eme ligne--><br />
<tr><td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K733013">K733013</a></p></td><br />
<td><p class="texte">P<sub>veg</sub> (<a href="http://parts.igem.org/Part:BBa_K316001">K316001</a>) + RBS-SpoVG (<a href="http://parts.igem.org/Part:BBa_K143021">K143021</a>) : Constitutive promotor P<sub>veg</sub> and strong RBS for <i>B. subtilis</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 7H</p></td><br />
<td><p class="texte">Fungicides (EcAMP)</p></td><br />
</tr><br />
<!--3eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823003">K823003</a></p></td><br />
<td><p class="texte">P<sub>veg</sub> : strong, constitutive promotor of <i>B. subtilis</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 20G</p></td><br />
<td><p class="texte">Fungicides (D4E1 GAFP-1)<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--4eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823002">K823002</a></p></td><br />
<td><p class="texte">P<sub>lepA</sub> : constitutive promotor of <i>B. subtilis</i>.<br />
<br>It is the promotor of the lepA gene of <i>B. subtilis</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 20E</p></td><br />
<td><p class="texte">Fungicides (D4E1)<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--5eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K606061">K606061</a></p></td><br />
<td><p class="texte">SpoVG RBS for <i>B. subtilis</i><br />
<br>Strong ribosome binding site from subtilis. Works fine in <i>E. coli</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 15A</p></td><br />
<td><p class="texte">Optional</p></td><br />
</tr><br />
<!--6eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K316027">K316027</a></p></td><br />
<td><p class="texte"><i>B. subtilis</i> transformation vector with LacI, targets amyE locus.</p></td><br />
<td><p class="texte">2014 Kit plate 1, 2N</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--7eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_B0015">B0015</a></p></td><br />
<td><p class="texte">Double terminator (<a href="http://parts.igem.org/Part:BBa_B0010">BBa_B0010</a> + <a href="http://parts.igem.org/Part:BBa_B0012">BBa_B0012</a>)</p></td><br />
<td><p class="texte">2014 Kit plate 3, 3F</p></td><br />
<td><p class="texte">Fungicides (GAFP-1, EcAMP-1)</p></td><br />
</tr><br />
<!--8eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780000">K780000</a></p></td><br />
<td><p class="texte">Terminator for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
<td></td><br />
</tr><br />
<!--9eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780001">K780001</a></p></td><br />
<td><p class="texte">Strong RBS for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
<td></td><br />
</tr><br />
<!--10eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780002">K780002</a></p></td><br />
<td><p class="texte">Strong RBS for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
<td></td><br />
</tr><br />
<!--11eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780003">K780003</a></p></td><br />
<td><p class="texte">Strong constitutive promotor for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--12eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K1162001">K1162001</a></p></td><br />
<td><p class="texte">EcAMP-1</p></td><br />
<td><p class="texte">Sent by the Utah State iGEM team</p></td><br />
<td><p class="texte">Fungicides (EcAMP-1)</p></td><br />
</tr><br />
<!--13eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823023">K823023</a></p></td><br />
<td><p class="texte">Empty backbone vector pSB<sub>BS</sub>1C for integration into <i>B. subtilis</i><br />
<br>It integrates in the amyE locus</p></td><br />
<td><p class="texte">Sent by the Munich iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td></tr><br />
<!--14eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823022">K823022</a></p></td><br />
<td><p class="texte">Empty backbone vector pSB<sub>BS</sub>4S for integration into<i>B. subtilis</i><br />
<br>It integrates in the thrC locus</p></td><br />
<td><p class="texte">Sent by the Munich iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--15eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823021">K823021</a></p></td><br />
<td><p class="texte">Reporter vector pSB<sub>BS</sub>1C-lacZ for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Munich iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td></tr><br />
</table></center><br />
</p><br />
<br />
<br><br />
<br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Experimental results<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Result/achievement" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Achievement</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Result/partsTeam:Toulouse/Result/parts2014-10-13T22:03:10Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#5a6060; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.title4{color:#5a6060; font-family:'Open Sans'; font-weight:700; font-size:15px; margin:0 0 20px 0; border:none;}<br />
<br />
.title5{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:14px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify; }<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify; }<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/9/95/Toulouse_Home_2.JPG') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
table {<br />
border-width:1px; <br />
border-style:solid; <br />
border-color:black;<br />
width:100%;<br />
}<br />
<br />
td {<br />
text-align:center;<br />
vertical-align:middle;<br />
border-width:1px; <br />
border-style:solid; <br />
border-color:black;<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Parts</h2><br />
<p>What did we send to the Registry?</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Parts</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<br />
<br />
<br />
<p class="texte">As the chassis <i>B. subtilis</i> is not as used as <i>E. coli</i>, <br />
it was sometimes hard for us to get or find desired parts.<br><br />
However, in collaboration with other iGEM teams and with a synthesis compagny, we managed to obtain or make the following parts:<br><br />
</p><br />
<br />
<p class="title1">Submitted parts</p><br />
<br />
<p class="texte"><br />
We have deposed 16 new BioBrick parts to the Registry. <br />
All of them were cloned into the standard plasmid pSB1C3, tested and sequenced.<br><br />
<!--à vérifier et confirmer--><br />
</p><br />
<br />
<br />
<p class="title2">Chemotaxis</p><br />
<br />
<!--K1364000 : RBS + Chimio + terminator--><br />
<br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a>:N-acetylglucosamine based chemotaxis for <i>Bacillus subtilis</i><br></p><br />
<p class="texte">This part is designed to enable a N-acetylglucosamine <br />
based chemotaxis in <i>Bacillus subtilis</i>.<br />
<p class="title4">Design</p><br />
<p class="texte">The encoded protein is a chimera of two proteins:<br><br />
- the methyl accepting Chemotaxis protein (McpA from <i>Bacillus subtilis</i> which is required for taxis towards glucose.<br><br />
- the N-acetylglucosamine regulated methyl-accepting chemotaxis protein from <i>Vibrio cholerae</i> (VCD).<br><br />
<br><br />
The chimeric protein contains the intracellular domains of McpA from <i>Bacillus subtilis</i> to enable the transduction of the signal: <br />
these domains correspond to the amino acids 1-37 and 282-610. <br />
The extracellular domain of VCD is inserted between the intracellular regions of McpA <br />
to sense N-acetylglucosamine and corresponds to the amino acids 31-310.<br></p><br />
<img src="https://static.igem.org/mediawiki/2014/a/a2/BBa_K1364000.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translation unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a><br></p><br />
<!--à compléter--><br />
</p><br />
<br />
<br><br />
<br />
<!--K1364004 : Promotor Pveg + Chimio--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364004">BBa_K1364004</a>: P<sub>veg</sub> + N-acetylatedglucosamine based chemotaxis for <i>B. subtilis</i> <br />
<br><br />
<p class="texte">This part is designed to enable a N-acetylglucosamine based chemotaxis in <i>Bacillus subtilis</i>.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This expression cassette is designed for the expression of an antifungal peptide, <br />
D4E1 and for its secretion in <i>Bacillus subtilis</i>.<br />
It is composed of the strong, constitutive promotor of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
strong RBS for <i>B. subtilis</i> (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of D4E1<br />
and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>)</p><br />
<img src="https://static.igem.org/mediawiki/2014/3/35/BBa_K1364004.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a><br><br />
</p><br />
<br />
<br><br />
<br />
<p class="title2">Binding</p><br />
<br />
<!--K1364001 : RBS + Binding + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364005">BBa_K1364005</a>: P<sub>veg</sub> + Chitin Binding Cell Wall protein</p><br />
<p class="texte">This part is designed to enable the binding of <i>Bacillus subtilis</i> to the fungi wall made of chitin.<p/><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Cell Wall Binding (CWB) sequence <br />
and a Chitin Binding Domain (CBD) linked with a 6 amino acids linker.<br><br />
<br><br />
The CWB domain has been extracted from LytC (<a href="http://parts.igem.org/Part:BBa_K316030">BBa_K316030</a>) of the iGEM 2010 Imperial College team.<br><br />
The CBD is composed of the Domain 4 of the GbpA protein of <i>Vibrio cholerae</i>,<br />
a protein reported to mediate bacterial attachment to chitin. <br />
It has been shown that the Domain 4 was essential to bind chitin.</p><br />
<img src="https://static.igem.org/mediawiki/2014/f/f5/BBa_K1364005.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested with chitin beeds. (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Binding module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br><br />
</p><br />
<br />
<br><br />
<br />
<br />
<p class="title2">Fungicides</p><br />
<p class="title3">GAFP-1</p><br />
<br />
<br><br />
<br />
<!--K1364002 : RBS + GAFP-1--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a>: RBS - Antifungal GAFP-1</p><br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>) and<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/5/5f/BBa_K1364002.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br></p><br />
<br><br />
<br />
<!--K1364007 : RBS + GAFP-1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364007">BBa_K1364007</a>: RBS + Antifungal GAFP-1 + Double terminator</p><br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator <a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>.<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/42/BBa_K1364007.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite part</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br></p><br />
<br><br />
<br />
<!--K13640008 : Pveg + RBS + GAFP-1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364008">BBa_K1364008</a>: P<sub>veg</sub> - strong RBS - Antifungal GAFP-1 - Double terminator</p><br />
<p class="texte">This expression cassette is designed for the expression and secretion of the <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin. GAFP-1 is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the strong, constitutive promoter of <i>Bacillus subtilis</i> P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>,<br />
strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>,<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator <a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>.<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/6/67/BBa_K1364008.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br><br />
</p><br />
<br />
<br><br />
<br />
<p class="title3">D4E1</p><br />
<br />
<br><br />
<br />
<!--K1364003 : RBS + D4E1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a>:RBS - Antifungal D4E1 - Double terminator</p><br />
<p class="texte">D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>,<br />
the open reading frame of D4E1 and <br />
a Double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
This part was optimized for the expression and its secretion in <i>Bacillus subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/b/bb/BBa_K1364003.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
<br />
<br />
<br><br />
<br />
<!--K1364009 : Pveg + RBS + D4E1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364009">BBa_K1364009</a>:P<sub>veg</sub> - RBS - Antifungal D4E1 - Double Terminator</p><br />
<p class="texte"><br />
D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the strong, constitutive promotor of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
a strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of D4E1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br />
This part was optimized for the expression and its secretion in <i>Bacillus subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/47/BBa_K1364009.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
<br><br />
<br />
<p class="title3">EcAMP-1</p><br />
<br />
<!--K1364010 : Pveg RBS + EcAMP --><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364010">BBa_K1364010</a>: P<sub>veg</sub> - SpoVG - Antifungal EcAMP-1</p><br />
<p class="texte"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>B. subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">K733013</a>) and<br />
the open reading frame of EcAMP-1.<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/5/5f/BBa_K1364010.png"><br />
<p class="title4">Type</p><br />
<p class="texte"> Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte"><!--à compléter : demander à emeline--></p><br />
<br />
<br><br />
<br />
<!--K1364011 : Pveg RBS + EcAMP + Double term--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364011">BBa_K1364011</a>: P<sub>veg</sub> - SpoVG - Antifungal EcAMP-1 - Double terminator</p><br />
<p class="texte"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>B. subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">K733013</a>),<br />
the open reading frame of EcAMP-1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/7/74/BBa_K1364011.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte"><!--à compléter : demander à emeline--></p><br />
<br />
<br><br />
<br />
<p class="title3">Fungicide operons</p><br />
<br />
<!--K1364013 : Pveg + RBS + GAFP + RBS + D4E1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364013">BBa_K1364013</a>: P<sub>veg</sub> - SpoVG - Antifungal EcAMP-1 - Double terminator</p><br />
<p class="texte"><br />
<p class="title4">Design</p><br />
<p class="texte">This part is designed for the co-expression of two different peptides <br />
with anti-fungal activities : D4E1 and GAFP-1. <br />
It is composed of the strong, constitutive promotor of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>) <br />
and the translation unit with GAFP-1 and D4E1 (BBa_K1364012).</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/BBa_K1364013.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
See <a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a> and <a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a>.</p><br />
<br><br />
<p class="title2">Basic tools</p><br />
<br />
<!--K1364015--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364015">BBa_K1364015</a>: P<sub>veg</sub> + RFP</p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--K1364016--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364016">BBa_K1364016</a>: P<sub>lepA</sub> + RFP</p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--K1364017--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364017">BBa_K1364017</a>: P<sub>lepA</sub> + RBS SpoVG</p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--K1364019--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364019">BBa_K1364019</a>: P<sub>veg</sub> + RBS + Antifungal EcAMP + Double terminator</p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--K1364021--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364021">BBa_K1364021</a>: Integrative plasmid for <i>Bacillus subtilis</i></p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--SOUS-TITRE--><br />
<p class="title1">Used parts</p><br />
<br />
<br><br />
<br />
<!--Table--><br />
<p class="texte"><center><br />
<table border="1" width="100%" height="20%"><br />
<!--1ere ligne--><br />
<tr><td><p class="texte"><b>BioBrick</b></p></td><br />
<td><p class="texte"><b>Description</b></p></td><br />
<td><p class="texte"><b>Origine</b></p></td><br />
<td><p class="texte"><b>Module(s)</b></p></td></tr><br />
<!--2eme ligne--><br />
<tr><td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K733013">K733013</a></p></td><br />
<td><p class="texte">P<sub>veg</sub> (<a href="http://parts.igem.org/Part:BBa_K316001">K316001</a>) + RBS-SpoVG (<a href="http://parts.igem.org/Part:BBa_K143021">K143021</a>) : Constitutive promotor P<sub>veg</sub> and strong RBS for <i>B. subtilis</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 7H</p></td><br />
<td><p class="texte">Fungicides (EcAMP)</p></td><br />
</tr><br />
<!--3eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823003">K823003</a></p></td><br />
<td><p class="texte">P<sub>veg</sub> : strong, constitutive promotor of <i>B. subtilis</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 20G</p></td><br />
<td><p class="texte">Fungicides (D4E1 GAFP-1)<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--4eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823002">K823002</a></p></td><br />
<td><p class="texte">P<sub>lepA</sub> : constitutive promotor of <i>B. subtilis</i>.<br />
<br>It is the promotor of the lepA gene of <i>B. subtilis</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 20E</p></td><br />
<td><p class="texte">Fungicides (D4E1)<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--5eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K606061">K606061</a></p></td><br />
<td><p class="texte">SpoVG RBS for <i>B. subtilis</i><br />
<br>Strong ribosome binding site from subtilis. Works fine in <i>E. coli</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 15A</p></td><br />
<td><p class="texte">Optional</p></td><br />
</tr><br />
<!--6eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K316027">K316027</a></p></td><br />
<td><p class="texte"><i>B. subtilis</i> transformation vector with LacI, targets amyE locus.</p></td><br />
<td><p class="texte">2014 Kit plate 1, 2N</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--7eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_B0015">B0015</a></p></td><br />
<td><p class="texte">Double terminator (<a href="http://parts.igem.org/Part:BBa_B0010">BBa_B0010</a> + <a href="http://parts.igem.org/Part:BBa_B0012">BBa_B0012</a>)</p></td><br />
<td><p class="texte">2014 Kit plate 3, 3F</p></td><br />
<td><p class="texte">Fungicides (GAFP-1, EcAMP-1)</p></td><br />
</tr><br />
<!--8eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780000">K780000</a></p></td><br />
<td><p class="texte">Terminator for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
<td></td><br />
</tr><br />
<!--9eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780001">K780001</a></p></td><br />
<td><p class="texte">Strong RBS for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
<td></td><br />
</tr><br />
<!--10eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780002">K780002</a></p></td><br />
<td><p class="texte">Strong RBS for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
<td></td><br />
</tr><br />
<!--11eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780003">K780003</a></p></td><br />
<td><p class="texte">Strong constitutive promotor for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--12eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K1162001">K1162001</a></p></td><br />
<td><p class="texte">EcAMP-1</p></td><br />
<td><p class="texte">Sent by the Utah State iGEM team</p></td><br />
<td><p class="texte">Fungicides (EcAMP-1)</p></td><br />
</tr><br />
<!--13eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823023">K823023</a></p></td><br />
<td><p class="texte">Empty backbone vector pSB<sub>BS</sub>1C for integration into <i>B. subtilis</i><br />
<br>It integrates in the amyE locus</p></td><br />
<td><p class="texte">Sent by the Munich iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td></tr><br />
<!--14eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823022">K823022</a></p></td><br />
<td><p class="texte">Empty backbone vector pSB<sub>BS</sub>4S for integration into<i>B. subtilis</i><br />
<br>It integrates in the thrC locus</p></td><br />
<td><p class="texte">Sent by the Munich iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--15eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823021">K823021</a></p></td><br />
<td><p class="texte">Reporter vector pSB<sub>BS</sub>1C-lacZ for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Munich iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td></tr><br />
</table></center><br />
</p><br />
<br />
<br><br />
<br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Experimental results<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Result/achievement" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Achievement</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Result/partsTeam:Toulouse/Result/parts2014-10-13T19:12:00Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#5a6060; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.title4{color:#5a6060; font-family:'Open Sans'; font-weight:700; font-size:15px; margin:0 0 20px 0; border:none;}<br />
<br />
.title5{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:14px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify; }<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify; }<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/9/95/Toulouse_Home_2.JPG') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
table {<br />
border-width:1px; <br />
border-style:solid; <br />
border-color:black;<br />
width:100%;<br />
}<br />
<br />
td {<br />
text-align:center;<br />
vertical-align:middle;<br />
border-width:1px; <br />
border-style:solid; <br />
border-color:black;<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Parts</h2><br />
<p>What did we send to the Registry?</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Parts</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<br />
<br />
<br />
<p class="texte">As the chassis <i>B. subtilis</i> is not as used as <i>E. coli</i>, <br />
it was sometimes hard for us to get or find desired parts.<br><br />
However, in collaboration with other iGEM teams and with a synthesis compagny, we managed to obtain or make the following parts:<br><br />
</p><br />
<br />
<p class="title1">Submitted parts</p><br />
<br />
<p class="texte"><br />
We have deposed 16 new BioBrick parts to the Registry. <br />
All of them were cloned into the standard plasmid pSB1C3, tested and sequenced.<br><br />
<!--à vérifier et confirmer--><br />
</p><br />
<br />
<br />
<p class="title2">Chemotaxis</p><br />
<br />
<!--K1364000 : RBS + Chimio + terminator--><br />
<br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a>:N-acetylglucosamine based chemotaxis for <i>Bacillus subtilis</i><br></p><br />
<p class="texte">This part is designed to enable a N-acetylglucosamine <br />
based chemotaxis in <i>Bacillus subtilis</i>.<br />
<p class="title4">Design:</p><br />
<p class="texte">The encoded protein is a chimera of two proteins:<br><br />
- the methyl accepting Chemotaxis protein (McpA from <i>Bacillus subtilis</i> which is required for taxis towards glucose.<br><br />
- the N-acetylglucosamine regulated methyl-accepting chemotaxis protein from <i>Vibrio cholerae</i> (VCD).<br><br />
<br><br />
The chimeric protein contains the intracellular domains of McpA from <i>Bacillus subtilis</i> to enable the transduction of the signal: <br />
these domains correspond to the amino acids 1-37 and 282-610. <br />
The extracellular domain of VCD is inserted between the intracellular regions of McpA <br />
to sense N-acetylglucosamine and corresponds to the amino acids 31-310.<br></p><br />
<img src="https://static.igem.org/mediawiki/2014/a/a2/BBa_K1364000.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translation unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a><br></p><br />
<!--à compléter--><br />
</p><br />
<br />
<br><br />
<br />
<!--K1364004 : Promotor Pveg + Chimio--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364004">BBa_K1364004</a>: P<sub>veg</sub> + N-acetylatedglucosamine based chemotaxis for <i>B. subtilis</i> <br />
<br><br />
<p class="texte">This part is designed to enable a N-acetylglucosamine based chemotaxis in <i>Bacillus subtilis</i>.</p><br />
<p class="title4">Design:</p><br />
<p class="texte">This expression cassette is designed for the expression of an antifungal peptide, <br />
D4E1 and for its secretion in <i>Bacillus subtilis</i>.<br />
It is composed of the strong, constitutive promotor of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
strong RBS for <i>B. subtilis</i> (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of D4E1<br />
and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>)</p><br />
<img src="https://static.igem.org/mediawiki/2014/3/35/BBa_K1364004.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a><br><br />
</p><br />
<br />
<br><br />
<br />
<p class="title2">Binding</p><br />
<br />
<!--K1364001 : RBS + Binding + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364005">BBa_K1364005</a>: P<sub>veg</sub> + Chitin Binding Cell Wall protein</p><br />
<p class="texte">This part is designed to enable the binding of <i>Bacillus subtilis</i> to the fungi wall made of chitin.<p/><br />
<p class="title4">Design:</p><br />
<p class="texte">This part is composed of a Cell Wall Binding (CWB) sequence <br />
and a Chitin Binding Domain (CBD) linked with a 6 amino acids linker.<br><br />
<br><br />
The CWB domain has been extracted from LytC (<a href="http://parts.igem.org/Part:BBa_K316030">BBa_K316030</a>) of the iGEM 2010 Imperial College team.<br><br />
The CBD is composed of the Domain 4 of the GbpA protein of <i>Vibrio cholerae</i>,<br />
a protein reported to mediate bacterial attachment to chitin. <br />
It has been shown that the Domain 4 was essential to bind chitin.</p><br />
<img src="https://static.igem.org/mediawiki/2014/f/f5/BBa_K1364005.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested with chitin beeds. (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Binding module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br><br />
</p><br />
<br />
<br><br />
<br />
<br />
<p class="title2">Fungicides</p><br />
<p class="title3">GAFP-1</p><br />
<br />
<br><br />
<br />
<!--K1364002 : RBS + GAFP-1--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a>: RBS - Antifungal GAFP-1</p><br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design:</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>) and<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/5/5f/BBa_K1364002.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br></p><br />
<br><br />
<br />
<!--K1364007 : RBS + GAFP-1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364007">BBa_K1364007</a>: RBS + Antifungal GAFP-1 + Double terminator</p><br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design:</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator <a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>.<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/42/BBa_K1364007.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite part</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br></p><br />
<br><br />
<br />
<!--K13640008 : Pveg + RBS + GAFP-1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364008">BBa_K1364008</a>: P<sub>veg</sub> - strong RBS - Antifungal GAFP-1 - Double terminator</p><br />
<p class="texte">This expression cassette is designed for the expression and secretion of the <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin. GAFP-1 is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design:</p><br />
<p class="texte">This part is composed of the strong, constitutive promoter of <i>Bacillus subtilis</i> P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>,<br />
strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>,<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator <a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>.<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/6/67/BBa_K1364008.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br><br />
</p><br />
<br />
<br><br />
<br />
<p class="title3">D4E1</p><br />
<br />
<br><br />
<br />
<!--K1364003 : RBS + D4E1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a>:RBS - Antifungal D4E1 - Double terminator</p><br />
<p class="texte">D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design:</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>,<br />
the open reading frame of D4E1 and <br />
a Double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
This part was optimized for the expression and its secretion in <i>Bacillus subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/b/bb/BBa_K1364003.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
<br />
<br />
<br><br />
<br />
<!--K1364009 : Pveg + RBS + D4E1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364009">BBa_K1364009</a>:P<sub>veg</sub> - RBS - Antifungal D4E1 - Double Terminator</p><br />
<p class="texte"><br />
D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design:</p><br />
<p class="texte">This part is composed of the strong, constitutive promotor of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
a strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of D4E1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br />
This part was optimized for the expression and its secretion in <i>Bacillus subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/47/BBa_K1364009.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
<br><br />
<br />
<p class="title3">EcAMP-1</p><br />
<br />
<!--K1364010 : Pveg RBS + EcAMP --><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364010">BBa_K1364010</a>: P<sub>veg</sub> - SpoVG - Antifungal EcAMP-1</p><br />
<p class="texte"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design:</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>B. subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">K733013</a>) and<br />
the open reading frame of EcAMP-1.<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/5/5f/BBa_K1364010.png"><br />
<p class="title4">Type</p><br />
<p class="texte"> Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte"><!--à compléter : demander à emeline--></p><br />
<br />
<br><br />
<br />
<!--K1364011 : Pveg RBS + EcAMP + Double term--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364011">BBa_K1364011</a>: P<sub>veg</sub> - SpoVG - Antifungal EcAMP-1 - Double terminator</p><br />
<p class="texte"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design:</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>B. subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">K733013</a>),<br />
the open reading frame of EcAMP-1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/7/74/BBa_K1364011.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte"><!--à compléter : demander à emeline--></p><br />
<br />
<br><br />
<br />
<p class="title3">Fungicide operons</p><br />
<br />
<!--K1364013 : Pveg + RBS + GAFP + RBS + D4E1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364013">BBa_K1364013</a>: P<sub>veg</sub> - SpoVG - Antifungal EcAMP-1 - Double terminator</p><br />
<p class="texte"><br />
<p class="title4">Design:</p><br />
<p class="texte">This part is designed for the co-expression of two different peptides <br />
with anti-fungal activities : D4E1 and GAFP-1. <br />
It is composed of the strong, constitutive promotor of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>) <br />
and the translation unit with GAFP-1 and D4E1 (BBa_K1364012).</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/BBa_K1364013.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
See <a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a> and <a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a>.</p><br />
<br><br />
<p class="title2">Basic tools</p><br />
<br />
<!--K1364015--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364015">BBa_K1364015</a>: P<sub>veg</sub> + RFP</p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design:</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--K1364016--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364016">BBa_K1364016</a>: P<sub>lepA</sub> + RFP</p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design:</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--K1364017--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364017">BBa_K1364017</a>: P<sub>lepA</sub> + RBS SpoVG</p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design:</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--K1364019--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364019">BBa_K1364019</a>: P<sub>veg</sub> + RBS + Antifungal EcAMP + Double terminator</p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design:</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--K1364021--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364021">BBa_K1364021</a>: Integrative plasmid for <i>Bacillus subtilis</i></p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design:</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--SOUS-TITRE--><br />
<p class="title1">Used parts</p><br />
<br />
<br><br />
<br />
<!--Table--><br />
<p class="texte"><center><br />
<table border="1" width="100%" height="20%"><br />
<!--1ere ligne--><br />
<tr><td><p class="texte"><b>BioBrick</b></p></td><br />
<td><p class="texte"><b>Description</b></p></td><br />
<td><p class="texte"><b>Origine</b></p></td><br />
<td><p class="texte"><b>Module(s)</b></p></td></tr><br />
<!--2eme ligne--><br />
<tr><td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K733013">K733013</a></p></td><br />
<td><p class="texte">P<sub>veg</sub> (<a href="http://parts.igem.org/Part:BBa_K316001">K316001</a>) + RBS-SpoVG (<a href="http://parts.igem.org/Part:BBa_K143021">K143021</a>) : Constitutive promotor P<sub>veg</sub> and strong RBS for <i>B. subtilis</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 7H</p></td><br />
<td><p class="texte">Fungicides (EcAMP)</p></td><br />
</tr><br />
<!--3eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823003">K823003</a></p></td><br />
<td><p class="texte">P<sub>veg</sub> : strong, constitutive promotor of <i>B. subtilis</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 20G</p></td><br />
<td><p class="texte">Fungicides (D4E1 GAFP-1)<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--4eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823002">K823002</a></p></td><br />
<td><p class="texte">P<sub>lepA</sub> : constitutive promotor of <i>B. subtilis</i>.<br />
<br>It is the promotor of the lepA gene of <i>B. subtilis</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 20E</p></td><br />
<td><p class="texte">Fungicides (D4E1)<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--5eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K606061">K606061</a></p></td><br />
<td><p class="texte">SpoVG RBS for <i>B. subtilis</i><br />
<br>Strong ribosome binding site from subtilis. Works fine in <i>E. coli</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 15A</p></td><br />
<td><p class="texte">Optional</p></td><br />
</tr><br />
<!--6eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K316027">K316027</a></p></td><br />
<td><p class="texte"><i>B. subtilis</i> transformation vector with LacI, targets amyE locus.</p></td><br />
<td><p class="texte">2014 Kit plate 1, 2N</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--7eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_B0015">B0015</a></p></td><br />
<td><p class="texte">Double terminator (<a href="http://parts.igem.org/Part:BBa_B0010">BBa_B0010</a> + <a href="http://parts.igem.org/Part:BBa_B0012">BBa_B0012</a>)</p></td><br />
<td><p class="texte">2014 Kit plate 3, 3F</p></td><br />
<td><p class="texte">Fungicides (GAFP-1, EcAMP-1)</p></td><br />
</tr><br />
<!--8eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780000">K780000</a></p></td><br />
<td><p class="texte">Terminator for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
<td></td><br />
</tr><br />
<!--9eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780001">K780001</a></p></td><br />
<td><p class="texte">Strong RBS for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
<td></td><br />
</tr><br />
<!--10eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780002">K780002</a></p></td><br />
<td><p class="texte">Strong RBS for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
<td></td><br />
</tr><br />
<!--11eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780003">K780003</a></p></td><br />
<td><p class="texte">Strong constitutive promotor for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--12eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K1162001">K1162001</a></p></td><br />
<td><p class="texte">EcAMP-1</p></td><br />
<td><p class="texte">Sent by the Utah State iGEM team</p></td><br />
<td><p class="texte">Fungicides (EcAMP-1)</p></td><br />
</tr><br />
<!--13eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823023">K823023</a></p></td><br />
<td><p class="texte">Empty backbone vector pSB<sub>BS</sub>1C for integration into <i>B. subtilis</i><br />
<br>It integrates in the amyE locus</p></td><br />
<td><p class="texte">Sent by the Munich iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td></tr><br />
<!--14eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823022">K823022</a></p></td><br />
<td><p class="texte">Empty backbone vector pSB<sub>BS</sub>4S for integration into<i>B. subtilis</i><br />
<br>It integrates in the thrC locus</p></td><br />
<td><p class="texte">Sent by the Munich iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--15eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823021">K823021</a></p></td><br />
<td><p class="texte">Reporter vector pSB<sub>BS</sub>1C-lacZ for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Munich iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td></tr><br />
</table></center><br />
</p><br />
<br />
<br><br />
<br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Experimental results<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Result/achievement" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Achievement</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Result/partsTeam:Toulouse/Result/parts2014-10-13T18:19:18Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#5a6060; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.title4{color:#5a6060; font-family:'Open Sans'; font-weight:700; font-size:15px; margin:0 0 20px 0; border:none;}<br />
<br />
.title5{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:14px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify; }<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify; }<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/9/95/Toulouse_Home_2.JPG') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
table {<br />
border-width:1px; <br />
border-style:solid; <br />
border-color:black;<br />
width:100%;<br />
}<br />
<br />
td {<br />
text-align:center;<br />
vertical-align:middle;<br />
border-width:1px; <br />
border-style:solid; <br />
border-color:black;<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Parts</h2><br />
<p>What did we send to the Registry?</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Parts</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<br />
<br />
<br />
<p class="texte">As the chassis <i>B. subtilis</i> is not as used as <i>E. coli</i>, <br />
it was sometimes hard for us to get or find desired parts.<br><br />
However, with the collaboration of other iGEM and a synthesis compagny, we managed to obtain or make the following parts:<br><br />
</p><br />
<br />
<p class="title1">Submitted parts</p><br />
<br />
<p class="texte"><br />
We have deposed 16 new BioBrick parts to the Registry. <br />
All of them were cloned into the standard plasmid pSB1C3, tested and sequenced.<br><br />
<!--à vérifier et confirmer--><br />
</p><br />
<br />
<br />
<p class="title2">Chemotaxis</p><br />
<br />
<!--K1364000 : RBS + Chimio + terminator--><br />
<br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364000">BBa_K1364000</a>:N-acetylglucosamine based chemotaxis for <i>Bacillus subtilis</i><br></p><br />
<p class="texte">This part is designed to enable a N-acetylglucosamine <br />
based chemotaxis in <i>Bacillus subtilis</i>.<br />
<p class="title4">Design:</p><br />
<p class="texte">The encoded protein is a chimera of two proteins:<br><br />
- the methyl accepting Chemotaxis protein (McpA from <i>Bacillus subtilis</i> which is required for taxis towards glucose.<br><br />
- the N-acetylglucosamine regulated methyl-accepting chemotaxis protein from <i>Vibrio cholerae</i> (VCD).<br><br />
<br><br />
The chimeric protein contains the intracellular domains of McpA from <i>Bacillus subtilis</i> to enable the transduction of the signal: <br />
these domains correspond to the amino acids 1-37 and 282-610. <br />
The extracellular domain of VCD is inserted between the intracellular regions of McpA <br />
to sense N-acetylglucosamine and corresponds to the amino acids 31-310.<br></p><br />
<img src="https://static.igem.org/mediawiki/2014/a/a2/BBa_K1364000.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translation unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a><br></p><br />
<!--à compléter--><br />
</p><br />
<br />
<br><br />
<br />
<!--K1364004 : Promotor Pveg + Chimio--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364004">BBa_K1364004</a>: P<sub>veg</sub> + N-acetylatedglucosamine based chemotaxis for <i>B. subtilis</i> <br />
<br><br />
<p class="texte">This part is designed to enable a N-acetylglucosamine based chemotaxis in <i>Bacillus subtilis</i>.</p><br />
<p class="title4">Design:</p><br />
<p class="texte">This expression cassette is designed for the expression of an antifungal peptide, <br />
D4E1 and for its secretion in <i>Bacillus subtilis</i>.<br />
It is composed of the strong, constitutive promotor of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
strong RBS for <i>B. subtilis</i> (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of D4E1<br />
and a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>)</p><br />
<img src="https://static.igem.org/mediawiki/2014/3/35/BBa_K1364004.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte">Uniprot:<br><br />
- <a href="http://www.uniprot.org/uniprot/P39214">McpA</a><br><br />
- <a href="http://www.uniprot.org/uniprot/C3NYT2">N-acetylglucosamine regulated methyl-accepting chemotaxis protein</a><br><br />
</p><br />
<br />
<br><br />
<br />
<p class="title2">Binding</p><br />
<br />
<!--K1364001 : RBS + Binding + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364005">BBa_K1364005</a>: P<sub>veg</sub> + Chitin Binding Cell Wall protein</p><br />
<p class="texte">This part is designed to enable the binding of <i>Bacillus subtilis</i> to the fungi wall made of chitin.<p/><br />
<p class="title4">Design:</p><br />
<p class="texte">This part is composed of a Cell Wall Binding (CWB) sequence <br />
and a Chitin Binding Domain (CBD) linked with a 6 amino acids linker.<br><br />
<br><br />
The CWB domain has been extracted from LytC (<a href="http://parts.igem.org/Part:BBa_K316030">BBa_K316030</a>) of the iGEM 2010 Imperial College team.<br><br />
The CBD is composed of the Domain 4 of the GbpA protein of <i>Vibrio cholerae</i>,<br />
a protein reported to mediate bacterial attachment to chitin. <br />
It has been shown that the Domain 4 was essential to bind chitin.</p><br />
<img src="https://static.igem.org/mediawiki/2014/f/f5/BBa_K1364005.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested with chitin beeds. (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Binding module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br><br />
</p><br />
<br />
<br><br />
<br />
<br />
<p class="title2">Fungicides</p><br />
<p class="title3">GAFP-1</p><br />
<br />
<br><br />
<br />
<!--K1364002 : RBS + GAFP-1--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a>: RBS - Antifungal GAFP-1</p><br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design:</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>) and<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/5/5f/BBa_K1364002.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br></p><br />
<br><br />
<br />
<!--K1364007 : RBS + GAFP-1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364007">BBa_K1364007</a>: RBS + Antifungal GAFP-1 + Double terminator</p><br />
<p class="texte">The <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin, is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design:</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator <a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>.<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/42/BBa_K1364007.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Composite part</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br></p><br />
<br><br />
<br />
<!--K13640008 : Pveg + RBS + GAFP-1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364008">BBa_K1364008</a>: P<sub>veg</sub> - strong RBS - Antifungal GAFP-1 - Double terminator</p><br />
<p class="texte">This expression cassette is designed for the expression and secretion of the <i>Gastrodia</i> anti-fungal protein(GAFP-1), also known as gastrodianin. GAFP-1 is a mannose and chitin binding lectin<br />
originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years.<br />
GAFP-1 is composed of 15 amino acids LDSLSFSYNNFEEDD and is able to inhibit the growth of multiples species of plant pathogenic fungi.</p><br />
<p class="title4">Design:</p><br />
<p class="texte">This part is composed of the strong, constitutive promoter of <i>Bacillus subtilis</i> P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>,<br />
strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>,<br />
the open reading frame of the <i>Gastrodia</i> anti-fungal protein 1 (GAFP-1) <br />
and a double terminator <a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>.<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
The codon optimization was made thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/6/67/BBa_K1364008.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, et al. (2012)<br><br />
- The Vibrio cholerae Colonization Factor GbpA Possesses a Modular Structure that Governs Binding to Different Host Surfaces. PLoS Pathog 8(1): e1002373. doi:10.1371/journal.ppat.1002373<br><br />
</p><br />
<br />
<br><br />
<br />
<p class="title3">D4E1</p><br />
<br />
<br><br />
<br />
<!--K1364003 : RBS + D4E1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a>:RBS - Antifungal D4E1 - Double terminator</p><br />
<p class="texte">D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design:</p><br />
<p class="texte">This part is composed of a Strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>,<br />
the open reading frame of D4E1 and <br />
a Double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>)<br />
optimized for its expression and its secretion in <i>Bacillus subtilis</i>.<br />
This part was optimized for the expression and its secretion in <i>Bacillus subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/b/bb/BBa_K1364003.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
<br />
<br />
<br><br />
<br />
<!--K1364009 : Pveg + RBS + D4E1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364009">BBa_K1364009</a>:P<sub>veg</sub> - RBS - Antifungal D4E1 - Double Terminator</p><br />
<p class="texte"><br />
D4E1 is a linear synthetic peptide of 17 amino acids <br />
which has shown to have antifungal activities <br />
by complexing with a sterol present in conodial wall of a varety of fungi.</p><br />
<p class="title4">Design:</p><br />
<p class="texte">This part is composed of the strong, constitutive promotor of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>),<br />
a strong RBS (<a href="http://parts.igem.org/Part:BBa_K780002">K780002</a>),<br />
the open reading frame of D4E1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br />
This part was optimized for the expression and its secretion in <i>Bacillus subtilis </i><br />
thanks to the DNA 2.0 software program.</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/47/BBa_K1364009.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Translational unit</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
<p class="texte">- De Lucca AJ, Bland JM, Grimm C, Jacks TJ, Cary JW, Jaynes JM, Cleveland TE, Walsh TJ. Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.<br><br />
- Can J Microbiol. 1998 Jun;44(6):514-20.</p><br />
<br><br />
<br />
<p class="title3">EcAMP-1</p><br />
<br />
<!--K1364010 : Pveg RBS + EcAMP --><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364010">BBa_K1364010</a>: P<sub>veg</sub> - SpoVG - Antifungal EcAMP-1</p><br />
<p class="texte"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design:</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>B. subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">K733013</a>) and<br />
the open reading frame of EcAMP-1.<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/5/5f/BBa_K1364010.png"><br />
<p class="title4">Type</p><br />
<p class="texte"> Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte"><!--à compléter : demander à emeline--></p><br />
<br />
<br><br />
<br />
<!--K1364011 : Pveg RBS + EcAMP + Double term--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364011">BBa_K1364011</a>: P<sub>veg</sub> - SpoVG - Antifungal EcAMP-1 - Double terminator</p><br />
<p class="texte"><br />
EcAMP-1 is an antimicrobial peptide of 37 amino acids <br />
originated from the specie <i>Echinochloa crus-galli</i>, <br />
a type of wild grass. <br />
This peptide has a particular structure : it is helical because of two disulfide bonds.<br><br />
EcAMP-1 has shown to have antifungal activities. Its mode of action may be the prevention of hyphae elongation.<br><br />
This part was added to the Registry by the iGEM Utah State team in 2013.</p><br />
<p class="title4">Design:</p><br />
<p class="texte">This part is composed of the constitutive promoter P<sub>veg</sub> and<br />
strong RBS for <i>B. subtilis</i><br />
(<a href="http://parts.igem.org/Part:BBa_K733013">K733013</a>),<br />
the open reading frame of EcAMP-1 and <br />
a double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">B0015</a>).<br><br />
The EcAMP-1 part was codon optimized for <i>E. coli</i> by the iGEM Utah State team<br />
and thanks to the Life Technologies GeneArt software program. </p><br />
<img src="https://static.igem.org/mediawiki/2014/7/74/BBa_K1364011.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part is not tested yet.</p><br />
<p class="title4">References</p><br />
<p class="texte"><!--à compléter : demander à emeline--></p><br />
<br />
<br><br />
<br />
<p class="title3">Fungicide operons</p><br />
<br />
<!--K1364013 : Pveg + RBS + GAFP + RBS + D4E1 + Terminator--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364013">BBa_K1364013</a>: P<sub>veg</sub> - SpoVG - Antifungal EcAMP-1 - Double terminator</p><br />
<p class="texte"><br />
<p class="title4">Design:</p><br />
<p class="texte">This part is designed for the co-expression of two different peptides <br />
with anti-fungal activities : D4E1 and GAFP-1. <br />
It is composed of the strong, constitutive promotor of <i>B. subtilis</i> <br />
P<sub>veg</sub> (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K823003">K823003</a>) <br />
and the translation unit with GAFP-1 and D4E1 (BBa_K1364012).</p><br />
<img src="https://static.igem.org/mediawiki/2014/4/40/BBa_K1364013.png"><br />
<p class="title4">Type</p><br />
<p class="texte">Generator</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was tested on the fungi <i>Trichoderma reesei</i> (See <a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results">Fungicides module</a>)</p><br />
<p class="title4">References</p><br />
See <a href="http://parts.igem.org/Part:BBa_K1364002">BBa_K1364002</a> and <a href="http://parts.igem.org/Part:BBa_K1364003">BBa_K1364003</a>.</p><br />
<br><br />
<p class="title2">Basic tools</p><br />
<br />
<!--K1364015--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364015">BBa_K1364015</a>: P<sub>veg</sub> + RFP</p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design:</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--K1364016--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364016">BBa_K1364016</a>: P<sub>lepA</sub> + RFP</p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design:</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Reporter</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--K1364017--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364017">BBa_K1364017</a>: P<sub>lepA</sub> + RBS SpoVG</p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design:</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--K1364019--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364019">BBa_K1364019</a>: P<sub>veg</sub> + RBS + Antifungal EcAMP + Double terminator</p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design:</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--K1364021--><br />
<p class="title3"><br />
- <a href="http://parts.igem.org/Part:BBa_K1364021">BBa_K1364021</a>: Integrative plasmid for <i>Bacillus subtilis</i></p><br />
<p class="texte"><!--Compléter--><br />
</p><br />
<p class="title4">Design:</p><br />
<p class="texte">Coming soon! </p><br />
<p class="title4">Type</p><br />
<p class="texte">Composite</p><br />
<p class="title4">Tests</p><br />
<p class="texte">This part was not tested yet</p><br />
<p class="title4">References</p><br />
<p class="texte">Coming soon!</p><br />
<br />
<br><br />
<br />
<!--SOUS-TITRE--><br />
<p class="title1">Used parts</p><br />
<br />
<br><br />
<br />
<!--Table--><br />
<p class="texte"><center><br />
<table border="1" width="100%" height="20%"><br />
<!--1ere ligne--><br />
<tr><td><p class="texte"><b>BioBrick</b></p></td><br />
<td><p class="texte"><b>Description</b></p></td><br />
<td><p class="texte"><b>Origine</b></p></td><br />
<td><p class="texte"><b>Module(s)</b></p></td></tr><br />
<!--2eme ligne--><br />
<tr><td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K733013">K733013</a></p></td><br />
<td><p class="texte">P<sub>veg</sub> (<a href="http://parts.igem.org/Part:BBa_K316001">K316001</a>) + RBS-SpoVG (<a href="http://parts.igem.org/Part:BBa_K143021">K143021</a>) : Constitutive promotor P<sub>veg</sub> and strong RBS for <i>B. subtilis</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 7H</p></td><br />
<td><p class="texte">Fungicides (EcAMP)</p></td><br />
</tr><br />
<!--3eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823003">K823003</a></p></td><br />
<td><p class="texte">P<sub>veg</sub> : strong, constitutive promotor of <i>B. subtilis</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 20G</p></td><br />
<td><p class="texte">Fungicides (D4E1 GAFP-1)<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--4eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823002">K823002</a></p></td><br />
<td><p class="texte">P<sub>lepA</sub> : constitutive promotor of <i>B. subtilis</i>.<br />
<br>It is the promotor of the lepA gene of <i>B. subtilis</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 20E</p></td><br />
<td><p class="texte">Fungicides (D4E1)<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--5eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K606061">K606061</a></p></td><br />
<td><p class="texte">SpoVG RBS for <i>B. subtilis</i><br />
<br>Strong ribosome binding site from subtilis. Works fine in <i>E. coli</i></p></td><br />
<td><p class="texte">2014 Kit plate 1, 15A</p></td><br />
<td><p class="texte">Optional</p></td><br />
</tr><br />
<!--6eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K316027">K316027</a></p></td><br />
<td><p class="texte"><i>B. subtilis</i> transformation vector with LacI, targets amyE locus.</p></td><br />
<td><p class="texte">2014 Kit plate 1, 2N</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--7eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_B0015">B0015</a></p></td><br />
<td><p class="texte">Double terminator (<a href="http://parts.igem.org/Part:BBa_B0010">BBa_B0010</a> + <a href="http://parts.igem.org/Part:BBa_B0012">BBa_B0012</a>)</p></td><br />
<td><p class="texte">2014 Kit plate 3, 3F</p></td><br />
<td><p class="texte">Fungicides (GAFP-1, EcAMP-1)</p></td><br />
</tr><br />
<!--8eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780000">K780000</a></p></td><br />
<td><p class="texte">Terminator for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
<td></td><br />
</tr><br />
<!--9eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780001">K780001</a></p></td><br />
<td><p class="texte">Strong RBS for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
<td></td><br />
</tr><br />
<!--10eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780002">K780002</a></p></td><br />
<td><p class="texte">Strong RBS for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
<td></td><br />
</tr><br />
<!--11eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K780003">K780003</a></p></td><br />
<td><p class="texte">Strong constitutive promotor for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Warsaw iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--12eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K1162001">K1162001</a></p></td><br />
<td><p class="texte">EcAMP-1</p></td><br />
<td><p class="texte">Sent by the Utah State iGEM team</p></td><br />
<td><p class="texte">Fungicides (EcAMP-1)</p></td><br />
</tr><br />
<!--13eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823023">K823023</a></p></td><br />
<td><p class="texte">Empty backbone vector pSB<sub>BS</sub>1C for integration into <i>B. subtilis</i><br />
<br>It integrates in the amyE locus</p></td><br />
<td><p class="texte">Sent by the Munich iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td></tr><br />
<!--14eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823022">K823022</a></p></td><br />
<td><p class="texte">Empty backbone vector pSB<sub>BS</sub>4S for integration into<i>B. subtilis</i><br />
<br>It integrates in the thrC locus</p></td><br />
<td><p class="texte">Sent by the Munich iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td><br />
</tr><br />
<!--15eme ligne--><br />
<tr><br />
<td><p class="texte"><a href="http://parts.igem.org/Part:BBa_K823021">K823021</a></p></td><br />
<td><p class="texte">Reporter vector pSB<sub>BS</sub>1C-lacZ for <i>B. subtilis</i></p></td><br />
<td><p class="texte">Sent by the Munich iGEM team</p></td><br />
<td><p class="texte">Fungicides<br>Chemotaxis<br>Binding</p></td></tr><br />
</table></center><br />
</p><br />
<br />
<br><br />
<br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Result/experimental-results" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Experimental results<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Result/achievement" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Achievement</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Result/experimental-resultsTeam:Toulouse/Result/experimental-results2014-10-13T17:55:51Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#5a6060; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify; }<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify; }<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/f/f7/Results_photo_tlse.png') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
p.legend{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: center;}<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Experimental results</h2><br />
<p> Are our modules functionnal? </p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Experimental results</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<p class="title1">Chemotaxis<br />
</p><br />
<p class="texte">For this module, we had to try several tests to prove the existence of chemotaxis in <i>Bacillus subtilis</i> wild type (WT) strain and SubtiTree bacterium towards N-Acetylglucosamine.<br />
</p><br />
<br />
<p class="title2">1. Plug in Pond system<br />
</p><br />
<p class="texte">Coming soon! <!--Florie--><br />
</p><br />
<br />
<p class="title2">2. Capillary test between two tubes also called the tubes test<br />
</p><br />
<p class="texte">After the experiment of the plug in pond, we decided to construct a system by welding two Eppendorf tubes with a capillary thanks to an electric burner.</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/f/fb/Chemotaxis_-_eppendorf.png"></center><br />
<p class="legend">Figure 1 : Photography of the first tubes system</p><br />
<br />
<p class="texte">We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin towards water. However this construction had a leakage next to the weld seam that we could not stop. <br />
Thus, the Toulouse iGEM Team asked the help from the glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/Chemotaxis_-_tubes.png"><center><br />
<p class="legend">Figure 2 : Scheme of the tubes system</p><br />
<br />
<p class="texte">As we did previously, we tested this new system with fuchsin. This experiment was made with WT <i>Bacillus subtilis</i> and N-Acetylglucosamine.<br />
<br><br><br />
<i>NB: We could not see the diffusion from one tube to the other. We made the hypothesis that it was not visible by sight because of by the small diameter of the capillary. <br />
</i><br><br />
<br><br />
The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:<br><br />
- The first step was the addition of Wash Buffer until the capillary was full to avoid the presence of air bubbles which could lead to diffusion problems.<br><br />
- Then, the tube 2 was plugged with the thumb while another person was adding the bacteria solution of WT Bacillus subtilis in the tube 1. <br><br />
- The tube 1 was also plugged and only after the thumb could be removed of the tube 2. <br><br />
- In the same way, the N-Acetylglucosamine was added in the tube 2. <br><br />
- The same process was made with a xylose positive control.<br><br />
<br><br />
<i>NB: According to the article Chemotaxis towards sugars by </i>Bacillus subtilis, (George W. Ordal et al., 1979), <i>glucose and xylose have the same attractant power. We prefer a positive control instead of a negative because we were not sure that this system was efficient.</i><br><br />
<br><br />
- The system was kept straight for 2hours. Every 40 minutes, we took a sample of each tube and spread it on an agar plate (dilution 1/1,000).</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/1/1b/Chemotaxis_-_tubes_photo.png"></center><br />
<p class="legend">Figure 3 : Photography of the tubes system</p><br />
<br />
<br />
<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement and the time was too short. We did not find any information in the literature.<br><br />
As we did not have the time to optimize this protocol we preferred using the protocol of the Imperial college iGEM team 2011: the tips capillary test.<br><br />
</p><br />
<br />
<p class="title2">3. Tips capillary system</p><br />
<p class="title3">First tips capillary system</p><br />
<p class="texte">This protocol comes from Imperial College iGEM team 2011 and was adapted by our team in several steps (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols">chemotaxis protocol</a>).<br><br />
<br><br />
First of all, parafilm was used to close the tips:<br><br />
- 15µL of each chemo-attractant was then pipetted. <br><br />
- The tips with the pipette were then put on a piece of parafilm and the pipette was removed from the tip.<br><br />
- The tip was sealed with a piece of parafilm. By this way, the sterility can be assured and the liquid stays inside the tip. <br><br />
- To finish, the level of the solution in the tip was marked.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/9/94/Chemotaxis_-_tip.png"></center><br />
<p class="legend">Figure 4 : Sealing of a tip with parafilm</p><br />
<br />
<p class="texte">- After all the chemo-attractants were added in the tips, we put them on a green base to carry them. The whole process can be seen on Figure 5.<br><br />
- Each tip was put in 300 µL of a bacteria solution in the wells of an Elisa plate.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/0/05/Chemotaxis_-_tip_and_support.png"></center><br />
<p class="legend">Figure 5 : First tips capillary system</p><br />
<br />
<p class="texte"><i>NB: the yellow carton was used to stabilize the system and keep it straight.</i><br><br />
<br><br />
- After one hour, the tips were removed from the bacteria solutions and the content of the tips was observed with Thoma cell under the microscope.<br><br />
<br><br />
We had several problems with this system:<br><br />
- The liquid level decreased during the experiment and we did not have enough liquid to fill the Thoma cell. Thus, it was not possible to count.<br><br />
- The bacteria were moving and therefore, we could not proceed to a bacteria count.<br><br />
<br><br />
Regarding these observations we decided to spread the tips content on agar plate instead of using Thoma cell and microscopy.<br><br />
<p class="title3">Second tips capillary system<br />
</p><br />
<p class="texte"And then the revolution came! We found a multichannel pipette. The same protocol was performed except that the parafilm was used to avoid the air entrance between the tips and the pipette and therefore the loss of liquid.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/e/e4/Chemotaxis_-_pipette.png"></center><br />
<p class="legend">Figure 6 : Second tips capillary system</p><br />
<br />
<p class="title3">Improvement of the second tips capillary system</p><br />
<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></center><br />
<p class="legend">Figure 7 : Improvement of the second tips capillary system</p><br />
<br />
<p class="texte"><b>At that point, the protocol was approved and the final test could finally start! :-)</b><br><br />
<br><br />
There was just one tiny problem… we did not have our optimized bacterium with the chemotaxis gene… That is why we concentrated our efforts on WT Bacillus subtilis strain.<br><br />
<br><br />
The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolarity bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br><br />
<br><br />
The experiment was conducted with fuchsin as a negative control and was tested with different positive controls: glucose (25mM) and xylose (25mM).<br><br />
<br><br />
We obtained the following result with NAG at different concentrations: 25mM, 250mM and 500mM. The tested strain was <i>Bacillus subtilis </i>168:<br><br />
<br></p><br />
<center><br />
<table align="center"><br />
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/8/8c/Chemotaxis_-_results_fuch.png"></td><br />
<td align=center><img src="https://static.igem.org/mediawiki/2014/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr><br />
<tr><td align=center><p class="legend">Figure 8 : Fuchsin - negative control (dilution 1/50)</p></td><br />
<td align=center><p class="legend">Figure 9 : NAG (25mM) (dilution 1/50)</p></td></tr><br />
</table></center><br><br />
<p class="texte">The average number of colonies with the negative control is 121. On the contrary, a cell layer is observed for the NAG plates with every concentration.<br><br />
<br><br />
Thus, we assumed that WT <i>Bacillus subtilis</i> was more attracted by NAG than fuchsin. Indeed we can neglect the bacterial growth because the test only lasts one hour. We also neglect diffusion and osmolality phenomena for the previous reasons. <br><br />
<br><br />
Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells which probably happened to our results.<br><br />
<br><br />
<b><p class="texte">This incredible discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><br><br />
<br><br />
However, our team did not give up on synthetic biology and on our strength! Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.<br><br />
<br><br />
We finally used galactose (25mM) as a negative control. The article Chemotaxis towards sugars by <i>Bacillus subtilis</i> (<i>George W. Ordal et al., 1979</i>) proved that it was a poor attractant.<br><br />
<br><br />
We made our tests again with this new molecule and glucose (25mM) as positive control.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/8/86/Chemotaxis_-_final_results.png"></center><br />
<p class="legend">Figure 10 : Final results (dilution : 1/10,000)</p><br />
<br />
<p class="texte"><i>NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.</i><br><br />
<br><br />
<b><p class="texte">Our results are not statistically significant however this result has been proved in literature.</p></b><br></p><br />
<br />
</br><br />
<br />
<p class="title1">Binding module</p> <br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Purpose</p><br />
<p class="texte">The first experiment deals with the culture conditions to see if <i>Bacillus subtilis</i> can resist to a low temperature and with the CBB buffer. To do that, several bacterial concentrations have been tested starting with an OD of 0.1 and diluting this solution to get estimated ODs of 0.05, 0.025, 0.01. These different <i>Bacillus subtilis</i> solutions were incubated 1 hour at 4°C with 500µL of CBB or water. Finally a cell count on Thoma cell counting chamber was performed.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The bacterial solutions could not be counted because of two main problems: the too high number of bacteria with the 0.1 OD or the too low number of bacteria with the 0.01 OD. Thus, the study is mostly focused on the intermediate values (Figure 1).<br />
<br/>First of all, a same cell concentration can be noticed with the presence of CBB or water with estimated ODs of 0.05 or 0.025. Moreover, twice less cells can be found in the lowest concentrations in bacteria comparing to the 0.05 OD concentration which is in agreement with the dilution ratio. <br />
<br/>Thus, the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C do not harm the cell surviving.<br />
</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/c/ce/Graphe_binding_1.png" width="45%"></center><br />
<br />
</br><br />
<p class="texte">Figure 1: CBB presence has no effect on bacteria. The bacterial concentration was measured regarding <span style="color:#0000FF">the presence</span> or <span style="color:#FF0000">the absence </span>of CBB for the observed OD (0.1) or estimated ODs (0.05, 0.025, 0.01).<br />
</p><br />
<br />
<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">Transformed <i>Bacillus subtilis</i> with the binding module is able to produce a protein composed of the bacterial peptidoglycan bonding of LycT and the GbpA 4th domain of <i>Vibrio cholerae</i> allowing the chitin bonding. The synthetic bacterium is put with special beads composed of the polymer miming the fungal pathogen wall. After several washes, bacteria specifically attached to the chitin are put on plates and counted.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The first observation is that both bacterial solutions of wild type <i>Bacillus subtilis</i> and SubtiTree have the same concentration : 105 bacteria/mL (Figure 2). Even though there is no significant difference between both strains after the first wash, the second wash has a major effect since it allows 40 times more Wild Type bacteria to come off the beads. This result correlates with the number of bacteria binded to the beads for the synthetic strain with the binding module. <br />
<br/>Thus, the binding system seems to function correctly and leads to the bacterial attachment on the chitin.</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ea/Graphe_binding_2.png" width="60%"></center><br />
</br><br />
<br />
<p class="texte">Figure 2: Attachment of <i>Bacillus subtilis</i> with binding module to chitin. <span style="color:#0000FF">The WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentration has been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p<0.05.</p><br />
<br />
<p class="title2">3. Microscopic observations</p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">We want to observe the SubtiTree's binding on beads coated with chitin. In order to perform a 3D reconstruction showing this interaction, we use confocal laser scanning microscope. Through the use of a fluorochrome (Syto9), we can highlight the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determine the minimum threshold to remove the background noise and the natural fluorescence.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">First, we note the great bacterial presence on the surface of beads coated with chitin. These images seem to highlight their interactions.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/archive/5/53/20141013073044!Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="texte">Figure ***: <br />
</p><br />
<br />
<p class="texte">Using ImageJ software, we are able to create 3D pictures and movies of those comments. </br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/53/Photo_billes_microscopie.png" width="45%"><iframe width="420" height="315" src="//www.youtube.com/embed/G3xpYnkUr3o" frameborder="0" allowfullscreen></iframe></center><br />
</br><br />
<p class="texte">Figure ***: <br />
</p><br />
<br />
<p class="texte">Finally we want to observe the bacteria after the second wash. When our bacterium has the binding module, results suggest a lower number of bacteria in the washing solution. SubtiTree is retained by the beads.</br><br />
Finally, overall results are consistent with the presence of functional binding system.</p><br />
<br />
<br />
<p class="title1">Fungicides module<br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Tests with commercial peptides and controls</p><br />
<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12.5µM, 25µM, 100µM). These tests were performed on different fungal strains sharing the same phylum with <i>Ceratocystis Platani</i>.<br />
As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus.</br><br />
After several days at 30°C, the PDA (Potato Dextrose Agar) plates couvered with fungus and commercial peptides were analyzed.</p></p><br />
<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i>. Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions.As positive control, a well-known chemical fungicide was used: the Copper Sulfate. The inhibition of the fungal growth was complete at 20mg/ml, and at 10mg/ml a darker halo appeared around the pad filled with Copper Sulfate as we can see on the figure below. This corresponds to a sporing halo in response to the stress generated by the fungicide.<br />
</p><br />
<br />
</br><br />
<br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/a/a8/Prelim_tests_fung.jpg"><br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/f/f8/Controls_fung.jpg"><br />
<br />
<br />
</br><br />
<br />
<p class="texte">Given these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth. Following these tests, new conditions were adopted in order not to encourage too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees: a 'sap-like' medium was elaborated. The incubations were then carried at room temperature.<br />
</p><br />
<br />
<p class="title2">2. Test with SubtiTree<p/><br />
<br />
<p class="texte">In order to test <i>Bacillus subtilis</i> mutants, it was essential to find the right balance between the fungal growth and the bacterial one. This condition was necessary to get a high concentration of peptides. In our genetic constructions, these peptides are designed to be exported in the extracellular medium.</br><br />
</br><br />
The transformed <i>Bacillus subtilis</i> strains grew at 37°C during 72h and were tested. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of <i>Trichoderma reesei</i>'s growth was clearly observable for the strain expressing D4E1 gene. The inhibition was even more noticeable with the strain carrying the operon GAFP-1 + D4E1 (see the photos below).</br><br />
However, no effect was detected for the strain expressing the GAFP-1 gene, supposing a synergistic effect between these two peptides.</br><br />
Regarding EcAMP and the triple-fungicides operon, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP and in addition to that, sequencing results of these constructs showed some differences with the original designed sequence.<br />
<p class="texte">Inhibition halos are not visible with supernatants, probably because of their low concentrations in the extracellular medium. <br />
Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus <i>Aspergillus brasiliansis</i>. This effect is comparable to the one previously noted with low concentration of sulfate copper. </br><br />
</p><br />
<br />
</br><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/c/c2/Resultfong.jpg"> Figure X<br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/9/92/Results_fong_2.jpg"> Figure X<br />
</br><br />
<br />
<p class="texte"><br />
The choice of our chassis appears to be optimal as we noted that wild type <i>Bacillus subtilis</i> disturbs the hyphae growth of the fungi. Some strains of <i>Bacillus subtilis</i> (qst 713) are already used as Biofungicides for use on several minor crops to treat a variety of plant diseases and fungal pathogens.</br><br />
After this set of experiments, the strains expressing D4E1 and expressing GAFP-1 + D4E1 have shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b> we decided to tests these strains in model plants </b> : <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br> These tests were performed in the National Institute for the Agronomic Research by experts in this domain. <br />
<br />
<br />
<p class="title2">3. <i> In planta </i> tests with SubtiTree<p/><br />
<br />
<div style="float:left; width:215px;"><br />
<img style="width:215px;" img src="https://static.igem.org/mediawiki/parts/a/af/In_planta.jpg" style="margin-top:5px" /><br />
</div> <br />
<div style="float: right; width:700px; margin-left:44px;"> <br />
<p class="texte"><br />
The goal of the project is to introduce the trasnformed bacteria in a diseased tree. So it is necessary to perform <i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous set of experiments. </br><br />
SubtiTree is first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) is placed on the leaves. </br><br />
These tests were made in association with Sylvain Raffaële and Marielle Barascud of the National Institute for the Agronomic Research laboratory. </br><br />
</p> </div> <br />
<br />
<br />
<br />
<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves can be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants don't have deleterious effects.</br><br />
Without proper treatment, the drop of the pyhtopathogenic fungus on <i>Nicotiana benthamiana</i>'s leaves causes a necrosis halo which can be measured after 40h. The lesion size and the number of inoculated sites seem reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike with the WT bacterium. A second set of experiments is expected to be more statistically precise.</br><br />
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br><br />
<br />
We can therefore conclude that when SubtiTree is in plant physiological conditions, <b> it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves' necrosis </b>.<br />
</p><br />
<br />
<img style="width:860px;" img src="https://static.igem.org/mediawiki/parts/f/f1/Results_d4%2B_gafp1.jpg" <br />
<br />
<br />
<p class="texte"> <br><br />
Thanks to the diversity of anti-fungal peptides, this strategy can be adapted to different types of diseases, with different degree of specifity, etc.<br />
</p><br />
<br />
<br />
<br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="" class="page-nav-right" style="min-width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;"><br />
<br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Result/parts" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Parts</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Result/experimental-resultsTeam:Toulouse/Result/experimental-results2014-10-13T15:38:23Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#5a6060; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify; }<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify; }<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/f/f7/Results_photo_tlse.png') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
p.legend{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: center;}<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Experimental results</h2><br />
<p> Are our modules functionnal? </p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Experimental results</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<p class="title1">Chemotaxis<br />
</p><br />
<p class="texte">For this module, we had to try several tests to prove the existence of chemotaxis in <i>Bacillus subtilis</i> wild type (WT) strain and SubtiTree bacterium towards N-Acetylglucosamine.<br />
</p><br />
<br />
<p class="title2">1. Plug in Pond system<br />
</p><br />
<p class="texte">Coming soon! <!--Florie--><br />
</p><br />
<br />
<p class="title2">2. Capillary test between two tubes also called the tubes test<br />
</p><br />
<p class="texte">After the experiment of the plug in pond, we decided to construct a system by welding two Eppendorf tubes with a capillary thanks to an electric burner.</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/f/fb/Chemotaxis_-_eppendorf.png"></center><br />
<p class="legend">Figure 1 : Photography of the first tubes system</p><br />
<br />
<p class="texte">We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin towards water. However this construction had a leakage next to the weld seam that we could not stop. <br />
Thus, the Toulouse iGEM Team asked the help from the glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/Chemotaxis_-_tubes.png"><center><br />
<p class="legend">Figure 2 : Scheme of the tubes system</p><br />
<br />
<p class="texte">As we did previously, we tested this new system with fuchsin. This experiment was made with WT <i>Bacillus subtilis</i> and N-Acetylglucosamine.<br />
<br><br><br />
<i>NB: We could not see the diffusion from one tube to the other. We made the hypothesis that it was not visible by sight because of by the small diameter of the capillary. <br />
</i><br><br />
<br><br />
The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:<br><br />
- The first step was the addition of Wash Buffer until the capillary was full to avoid the presence of air bubbles which could lead to diffusion problems.<br><br />
- Then, the tube 2 was plugged with the thumb while another person was adding the bacteria solution of WT Bacillus subtilis in the tube 1. <br><br />
- The tube 1 was also plugged and only after the thumb could be removed of the tube 2. <br><br />
- In the same way, the N-Acetylglucosamine was added in the tube 2. <br><br />
- The same process was made with a xylose positive control.<br><br />
<br><br />
<i>NB: According to the article Chemotaxis towards sugars by </i>Bacillus subtilis, (George W. Ordal et al., 1979), <i>glucose and xylose have the same attractant power. We prefer a positive control instead of a negative because we were not sure that this system was efficient.</i><br><br />
<br><br />
- The system was kept straight for 2hours. Every 40 minutes, we took a sample of each tube and spread it on an agar plate (dilution 1/1,000).</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/1/1b/Chemotaxis_-_tubes_photo.png"></center><br />
<p class="legend">Figure 3 : Photography of the tubes system</p><br />
<br />
<br />
<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement and the time was too short. We did not find any information in the literature.<br><br />
As we did not have the time to optimize this protocol we preferred using the protocol of the Imperial college iGEM team 2011: the tips capillary test.<br><br />
</p><br />
<br />
<p class="title2">3. Tips capillary system</p><br />
<p class="title3">First tips capillary system</p><br />
<p class="texte">This protocol comes from Imperial College iGEM team 2011 and was adapted by our team in several steps (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols">chemotaxis protocol</a>).<br><br />
<br><br />
First of all, parafilm was used to close the tips:<br><br />
- 15µL of each chemo-attractant was then pipetted. <br><br />
- The tips with the pipette were then put on a piece of parafilm and the pipette was removed from the tip.<br><br />
- The tip was sealed with a piece of parafilm. By this way, the sterility can be assured and the liquid stays inside the tip. <br><br />
- To finish, the level of the solution in the tip was marked.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/9/94/Chemotaxis_-_tip.png"></center><br />
<p class="legend">Figure 4 : Sealing of a tip with parafilm</p><br />
<br />
<p class="texte">- After all the chemo-attractants were added in the tips, we put them on a green base to carry them. The whole process can be seen on Figure 5.<br><br />
- Each tip was put in 300 µL of a bacteria solution in the wells of an Elisa plate.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/0/05/Chemotaxis_-_tip_and_support.png"></center><br />
<p class="legend">Figure 5 : First tips capillary system</p><br />
<br />
<p class="texte"><i>NB: the yellow carton was used to stabilize the system and keep it straight.</i><br><br />
<br><br />
- After one hour, the tips were removed from the bacteria solutions and the content of the tips was observed with Thoma cell under the microscope.<br><br />
<br><br />
We had several problems with this system:<br><br />
- The liquid level decreased during the experiment and we did not have enough liquid to fill the Thoma cell. Thus, it was not possible to count.<br><br />
- The bacteria were moving and therefore, we could not proceed to a bacteria count.<br><br />
<br><br />
Regarding these observations we decided to spread the tips content on agar plate instead of using Thoma cell and microscopy.<br><br />
<p class="title3">Second tips capillary system<br />
</p><br />
<p class="texte"And then the revolution came! We found a multichannel pipette. The same protocol was performed except that the parafilm was used to avoid the air entrance between the tips and the pipette and therefore the loss of liquid.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/e/e4/Chemotaxis_-_pipette.png"></center><br />
<p class="legend">Figure 6 : Second tips capillary system</p><br />
<br />
<p class="title3">Improvement of the second tips capillary system</p><br />
<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></center><br />
<p class="legend">Figure 7 : Improvement of the second tips capillary system</p><br />
<br />
<p class="texte"><b>At that point, the protocol was approved and the final test could finally start! :-)</b><br><br />
<br><br />
There was just one tiny problem… we did not have our optimized bacterium with the chemotaxis gene… That is why we concentrated our efforts on WT Bacillus subtilis strain.<br><br />
<br><br />
The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolarity bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br><br />
<br><br />
The experiment was conducted with fuchsin as a negative control and was tested with different positive controls: glucose (25mM) and xylose (25mM).<br><br />
<br><br />
We obtained the following result with NAG at different concentrations: 25mM, 250mM and 500mM. The tested strain was <i>Bacillus subtilis </i>168:<br><br />
<br></p><br />
<center><br />
<table align="center"><br />
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/8/8c/Chemotaxis_-_results_fuch.png"></td><br />
<td align=center><img src="https://static.igem.org/mediawiki/2014/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr><br />
<tr><td align=center><p class="legend">Figure 8 : Fuchsin - negative control (dilution 1/50)</p></td><br />
<td align=center><p class="legend">Figure 9 : NAG (25mM) (dilution 1/50)</p></td></tr><br />
</table></center><br><br />
<p class="texte">The average number of colonies with the negative control is 121. On the contrary, a cell layer is observed for the NAG plates with every concentration.<br><br />
<br><br />
Thus, we assumed that WT <i>Bacillus subtilis</i> was more attracted by NAG than fuchsin. Indeed we can neglect the bacterial growth because the test only lasts one hour. We also neglect diffusion and osmolality phenomena for the previous reasons. <br><br />
<br><br />
Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells which probably happened to our results.<br><br />
<br><br />
<b><p class="texte">This incredible discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><br><br />
<br><br />
However, our team did not give up on synthetic biology and on our strength! Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.<br><br />
<br><br />
We finally used galactose (25mM) as a negative control. The article Chemotaxis towards sugars by <i>Bacillus subtilis</i> (<i>George W. Ordal et al., 1979</i>) proved that it was a poor attractant.<br><br />
<br><br />
We made our tests again with this new molecule and glucose (25mM) as positive control.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/8/86/Chemotaxis_-_final_results.png"></center><br />
<p class="legend">Figure 10 : Final results (dilution : 1/10,000)</p><br />
<br />
<p class="texte"><i>NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.</i><br><br />
<br><br />
<b><p class="texte">Our results are not statistically significant however this result has been proved in literature.</p></b><br></p><br />
<br />
</br><br />
<br />
<p class="title1">Binding module</p> <br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Purpose</p><br />
<p class="texte">The first experiment deals with the culture conditions to see if <i>Bacillus subtilis</i> can resist to a low temperature and with the CBB buffer. To do that, several bacterial concentrations have been tested starting with an OD of 0.1 and diluting this solution to get estimated ODs of 0.05, 0.025, 0.01. These different <i>Bacillus subtilis</i> solutions were incubated 1 hour at 4°C with 500µL of CBB or water. Finally a cell count on Thoma cell counting chamber was performed.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The bacterial solutions could not be counted because of two main problems: the too high number of bacteria with the 0.1 OD or the too low number of bacteria with the 0.01 OD. Thus, the study is mostly focused on the intermediate values (Figure 1).<br />
<br/>First of all, a same cell concentration can be noticed with the presence of CBB or water with estimated ODs of 0.05 or 0.025. Moreover, twice less cells can be found in the lowest concentrations in bacteria comparing to the 0.05 OD concentration which is in agreement with the dilution ratio. <br />
<br/>Thus, the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C do not harm the cell surviving.<br />
</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/c/ce/Graphe_binding_1.png" width="45%"></center><br />
<br />
</br><br />
<p class="texte">Figure 1: CBB presence has no effect on bacteria. The bacterial concentration was measured regarding <span style="color:#0000FF">the presence</span> or <span style="color:#FF0000">the absence </span>of CBB for the observed OD (0.1) or estimated ODs (0.05, 0.025, 0.01).<br />
</p><br />
<br />
<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">Transformed <i>Bacillus subtilis</i> with the binding module is able to produce a protein composed of the bacterial peptidoglycan bonding of LycT and the GbpA 4th domain of <i>Vibrio cholerae</i> allowing the chitin bonding. The synthetic bacterium is put with special beads composed of the polymer miming the fungal pathogen wall. After several washes, bacteria specifically attached to the chitin are put on plates and counted.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The first observation is that both bacterial solutions of wild type <i>Bacillus subtilis</i> and SubtiTree have the same concentration : 105 bacteria/mL (Figure 2). Even though there is no significant difference between both strains after the first wash, the second wash has a major effect since it allows 40 times more Wild Type bacteria to come off the beads. This result correlates with the number of bacteria binded to the beads for the synthetic strain with the binding module. <br />
<br/>Thus, the binding system seems to function correctly and leads to the bacterial attachment on the chitin.</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ea/Graphe_binding_2.png" width="60%"></center><br />
</br><br />
<br />
<p class="texte">Figure 2: Attachment of <i>Bacillus subtilis</i> with binding module to chitin. <span style="color:#0000FF">The WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentration has been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p<0.05.</p><br />
<br />
<p class="title2">3. Microscopic observations</p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">We want to observe the SubtiTree's binding on beads coated with chitin. In order to perform a 3D reconstruction showing this interaction, we use confocal laser scanning microscope. Through the use of a fluorochrome (Syto9), we can highlight the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determine the minimum threshold to remove the background noise and the natural fluorescence.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">First, we note the great bacterial presence on the surface of beads coated with chitin. These images seem to highlight their interactions.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/archive/5/53/20141013073044!Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="texte">Figure ***: <br />
</p><br />
<br />
<p class="texte">Using ImageJ software, we are able to create 3D pictures and movies of those comments. </br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/53/Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="texte">Figure ***: <br />
</p><br />
<br />
<p class="texte">Finally we want to observe the bacteria after the second wash. When our bacterium has the binding module, results suggest a lower number of bacteria in the washing solution. SubtiTree is retained by the beads.</br><br />
Finally, overall results are consistent with the presence of functional binding system.</p><br />
<br />
<br />
<p class="title1">Fungicides module<br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Tests with commercial peptides and controls</p><br />
<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12.5µM, 25µM, 100µM). These tests were performed on different fungal strains sharing the same phylum with <i>Ceratocystis Platani</i>.<br />
As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus.</br><br />
After several days at 30°C, the PDA (Potato Dextrose Agar) plates couvered with fungus and commercial peptides were analyzed.</p></p><br />
<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i>. Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions.As positive control, a well-known chemical fungicide was used: the Copper Sulfate. The inhibition of the fungal growth was complete at 20mg/ml, and at 10mg/ml a darker halo appeared around the pad filled with Copper Sulfate as we can see on the figure below. This corresponds to a sporing halo in response to the stress generated by the fungicide.<br />
</p><br />
<br />
</br><br />
<br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/a/a8/Prelim_tests_fung.jpg"><br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/f/f8/Controls_fung.jpg"><br />
<br />
<br />
</br><br />
<br />
<p class="texte">Given these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth. Following these tests, new conditions were adopted in order not to encourage too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees: a 'sap-like' medium was elaborated. The incubations were then carried at room temperature.<br />
</p><br />
<br />
<p class="title2">2. Test with SubtiTree<p/><br />
<br />
<p class="texte">In order to test <i>Bacillus subtilis</i> mutants, it was essential to find the right balance between the fungal growth and the bacterial one. This condition was necessary to get a high concentration of peptides. In our genetic constructions, these peptides are designed to be exported in the extracellular medium.</br><br />
</br><br />
The transformed <i>Bacillus subtilis</i> strains were grown at 37°C during 72h and tested. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of <i>Trichoderma reesei</i>'s growth was clearly observable for the strain expressing D4E1 gene. The inhibition was even more noticeable with the strain carrying the operon GAFP-1 + D4E1 (see the photos below).</br><br />
However, no effect was detected for the strain expressing the GAFP-1 gene, supposing a synergistic effect between these two peptides.</br><br />
Regarding EcAMP and the triple-fungicides operon, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP and in addition to that, sequencing results of these constructs showed some differences with the original designed sequence.<br />
<p class="texte">Inhibition halos are not visible with supernatants, probably because of their low concentrations in the extracellular medium. <br />
Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus <i>Aspergillus brasiliansis</i>. This effect is comparable to the one previously noted with low concentration of sulfate copper. </br><br />
</p><br />
<br />
</br><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/c/c2/Resultfong.jpg"> Figure X<br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/9/92/Results_fong_2.jpg"> Figure X<br />
</br><br />
<br />
<p class="texte"><br />
The choice of our chassis appears to be optimal as we noted that wild type <i>Bacillus subtilis</i> disturbs the hyphae growth of the fungi. Some strains of <i>Bacillus subtilis</i> (qst 713) are already used as Biofungicides for use on several minor crops to treat a variety of plant diseases and fungal pathogens.</br><br />
After this set of experiments, the strains expressing D4E1 and expressing GAFP-1 + D4E1 have shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b> we decided to tests these strains in model plants </b> : <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br> These tests were performed in the National Institute for the Agronomic Research by experts in this domain. <br />
<br />
<br />
<p class="title2">3. <i> In planta </i> tests with SubtiTree<p/><br />
<br />
<div style="float:left; width:215px;"><br />
<img style="width:215px;" img src="https://static.igem.org/mediawiki/parts/a/af/In_planta.jpg" style="margin-top:5px" /><br />
</div> <br />
<div style="float: right; width:700px; margin-left:44px;"> <br />
<p class="texte"><br />
The goal of the project is to introduce the trasnformed bacteria in a diseased tree. So it is necessary to perform <i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous set of experiments. </br><br />
SubtiTree is first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) is placed on the leaves. </br><br />
These tests were made in association with Sylvain Raffaële and Marielle Barascud of the National Institute for the Agronomic Research laboratory. </br><br />
</p> </div> <br />
<br />
<br />
<br />
<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves can be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants don't have deleterious effects.</br><br />
Without proper treatment, the drop of the pyhtopathogenic fungus on <i>Nicotiana benthamiana</i>'s leaves causes a necrosis halo which can be measured after 40h. The lesion size and the number of inoculated sites seem reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike with the WT bacterium. A second set of experiments is expected to be more statistically precise.</br><br />
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br><br />
<br />
We can therefore conclude that when SubtiTree is in plant physiological conditions, <b> it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves' necrosis </b>.<br />
</p><br />
<br />
TABLEAU DE RESULTAT<br />
<br />
<p class="texte"><br />
Thanks to the diversity of anti-fungal peptides, this strategy can be adapted to different types of diseases, with different degree of specifity, etc.<br />
</p><br />
<br />
<br />
<br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="" class="page-nav-right" style="min-width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;"><br />
<br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Result/parts" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Parts</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Result/experimental-resultsTeam:Toulouse/Result/experimental-results2014-10-13T15:17:16Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#5a6060; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify; }<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify; }<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/f/f7/Results_photo_tlse.png') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
p.legend{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: center;}<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Experimental results</h2><br />
<p> Are our modules functionnal? </p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Experimental results</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<p class="title1">Chemotaxis<br />
</p><br />
<p class="texte">For this module, we had to try several tests to prove the existence of chemotaxis in <i>Bacillus subtilis</i> wild type (WT) strain and SubtiTree bacterium towards N-Acetylglucosamine.<br />
</p><br />
<br />
<p class="title2">1. Plug in Pond system<br />
</p><br />
<p class="texte">Coming soon! <!--Florie--><br />
</p><br />
<br />
<p class="title2">2. Capillary test between two tubes also called the tubes test<br />
</p><br />
<p class="texte">After the experiment of the plug in pond, we decided to construct a system by welding two Eppendorf tubes with a capillary thanks to an electric burner.</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/f/fb/Chemotaxis_-_eppendorf.png"></center><br />
<p class="legend">Figure 1 : Photography of the first tubes system</p><br />
<br />
<p class="texte">We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin towards water. However this construction had a leakage next to the weld seam that we could not stop. <br />
Thus, the Toulouse iGEM Team asked the help from the glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/Chemotaxis_-_tubes.png"><center><br />
<p class="legend">Figure 2 : Scheme of the tubes system</p><br />
<br />
<p class="texte">As we did previously, we tested this new system with fuchsin. This experiment was made with WT <i>Bacillus subtilis</i> and N-Acetylglucosamine.<br />
<br><br><br />
<i>NB: We could not see the diffusion from one tube to the other. We made the hypothesis that it was not visible by sight because of by the small diameter of the capillary. <br />
</i><br><br />
<br><br />
The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:<br><br />
- The first step was the addition of Wash Buffer until the capillary was full to avoid the presence of air bubbles which could lead to diffusion problems.<br><br />
- Then, the tube 2 was plugged with the thumb while another person was adding the bacteria solution of WT Bacillus subtilis in the tube 1. <br><br />
- The tube 1 was also plugged and only after the thumb could be removed of the tube 2. <br><br />
- In the same way, the N-Acetylglucosamine was added in the tube 2. <br><br />
- The same process was made with a xylose positive control.<br><br />
<br><br />
<i>NB: According to the article Chemotaxis towards sugars by </i>Bacillus subtilis, (George W. Ordal et al., 1979), <i>glucose and xylose have the same attractant power. We prefer a positive control instead of a negative because we were not sure that this system was efficient.</i><br><br />
<br><br />
- The system was kept straight for 2hours. Every 40 minutes, we took a sample of each tube and spread it on an agar plate (dilution 1/1,000).</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/1/1b/Chemotaxis_-_tubes_photo.png"></center><br />
<p class="legend">Figure 3 : Photography of the tubes system</p><br />
<br />
<br />
<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement and the time was too short. We did not find any information in the literature.<br><br />
As we did not have the time to optimize this protocol we preferred using the protocol of the Imperial college iGEM team 2011: the tips capillary test.<br><br />
</p><br />
<br />
<p class="title2">3. Tips capillary system</p><br />
<p class="title3">First tips capillary system</p><br />
<p class="texte">This protocol comes from Imperial College iGEM team 2011 and was adapted by our team in several steps (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols">chemotaxis protocol</a>).<br><br />
<br><br />
First of all, parafilm was used to close the tips:<br><br />
- 15µL of each chemo-attractant was then pipetted. <br><br />
- The tips with the pipette were then put on a piece of parafilm and the pipette was removed from the tip.<br><br />
- The tip was sealed with a piece of parafilm. By this way, the sterility can be assured and the liquid stays inside the tip. <br><br />
- To finish, the level of the solution in the tip was marked.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/9/94/Chemotaxis_-_tip.png"></center><br />
<p class="legend">Figure 4 : Sealing of a tip with parafilm</p><br />
<br />
<p class="texte">- After all the chemo-attractants were added in the tips, we put them on a green base to carry them. The whole process can be seen on Figure 5.<br><br />
- Each tip was put in 300 µL of a bacteria solution in the wells of an Elisa plate.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/0/05/Chemotaxis_-_tip_and_support.png"></center><br />
<p class="legend">Figure 5 : First tips capillary system</p><br />
<br />
<p class="texte"><i>NB: the yellow carton was used to stabilize the system and keep it straight.</i><br><br />
<br><br />
- After one hour, the tips were removed from the bacteria solutions and the content of the tips was observed with Thoma cell under the microscope.<br><br />
<br><br />
We had several problems with this system:<br><br />
- The liquid level decreased during the experiment and we did not have enough liquid to fill the Thoma cell. Thus, it was not possible to count.<br><br />
- The bacteria were moving and therefore, we could not proceed to a bacteria count.<br><br />
<br><br />
Regarding these observations we decided to spread the tips content on agar plate instead of using Thoma cell and microscopy.<br><br />
<p class="title3">Second tips capillary system<br />
</p><br />
<p class="texte"And then the revolution came! We found a multichannel pipette. The same protocol was performed except that the parafilm was used to avoid the air entrance between the tips and the pipette and therefore the loss of liquid.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/e/e4/Chemotaxis_-_pipette.png"></center><br />
<p class="legend">Figure 6 : Second tips capillary system</p><br />
<br />
<p class="title3">Improvement of the second tips capillary system</p><br />
<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></center><br />
<p class="legend">Figure 7 : Improvement of the second tips capillary system</p><br />
<br />
<p class="texte"><b>At that point, the protocol was approved and the final test could finally start! :-)</b><br><br />
<br><br />
There was just one tiny problem… we did not have our optimized bacterium with the chemotaxis gene… That is why we concentrated our efforts on WT Bacillus subtilis strain.<br><br />
<br><br />
The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolarity bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br><br />
<br><br />
The experiment was conducted with fuchsin as a negative control and was tested with different positive controls: glucose (25mM) and xylose (25mM).<br><br />
<br><br />
We obtained the following result with NAG at different concentrations: 25mM, 250mM and 500mM. The tested strain was <i>Bacillus subtilis </i>168:<br><br />
<br></p><br />
<center><br />
<table align="center"><br />
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/8/8c/Chemotaxis_-_results_fuch.png"></td><br />
<td align=center><img src="https://static.igem.org/mediawiki/2014/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr><br />
<tr><td align=center><p class="legend">Figure 8 : Fuchsin - negative control (dilution 1/50)</p></td><br />
<td align=center><p class="legend">Figure 9 : NAG (25mM) (dilution 1/50)</p></td></tr><br />
</table></center><br><br />
<p class="texte">The average number of colonies with the negative control is 121. On the contrary, a cell layer is observed for the NAG plates with every concentration.<br><br />
<br><br />
Thus, we assumed that WT <i>Bacillus subtilis</i> was more attracted by NAG than fuchsin. Indeed we can neglect the bacterial growth because the test only lasts one hour. We also neglect diffusion and osmolality phenomena for the previous reasons. <br><br />
<br><br />
Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells which probably happened to our results.<br><br />
<br><br />
<b><p class="texte">This incredible discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><br><br />
<br><br />
However, our team did not give up on synthetic biology and on our strength! Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.<br><br />
<br><br />
We finally used galactose (25mM) as a negative control. The article Chemotaxis towards sugars by <i>Bacillus subtilis</i> (<i>George W. Ordal et al., 1979</i>) proved that it was a poor attractant.<br><br />
<br><br />
We made our tests again with this new molecule and glucose (25mM) as positive control.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/8/86/Chemotaxis_-_final_results.png"></center><br />
<p class="legend">Figure 10 : Final results (dilution : 1/10,000)</p><br />
<br />
<p class="texte"><i>NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.</i><br><br />
<br><br />
<b><p class="texte">Our results are not statistically significant however this result has been proved in literature.</p></b><br></p><br />
<br />
</br><br />
<br />
<p class="title1">Binding module</p> <br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Purpose</p><br />
<p class="texte">The first experiment deals with the culture conditions to see if <i>Bacillus subtilis</i> can resist to a low temperature and with the CBB buffer. To do that, several bacterial concentrations have been tested starting with an OD of 0.1 and diluting this solution to get estimated ODs of 0.05, 0.025, 0.01. These different <i>Bacillus subtilis</i> solutions were incubated 1 hour at 4°C with 500µL of CBB or water. Finally a cell count on Thoma cell counting chamber was performed.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The bacterial solutions could not be counted because of two main problems: the too high number of bacteria with the 0.1 OD or the too low number of bacteria with the 0.01 OD. Thus, the study is mostly focused on the intermediate values (Figure 1).<br />
<br/>First of all, a same cell concentration can be noticed with the presence of CBB or water with estimated ODs of 0.05 or 0.025. Moreover, twice less cells can be found in the lowest concentrations in bacteria comparing to the 0.05 OD concentration which is in agreement with the dilution ratio. <br />
<br/>Thus, the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C do not harm the cell surviving.<br />
</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/c/ce/Graphe_binding_1.png" width="45%"></center><br />
<br />
</br><br />
<p class="texte">Figure 1: CBB presence has no effect on bacteria. The bacterial concentration was measured regarding <span style="color:#0000FF">the presence</span> or <span style="color:#FF0000">the absence </span>of CBB for the observed OD (0.1) or estimated ODs (0.05, 0.025, 0.01).<br />
</p><br />
<br />
<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">Transformed <i>Bacillus subtilis</i> with the binding module is able to produce a protein composed of the bacterial peptidoglycan bonding of LycT and the GbpA 4th domain of <i>Vibrio cholerae</i> allowing the chitin bonding. The synthetic bacterium is put with special beads composed of the polymer miming the fungal pathogen wall. After several washes, bacteria specifically attached to the chitin are put on plates and counted.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The first observation is that both bacterial solutions of wild type <i>Bacillus subtilis</i> and SubtiTree have the same concentration : 105 bacteria/mL (Figure 2). Even though there is no significant difference between both strains after the first wash, the second wash has a major effect since it allows 40 times more Wild Type bacteria to come off the beads. This result correlates with the number of bacteria binded to the beads for the synthetic strain with the binding module. <br />
<br/>Thus, the binding system seems to function correctly and leads to the bacterial attachment on the chitin.</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ea/Graphe_binding_2.png" width="60%"></center><br />
</br><br />
<br />
<p class="texte">Figure 2: Attachment of <i>Bacillus subtilis</i> with binding module to chitin. <span style="color:#0000FF">The WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentration has been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p < 0.05.</p><br />
<br />
<p class="title2">3. Microscopic observations</p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">We want to observe the SubtiTree's binding on beads coated with chitin. In order to perform a 3D reconstruction showing this interaction, we use confocal laser scanning microscope. Through the use of a fluorochrome (Syto9), we can highlight the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determine the minimum threshold to remove the background noise and the natural fluorescence.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">First, we note the great bacterial presence on the surface of beads coated with chitin. These images seem to highlight their interactions.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/archive/5/53/20141013073044!Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="texte">Figure ***: <br />
</p><br />
<br />
<p class="texte">Using ImageJ software, we are able to create 3D pictures and movies of those comments. </br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/53/Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="texte">Figure ***: <br />
</p><br />
<br />
<p class="texte">Finally we want to observe the bacteria after the second wash. When our bacterium has the binding module, results suggest a lower number of bacteria in the washing solution. SubtiTree is retained by the beads.</br><br />
Finally, overall results are consistent with the presence of functional binding system.</p><br />
<br />
<br />
<p class="title1">Fungicides module<br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Tests with commercial peptides and controls</p><br />
<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12,5µM/25µM/100µM). These tests were performed on different fungal strains sharing the same phylum with <i>Ceratocystis Platani</i>.<br />
As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus.</br><br />
After several days at 30°C, the PDA (Potato Dextrose Agar) plates couvered with fungus and commercial peptides were analyzed.</p></p><br />
<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i>. Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions.As positive control, a well-known chemical fungicide was used: the Copper Sulfate. The inhibition of the fungal growth was complete at 20mg/ml, and at 10mg/ml a darker halo appeared around the pad filled with Copper Sulfate as we can see on the figure below. This corresponds to a sporing halo in response to the stress generated by the fungicide.<br />
</p><br />
<br />
</br><br />
<br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/a/a8/Prelim_tests_fung.jpg"><br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/f/f8/Controls_fung.jpg"><br />
<br />
<br />
</br><br />
<br />
<p class="texte">Given these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth. Following these tests, new conditions were adopted in order not to encourage too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees: a 'sap-like' medium was elaborated. The incubations were then carried at room temperature.<br />
</p><br />
<br />
<p class="title2">2. Test with SubtiTree<p/><br />
<br />
<p class="texte">In order to test <i>Bacillus subtilis</i> mutants, it was essential to find the right balance between the fungal growth and the bacterial one. This condition was necessary to get a high concentration of peptides. In our genetic constructions, these peptides are designed to be exported in the extracellular medium.</br><br />
</br><br />
The transformed <i>Bacillus subtilis</i> strains were grown at 37°C during 72h and tested. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of <i>Trichoderma reesei</i>'s growth was clearly observable for the strain expressing D4E1 gene. The inhibition was even more noticeable with the strain carrying the operon GAFP-1 + D4E1 (see the photos below).</br><br />
However, no effect was detected for the strain expressing the GAFP-1 gene, supposing a synergistic effect between these two peptides.</br><br />
Regarding EcAMP and the triple-fungicides operon, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP and in addition to that, sequencing results of these constructs showed some differences with the original designed sequence.<br />
<p class="texte">Inhibition halos are not visible with supernatants, probably because of their low concentrations in the extracellular medium. <br />
Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus <i>Aspergillus brasiliansis</i>. This effect is comparable to the one previously noted with low concentration of sulfate copper. </br><br />
</p><br />
<br />
</br><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/c/c2/Resultfong.jpg"> Figure X<br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/9/92/Results_fong_2.jpg"> Figure X<br />
</br><br />
<br />
<p class="texte"><br />
The choice of our chassis appears to be optimal as we noted that wild type <i>Bacillus subtilis</i> disturbs the hyphae growth of the fungi. Some strains of <i>Bacillus subtilis</i> (qst 713) are already used as Biofungicides for use on several minor crops to treat a variety of plant diseases and fungal pathogens.</br><br />
After this set of experiments, the strains expressing D4E1 and expressing GAFP-1 + D4E1 have shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b> we decided to tests these strains in model plants </b> : <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br> These tests were performed in the National Institute for the Agronomic Research by experts in this domain. <br />
<br />
<br />
<p class="title2">3. <i> In planta </i> tests with SubtiTree<p/><br />
<br />
<div style="float:left; width:215px;"><br />
<img style="width:215px;" img src="https://static.igem.org/mediawiki/parts/a/af/In_planta.jpg" style="margin-top:5px" /><br />
</div> <br />
<div style="float: right; width:700px; margin-left:44px;"> <br />
<p class="texte"><br />
The goal of the project is to introduce the trasnformed bacteria in a diseased tree. So it is necessary to perform <i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous set of experiments. </br><br />
SubtiTree is first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) is placed on the leaves. </br><br />
These tests were made in association with Sylvain Raffaële and Marielle Barascud of the National Institute for the Agronomic Research laboratory. </br><br />
</p> </div> <br />
<br />
<br />
<br />
<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves can be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants don't have deleterious effects.</br><br />
Without proper treatment, the drop of the pyhtopathogenic fungus on <i>Nicotiana benthamiana</i>'s leaves causes a necrosis halo which can be measured after 40h. The lesion size and the number of inoculated sites seem reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike with the WT bacterium. A second set of experiments is expected to be more statistically precise.</br><br />
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br><br />
<br />
We can therefore conclude that when SubtiTree is in plant physiological conditions, <b> it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves' necrosis </b>.<br />
</p><br />
<br />
TABLEAU DE RESULTAT<br />
<br />
<p class="texte"><br />
Thanks to the diversity of anti-fungal peptides, this strategy can be adapted to different types of diseases, with different degree of specifity, etc.<br />
</p><br />
<br />
<br />
<br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="" class="page-nav-right" style="min-width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;"><br />
<br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Result/parts" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Parts</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Result/experimental-resultsTeam:Toulouse/Result/experimental-results2014-10-13T15:11:30Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#5a6060; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify; }<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify; }<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/f/f7/Results_photo_tlse.png') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
p.legend{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: center;}<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Experimental results</h2><br />
<p> Are our modules functionnal? </p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Experimental results</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<p class="title1">Chemotaxis<br />
</p><br />
<p class="texte">For this module, we had to try several tests to prove the existence of chemotaxis in <i>Bacillus subtilis</i> wild type (WT) strain and SubtiTree bacterium towards N-Acetylglucosamine.<br />
</p><br />
<br />
<p class="title2">1. Plug in Pond system<br />
</p><br />
<p class="texte">Coming soon! <!--Florie--><br />
</p><br />
<br />
<p class="title2">2. Capillary test between two tubes also called the tubes test<br />
</p><br />
<p class="texte">After the experiment of the plug in pond, we decided to construct a system by welding two Eppendorf tubes with a capillary thanks to an electric burner.</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/f/fb/Chemotaxis_-_eppendorf.png"></center><br />
<p class="legend">Figure 1 : Photography of the first tubes system</p><br />
<br />
<p class="texte">We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin towards water. However this construction had a leakage next to the weld seam that we could not stop. <br />
Thus, the Toulouse iGEM Team asked the help from the glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/Chemotaxis_-_tubes.png"><center><br />
<p class="legend">Figure 2 : Scheme of the tubes system</p><br />
<br />
<p class="texte">As we did previously, we tested this new system with fuchsin. This experiment was made with WT <i>Bacillus subtilis</i> and N-Acetylglucosamine.<br />
<br><br><br />
<i>NB: We could not see the diffusion from one tube to the other. We made the hypothesis that it was not visible by sight because of by the small diameter of the capillary. <br />
</i><br><br />
<br><br />
The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:<br><br />
- The first step was the addition of Wash Buffer until the capillary was full to avoid the presence of air bubbles which could lead to diffusion problems.<br><br />
- Then, the tube 2 was plugged with the thumb while another person was adding the bacteria solution of WT Bacillus subtilis in the tube 1. <br><br />
- The tube 1 was also plugged and only after the thumb could be removed of the tube 2. <br><br />
- In the same way, the N-Acetylglucosamine was added in the tube 2. <br><br />
- The same process was made with a xylose positive control.<br><br />
<br><br />
<i>NB: According to the article Chemotaxis towards sugars by </i>Bacillus subtilis, (George W. Ordal et al., 1979), <i>glucose and xylose have the same attractant power. We prefer a positive control instead of a negative because we were not sure that this system was efficient.</i><br><br />
<br><br />
- The system was kept straight for 2hours. Every 40 minutes, we took a sample of each tube and spread it on an agar plate (dilution 1/1,000).</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/1/1b/Chemotaxis_-_tubes_photo.png"></center><br />
<p class="legend">Figure 3 : Photography of the tubes system</p><br />
<br />
<br />
<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement and the time was too short. We did not find any information in the literature.<br><br />
As we did not have the time to optimize this protocol we preferred using the protocol of the Imperial college iGEM team 2011: the tips capillary test.<br><br />
</p><br />
<br />
<p class="title2">3. Tips capillary system</p><br />
<p class="title3">First tips capillary system</p><br />
<p class="texte">This protocol comes from Imperial College iGEM team 2011 and was adapted by our team in several steps (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols">chemotaxis protocol</a>).<br><br />
<br><br />
First of all, parafilm was used to close the tips:<br><br />
- 15µL of each chemo-attractant was then pipetted. <br><br />
- The tips with the pipette were then put on a piece of parafilm and the pipette was removed from the tip.<br><br />
- The tip was sealed with a piece of parafilm. By this way, the sterility can be assured and the liquid stays inside the tip. <br><br />
- To finish, the level of the solution in the tip was marked.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/9/94/Chemotaxis_-_tip.png"></center><br />
<p class="legend">Figure 4 : Sealing of a tip with parafilm</p><br />
<br />
<p class="texte">- After all the chemo-attractants were added in the tips, we put them on a green base to carry them. The whole process can be seen on Figure 5.<br><br />
- Each tip was put in 300 µL of a bacteria solution in the wells of an Elisa plate.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/0/05/Chemotaxis_-_tip_and_support.png"></center><br />
<p class="legend">Figure 5 : First tips capillary system</p><br />
<br />
<p class="texte"><i>NB: the yellow carton was used to stabilize the system and keep it straight.</i><br><br />
<br><br />
- After one hour, the tips were removed from the bacteria solutions and the content of the tips was observed with Thoma cell under the microscope.<br><br />
<br><br />
We had several problems with this system:<br><br />
- The liquid level decreased during the experiment and we did not have enough liquid to fill the Thoma cell. Thus, it was not possible to count.<br><br />
- The bacteria were moving and therefore, we could not proceed to a bacteria count.<br><br />
<br><br />
Regarding these observations we decided to spread the tips content on agar plate instead of using Thoma cell and microscopy.<br><br />
<p class="title3">Second tips capillary system<br />
</p><br />
<p class="texte"And then the revolution came! We found a multichannel pipette. The same protocol was performed except that the parafilm was used to avoid the air entrance between the tips and the pipette and therefore the loss of liquid.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/e/e4/Chemotaxis_-_pipette.png"></center><br />
<p class="legend">Figure 6 : Second tips capillary system</p><br />
<br />
<p class="title3">Improvement of the second tips capillary system</p><br />
<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></center><br />
<p class="legend">Figure 7 : Improvement of the second tips capillary system</p><br />
<br />
<p class="texte"><b>At that point, the protocol was approved and the final test could finally start! :-)</b><br><br />
<br><br />
There was just one tiny problem… we did not have our optimized bacterium with the chemotaxis gene… That is why we concentrated our efforts on WT Bacillus subtilis strain.<br><br />
<br><br />
The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolarity bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br><br />
<br><br />
The experiment was conducted with fuchsin as a negative control and was tested with different positive controls: glucose (25mM) and xylose (25mM).<br><br />
<br><br />
We obtained the following result with NAG at different concentrations: 25mM, 250mM and 500mM. The tested strain was Bacillus subtilis 168:<br><br />
<br></p><br />
<center><br />
<table align="center"><br />
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/8/8c/Chemotaxis_-_results_fuch.png"></td><br />
<td align=center><img src="https://static.igem.org/mediawiki/2014/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr><br />
<tr><td align=center><p class="legend">Figure 8 : Fuchsin - negative control (dilution 1/50)</p></td><br />
<td align=center><p class="legend">Figure 9 : NAG (25mM) (dilution 1/50)</p></td></tr><br />
</table></center><br><br />
<p class="texte">The average number of colonies with the negative control is 121. On the contrary, a cell layer is observed for the NAG plates with every concentration.<br><br />
<br><br />
Thus, we assumed that WT <i>Bacillus subtilis</i> was more attracted by NAG than fuchsin. Indeed we can neglect the bacterial growth because the test only lasts one hour. We also neglect diffusion and osmolality phenomena for the previous reasons. <br><br />
<br><br />
Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells which probably happened to our results.<br><br />
<br><br />
<b><p class="texte">This incredible discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><br><br />
<br><br />
However, our team did not give up on synthetic biology and on our strength! Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.<br><br />
<br><br />
We finally used galactose (25mM) as a negative control. The article Chemotaxis towards sugars by <i>Bacillus subtilis</i> (<i>George W. Ordal et al., 1979</i>) proved that it was a poor attractant.<br><br />
<br><br />
We made our tests again with this new molecule and glucose (25mM) as positive control.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/8/86/Chemotaxis_-_final_results.png"></center><br />
<p class="legend">Figure 10 : Final results (dilution : 1/10,000)</p><br />
<br />
<p class="texte"><i>NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.</i><br><br />
<br><br />
<b><p class="texte">Our results are not statistically significant however this result has been proved in literature.</p></b><br></p><br />
<br />
</br><br />
<br />
<p class="title1">Binding module</p> <br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Purpose</p><br />
<p class="texte">The first experiment deals with the culture conditions to see if <i>Bacillus subtilis</i> can resist to a low temperature and with the CBB buffer. To do that, several bacterial concentrations have been tested starting with an OD of 0.1 and diluting this solution to get estimated ODs of 0.05, 0.025, 0.01. These different <i>Bacillus subtilis</i> solutions were incubated 1 hour at 4°C with 500µL of CBB or water. Finally a cell count on Thoma cell counting chamber was performed.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The bacterial solutions could not be counted because of two main problems: the too high number of bacteria with the 0.1 OD or the too low number of bacteria with the 0.01 OD. Thus, the study is mostly focused on the intermediate values (Figure 1).<br />
<br/>First of all, a same cell concentration can be noticed with the presence of CBB or water with estimated ODs of 0.05 or 0.025. Moreover, twice less cells can be found in the lowest concentrations in bacteria comparing to the 0.05 OD concentration which is in agreement with the dilution ratio. <br />
<br/>Thus, the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C do not harm the cell surviving.<br />
</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/c/ce/Graphe_binding_1.png" width="45%"></center><br />
<br />
</br><br />
<p class="texte">Figure 1: CBB presence has no effect on bacteria. The bacterial concentration was measured regarding <span style="color:#0000FF">the presence</span> or <span style="color:#FF0000">the absence </span>of CBB for the observed OD (0.1) or estimated ODs (0.05, 0.025, 0.01).<br />
</p><br />
<br />
<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">Transformed <i>Bacillus subtilis</i> with the binding module is able to produce a protein composed of the bacterial peptidoglycan bonding of LycT and the GbpA 4th domain of <i>Vibrio cholerae</i> allowing the chitin bonding. The synthetic bacterium is put with special beads composed of the polymer miming the fungal pathogen wall. After several washes, bacteria specifically attached to the chitin are put on plates and counted.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The first observation is that both bacterial solutions of wild type <i>Bacillus subtilis</i> and SubtiTree have the same concentration : 105 bacteria/mL (Figure 2). Even though there is no significant difference between both strains after the first wash, the second wash has a major effect since it allows 40 times more Wild Type bacteria to come off the beads. This result correlates with the number of bacteria binded to the beads for the synthetic strain with the binding module. <br />
<br/>Thus, the binding system seems to function correctly and leads to the bacterial attachment on the chitin.</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ea/Graphe_binding_2.png" width="60%"></center><br />
</br><br />
<br />
<p class="texte">Figure 2: Attachment of <i>Bacillus subtilis</i> with binding module to chitin. <span style="color:#0000FF">The WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentration has been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p < 0.05.</p><br />
<br />
<p class="title2">3. Microscopic observations</p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">We want to observe the SubtiTree's binding on beads coated with chitin. In order to perform a 3D reconstruction showing this interaction, we use confocal laser scanning microscope. Through the use of a fluorochrome (Syto9), we can highlight the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determine the minimum threshold to remove the background noise and the natural fluorescence.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">First, we note the great bacterial presence on the surface of beads coated with chitin. These images seem to highlight their interactions.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/archive/5/53/20141013073044!Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="texte">Figure ***: <br />
</p><br />
<br />
<p class="texte">Using ImageJ software, we are able to create 3D pictures and movies of those comments. </br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/53/Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="texte">Figure ***: <br />
</p><br />
<br />
<p class="texte">Finally we want to observe the bacteria after the second wash. When our bacterium has the binding module, results suggest a lower number of bacteria in the washing solution. SubtiTree is retained by the beads.</br><br />
Finally, overall results are consistent with the presence of functional binding system.</p><br />
<br />
<br />
<p class="title1">Fungicides module<br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Tests with commercial peptides and controls</p><br />
<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12,5µM/25µM/100µM). These tests were performed on different fungal strains sharing the same phylum with <i>Ceratocystis Platani</i>.<br />
As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus.</br><br />
After several days at 30°C, the PDA (Potato Dextrose Agar) plates couvered with fungus and commercial peptides were analyzed.</p></p><br />
<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i>. Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions.As positive control, a well-known chemical fungicide was used: the Copper Sulfate. The inhibition of the fungal growth was complete at 20mg/ml, and at 10mg/ml a darker halo appeared around the pad filled with Copper Sulfate as we can see on the figure below. This corresponds to a sporing halo in response to the stress generated by the fungicide.<br />
</p><br />
<br />
</br><br />
<br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/a/a8/Prelim_tests_fung.jpg"><br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/f/f8/Controls_fung.jpg"><br />
<br />
<br />
</br><br />
<br />
<p class="texte">Given these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth. Following these tests, new conditions were adopted in order not to encourage too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees: a 'sap-like' medium was elaborated. The incubations were then carried at room temperature.<br />
</p><br />
<br />
<p class="title2">2. Test with SubtiTree<p/><br />
<br />
<p class="texte">In order to test <i>Bacillus subtilis</i> mutants, it was essential to find the right balance between the fungal growth and the bacterial one. This condition was necessary to get a high concentration of peptides. In our genetic constructions, these peptides are designed to be exported in the extracellular medium.</br><br />
</br><br />
The transformed <i>Bacillus subtilis</i> strains were grown at 37°C during 72h and tested. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of <i>Trichoderma reesei</i>'s growth was clearly observable for the strain expressing D4E1 gene. The inhibition was even more noticeable with the strain carrying the operon GAFP-1 + D4E1 (see the photos below).</br><br />
However, no effect was detected for the strain expressing the GAFP-1 gene, supposing a synergistic effect between these two peptides.</br><br />
Regarding EcAMP and the triple-fungicides operon, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP and in addition to that, sequencing results of these constructs showed some differences with the original designed sequence.<br />
<p class="texte">Inhibition halos are not visible with supernatants, probably because of their low concentrations in the extracellular medium. <br />
Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus <i>Aspergillus brasiliansis</i>. This effect is comparable to the one previously noted with low concentration of sulfate copper. </br><br />
</p><br />
<br />
</br><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/c/c2/Resultfong.jpg"> Figure X<br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/9/92/Results_fong_2.jpg"> Figure X<br />
</br><br />
<br />
<p class="texte"><br />
The choice of our chassis appears to be optimal as we noted that wild type <i>Bacillus subtilis</i> disturbs the hyphae growth of the fungi. Some strains of <i>Bacillus subtilis</i> (qst 713) are already used as Biofungicides for use on several minor crops to treat a variety of plant diseases and fungal pathogens.</br><br />
After this set of experiments, the strains expressing D4E1 and expressing GAFP-1 + D4E1 have shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b> we decided to tests these strains in model plants </b> : <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br> These tests were performed in the National Institute for the Agronomic Research by experts in this domain. <br />
<br />
<br />
<p class="title2">3. <i> In planta </i> tests with SubtiTree<p/><br />
<br />
<div style="float:left; width:215px;"><br />
<img style="width:215px;" img src="https://static.igem.org/mediawiki/parts/a/af/In_planta.jpg" style="margin-top:5px" /><br />
</div> <br />
<div style="float: right; width:700px; margin-left:44px;"> <br />
<p class="texte"><br />
The goal of the project is to introduce the trasnformed bacteria in a diseased tree. So it is necessary to perform <i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous set of experiments. </br><br />
SubtiTree is first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) is placed on the leaves. </br><br />
These tests were made in association with Sylvain Raffaële and Marielle Barascud of the National Institute for the Agronomic Research laboratory. </br><br />
</p> </div> <br />
<br />
<br />
<br />
<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves can be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants don't have deleterious effects.</br><br />
Without proper treatment, the drop of the pyhtopathogenic fungus on <i>Nicotiana benthamiana</i>'s leaves causes a necrosis halo which can be measured after 40h. The lesion size and the number of inoculated sites seem reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike with the WT bacterium. A second set of experiments is expected to be more statistically precise.</br><br />
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br><br />
<br />
We can therefore conclude that when SubtiTree is in plant physiological conditions, <b> it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves' necrosis </b>.<br />
</p><br />
<br />
TABLEAU DE RESULTAT<br />
<br />
<p class="texte"><br />
Thanks to the diversity of anti-fungal peptides, this strategy can be adapted to different types of diseases, with different degree of specifity, etc.<br />
</p><br />
<br />
<br />
<br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="" class="page-nav-right" style="min-width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;"><br />
<br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Result/parts" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Parts</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Result/experimental-resultsTeam:Toulouse/Result/experimental-results2014-10-13T15:10:59Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#5a6060; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify; }<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify; }<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/f/f7/Results_photo_tlse.png') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
p.legend{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: center;}<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Experimental results</h2><br />
<p> Are our modules functionnal? </p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Experimental results</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<p class="title1">Chemotaxis<br />
</p><br />
<p class="texte">For this module, we had to try several tests to prove the existence of chemotaxis in <i>Bacillus subtilis</i> wild type (WT) strain and SubtiTree bacterium towards N-Acetylglucosamine.<br />
</p><br />
<br />
<p class="title2">1. Plug in Pond system<br />
</p><br />
<p class="texte">Coming soon! <!--Florie--><br />
</p><br />
<br />
<p class="title2">2. Capillary test between two tubes also called the tubes test<br />
</p><br />
<p class="texte">After the experiment of the plug in pond, we decided to construct a system by welding two Eppendorf tubes with a capillary thanks to an electric burner.</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/f/fb/Chemotaxis_-_eppendorf.png"></center><br />
<p class="legend">Figure 1 : Photography of the first tubes system</p><br />
<br />
<p class="texte">We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin towards water. However this construction had a leakage next to the weld seam that we could not stop. <br />
Thus, the Toulouse iGEM Team asked the help from the glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/Chemotaxis_-_tubes.png"><center><br />
<p class="legend">Figure 2 : Scheme of the tubes system</p><br />
<br />
<p class="texte">As we did previously, we tested this new system with fuchsin. This experiment was made with WT <i>Bacillus subtilis</i> and N-Acetylglucosamine.<br />
<br><br><br />
<i>NB: We could not see the diffusion from one tube to the other. We made the hypothesis that it was not visible by sight because of by the small diameter of the capillary. <br />
</i><br><br />
<br><br />
The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:<br><br />
- The first step was the addition of Wash Buffer until the capillary was full to avoid the presence of air bubbles which could lead to diffusion problems.<br><br />
- Then, the tube 2 was plugged with the thumb while another person was adding the bacteria solution of WT Bacillus subtilis in the tube 1. <br><br />
- The tube 1 was also plugged and only after the thumb could be removed of the tube 2. <br><br />
- In the same way, the N-Acetylglucosamine was added in the tube 2. <br><br />
- The same process was made with a xylose positive control.<br><br />
<br><br />
<i>NB: According to the article Chemotaxis towards sugars by </i>Bacillus subtilis, (<i>George W. Ordal et al., 1979</i>), <i>glucose and xylose have the same attractant power. We prefer a positive control instead of a negative because we were not sure that this system was efficient.</i><br><br />
<br><br />
- The system was kept straight for 2hours. Every 40 minutes, we took a sample of each tube and spread it on an agar plate (dilution 1/1,000).</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/1/1b/Chemotaxis_-_tubes_photo.png"></center><br />
<p class="legend">Figure 3 : Photography of the tubes system</p><br />
<br />
<br />
<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement and the time was too short. We did not find any information in the literature.<br><br />
As we did not have the time to optimize this protocol we preferred using the protocol of the Imperial college iGEM team 2011: the tips capillary test.<br><br />
</p><br />
<br />
<p class="title2">3. Tips capillary system</p><br />
<p class="title3">First tips capillary system</p><br />
<p class="texte">This protocol comes from Imperial College iGEM team 2011 and was adapted by our team in several steps (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols">chemotaxis protocol</a>).<br><br />
<br><br />
First of all, parafilm was used to close the tips:<br><br />
- 15µL of each chemo-attractant was then pipetted. <br><br />
- The tips with the pipette were then put on a piece of parafilm and the pipette was removed from the tip.<br><br />
- The tip was sealed with a piece of parafilm. By this way, the sterility can be assured and the liquid stays inside the tip. <br><br />
- To finish, the level of the solution in the tip was marked.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/9/94/Chemotaxis_-_tip.png"></center><br />
<p class="legend">Figure 4 : Sealing of a tip with parafilm</p><br />
<br />
<p class="texte">- After all the chemo-attractants were added in the tips, we put them on a green base to carry them. The whole process can be seen on Figure 5.<br><br />
- Each tip was put in 300 µL of a bacteria solution in the wells of an Elisa plate.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/0/05/Chemotaxis_-_tip_and_support.png"></center><br />
<p class="legend">Figure 5 : First tips capillary system</p><br />
<br />
<p class="texte"><i>NB: the yellow carton was used to stabilize the system and keep it straight.</i><br><br />
<br><br />
- After one hour, the tips were removed from the bacteria solutions and the content of the tips was observed with Thoma cell under the microscope.<br><br />
<br><br />
We had several problems with this system:<br><br />
- The liquid level decreased during the experiment and we did not have enough liquid to fill the Thoma cell. Thus, it was not possible to count.<br><br />
- The bacteria were moving and therefore, we could not proceed to a bacteria count.<br><br />
<br><br />
Regarding these observations we decided to spread the tips content on agar plate instead of using Thoma cell and microscopy.<br><br />
<p class="title3">Second tips capillary system<br />
</p><br />
<p class="texte"And then the revolution came! We found a multichannel pipette. The same protocol was performed except that the parafilm was used to avoid the air entrance between the tips and the pipette and therefore the loss of liquid.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/e/e4/Chemotaxis_-_pipette.png"></center><br />
<p class="legend">Figure 6 : Second tips capillary system</p><br />
<br />
<p class="title3">Improvement of the second tips capillary system</p><br />
<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></center><br />
<p class="legend">Figure 7 : Improvement of the second tips capillary system</p><br />
<br />
<p class="texte"><b>At that point, the protocol was approved and the final test could finally start! :-)</b><br><br />
<br><br />
There was just one tiny problem… we did not have our optimized bacterium with the chemotaxis gene… That is why we concentrated our efforts on WT Bacillus subtilis strain.<br><br />
<br><br />
The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolarity bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br><br />
<br><br />
The experiment was conducted with fuchsin as a negative control and was tested with different positive controls: glucose (25mM) and xylose (25mM).<br><br />
<br><br />
We obtained the following result with NAG at different concentrations: 25mM, 250mM and 500mM. The tested strain was Bacillus subtilis 168:<br><br />
<br></p><br />
<center><br />
<table align="center"><br />
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/8/8c/Chemotaxis_-_results_fuch.png"></td><br />
<td align=center><img src="https://static.igem.org/mediawiki/2014/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr><br />
<tr><td align=center><p class="legend">Figure 8 : Fuchsin - negative control (dilution 1/50)</p></td><br />
<td align=center><p class="legend">Figure 9 : NAG (25mM) (dilution 1/50)</p></td></tr><br />
</table></center><br><br />
<p class="texte">The average number of colonies with the negative control is 121. On the contrary, a cell layer is observed for the NAG plates with every concentration.<br><br />
<br><br />
Thus, we assumed that WT <i>Bacillus subtilis</i> was more attracted by NAG than fuchsin. Indeed we can neglect the bacterial growth because the test only lasts one hour. We also neglect diffusion and osmolality phenomena for the previous reasons. <br><br />
<br><br />
Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells which probably happened to our results.<br><br />
<br><br />
<b><p class="texte">This incredible discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><br><br />
<br><br />
However, our team did not give up on synthetic biology and on our strength! Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.<br><br />
<br><br />
We finally used galactose (25mM) as a negative control. The article Chemotaxis towards sugars by <i>Bacillus subtilis</i> (<i>George W. Ordal et al., 1979</i>) proved that it was a poor attractant.<br><br />
<br><br />
We made our tests again with this new molecule and glucose (25mM) as positive control.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/8/86/Chemotaxis_-_final_results.png"></center><br />
<p class="legend">Figure 10 : Final results (dilution : 1/10,000)</p><br />
<br />
<p class="texte"><i>NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.</i><br><br />
<br><br />
<b><p class="texte">Our results are not statistically significant however this result has been proved in literature.</p></b><br></p><br />
<br />
</br><br />
<br />
<p class="title1">Binding module</p> <br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Purpose</p><br />
<p class="texte">The first experiment deals with the culture conditions to see if <i>Bacillus subtilis</i> can resist to a low temperature and with the CBB buffer. To do that, several bacterial concentrations have been tested starting with an OD of 0.1 and diluting this solution to get estimated ODs of 0.05, 0.025, 0.01. These different <i>Bacillus subtilis</i> solutions were incubated 1 hour at 4°C with 500µL of CBB or water. Finally a cell count on Thoma cell counting chamber was performed.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The bacterial solutions could not be counted because of two main problems: the too high number of bacteria with the 0.1 OD or the too low number of bacteria with the 0.01 OD. Thus, the study is mostly focused on the intermediate values (Figure 1).<br />
<br/>First of all, a same cell concentration can be noticed with the presence of CBB or water with estimated ODs of 0.05 or 0.025. Moreover, twice less cells can be found in the lowest concentrations in bacteria comparing to the 0.05 OD concentration which is in agreement with the dilution ratio. <br />
<br/>Thus, the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C do not harm the cell surviving.<br />
</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/c/ce/Graphe_binding_1.png" width="45%"></center><br />
<br />
</br><br />
<p class="texte">Figure 1: CBB presence has no effect on bacteria. The bacterial concentration was measured regarding <span style="color:#0000FF">the presence</span> or <span style="color:#FF0000">the absence </span>of CBB for the observed OD (0.1) or estimated ODs (0.05, 0.025, 0.01).<br />
</p><br />
<br />
<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">Transformed <i>Bacillus subtilis</i> with the binding module is able to produce a protein composed of the bacterial peptidoglycan bonding of LycT and the GbpA 4th domain of <i>Vibrio cholerae</i> allowing the chitin bonding. The synthetic bacterium is put with special beads composed of the polymer miming the fungal pathogen wall. After several washes, bacteria specifically attached to the chitin are put on plates and counted.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The first observation is that both bacterial solutions of wild type <i>Bacillus subtilis</i> and SubtiTree have the same concentration : 105 bacteria/mL (Figure 2). Even though there is no significant difference between both strains after the first wash, the second wash has a major effect since it allows 40 times more Wild Type bacteria to come off the beads. This result correlates with the number of bacteria binded to the beads for the synthetic strain with the binding module. <br />
<br/>Thus, the binding system seems to function correctly and leads to the bacterial attachment on the chitin.</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ea/Graphe_binding_2.png" width="60%"></center><br />
</br><br />
<br />
<p class="texte">Figure 2: Attachment of <i>Bacillus subtilis</i> with binding module to chitin. <span style="color:#0000FF">The WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentration has been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p < 0.05.</p><br />
<br />
<p class="title2">3. Microscopic observations</p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">We want to observe the SubtiTree's binding on beads coated with chitin. In order to perform a 3D reconstruction showing this interaction, we use confocal laser scanning microscope. Through the use of a fluorochrome (Syto9), we can highlight the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determine the minimum threshold to remove the background noise and the natural fluorescence.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">First, we note the great bacterial presence on the surface of beads coated with chitin. These images seem to highlight their interactions.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/archive/5/53/20141013073044!Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="texte">Figure ***: <br />
</p><br />
<br />
<p class="texte">Using ImageJ software, we are able to create 3D pictures and movies of those comments. </br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/53/Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="texte">Figure ***: <br />
</p><br />
<br />
<p class="texte">Finally we want to observe the bacteria after the second wash. When our bacterium has the binding module, results suggest a lower number of bacteria in the washing solution. SubtiTree is retained by the beads.</br><br />
Finally, overall results are consistent with the presence of functional binding system.</p><br />
<br />
<br />
<p class="title1">Fungicides module<br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Tests with commercial peptides and controls</p><br />
<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12,5µM/25µM/100µM). These tests were performed on different fungal strains sharing the same phylum with <i>Ceratocystis Platani</i>.<br />
As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus.</br><br />
After several days at 30°C, the PDA (Potato Dextrose Agar) plates couvered with fungus and commercial peptides were analyzed.</p></p><br />
<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i>. Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions.As positive control, a well-known chemical fungicide was used: the Copper Sulfate. The inhibition of the fungal growth was complete at 20mg/ml, and at 10mg/ml a darker halo appeared around the pad filled with Copper Sulfate as we can see on the figure below. This corresponds to a sporing halo in response to the stress generated by the fungicide.<br />
</p><br />
<br />
</br><br />
<br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/a/a8/Prelim_tests_fung.jpg"><br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/f/f8/Controls_fung.jpg"><br />
<br />
<br />
</br><br />
<br />
<p class="texte">Given these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth. Following these tests, new conditions were adopted in order not to encourage too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees: a 'sap-like' medium was elaborated. The incubations were then carried at room temperature.<br />
</p><br />
<br />
<p class="title2">2. Test with SubtiTree<p/><br />
<br />
<p class="texte">In order to test <i>Bacillus subtilis</i> mutants, it was essential to find the right balance between the fungal growth and the bacterial one. This condition was necessary to get a high concentration of peptides. In our genetic constructions, these peptides are designed to be exported in the extracellular medium.</br><br />
</br><br />
The transformed <i>Bacillus subtilis</i> strains were grown at 37°C during 72h and tested. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of <i>Trichoderma reesei</i>'s growth was clearly observable for the strain expressing D4E1 gene. The inhibition was even more noticeable with the strain carrying the operon GAFP-1 + D4E1 (see the photos below).</br><br />
However, no effect was detected for the strain expressing the GAFP-1 gene, supposing a synergistic effect between these two peptides.</br><br />
Regarding EcAMP and the triple-fungicides operon, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP and in addition to that, sequencing results of these constructs showed some differences with the original designed sequence.<br />
<p class="texte">Inhibition halos are not visible with supernatants, probably because of their low concentrations in the extracellular medium. <br />
Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus <i>Aspergillus brasiliansis</i>. This effect is comparable to the one previously noted with low concentration of sulfate copper. </br><br />
</p><br />
<br />
</br><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/c/c2/Resultfong.jpg"> Figure X<br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/9/92/Results_fong_2.jpg"> Figure X<br />
</br><br />
<br />
<p class="texte"><br />
The choice of our chassis appears to be optimal as we noted that wild type <i>Bacillus subtilis</i> disturbs the hyphae growth of the fungi. Some strains of <i>Bacillus subtilis</i> (qst 713) are already used as Biofungicides for use on several minor crops to treat a variety of plant diseases and fungal pathogens.</br><br />
After this set of experiments, the strains expressing D4E1 and expressing GAFP-1 + D4E1 have shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b> we decided to tests these strains in model plants </b> : <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br> These tests were performed in the National Institute for the Agronomic Research by experts in this domain. <br />
<br />
<br />
<p class="title2">3. <i> In planta </i> tests with SubtiTree<p/><br />
<br />
<div style="float:left; width:215px;"><br />
<img style="width:215px;" img src="https://static.igem.org/mediawiki/parts/a/af/In_planta.jpg" style="margin-top:5px" /><br />
</div> <br />
<div style="float: right; width:700px; margin-left:44px;"> <br />
<p class="texte"><br />
The goal of the project is to introduce the trasnformed bacteria in a diseased tree. So it is necessary to perform <i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous set of experiments. </br><br />
SubtiTree is first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) is placed on the leaves. </br><br />
These tests were made in association with Sylvain Raffaële and Marielle Barascud of the National Institute for the Agronomic Research laboratory. </br><br />
</p> </div> <br />
<br />
<br />
<br />
<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves can be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants don't have deleterious effects.</br><br />
Without proper treatment, the drop of the pyhtopathogenic fungus on <i>Nicotiana benthamiana</i>'s leaves causes a necrosis halo which can be measured after 40h. The lesion size and the number of inoculated sites seem reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike with the WT bacterium. A second set of experiments is expected to be more statistically precise.</br><br />
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br><br />
<br />
We can therefore conclude that when SubtiTree is in plant physiological conditions, <b> it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves' necrosis </b>.<br />
</p><br />
<br />
TABLEAU DE RESULTAT<br />
<br />
<p class="texte"><br />
Thanks to the diversity of anti-fungal peptides, this strategy can be adapted to different types of diseases, with different degree of specifity, etc.<br />
</p><br />
<br />
<br />
<br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="" class="page-nav-right" style="min-width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;"><br />
<br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Result/parts" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Parts</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Result/experimental-resultsTeam:Toulouse/Result/experimental-results2014-10-13T15:08:44Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#5a6060; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify; }<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify; }<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/f/f7/Results_photo_tlse.png') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
p.legend{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: center;}<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Experimental results</h2><br />
<p> Are our modules functionnal? </p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Experimental results</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<p class="title1">Chemotaxis<br />
</p><br />
<p class="texte">For this module, we had to try several tests to prove the existence of chemotaxis in <i>Bacillus subtilis</i> wild type (WT) strain and SubtiTree bacterium towards N-Acetylglucosamine.<br />
</p><br />
<br />
<p class="title2">1. Plug in Pond system<br />
</p><br />
<p class="texte">Coming soon! <!--Florie--><br />
</p><br />
<br />
<p class="title2">2. Capillary test between two tubes also called the tubes test<br />
</p><br />
<p class="texte">After the experiment of the plug in pond, we decided to construct a system by welding two Eppendorf tubes with a capillary thanks to an electric burner.</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/f/fb/Chemotaxis_-_eppendorf.png"></center><br />
<p class="legend">Figure 1 : Photography of the first tubes system</p><br />
<br />
<p class="texte">We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin towards water. However this construction had a leakage next to the weld seam that we could not stop. <br />
Thus, the Toulouse iGEM Team asked the help from the glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/Chemotaxis_-_tubes.png"><center><br />
<p class="legend">Figure 2 : Scheme of the tubes system</p><br />
<br />
<p class="texte">As we did previously, we tested this new system with fuchsin. This experiment was made with WT <i>Bacillus subtilis</i> and N-Acetylglucosamine.<br />
<br><br><br />
<i>NB: We could not see the diffusion from one tube to the other. We made the hypothesis that it was not visible by sight because of by the small diameter of the capillary. <br />
</i><br><br />
<br><br />
The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:<br><br />
- The first step was the addition of Wash Buffer until the capillary was full to avoid the presence of air bubbles which could lead to diffusion problems.<br><br />
- Then, the tube 2 was plugged with the thumb while another person was adding the bacteria solution of WT Bacillus subtilis in the tube 1. <br><br />
- The tube 1 was also plugged and only after the thumb could be removed of the tube 2. <br><br />
- In the same way, the N-Acetylglucosamine was added in the tube 2. <br><br />
- The same process was made with a xylose positive control.<br><br />
<br><br />
<i>NB: According to the article Chemotaxis towards sugars by Bacillus subtilis, (<i>George W. Ordal et al., 1979</i>), glucose and xylose have the same attractant power. We prefer a positive control instead of a negative because we were not sure that this system was efficient.</i><br><br />
<br><br />
- The system was kept straight for 2hours. Every 40 minutes, we took a sample of each tube and spread it on an agar plate (dilution 1/1,000).</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/1/1b/Chemotaxis_-_tubes_photo.png"></center><br />
<p class="legend">Figure 3 : Photography of the tubes system</p><br />
<br />
<br />
<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement and the time was too short. We did not find any information in the literature.<br><br />
As we did not have the time to optimize this protocol we preferred using the protocol of the Imperial college iGEM team 2011: the tips capillary test.<br><br />
</p><br />
<br />
<p class="title2">3. Tips capillary system</p><br />
<p class="title3">First tips capillary system</p><br />
<p class="texte">This protocol comes from Imperial College iGEM team 2011 and was adapted by our team in several steps (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols">chemotaxis protocol</a>).<br><br />
<br><br />
First of all, parafilm was used to close the tips:<br><br />
- 15µL of each chemo-attractant was then pipetted. <br><br />
- The tips with the pipette were then put on a piece of parafilm and the pipette was removed from the tip.<br><br />
- The tip was sealed with a piece of parafilm. By this way, the sterility can be assured and the liquid stays inside the tip. <br><br />
- To finish, the level of the solution in the tip was marked.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/9/94/Chemotaxis_-_tip.png"></center><br />
<p class="legend">Figure 4 : Sealing of a tip with parafilm</p><br />
<br />
<p class="texte">- After all the chemo-attractants were added in the tips, we put them on a green base to carry them. The whole process can be seen on Figure 5.<br><br />
- Each tip was put in 300 µL of a bacteria solution in the wells of an Elisa plate.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/0/05/Chemotaxis_-_tip_and_support.png"></center><br />
<p class="legend">Figure 5 : First tips capillary system</p><br />
<br />
<p class="texte"><i>NB: the yellow carton was used to stabilize the system and keep it straight.</i><br><br />
<br><br />
- After one hour, the tips were removed from the bacteria solutions and the content of the tips was observed with Thoma cell under the microscope.<br><br />
<br><br />
We had several problems with this system:<br><br />
- The liquid level decreased during the experiment and we did not have enough liquid to fill the Thoma cell. Thus, it was not possible to count.<br><br />
- The bacteria were moving and therefore, we could not proceed to a bacteria count.<br><br />
<br><br />
Regarding these observations we decided to spread the tips content on agar plate instead of using Thoma cell and microscopy.<br><br />
<p class="title3">Second tips capillary system<br />
</p><br />
<p class="texte"And then the revolution came! We found a multichannel pipette. The same protocol was performed except that the parafilm was used to avoid the air entrance between the tips and the pipette and therefore the loss of liquid.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/e/e4/Chemotaxis_-_pipette.png"></center><br />
<p class="legend">Figure 6 : Second tips capillary system</p><br />
<br />
<p class="title3">Improvement of the second tips capillary system</p><br />
<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></center><br />
<p class="legend">Figure 7 : Improvement of the second tips capillary system</p><br />
<br />
<p class="texte"><b>At that point, the protocol was approved and the final test could finally start! :-)</b><br><br />
<br><br />
There was just one tiny problem… we did not have our optimized bacterium with the chemotaxis gene… That is why we concentrated our efforts on WT Bacillus subtilis strain.<br><br />
<br><br />
The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolarity bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br><br />
<br><br />
The experiment was conducted with fuchsin as a negative control and was tested with different positive controls: glucose (25mM) and xylose (25mM).<br><br />
<br><br />
We obtained the following result with NAG at different concentrations: 25mM, 250mM and 500mM. The tested strain was Bacillus subtilis 168:<br><br />
<br></p><br />
<center><br />
<table align="center"><br />
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/8/8c/Chemotaxis_-_results_fuch.png"></td><br />
<td align=center><img src="https://static.igem.org/mediawiki/2014/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr><br />
<tr><td align=center><p class="legend">Figure 8 : Fuchsin - negative control (dilution 1/50)</p></td><br />
<td align=center><p class="legend">Figure 9 : NAG (25mM) (dilution 1/50)</p></td></tr><br />
</table></center><br><br />
<p class="texte">The average number of colonies with the negative control is 121. On the contrary, a cell layer is observed for the NAG plates with every concentration.<br><br />
<br><br />
Thus, we assumed that WT <i>Bacillus subtilis</i> was more attracted by NAG than fuchsin. Indeed we can neglect the bacterial growth because the test only lasts one hour. We also neglect diffusion and osmolality phenomena for the previous reasons. <br><br />
<br><br />
Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells which probably happened to our results.<br><br />
<br><br />
<b><p class="texte">This incredible discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><br><br />
<br><br />
However, our team did not give up on synthetic biology and on our strength! Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.<br><br />
<br><br />
We finally used galactose (25mM) as a negative control. The article Chemotaxis towards sugars by <i>Bacillus subtilis</i> (<i>George W. Ordal et al., 1979</i>) proved that it was a poor attractant.<br><br />
<br><br />
We made our tests again with this new molecule and glucose (25mM) as positive control.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/8/86/Chemotaxis_-_final_results.png"></center><br />
<p class="legend">Figure 10 : Final results (dilution : 1/10,000)</p><br />
<br />
<p class="texte"><i>NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.</i><br><br />
<br><br />
<b><p class="texte">Our results are not statistically significant however this result has been proved in literature.</p></b><br></p><br />
<br />
</br><br />
<br />
<p class="title1">Binding module</p> <br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Purpose</p><br />
<p class="texte">The first experiment deals with the culture conditions to see if <i>Bacillus subtilis</i> can resist to a low temperature and with the CBB buffer. To do that, several bacterial concentrations have been tested starting with an OD of 0.1 and diluting this solution to get estimated ODs of 0.05, 0.025, 0.01. These different <i>Bacillus subtilis</i> solutions were incubated 1 hour at 4°C with 500µL of CBB or water. Finally a cell count on Thoma cell counting chamber was performed.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The bacterial solutions could not be counted because of two main problems: the too high number of bacteria with the 0.1 OD or the too low number of bacteria with the 0.01 OD. Thus, the study is mostly focused on the intermediate values (Figure 1).<br />
<br/>First of all, a same cell concentration can be noticed with the presence of CBB or water with estimated ODs of 0.05 or 0.025. Moreover, twice less cells can be found in the lowest concentrations in bacteria comparing to the 0.05 OD concentration which is in agreement with the dilution ratio. <br />
<br/>Thus, the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C do not harm the cell surviving.<br />
</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/c/ce/Graphe_binding_1.png" width="45%"></center><br />
<br />
</br><br />
<p class="texte">Figure 1: CBB presence has no effect on bacteria. The bacterial concentration was measured regarding <span style="color:#0000FF">the presence</span> or <span style="color:#FF0000">the absence </span>of CBB for the observed OD (0.1) or estimated ODs (0.05, 0.025, 0.01).<br />
</p><br />
<br />
<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">Transformed <i>Bacillus subtilis</i> with the binding module is able to produce a protein composed of the bacterial peptidoglycan bonding of LycT and the GbpA 4th domain of <i>Vibrio cholerae</i> allowing the chitin bonding. The synthetic bacterium is put with special beads composed of the polymer miming the fungal pathogen wall. After several washes, bacteria specifically attached to the chitin are put on plates and counted.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The first observation is that both bacterial solutions of wild type <i>Bacillus subtilis</i> and SubtiTree have the same concentration : 105 bacteria/mL (Figure 2). Even though there is no significant difference between both strains after the first wash, the second wash has a major effect since it allows 40 times more Wild Type bacteria to come off the beads. This result correlates with the number of bacteria binded to the beads for the synthetic strain with the binding module. <br />
<br/>Thus, the binding system seems to function correctly and leads to the bacterial attachment on the chitin.</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ea/Graphe_binding_2.png" width="60%"></center><br />
</br><br />
<br />
<p class="texte">Figure 2: Attachment of <i>Bacillus subtilis</i> with binding module to chitin. <span style="color:#0000FF">The WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentration has been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p < 0.05.</p><br />
<br />
<p class="title2">3. Microscopic observations</p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">We want to observe the SubtiTree's binding on beads coated with chitin. In order to perform a 3D reconstruction showing this interaction, we use confocal laser scanning microscope. Through the use of a fluorochrome (Syto9), we can highlight the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determine the minimum threshold to remove the background noise and the natural fluorescence.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">First, we note the great bacterial presence on the surface of beads coated with chitin. These images seem to highlight their interactions.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/archive/5/53/20141013073044!Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="texte">Figure ***: <br />
</p><br />
<br />
<p class="texte">Using ImageJ software, we are able to create 3D pictures and movies of those comments. </br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/53/Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="texte">Figure ***: <br />
</p><br />
<br />
<p class="texte">Finally we want to observe the bacteria after the second wash. When our bacterium has the binding module, results suggest a lower number of bacteria in the washing solution. SubtiTree is retained by the beads.</br><br />
Finally, overall results are consistent with the presence of functional binding system.</p><br />
<br />
<br />
<p class="title1">Fungicides module<br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Tests with commercial peptides and controls</p><br />
<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12,5µM/25µM/100µM). These tests were performed on different fungal strains sharing the same phylum with <i>Ceratocystis Platani</i>.<br />
As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus.</br><br />
After several days at 30°C, the PDA (Potato Dextrose Agar) plates couvered with fungus and commercial peptides were analyzed.</p></p><br />
<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i>. Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions.As positive control, a well-known chemical fungicide was used: the Copper Sulfate. The inhibition of the fungal growth was complete at 20mg/ml, and at 10mg/ml a darker halo appeared around the pad filled with Copper Sulfate as we can see on the figure below. This corresponds to a sporing halo in response to the stress generated by the fungicide.<br />
</p><br />
<br />
</br><br />
<br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/a/a8/Prelim_tests_fung.jpg"><br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/f/f8/Controls_fung.jpg"><br />
<br />
<br />
</br><br />
<br />
<p class="texte">Given these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth. Following these tests, new conditions were adopted in order not to encourage too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees: a 'sap-like' medium was elaborated. The incubations were then carried at room temperature.<br />
</p><br />
<br />
<p class="title2">2. Test with SubtiTree<p/><br />
<br />
<p class="texte">In order to test <i>Bacillus subtilis</i> mutants, it was essential to find the right balance between the fungal growth and the bacterial one. This condition was necessary to get a high concentration of peptides. In our genetic constructions, these peptides are designed to be exported in the extracellular medium.</br><br />
</br><br />
The transformed <i>Bacillus subtilis</i> strains were grown at 37°C during 72h and tested. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of <i>Trichoderma reesei</i>'s growth was clearly observable for the strain expressing D4E1 gene. The inhibition was even more noticeable with the strain carrying the operon GAFP-1 + D4E1 (see the photos below).</br><br />
However, no effect was detected for the strain expressing the GAFP-1 gene, supposing a synergistic effect between these two peptides.</br><br />
Regarding EcAMP and the triple-fungicides operon, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP and in addition to that, sequencing results of these constructs showed some differences with the original designed sequence.<br />
<p class="texte">Inhibition halos are not visible with supernatants, probably because of their low concentrations in the extracellular medium. <br />
Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus <i>Aspergillus brasiliansis</i>. This effect is comparable to the one previously noted with low concentration of sulfate copper. </br><br />
</p><br />
<br />
</br><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/c/c2/Resultfong.jpg"> Figure X<br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/9/92/Results_fong_2.jpg"> Figure X<br />
</br><br />
<br />
<p class="texte"><br />
The choice of our chassis appears to be optimal as we noted that wild type <i>Bacillus subtilis</i> disturbs the hyphae growth of the fungi. Some strains of <i>Bacillus subtilis</i> (qst 713) are already used as Biofungicides for use on several minor crops to treat a variety of plant diseases and fungal pathogens.</br><br />
After this set of experiments, the strains expressing D4E1 and expressing GAFP-1 + D4E1 have shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b> we decided to tests these strains in model plants </b> : <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br> These tests were performed in the National Institute for the Agronomic Research by experts in this domain. <br />
<br />
<br />
<p class="title2">3. <i> In planta </i> tests with SubtiTree<p/><br />
<br />
<div style="float:left; width:215px;"><br />
<img style="width:215px;" img src="https://static.igem.org/mediawiki/parts/a/af/In_planta.jpg" style="margin-top:5px" /><br />
</div> <br />
<div style="float: right; width:700px; margin-left:44px;"> <br />
<p class="texte"><br />
The goal of the project is to introduce the trasnformed bacteria in a diseased tree. So it is necessary to perform <i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous set of experiments. </br><br />
SubtiTree is first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) is placed on the leaves. </br><br />
These tests were made in association with Sylvain Raffaële and Marielle Barascud of the National Institute for the Agronomic Research laboratory. </br><br />
</p> </div> <br />
<br />
<br />
<br />
<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves can be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants don't have deleterious effects.</br><br />
Without proper treatment, the drop of the pyhtopathogenic fungus on <i>Nicotiana benthamiana</i>'s leaves causes a necrosis halo which can be measured after 40h. The lesion size and the number of inoculated sites seem reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike with the WT bacterium. A second set of experiments is expected to be more statistically precise.</br><br />
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br><br />
<br />
We can therefore conclude that when SubtiTree is in plant physiological conditions, <b> it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves' necrosis </b>.<br />
</p><br />
<br />
TABLEAU DE RESULTAT<br />
<br />
<p class="texte"><br />
Thanks to the diversity of anti-fungal peptides, this strategy can be adapted to different types of diseases, with different degree of specifity, etc.<br />
</p><br />
<br />
<br />
<br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="" class="page-nav-right" style="min-width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;"><br />
<br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Result/parts" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Parts</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Result/experimental-resultsTeam:Toulouse/Result/experimental-results2014-10-13T15:05:50Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#5a6060; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify; }<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify; }<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/f/f7/Results_photo_tlse.png') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
p.legend{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: center;}<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Experimental results</h2><br />
<p> Are our modules functionnal? </p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Results&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Experimental results</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<p class="title1">Chemotaxis<br />
</p><br />
<p class="texte">For this module, we had to try several tests to prove the existence of chemotaxis in <i>Bacillus subtilis</i> wild type strain and SubtiTree bacterium towards N-Acetylglucosamine.<br />
</p><br />
<br />
<p class="title2">1. Plug in Pond system<br />
</p><br />
<p class="texte">Coming soon! <!--Florie--><br />
</p><br />
<br />
<p class="title2">2. Capillary test between two tubes also called the tubes test<br />
</p><br />
<p class="texte">After the experiment of the plug in pond, we decided to construct a system by welding two Eppendorf tubes with a capillary thanks to an electric burner.</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/f/fb/Chemotaxis_-_eppendorf.png"></center><br />
<p class="legend">Figure 1 : Photography of the first tubes system</p><br />
<br />
<p class="texte">We tested this system with a fuchsin dye and water and we were able to observe the diffusion of fuchsin towards water. However this construction had a leakage next to the weld seam that we could not stop. <br />
Thus, the Toulouse iGEM Team asked the help from the glass blower, Patrick Chekroun. He designed two systems composed of two tubes linked by a capillary.</p><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/2/2b/Chemotaxis_-_tubes.png"><center><br />
<p class="legend">Figure 2 : Scheme of the tubes system</p><br />
<br />
<p class="texte">As we did previously, we tested this new system with fuchsin. This experiment was made with WT Bacillus subtilis and N-Acetylglucosamine.<br />
<br><br><br />
<i>NB: We could not see the diffusion from one tube to the other. We made the hypothesis that it was not visible by sight because of by the small diameter of the capillary. <br />
</i><br><br />
<br><br />
The following strategy was used to avoid disturbance due to pressure and liquid movement through the capillary:<br><br />
- The first step was the addition of Wash Buffer until the capillary was full to avoid the presence of air bubbles which could lead to diffusion problems.<br><br />
- Then, the tube 2 was plugged with the thumb while another person was adding the bacteria solution of WT Bacillus subtilis in the tube 1. <br><br />
- The tube 1 was also plugged and only after the thumb could be removed of the tube 2. <br><br />
- In the same way, the N-Acetylglucosamine was added in the tube 2. <br><br />
- The same process was made with a xylose positive control.<br><br />
<br><br />
<i>NB: According to the article Chemotaxis towards sugars by Bacillus subtilis, (<i>George W. Ordal et al., 1979</i>), glucose and xylose have the same attractant power. We prefer a positive control instead of a negative because we were not sure that this system was efficient.</i><br><br />
<br><br />
- The system was kept straight for 2hours. Every 40 minutes, we took a sample of each tube and spread it on an agar plate (dilution 1/1,000).</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/1/1b/Chemotaxis_-_tubes_photo.png"></center><br />
<p class="legend">Figure 3 : Photography of the tubes system</p><br />
<br />
<br />
<p class="texte">Unfortunately, the dilution was too high to detect any chemotaxis movement and the time was too short. We did not find any information in the literature.<br><br />
As we did not have the time to optimize this protocol we preferred using the protocol of the Imperial college iGEM team 2011: the tips capillary test.<br><br />
</p><br />
<br />
<p class="title2">3. Tips capillary system</p><br />
<p class="title3">First tips capillary system</p><br />
<p class="texte">This protocol comes from Imperial College iGEM team 2011 and was adapted by our team in several steps (See <a href="https://2014.igem.org/Team:Toulouse/Notebook/Protocols">chemotaxis protocol</a>).<br><br />
<br><br />
First of all, parafilm was used to close the tips:<br><br />
- 15µL of each chemo-attractant was then pipetted. <br><br />
- The tips with the pipette were then put on a piece of parafilm and the pipette was removed from the tip.<br><br />
- The tip was sealed with a piece of parafilm. By this way, the sterility can be assured and the liquid stays inside the tip. <br><br />
- To finish, the level of the solution in the tip was marked.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/9/94/Chemotaxis_-_tip.png"></center><br />
<p class="legend">Figure 4 : Sealing of a tip with parafilm</p><br />
<br />
<p class="texte">- After all the chemo-attractants were added in the tips, we put them on a green base to carry them. The whole process can be seen on Figure 5.<br><br />
- Each tip was put in 300 µL of a bacteria solution in the wells of an Elisa plate.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/0/05/Chemotaxis_-_tip_and_support.png"></center><br />
<p class="legend">Figure 5 : First tips capillary system</p><br />
<br />
<p class="texte"><i>NB: the yellow carton was used to stabilize the system and keep it straight.</i><br><br />
<br><br />
- After one hour, the tips were removed from the bacteria solutions and the content of the tips was observed with Thoma cell under the microscope.<br><br />
<br><br />
We had several problems with this system:<br><br />
- The liquid level decreased during the experiment and we did not have enough liquid to fill the Thoma cell. Thus, it was not possible to count.<br><br />
- The bacteria were moving and therefore, we could not proceed to a bacteria count.<br><br />
<br><br />
Regarding these observations we decided to spread the tips content on agar plate instead of using Thoma cell and microscopy.<br><br />
<p class="title3">Second tips capillary system<br />
</p><br />
<p class="texte"And then the revolution came! We found a multichannel pipette. The same protocol was performed except that the parafilm was used to avoid the air entrance between the tips and the pipette and therefore the loss of liquid.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/e/e4/Chemotaxis_-_pipette.png"></center><br />
<p class="legend">Figure 6 : Second tips capillary system</p><br />
<br />
<p class="title3">Improvement of the second tips capillary system</p><br />
<p class="texte">However this system was not optimal it is why we decided to use blu tack instead of parafilm: <br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/4/42/Chemotaxis_-_pipette_and_blu_tack.png"></center><br />
<p class="legend">Figure 7 : Improvement of the second tips capillary system</p><br />
<br />
<p class="texte"><b>At that point, the protocol was approved and the final test could finally start! :-)</b><br><br />
<br><br />
There was just one tiny problem… we did not have our optimized bacterium with the chemotaxis gene… That is why we concentrated our efforts on WT Bacillus subtilis strain.<br><br />
<br><br />
The main goal was to find an optimized control and to analyze the eventual chemotaxis of the WT strain. To avoid osmolarity bias, we wanted to find a molecule which was non-attractant and with a similar molecular weight than the N-Acetylglucosamine (221.21 g/mol). Our first idea was to use fuchsin (Molecular weight: 337.85 g/mol).<br><br />
<br><br />
The experiment was conducted with fuchsin as a negative control and was tested with different positive controls: glucose (25mM) and xylose (25mM).<br><br />
<br><br />
We obtained the following result with NAG at different concentrations: 25mM, 250mM and 500mM. The tested strain was Bacillus subtilis 168:<br><br />
<br></p><br />
<center><br />
<table align="center"><br />
<tr><td align=center><img src="https://static.igem.org/mediawiki/2014/8/8c/Chemotaxis_-_results_fuch.png"></td><br />
<td align=center><img src="https://static.igem.org/mediawiki/2014/f/fd/Chemotaxis_-_results_fuchsin.png"></td></tr><br />
<tr><td align=center><p class="legend">Figure 8 : Fuchsin - negative control (dilution 1/50)</p></td><br />
<td align=center><p class="legend">Figure 9 : NAG (25mM) (dilution 1/50)</p></td></tr><br />
</table></center><br><br />
<p class="texte">The average number of colonies with the negative control is 121. On the contrary, a cell layer is observed for the NAG plates with every concentration.<br><br />
<br><br />
Thus, we assumed that WT <i>Bacillus subtilis</i> was more attracted by NAG than fuchsin. Indeed we can neglect the bacterial growth because the test only lasts one hour. We also neglect diffusion and osmolality phenomena for the previous reasons. <br><br />
<br><br />
Unfortunately for us we forgot one major effect… Can you believe that fuchsin solution contains about 15% of ethanol?!!! This concentration can lead to the death of some cells which probably happened to our results.<br><br />
<br><br />
<b><p class="texte">This incredible discovery destroyed all of our hopes about the God of chemotaxis! :-(</b><br><br />
<br><br />
However, our team did not give up on synthetic biology and on our strength! Indeed, after days of disappointment and no time left for lab work, we raised from ashes and tried to find another negative control.<br><br />
<br><br />
We finally used galactose (25mM) as a negative control. The article Chemotaxis towards sugars by <i>Bacillus subtilis</i> (<i>George W. Ordal et al., 1979</i>) proved that it was a poor attractant.<br><br />
<br><br />
We made our tests again with this new molecule and glucose (25mM) as positive control.<br></p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2014/8/86/Chemotaxis_-_final_results.png"></center><br />
<p class="legend">Figure 10 : Final results (dilution : 1/10,000)</p><br />
<br />
<p class="texte"><i>NB: It was our last experiment. Unfortunately we were running out of time and we could not do much more test. We would like to do the experiment with a lower dilution and repeat it several times.</i><br><br />
<br><br />
<b><p class="texte">Our results are not statistically significant however this result has been proved in literature.</p></b><br></p><br />
<br />
</br><br />
<br />
<p class="title1">Binding module</p> <br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Purpose</p><br />
<p class="texte">The first experiment deals with the culture conditions to see if <i>Bacillus subtilis</i> can resist to a low temperature and with the CBB buffer. To do that, several bacterial concentrations have been tested starting with an OD of 0.1 and diluting this solution to get estimated ODs of 0.05, 0.025, 0.01. These different <i>Bacillus subtilis</i> solutions were incubated 1 hour at 4°C with 500µL of CBB or water. Finally a cell count on Thoma cell counting chamber was performed.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The bacterial solutions could not be counted because of two main problems: the too high number of bacteria with the 0.1 OD or the too low number of bacteria with the 0.01 OD. Thus, the study is mostly focused on the intermediate values (Figure 1).<br />
<br/>First of all, a same cell concentration can be noticed with the presence of CBB or water with estimated ODs of 0.05 or 0.025. Moreover, twice less cells can be found in the lowest concentrations in bacteria comparing to the 0.05 OD concentration which is in agreement with the dilution ratio. <br />
<br/>Thus, the experimental conditions regarding the presence of CBB and the incubation temperature at 4°C do not harm the cell surviving.<br />
</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/c/ce/Graphe_binding_1.png" width="45%"></center><br />
<br />
</br><br />
<p class="texte">Figure 1: CBB presence has no effect on bacteria. The bacterial concentration was measured regarding <span style="color:#0000FF">the presence</span> or <span style="color:#FF0000">the absence </span>of CBB for the observed OD (0.1) or estimated ODs (0.05, 0.025, 0.01).<br />
</p><br />
<br />
<p class="title2">2. Binding test using engineered <i>B. subtilis</i></p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">Transformed <i>Bacillus subtilis</i> with the binding module is able to produce a protein composed of the bacterial peptidoglycan bonding of LycT and the GbpA 4th domain of <i>Vibrio cholerae</i> allowing the chitin bonding. The synthetic bacterium is put with special beads composed of the polymer miming the fungal pathogen wall. After several washes, bacteria specifically attached to the chitin are put on plates and counted.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">The first observation is that both bacterial solutions of wild type <i>Bacillus subtilis</i> and SubtiTree have the same concentration : 105 bacteria/mL (Figure 2). Even though there is no significant difference between both strains after the first wash, the second wash has a major effect since it allows 40 times more Wild Type bacteria to come off the beads. This result correlates with the number of bacteria binded to the beads for the synthetic strain with the binding module. <br />
<br/>Thus, the binding system seems to function correctly and leads to the bacterial attachment on the chitin.</p><br />
<br />
</br><br />
<center><img src="https://static.igem.org/mediawiki/2014/e/ea/Graphe_binding_2.png" width="60%"></center><br />
</br><br />
<br />
<p class="texte">Figure 2: Attachment of <i>Bacillus subtilis</i> with binding module to chitin. <span style="color:#0000FF">The WT bacteria</span> or <span style="color:#FF0000">the bacteria with the binding system</span> concentration has been determined during the different steps of the binding test. The stars represent a significant difference observed with a Student test with p < 0.05.</p><br />
<br />
<p class="title2">3. Microscopic observations</p><br />
<br />
<p class="title3">Purpose</p><br />
<p class="texte">We want to observe the SubtiTree's binding on beads coated with chitin. In order to perform a 3D reconstruction showing this interaction, we use confocal laser scanning microscope. Through the use of a fluorochrome (Syto9), we can highlight the presence of bacteria on the surface of the beads (individualized by phase-contrast). A first calibration step determine the minimum threshold to remove the background noise and the natural fluorescence.</p><br />
<br />
<p class="title3">Results</p><br />
<p class="texte">First, we note the great bacterial presence on the surface of beads coated with chitin. These images seem to highlight their interactions.</br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/archive/5/53/20141013073044!Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="texte">Figure ***: <br />
</p><br />
<br />
<p class="texte">Using ImageJ software, we are able to create 3D pictures and movies of those comments. </br></p><br />
<center><img src="https://static.igem.org/mediawiki/2014/5/53/Photo_billes_microscopie.png" width="45%"></center><br />
</br><br />
<p class="texte">Figure ***: <br />
</p><br />
<br />
<p class="texte">Finally we want to observe the bacteria after the second wash. When our bacterium has the binding module, results suggest a lower number of bacteria in the washing solution. SubtiTree is retained by the beads.</br><br />
Finally, overall results are consistent with the presence of functional binding system.</p><br />
<br />
<br />
<p class="title1">Fungicides module<br />
<p class="title2"> 1. Preliminary experiments</p><br />
<p class="title3">Tests with commercial peptides and controls</p><br />
<p class="texte">The first tests were accomplished with commercial GAFP-1 and D4E1 peptides at different concentrations (12,5µM/25µM/100µM). These tests were performed on different fungal strains sharing the same phylum with <i>Ceratocystis Platani</i>.<br />
As <i>Ceratocystis Platani</i> is pathogenic, we could not perform tests directly with this fungus.</br><br />
After several days at 30°C, the PDA (Potato Dextrose Agar) plates couvered with fungus and commercial peptides were analyzed.</p></p><br />
<p class="texte">An inhibition halo was noticeable with commercial D4E1 peptide at 100µM on <i>Aspergillus brasiliensis</i>. Less bright halos were also present with lower concentrations. Concerning commercial GAFP-1, we did not notice any effect in the tested conditions.As positive control, a well-known chemical fungicide was used: the Copper Sulfate. The inhibition of the fungal growth was complete at 20mg/ml, and at 10mg/ml a darker halo appeared around the pad filled with Copper Sulfate as we can see on the figure below. This corresponds to a sporing halo in response to the stress generated by the fungicide.<br />
</p><br />
<br />
</br><br />
<br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/a/a8/Prelim_tests_fung.jpg"><br />
<img style="width:450px; " src="https://static.igem.org/mediawiki/parts/f/f8/Controls_fung.jpg"><br />
<br />
<br />
</br><br />
<br />
<p class="texte">Given these results, we concluded that very high fungicide concentrations are required to inhibit the fungal growth. Following these tests, new conditions were adopted in order not to encourage too much fungal growth over bacterial growth. The culture medium was adjusted to fit our objective and to approximate the conditions found in the trees: a 'sap-like' medium was elaborated. The incubations were then carried at room temperature.<br />
</p><br />
<br />
<p class="title2">2. Test with SubtiTree<p/><br />
<br />
<p class="texte">In order to test <i>Bacillus subtilis</i> mutants, it was essential to find the right balance between the fungal growth and the bacterial one. This condition was necessary to get a high concentration of peptides. In our genetic constructions, these peptides are designed to be exported in the extracellular medium.</br><br />
</br><br />
The transformed <i>Bacillus subtilis</i> strains were grown at 37°C during 72h and tested. After centrifugation, the supernatant and the resuspended pellet were placed on pads and disposed on plates previously seeded with a defined number of conidia (see protocols to have more details). After several days at room temperature, an inhibition halo of <i>Trichoderma reesei</i>'s growth was clearly observable for the strain expressing D4E1 gene. The inhibition was even more noticeable with the strain carrying the operon GAFP-1 + D4E1 (see the photos below).</br><br />
However, no effect was detected for the strain expressing the GAFP-1 gene, supposing a synergistic effect between these two peptides.</br><br />
Regarding EcAMP and the triple-fungicides operon, no effect has been detected on the fungal growth. Several factors can explain these results: a number of post-transcriptional modifications are required to have a functional EcAMP and in addition to that, sequencing results of these constructs showed some differences with the original designed sequence.<br />
<p class="texte">Inhibition halos are not visible with supernatants, probably because of their low concentrations in the extracellular medium. <br />
Another effect was noted with the same strains expressing D4E1 and GAFP-1 + D4E1 on another fungus <i>Aspergillus brasiliansis</i>. This effect is comparable to the one previously noted with low concentration of sulfate copper. </br><br />
</p><br />
<br />
</br><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/c/c2/Resultfong.jpg"> Figure X<br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/parts/9/92/Results_fong_2.jpg"> Figure X<br />
</br><br />
<br />
<p class="texte"><br />
The choice of our chassis appears to be optimal as we noted that wild type <i>Bacillus subtilis</i> disturbs the hyphae growth of the fungi. Some strains of <i>Bacillus subtilis</i> (qst 713) are already used as Biofungicides for use on several minor crops to treat a variety of plant diseases and fungal pathogens.</br><br />
After this set of experiments, the strains expressing D4E1 and expressing GAFP-1 + D4E1 have shown to be the best candidates to play a major role in the fight against fungal diseases such as Canker stain. Keeping in mind our objective, <b> we decided to tests these strains in model plants </b> : <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>.</br> These tests were performed in the National Institute for the Agronomic Research by experts in this domain. <br />
<br />
<br />
<p class="title2">3. <i> In planta </i> tests with SubtiTree<p/><br />
<br />
<div style="float:left; width:215px;"><br />
<img style="width:215px;" img src="https://static.igem.org/mediawiki/parts/a/af/In_planta.jpg" style="margin-top:5px" /><br />
</div> <br />
<div style="float: right; width:700px; margin-left:44px;"> <br />
<p class="texte"><br />
The goal of the project is to introduce the trasnformed bacteria in a diseased tree. So it is necessary to perform <i> in planta </i> tests to judge the fungus-killing abilities of the two strains selected after the previous set of experiments. </br><br />
SubtiTree is first inoculated in two model plants (<i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>). After this step, a phytopathogenic fungus (<i>Sclerotinia sclerotiorum</i>) is placed on the leaves. </br><br />
These tests were made in association with Sylvain Raffaële and Marielle Barascud of the National Institute for the Agronomic Research laboratory. </br><br />
</p> </div> <br />
<br />
<br />
<br />
<p class="texte">Twenty-four hours after SubtiTree inoculation, no phenotypic modification of the leaves can be detected. We can conclude that our bacterium, its introduction and the fungicides production in plants don't have deleterious effects.</br><br />
Without proper treatment, the drop of the pyhtopathogenic fungus on <i>Nicotiana benthamiana</i>'s leaves causes a necrosis halo which can be measured after 40h. The lesion size and the number of inoculated sites seem reduced by <i>B. subtilis</i> expressing DE41 or GAFP1-D4E1, unlike with the WT bacterium. A second set of experiments is expected to be more statistically precise.</br><br />
We did not observe any significant results for <i>Arabidopsis thaliana</i> because of the use of two plants batches with different ages.</br><br />
<br />
We can therefore conclude that when SubtiTree is in plant physiological conditions, <b> it is harmless to the plant, and that the production of fungicides is effective, reducing the leaves' necrosis </b>.<br />
</p><br />
<br />
TABLEAU DE RESULTAT<br />
<br />
<p class="texte"><br />
Thanks to the diversity of anti-fungal peptides, this strategy can be adapted to different types of diseases, with different degree of specifity, etc.<br />
</p><br />
<br />
<br />
<br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="" class="page-nav-right" style="min-width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;"><br />
<br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Result/parts" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Parts</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/ModellingTeam:Toulouse/Modelling2014-10-13T14:49:40Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify;}<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify;}<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/d/de/Banniere.jpg') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
/*content*/<br />
<br />
.Title{<br />
text-shadow: 1px 3px 3px rgba(70, 156, 133, 0.75);<br />
color: #2CC23E;<br />
font-size: 50px;<br />
text-align: center;<br />
}<br />
<br />
.Sub_title{<br />
color: #2CC23E;<br />
font-size: 30px;<br />
}<br />
<br />
.Article {<br />
text-align: justify;<br />
text-indent:15px;<br />
}<br />
<br />
a.Link {<br />
font-size:10pt;<br />
color :#A4A4A4; <br />
text-decoration:none;<br />
}<br />
<br />
a.Link:hover{<br />
text-decoration:underline;}<br />
<br />
p.legend{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: center;}<br />
<br />
ol.list1{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px;}<br />
<br />
ul.list2{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px;}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Modelling</h2><br />
<p>To develop a predictive model</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Modelling</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<p class="texte"><br />
<br />
Modelling is a tool used to simplify and study systems. We can try to predict behavior with bibliographic information or information obtained from experiment.</br><br />
<br />
Our project focuses on the development of our engineered bacteria in tree. The bacterial growth in tree seems to be unknown, so we must infer <i>Bacillus subtilis</i> behavior.</p><br />
<br />
<p class="title1"><br />
Bacterial Growth<br />
</p><br />
<br />
<p class="title2"><br />
Aim<br />
</p><br />
<br />
<p class="texte"><br />
<i>Bacillus subtilis</i> is a trees endophyte strain. A study <b>[1]</b> showed that <i>Bacillus subtilis</i> could develop and fully colonize a tree, reaching a concentration of 10⁵ cells per gram of fresh plant. We need to know in which conditions the growth of <i>B. subtilis</i> is optimum in a tree and if the weather can stop its development during winter. So we decided to work on the <i>Bacillus subtilis</i> growth in function of the temperature during the year. Modeling bacterial growth in a tree section generates some difficulties, we need to know distance between two tree extremities (treetops and root) or the speed sap flow which can vary with temperatures during the day and seasons, cause of the type of sap (phloem, xylem). Furthermore a tree is not an homogeneous system, its roots, trunk and branch do not contain same amount of sap and wood. The average speed of the plane tree sap is 2.4m/h <b>[2]</b>, which means that in a day the sap will flow from one end to the other of a tree 30m. Tree is reduced to a bioreactor.<br />
</p><br />
<p class="texte"><br />
We make the following hypothesis:</p><br />
<ol class="list1"><br />
<li><br />
According to the publication of <b>Xianling Ji[1]</b>, after 6 months of <i>Bacillus subtilis</i> growth in a tree, bacteria cells reach a concentration of 10⁵ cells per gram of fresh plant. We assume that 10⁵ cells / g is the maximum concentration.<br />
</li><br />
<li><br />
The composition of the phloem is stable. There is no effect of depletion of the medium.<br />
</li><br />
<li><br />
Only temperature impact on bacterial growth.<br />
</li><br />
<li><br />
It is believed that there is no leakage of cells.<br />
</li><br />
</ol><br />
<br />
<br />
<p class="title2"><br />
Method<br />
</p><br />
<br />
<p class="texte"><br />
An assessment of the <i>Bacillus subtilis</i> growth in a similar sap, the birch sap <b>[3]</b> was performed in laboratory conditions with optimum growth medium for <i>Bacillus subtilis</i>. Thus, a growth rate μ opt. From this value we can extrapolate a growth curve as a function of temperature.<br />
For this we used to <b>cardinal temperature model [4]</b>: </p><br />
<br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/8/85/Formules_Rosso.png" alt="cardinal temperature model"></center><br />
<br />
<br />
<p class="texte"><br />
T: Temperature.</br><br />
µ_opt: optimal growth rate.</br><br />
µ: growth rate at T.</br><br />
T_max = max temperature supported by bacteria.</br><br />
T_min = min temperature supported by bacteria.</br><br />
T_opt = optimum temperature for the growth.</br></br><br />
<br />
Necessary parameters for this function are minimun temperature T_min and maximum temperature T_max, optimal temperature for the growth T_opt and optimal growth rate µ_opt.</br><br />
</br><br />
T_min: 10°C</br><br />
T_max: 52°C</br><br />
T_opt: 37°C</br><br />
µ_opt: 8.5968 cfu/d</br></br><br />
<br />
The optimal growth rate (µ_opt) is obtained experimentally with a similar birch sap environment.</br><br />
The growth rate is negative below 10°C (growth test performed at 10°C and 4°C under similar conditions for the measurement of μ_opt), survival rate after 24h was 0.3 % at 10°C and null at 4°C.<br />
Conditions apply:</p><br />
<br />
<p class="texte"> <br />
If<span style="color:#FFFFFF; font-family:'Open Sans'; font-size:14px;">__</span>| T<= 4°C -> µ = -1</br><br />
<span style="color:#FFFFFF; font-family:'Open Sans'; font-size:14px;">____</span>| 4°C<T<= 10°C -> µ = -0.97</br><br />
<span style="color:#FFFFFF; font-family:'Open Sans'; font-size:14px;">____</span>| T > 10°C -> µ = f(T) with f(T) egal to cardinal temperature model.</p><br />
<br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/b/b1/Plot_growth_rate.png" alt="Figure1"></center><br />
<p class="legend">Fig 1: bacterial growth (µ) as a function of temperature</p><br />
<br />
<p class="texte"> A logistic model developed by <b>Hiroshi Fujikawa [5]</b> is used to study bacterial growth.</p><br />
<br />
<p class="legend">General logistics formulas</p><br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/c/c3/Form_general_fonction.png" alt="General logistics formulas"></center><br />
<br />
<br />
<p class="texte"><br />
In our cases, µ depends on the temperature. N corresponds to the bacterial population, Nmin and Nmax are two asymptotes. Parameter "m" is a curvature parameter; larger is m, smaller is the curvature of the deceleration phase with the model. Parameter n is a parameter related to the period lag; larger is n, shorter is the period of lag. Nmin is slightly lower than N0, when N is small, close to Nmin, as the initial state (N is equal to N0), Nmin / N is almost equal to 1 so the term (1-(Nmin/N)) is less than 1, growth is very slow. If N decrease until reach Nmin the term (1-(Nmin/N)) is equal to 0 thus there can not be any growth. Similarly when N is equal to Nmax the term (1-(N/Nmax)) is equal to 0 and the growth is blocked.</br><br />
<br />
To overcome this we labor under two conditions , positive growth and negative growth, so two equations.This led to the writing of this model:</p><br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/f/f8/Form_part.png" alt="model"></center><br />
<br />
<br />
<br />
<br />
<p class="texte"><br />
with n = 1 and m = 0.5</br></br><br />
<br />
Term (1-(Nmin/N)) is not taken into account when there is growth and (1-(N/Nmax)) is not taken into account when there is bacterial decay.</br><br />
Meteorological records of the Toulouse region of 2011-2013 are used to do averages daily temperatures. Thus we can determine <i>B.subtilis</i> growth during a year on Toulouse. This values are obtained for each day by the average on the hightest and the lowest temperature.<br />
</br><br />
<br />
The density of green wood plane is about 650kg/m³. The average diameter of the trunks of the trees in question is about 0.80m and 15m high. This represents a volume of 30 m³ . The weight of the trunk is therefore 19,604kg .<br />
Added to this weight the weight of branches, twigs, leaves about 25% and about 15% of roots (source).<br />
</br><br />
The average weight of a plane tree is 27,446kg where in inoculated 10mL of bacterial culture at 10⁹cfu/mL, ie 10^10 bacterial cells. This represents 3.64x10² cfu/g of fresh plant (N0).<br />
</p><br />
<br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/5/53/Bacterial_growth.png" alt="Figure2"></center><br />
<p class="legend">Fig 2: (<span style="color:#000000; font-family:'Open Sans'; font-size:14px;">black</span>) Bacillus Subtilis growth curve during one year. (<span style="color:#FF0040; font-family:'Open Sans'; font-size:14px;">red</span>) average temperature. (<span style="color:#0101DF; font-family:'Open Sans'; font-size:14px;">blue</span>) threshold at 10 °C.</p><br />
<br />
<p class="texte"><br />
In our model, growth starts only from 10°C, which happens between March and April. This period seems to be suitable to put the strain in the tree. From December the temperature decreased below 4°C, threshold below which bacteria die.<br />
</p><br />
<br />
<p class="title2"><br />
Discussion<br />
</p><br />
<br />
<p class="texte"><br />
In practice, temperature variations are certainly lower in tree than outside, especially if roots extend very deep. Composition of the tree sap must also intervene in the growth rate, nutrient content of sap is also temperature dependent. The effects of the decrease of the temperature in winter also involves a fall of the sap and this must also be involved in the disappearance of our strain in the tree. The period of <i>Bacillus subtilis</i> growth is certainly affected by the change in temperature, the rise of sap, its composition variations can consequently slow the growth rate.<br />
<br />
The modeling work is done with the programming language 'R' script attached.<br />
</p><br />
<br />
<p class="title2"><br />
References<br />
</p><br />
<br />
<p class="texte"><br />
<ul class="list2"><br />
<li><br />
[1] Xianling Ji, Guobing Lu, Yingping Gai, Chengchao Zheng & Zhimei Mu (2008) Biological control against bacterial wilt and colonization of mulberry by an endophytic Bacillus subtilis strain. FEMS Microbiol Ecol 65: 565–573<br />
</li><br />
<li><br />
[2] A. Garnier(1977) Transfert de sève brute dans le tronc des arbres aspects méthodologiques et physiologiques. Ann. Sci. Foresi. 34 (1): 17-45<br />
</li><br />
<li><br />
[3] Heikki Kallio , Tuija Teerinen , Seija Ahtonen , Meri Suihko , Reino R. Linko (1989) Composition and properties of birch syrup (Betula pubescens). J. Agric. Food Chem 37 (1): 51–54<br />
</li><br />
<li><br />
[4] L. Rosso, J. R. Lobry & J. P. Flandrois (1992) AN Unexpected Correlation between Cardinal Temperatures of Microbial Growth Highlighted by a New Model. J. theor. Biol. 162 : 447-463<br />
</li><br />
<li><br />
[5] Hiroshi Fujikawa (2010), Development of a New Logistic Model for Microbial Growth in Foods. Biocontrol of Science Vol 15: 75-80<br />
</li><br />
</ul><br />
<br />
<p class="title2"><br />
Annexe<br />
</p><br />
<br />
script<br />
tableau des temperatures.<br />
<br />
</p><br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Spreading" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Spreading<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;"><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/ModellingTeam:Toulouse/Modelling2014-10-13T14:43:02Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify;}<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify;}<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/d/de/Banniere.jpg') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
/*content*/<br />
<br />
.Title{<br />
text-shadow: 1px 3px 3px rgba(70, 156, 133, 0.75);<br />
color: #2CC23E;<br />
font-size: 50px;<br />
text-align: center;<br />
}<br />
<br />
.Sub_title{<br />
color: #2CC23E;<br />
font-size: 30px;<br />
}<br />
<br />
.Article {<br />
text-align: justify;<br />
text-indent:15px;<br />
}<br />
<br />
a.Link {<br />
font-size:10pt;<br />
color :#A4A4A4; <br />
text-decoration:none;<br />
}<br />
<br />
a.Link:hover{<br />
text-decoration:underline;}<br />
<br />
p.legend{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: center;}<br />
<br />
ol.list1{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px;}<br />
<br />
ul.list2{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px;}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Modelling</h2><br />
<p>To develop a predictive model</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Modelling</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<p class="texte"><br />
<br />
Modelling is a tool used to simplify and study systems. We can try to predict behavior with bibliographic information or information obtained from experiment.</br><br />
<br />
Our project focuses on the development of our engineered bacteria in tree. The bacterial growth in tree seems to be unknown, so we must infer <i>Bacillus subtilis</i> behavior.</p><br />
<br />
<p class="title1"><br />
Bacterial Growth<br />
</p><br />
<br />
<p class="title2"><br />
Aim<br />
</p><br />
<br />
<p class="texte"><br />
<i>Bacillus subtilis</i> is a trees endophyte strain. A study <b>[1]</b> showed that <i>Bacillus subtilis</i> could develop and fully colonize a tree, reaching a concentration of 10⁵ cells per gram of fresh plant. We need to know in which conditions the growth of <i>B. subtilis</i> is optimum in a tree and if the weather can stop its development during winter. So we decided to work on the <i>Bacillus subtilis</i> growth in function of the temperature during the year. Modeling bacterial growth in a tree section generates some difficulties, we need to know distance between two tree extremities (treetops and root) or the speed sap flow which can vary with temperatures during the day and seasons, cause of the type of sap (phloem, xylem). Furthermore a tree is not an homogeneous system, its roots, trunk and branch do not contain same amount of sap and wood. The average speed of the plane tree sap is 2.4m/h <b>[2]</b>, which means that in a day the sap will flow from one end to the other of a tree 30m. Tree is reduced to a bioreactor.<br />
</p><br />
<p class="texte"><br />
We make the following hypothesis:</p><br />
<ol class="list1"><br />
<li><br />
According to the publication of <b>Xianling Ji[1]</b>, after 6 months of <i>Bacillus subtilis</i> growth in a tree, bacteria cells reach a concentration of 10⁵ cells per gram of fresh plant. We assume that 10⁵ cells / g is the maximum concentration.<br />
</li><br />
<li><br />
The composition of the phloem is stable. There is no effect of depletion of the medium.<br />
</li><br />
<li><br />
Only temperature impact on bacterial growth.<br />
</li><br />
<li><br />
It is believed that there is no leakage of cells.<br />
</li><br />
</ol><br />
<br />
<br />
<p class="title2"><br />
Method<br />
</p><br />
<br />
<p class="texte"><br />
An assessment of the <i>Bacillus subtilis</i> growth in a similar sap, the birch sap <b>[3]</b> was performed in laboratory conditions with optimum growth medium for <i>Bacillus subtilis</i>. Thus, a growth rate μ opt. From this value we can extrapolate a growth curve as a function of temperature.<br />
For this we used to <b>cardinal temperature model [4]</b>: </p><br />
<br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/8/85/Formules_Rosso.png" alt="cardinal temperature model"></center><br />
<br />
<br />
<p class="texte"><br />
T: Temperature.</br><br />
µ_opt: optimal growth rate.</br><br />
µ: growth rate at T.</br><br />
T_max = max temperature supported by bacteria.</br><br />
T_min = min temperature supported by bacteria.</br><br />
T_opt = optimum temperature for the growth.</br></br><br />
<br />
Necessary parameters for this function are minimun temperature T_min and maximum temperature T_max, optimal temperature for the growth T_opt and optimal growth rate µ_opt.</br><br />
</br><br />
T_min: 10°C</br><br />
T_max: 52°C</br><br />
T_opt: 37°C</br><br />
µ_opt: 8.5968 cfu/d</br></br><br />
<br />
The optimal growth rate (µ_opt) is obtained experimentally with a similar birch sap environment.</br><br />
The growth rate is negative below 10°C (growth test performed at 10°C and 4°C under similar conditions for the measurement of μ_opt), survival rate after 24h was 0.3 % at 10°C and null at 4°C.<br />
Conditions apply:</p><br />
<br />
<p class="texte"> <br />
If<span style="color:#FFFFFF; font-family:'Open Sans'; font-size:14px;">__</span>| T<= 4°C -> µ = -1</br><br />
<span style="color:#FFFFFF; font-family:'Open Sans'; font-size:14px;">____</span>| 4°C<T<= 10°C -> µ = -0.97</br><br />
<span style="color:#FFFFFF; font-family:'Open Sans'; font-size:14px;">____</span>| T > 10°C -> µ = f(T) with f(T) egal to cardinal temperature model.</p><br />
<br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/b/b1/Plot_growth_rate.png" alt="Figure1"></center><br />
<p class="legend">Fig 1: bacterial growth (µ) as a function of temperature</p><br />
<br />
<p class="texte"> A logistic model developed by <b>Hiroshi Fujikawa [5]</b> is used to study bacterial growth.</p><br />
<br />
<p class="legend">General logistics formulas</p><br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/c/c3/Form_general_fonction.png" alt="General logistics formulas"></center><br />
<br />
<br />
<p class="texte"><br />
In our cases, µ depends on the temperature. N corresponds to the bacterial population, Nmin and Nmax are two asymptotes. Parameter "m" is a curvature parameter; larger is m, smaller is the curvature of the deceleration phase with the model. Parameter n is a parameter related to the period lag; larger is n, shorter is the period of lag. Nmin is slightly lower than N0, when N is small, close to Nmin, as the initial state (N is equal to N0), Nmin / N is almost equal to 1 so the term (1-(Nmin/N)) is less than 1, growth is very slow. If N decrease until reach Nmin the term (1-(Nmin/N)) is equal to 0 thus there can not be any growth. Similarly when N is equal to Nmax the term (1-(N/Nmax)) is equal to 0 and the growth is blocked.</br><br />
<br />
To overcome this we labor under two conditions , positive growth and negative growth, so two equations.This led to the writing of this model:</p><br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/f/f8/Form_part.png" alt="model"></center><br />
<br />
<br />
<br />
<br />
<p class="texte"><br />
with n = 1 and m = 0.5</br></br><br />
<br />
Term (1-(Nmin/N)) is not taken into account when there is growth and (1-(N/Nmax)) is not taken into account when there is bacterial decay.</br><br />
Meteorological records of the Toulouse region of 2011-2013 are used to do averages daily temperatures. Thus we can determine <i>B.subtilis</i> growth during a year on Toulouse. This values are obtained for each day by the average on the hightest and the lowest temperature.<br />
</br><br />
<br />
The density of green wood plane is about 650kg / m³. The average diameter of the trunks of the trees in question is about 0.80m and 15m high. This represents a volume of 30 m³ . The weight of the trunk is therefore 19,604kg .<br />
Added to this weight the weight of branches, twigs, leaves about 25% and about 15% of roots (source).<br />
</br><br />
The average weight of a tree plane is 27,446kg where in inoculated 10mL of bacterial culture at 10⁹cfu/mL, ie 10^10 bacterial cells. This represents 3.64x10² cfu/g of fresh plant (N0).<br />
</p><br />
<br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/5/53/Bacterial_growth.png" alt="Figure2"></center><br />
<p class="legend">Fig 2: (<span style="color:#000000; font-family:'Open Sans'; font-size:14px;">black</span>) Bacillus Subtilis growth curve during one year. (<span style="color:#FF0040; font-family:'Open Sans'; font-size:14px;">red</span>) average temperature. (<span style="color:#0101DF; font-family:'Open Sans'; font-size:14px;">blue</span>) threshold at 10 °C.</p><br />
<br />
<p class="texte"><br />
In our model, growth starts only from 10°C, which happens between March and April. This period seems to be suitable to put the strain in the tree. From December the temperature decreased below 4°C, threshold below which bacteria die.<br />
</p><br />
<br />
<p class="title2"><br />
Discussion<br />
</p><br />
<br />
<p class="texte"><br />
In practice, temperature variations are certainly lower in tree than outside, especially if roots extend very deep. Composition of the tree sap must also intervene in the growth rate, nutrient content of sap is also temperature dependent. The effects of the decrease of the temperature in winter also involves a fall of the sap and this must also be involved in the disappearance of our strain in the tree. The period of <i>Bacillus subtilis</i> growth is certainly affected by the change in temperature, the rise of sap, its composition variations can consequently slow the growth rate.<br />
<br />
The modeling work is done with the programming language 'R' script attached.<br />
</p><br />
<br />
<p class="title2"><br />
References<br />
</p><br />
<br />
<p class="texte"><br />
<ul class="list2"><br />
<li><br />
[1] Xianling Ji, Guobing Lu, Yingping Gai, Chengchao Zheng & Zhimei Mu (2008) Biological control against bacterial wilt and colonization of mulberry by an endophytic Bacillus subtilis strain. FEMS Microbiol Ecol 65: 565–573<br />
</li><br />
<li><br />
[2] A. Garnier(1977) Transfert de sève brute dans le tronc des arbres aspects méthodologiques et physiologiques. Ann. Sci. Foresi. 34 (1): 17-45<br />
</li><br />
<li><br />
[3] Heikki Kallio , Tuija Teerinen , Seija Ahtonen , Meri Suihko , Reino R. Linko (1989) Composition and properties of birch syrup (Betula pubescens). J. Agric. Food Chem 37 (1): 51–54<br />
</li><br />
<li><br />
[4] L. Rosso, J. R. Lobry & J. P. Flandrois (1992) AN Unexpected Correlation between Cardinal Temperatures of Microbial Growth Highlighted by a New Model. J. theor. Biol. 162 : 447-463<br />
</li><br />
<li><br />
[5] Hiroshi Fujikawa (2010), Development of a New Logistic Model for Microbial Growth in Foods. Biocontrol of Science Vol 15: 75-80<br />
</li><br />
</ul><br />
<br />
<p class="title2"><br />
Annexe<br />
</p><br />
<br />
script<br />
tableau des temperatures.<br />
<br />
</p><br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Spreading" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Spreading<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;"><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/ModellingTeam:Toulouse/Modelling2014-10-13T14:39:55Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify;}<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify;}<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/d/de/Banniere.jpg') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
/*content*/<br />
<br />
.Title{<br />
text-shadow: 1px 3px 3px rgba(70, 156, 133, 0.75);<br />
color: #2CC23E;<br />
font-size: 50px;<br />
text-align: center;<br />
}<br />
<br />
.Sub_title{<br />
color: #2CC23E;<br />
font-size: 30px;<br />
}<br />
<br />
.Article {<br />
text-align: justify;<br />
text-indent:15px;<br />
}<br />
<br />
a.Link {<br />
font-size:10pt;<br />
color :#A4A4A4; <br />
text-decoration:none;<br />
}<br />
<br />
a.Link:hover{<br />
text-decoration:underline;}<br />
<br />
p.legend{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: center;}<br />
<br />
ol.list1{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px;}<br />
<br />
ul.list2{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px;}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Modelling</h2><br />
<p>To develop a predictive model</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Modelling</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<p class="texte"><br />
<br />
Modelling is a tool used to simplify and study systems. We can try to predict behavior with bibliographic information or information obtained from experiment.</br><br />
<br />
Our project focuses on the development of our engineered bacteria in tree. The bacterial growth in tree seems to be unknown, so we must infer <i>Bacillus subtilis</i> behavior.</p><br />
<br />
<p class="title1"><br />
Bacterial Growth<br />
</p><br />
<br />
<p class="title2"><br />
Aim<br />
</p><br />
<br />
<p class="texte"><br />
<i>Bacillus subtilis</i> is a trees endophyte strain. A study <b>[1]</b> showed that <i>Bacillus subtilis</i> could develop and fully colonize a tree, reaching a concentration of 10⁵ cells per gram of fresh plant. We need to know in which conditions the growth of <i>B. subtilis</i> is optimum in a tree and if the weather can stop its development during winter. So we decided to work on the <i>Bacillus subtilis</i> growth in function of the temperature during the year. Modeling bacterial growth in a tree section generates some difficulties, we need to know distance between two tree extremities (treetops and root) or the speed sap flow which can vary with temperatures during the day and seasons, cause of the type of sap (phloem, xylem). Furthermore a tree is not an homogeneous system, its roots, trunk and branch do not contain same amount of sap and wood. The average speed of the plane tree sap is 2.4m/h <b>[2]</b>, which means that in a day the sap will flow from one end to the other of a tree 30m. Tree is reduced to a bioreactor.<br />
</p><br />
<p class="texte"><br />
We make the following hypothesis:</p><br />
<ol class="list1"><br />
<li><br />
According to the publication of <b>Xianling Ji[1]</b>, after 6 months of <i>Bacillus subtilis</i> growth in a tree, bacteria cells reach a concentration of 10⁵ cells per gram of fresh plant. We assume that 10⁵ cells / g is the maximum concentration.<br />
</li><br />
<li><br />
The composition of the phloem is stable. There is no effect of depletion of the medium.<br />
</li><br />
<li><br />
Only temperature impact on bacterial growth.<br />
</li><br />
<li><br />
It is believed that there is no leakage of cells.<br />
</li><br />
</ol><br />
<br />
<br />
<p class="title2"><br />
Method<br />
</p><br />
<br />
<p class="texte"><br />
An assessment of the <i>Bacillus subtilis</i> growth in a similar sap, the birch sap <b>[3]</b> was performed in laboratory conditions with optimum growth medium for <i>Bacillus subtilis</i>. Thus, a growth rate μ opt. From this value we can extrapolate a growth curve as a function of temperature.<br />
For this we used to <b>cardinal temperature model [4]</b>: </p><br />
<br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/8/85/Formules_Rosso.png" alt="cardinal temperature model"></center><br />
<br />
<br />
<p class="texte"><br />
T: Temperature.</br><br />
µ_opt: optimal growth rate.</br><br />
µ: growth rate at T.</br><br />
T_max = max temperature supported by bacteria.</br><br />
T_min = min temperature supported by bacteria.</br><br />
T_opt = optimum temperature for the growth.</br></br><br />
<br />
Necessary parameters for this function are minimun temperature T_min and maximum temperature T_max, optimal temperature for the growth T_opt and optimal growth rate µ_opt.</br><br />
</br><br />
T_min: 10°C</br><br />
T_max: 52°C</br><br />
T_opt: 37°C</br><br />
µ_opt: 8.5968 cfu/d</br></br><br />
<br />
The optimal growth rate (µ_opt) is obtained experimentally with a similar birch sap environment.</br><br />
The growth rate is negative below 10°C (growth test performed at 10°C and 4°C under similar conditions for the measurement of μ_opt), survival rate after 24h was 0.3 % at 10°C and null at 4°C.<br />
Conditions apply:</p><br />
<br />
<p class="texte"> <br />
If<span style="color:#FFFFFF; font-family:'Open Sans'; font-size:14px;">__</span>| T<= 4°C -> µ = -1</br><br />
<span style="color:#FFFFFF; font-family:'Open Sans'; font-size:14px;">____</span>| 4°C<T<= 10°C -> µ = -0.97</br><br />
<span style="color:#FFFFFF; font-family:'Open Sans'; font-size:14px;">____</span>| T > 10°C -> µ = f(T) with f(T) egal to cardinal temperature model.</p><br />
<br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/b/b1/Plot_growth_rate.png" alt="Figure1"></center><br />
<p class="legend">Fig 1: bacterial growth (µ) as a function of temperature</p><br />
<br />
<p class="texte"> A logistic model developed by <b>Hiroshi Fujikawa [5]</b> is used to study bacterial growth.</p><br />
<br />
<p class="legend">General logistics formulas</p><br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/c/c3/Form_general_fonction.png" alt="General logistics formulas"></center><br />
<br />
<br />
<p class="texte"><br />
In our cases, µ depends on the temperature. N corresponds to the bacterial population, Nmin and Nmax are two asymptotes. Parameter "m" is a curvature parameter; larger is m, smaller is the curvature of the deceleration phase with the model. Parameter n is a parameter related to the period lag; larger is n, shorter is the period of lag. Nmin is slightly lower than N0, when N is small, close to Nmin, as the initial state (N is equal to N0), Nmin / N is almost equal to 1 so the term ( 1 - ( Nmin / N) ) is less than 1, growth is very slow. If N decrease until reach Nmin the term (1-(Nmin/N)) is equal to 0 thus there are can not be any growth. Similarly when N is equal to Nmax the term (1- (N / Nmax ) ) is equal to 0 and the growth is blocked.</br><br />
<br />
To overcome this we labor under two conditions , positive growth and negative growth, so two equations.This led to the writing of this model:</p><br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/f/f8/Form_part.png" alt="model"></center><br />
<br />
<br />
<br />
<br />
<p class="texte"><br />
with n = 1 and m = 0.5</br></br><br />
<br />
Term (1-(Nmin/N)) is not taken into account when there is growth and (1-(N/Nmax)) is not taken into account when there is bacterial decay.</br><br />
Meteorological records of the Toulouse region of 2011-2013 are used to do averages daily temperatures. Thus we can determine <i>B.subtilis</i> growth during a year on Toulouse. This values are obtained for each day by the average on the hightest and the lowest temperature.<br />
</br><br />
<br />
The density of green wood plane is about 650kg / m³. The average diameter of the trunks of the trees in question is about 0.80m and 15m high. This represents a volume of 30 m³ . The weight of the trunk is therefore 19,604kg .<br />
Added to this weight the weight of branches, twigs, leaves about 25% and about 15% of roots (source).<br />
</br><br />
The average weight of a tree plane is 27,446kg where in inoculated 10mL of bacterial culture at 10⁹cfu/mL, ie 10^10 bacterial cells. This represents 3.64x10² cfu/g of fresh plant (N0).<br />
</p><br />
<br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/5/53/Bacterial_growth.png" alt="Figure2"></center><br />
<p class="legend">Fig 2: (<span style="color:#000000; font-family:'Open Sans'; font-size:14px;">black</span>) Bacillus Subtilis growth curve during one year. (<span style="color:#FF0040; font-family:'Open Sans'; font-size:14px;">red</span>) average temperature. (<span style="color:#0101DF; font-family:'Open Sans'; font-size:14px;">blue</span>) threshold at 10 °C.</p><br />
<br />
<p class="texte"><br />
In our model, growth starts only from 10°C, which happens between March and April. This period seems to be suitable to put the strain in the tree. From December the temperature decreased below 4°C, threshold below which bacteria die.<br />
</p><br />
<br />
<p class="title2"><br />
Discussion<br />
</p><br />
<br />
<p class="texte"><br />
In practice, temperature variations are certainly lower in tree than outside, especially if roots extend very deep. Composition of the tree sap must also intervene in the growth rate, nutrient content of sap is also temperature dependent. The effects of the decrease of the temperature in winter also involves a fall of the sap and this must also be involved in the disappearance of our strain in the tree. The period of <i>Bacillus subtilis</i> growth is certainly affected by the change in temperature, the rise of sap, its composition variations can consequently slow the growth rate.<br />
<br />
The modeling work is done with the programming language 'R' script attached.<br />
</p><br />
<br />
<p class="title2"><br />
References<br />
</p><br />
<br />
<p class="texte"><br />
<ul class="list2"><br />
<li><br />
[1] Xianling Ji, Guobing Lu, Yingping Gai, Chengchao Zheng & Zhimei Mu (2008) Biological control against bacterial wilt and colonization of mulberry by an endophytic Bacillus subtilis strain. FEMS Microbiol Ecol 65: 565–573<br />
</li><br />
<li><br />
[2] A. Garnier(1977) Transfert de sève brute dans le tronc des arbres aspects méthodologiques et physiologiques. Ann. Sci. Foresi. 34 (1): 17-45<br />
</li><br />
<li><br />
[3] Heikki Kallio , Tuija Teerinen , Seija Ahtonen , Meri Suihko , Reino R. Linko (1989) Composition and properties of birch syrup (Betula pubescens). J. Agric. Food Chem 37 (1): 51–54<br />
</li><br />
<li><br />
[4] L. Rosso, J. R. Lobry & J. P. Flandrois (1992) AN Unexpected Correlation between Cardinal Temperatures of Microbial Growth Highlighted by a New Model. J. theor. Biol. 162 : 447-463<br />
</li><br />
<li><br />
[5] Hiroshi Fujikawa (2010), Development of a New Logistic Model for Microbial Growth in Foods. Biocontrol of Science Vol 15: 75-80<br />
</li><br />
</ul><br />
<br />
<p class="title2"><br />
Annexe<br />
</p><br />
<br />
script<br />
tableau des temperatures.<br />
<br />
</p><br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Spreading" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Spreading<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;"><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/ModellingTeam:Toulouse/Modelling2014-10-13T14:36:32Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify;}<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify;}<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/d/de/Banniere.jpg') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
/*content*/<br />
<br />
.Title{<br />
text-shadow: 1px 3px 3px rgba(70, 156, 133, 0.75);<br />
color: #2CC23E;<br />
font-size: 50px;<br />
text-align: center;<br />
}<br />
<br />
.Sub_title{<br />
color: #2CC23E;<br />
font-size: 30px;<br />
}<br />
<br />
.Article {<br />
text-align: justify;<br />
text-indent:15px;<br />
}<br />
<br />
a.Link {<br />
font-size:10pt;<br />
color :#A4A4A4; <br />
text-decoration:none;<br />
}<br />
<br />
a.Link:hover{<br />
text-decoration:underline;}<br />
<br />
p.legend{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: center;}<br />
<br />
ol.list1{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px;}<br />
<br />
ul.list2{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px;}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Modelling</h2><br />
<p>To develop a predictive model</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Modelling</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<p class="texte"><br />
<br />
Modelling is a tool used to simplify and study systems. We can try to predict behavior with bibliographic information or information obtained from experiment.</br><br />
<br />
Our project focuses on the development of our engineered bacteria in tree. The bacterial growth in tree seems to be unknown, so we must infer <i>Bacillus subtilis</i> behavior.</p><br />
<br />
<p class="title1"><br />
Bacterial Growth<br />
</p><br />
<br />
<p class="title2"><br />
Aim<br />
</p><br />
<br />
<p class="texte"><br />
<i>Bacillus subtilis</i> is a trees endophyte strain. A study <b>[1]</b> showed that <i>Bacillus subtilis</i> could develop and fully colonize a tree, reaching a concentration of 10⁵ cells per gram of fresh plant. We need to know in which conditions the growth of <i>B. subtilis</i> is optimum in a tree and if the weather can stop its development during winter. So we decided to work on the <i>Bacillus subtilis</i> growth in function of the temperature during the year. Modeling bacterial growth in a tree section generates some difficulties, we need to know distance between two tree extremities (treetops and root) or the speed sap flow which can vary with temperatures during the day and seasons, cause of the type of sap (phloem, xylem). Furthermore a tree is not an homogeneous system, its roots, trunk and branch do not contain same amount of sap and wood. The average speed of the plane tree sap is 2.4m/h <b>[2]</b>, which means that in a day the sap will flow from one end to the other of a tree 30m. Tree is reduced to a bioreactor.<br />
</p><br />
<p class="texte"><br />
We make the following hypothesis:</p><br />
<ol class="list1"><br />
<li><br />
According to the publication of <b>Xianling Ji[1]</b>, after 6 months of <i>Bacillus subtilis</i> growth in a tree, bacteria cells reach a concentration of 10⁵ cells per gram of fresh plant. We assume that 10⁵ cells / g is the maximum concentration.<br />
</li><br />
<li><br />
The composition of the phloem is stable. There is no effect of depletion of the medium.<br />
</li><br />
<li><br />
Only temperature impact on bacterial growth.<br />
</li><br />
<li><br />
It is believed that there is no leakage of cells.<br />
</li><br />
</ol><br />
<br />
<br />
<p class="title2"><br />
Method<br />
</p><br />
<br />
<p class="texte"><br />
An assessment of the <i>Bacillus subtilis</i> growth in a similar sap, the birch sap <b>[3]</b> was performed in laboratory conditions with optimum growth medium for <i>Bacillus subtilis</i>. Thus, a growth rate μ opt. From this value we can extrapolate a growth curve as a function of temperature.<br />
For this we used to <b>cardinal temperature model [4]</b>: </p><br />
<br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/8/85/Formules_Rosso.png" alt="cardinal temperature model"></center><br />
<br />
<br />
<p class="texte"><br />
T: Temperature.</br><br />
µ_opt: optimal growth rate.</br><br />
µ: growth rate at T.</br><br />
T_max = max temperature supported by bacteria.</br><br />
T_min = min temperature supported by bacteria.</br><br />
T_opt = optimum temperature for the growth.</br></br><br />
<br />
Necessary parameters for this function are minimun temperature T_min and maximum temperature T_max, optimal temperature for the growth T_opt and optimal growth rate µ_opt.</br><br />
</br><br />
T_min: 10°C</br><br />
T_max: 52°C</br><br />
T_opt: 37°C</br><br />
µ_opt: 8.5968 cfu/d</br></br><br />
<br />
The optimal growth rate (µ_opt) is obtained experimentally with a similar birch sap environment.</br><br />
The growth rate is negative below 10°C (growth test performed at 10°C and 4°C under similar conditions for the measurement of μ_opt), survival rate after 24h was 0.3 % at 10°C and null at 4°C.<br />
Conditions apply:</p><br />
<br />
<p class="texte"> <br />
If<span style="color:#FFFFFF; font-family:'Open Sans'; font-size:14px;">__</span>| T<= 4°C -> µ = -1</br><br />
<span style="color:#FFFFFF; font-family:'Open Sans'; font-size:14px;">____</span>| 4°C<T<= 10°C -> µ = -0.97</br><br />
<span style="color:#FFFFFF; font-family:'Open Sans'; font-size:14px;">____</span>| T > 10°C -> µ = f(T) with f(T) egal to cardinal temperature model.</p><br />
<br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/b/b1/Plot_growth_rate.png" alt="Figure1"></center><br />
<p class="legend">Fig 1: bacterial growth (µ) as a function of temperature</p><br />
<br />
<p class="texte"> A logistic model developed by <b>Hiroshi Fujikawa [5]</b> is used to study bacterial growth.</p><br />
<br />
<p class="legend">General logistics formulas</p><br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/c/c3/Form_general_fonction.png" alt="General logistics formulas"></center><br />
<br />
<br />
<p class="texte"><br />
In our cases, µ depending of the temperature. N corresponds to the bacterial population, Nmin and Nmax are two asymptotes.Parameter "m" is a curvature parameter; With a larger m, the curvature of the deceleration phase with the model is smaller. Parameter n is a parameter related to the period lag. With a larger n, the period of lag is shorter. Nmin is slightly lower than N0, when N is small, close to Nmin, as the initial state (N is equal to N0), Nmin / N is almost equal to 1 so the term ( 1 - ( Nmin / N) ) is less than 1, growth is very slow. If N decrease until reach Nmin the term (1-(Nmin/N)) is equal to 0 thus there are can not be any growth. Similarly when N is equal to Nmax the term (1- (N / Nmax ) ) is equal to 0 and the growth is blocked.</br><br />
<br />
To overcome this we labor under two conditions , positive growth and negative growth, so two equations.This led to the writing of this model:</p><br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/f/f8/Form_part.png" alt="model"></center><br />
<br />
<br />
<br />
<br />
<p class="texte"><br />
with n = 1 and m = 0.5</br></br><br />
<br />
Term (1-(Nmin/N)) is not taken into account when there is growth and (1-(N/Nmax)) is not taken into account when there is bacterial decay.</br><br />
Meteorological records of the Toulouse region of 2011-2013 are used to do averages daily temperatures. Thus we can determine <i>B.subtilis</i> growth during a year on Toulouse. This values are obtained for each day by the average on the hightest and the lowest temperature.<br />
</br><br />
<br />
The density of green wood plane is about 650kg / m³. The average diameter of the trunks of the trees in question is about 0.80m and 15m high. This represents a volume of 30 m³ . The weight of the trunk is therefore 19,604kg .<br />
Added to this weight the weight of branches, twigs, leaves about 25% and about 15% of roots (source).<br />
</br><br />
The average weight of a tree plane is 27,446kg where in inoculated 10mL of bacterial culture at 10⁹cfu/mL, ie 10^10 bacterial cells. This represents 3.64x10² cfu/g of fresh plant (N0).<br />
</p><br />
<br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/5/53/Bacterial_growth.png" alt="Figure2"></center><br />
<p class="legend">Fig 2: (<span style="color:#000000; font-family:'Open Sans'; font-size:14px;">black</span>) Bacillus Subtilis growth curve during one year. (<span style="color:#FF0040; font-family:'Open Sans'; font-size:14px;">red</span>) average temperature. (<span style="color:#0101DF; font-family:'Open Sans'; font-size:14px;">blue</span>) threshold at 10 °C.</p><br />
<br />
<p class="texte"><br />
In our model, growth starts only from 10°C, which happens between March and April. This period seems to be suitable to put the strain in the tree. From December the temperature decreased below 4°C, threshold below which bacteria die.<br />
</p><br />
<br />
<p class="title2"><br />
Discussion<br />
</p><br />
<br />
<p class="texte"><br />
In practice, temperature variations are certainly lower in tree than outside, especially if roots extend very deep. Composition of the tree sap must also intervene in the growth rate, nutrient content of sap is also temperature dependent. The effects of the decrease of the temperature in winter also involves a fall of the sap and this must also be involved in the disappearance of our strain in the tree. The period of <i>Bacillus subtilis</i> growth is certainly affected by the change in temperature, the rise of sap, its composition variations can consequently slow the growth rate.<br />
<br />
The modeling work is done with the programming language 'R' script attached.<br />
</p><br />
<br />
<p class="title2"><br />
References<br />
</p><br />
<br />
<p class="texte"><br />
<ul class="list2"><br />
<li><br />
[1] Xianling Ji, Guobing Lu, Yingping Gai, Chengchao Zheng & Zhimei Mu (2008) Biological control against bacterial wilt and colonization of mulberry by an endophytic Bacillus subtilis strain. FEMS Microbiol Ecol 65: 565–573<br />
</li><br />
<li><br />
[2] A. Garnier(1977) Transfert de sève brute dans le tronc des arbres aspects méthodologiques et physiologiques. Ann. Sci. Foresi. 34 (1): 17-45<br />
</li><br />
<li><br />
[3] Heikki Kallio , Tuija Teerinen , Seija Ahtonen , Meri Suihko , Reino R. Linko (1989) Composition and properties of birch syrup (Betula pubescens). J. Agric. Food Chem 37 (1): 51–54<br />
</li><br />
<li><br />
[4] L. Rosso, J. R. Lobry & J. P. Flandrois (1992) AN Unexpected Correlation between Cardinal Temperatures of Microbial Growth Highlighted by a New Model. J. theor. Biol. 162 : 447-463<br />
</li><br />
<li><br />
[5] Hiroshi Fujikawa (2010), Development of a New Logistic Model for Microbial Growth in Foods. Biocontrol of Science Vol 15: 75-80<br />
</li><br />
</ul><br />
<br />
<p class="title2"><br />
Annexe<br />
</p><br />
<br />
script<br />
tableau des temperatures.<br />
<br />
</p><br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Spreading" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Spreading<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;"><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/ModellingTeam:Toulouse/Modelling2014-10-13T14:34:37Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify;}<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify;}<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/d/de/Banniere.jpg') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
/*content*/<br />
<br />
.Title{<br />
text-shadow: 1px 3px 3px rgba(70, 156, 133, 0.75);<br />
color: #2CC23E;<br />
font-size: 50px;<br />
text-align: center;<br />
}<br />
<br />
.Sub_title{<br />
color: #2CC23E;<br />
font-size: 30px;<br />
}<br />
<br />
.Article {<br />
text-align: justify;<br />
text-indent:15px;<br />
}<br />
<br />
a.Link {<br />
font-size:10pt;<br />
color :#A4A4A4; <br />
text-decoration:none;<br />
}<br />
<br />
a.Link:hover{<br />
text-decoration:underline;}<br />
<br />
p.legend{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: center;}<br />
<br />
ol.list1{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px;}<br />
<br />
ul.list2{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px;}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Modelling</h2><br />
<p>To develop a predictive model</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Modelling</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<p class="texte"><br />
<br />
Modelling is a tool used to simplify and study systems. We can try to predict behavior with bibliographic information or information obtained from experiment.</br><br />
<br />
Our project focuses on the development of our engineered bacteria in tree. The bacterial growth in tree seems to be unknown, so we must infer <i>Bacillus subtilis</i> behavior.</p><br />
<br />
<p class="title1"><br />
Bacterial Growth<br />
</p><br />
<br />
<p class="title2"><br />
Aim<br />
</p><br />
<br />
<p class="texte"><br />
<i>Bacillus subtilis</i> is a trees endophyte strain. A study <b>[1]</b> showed that <i>Bacillus subtilis</i> could develop and fully colonize a tree, reaching a concentration of 10⁵ cells per gram of fresh plant. We need to know in which conditions the growth of <i>B. subtilis</i> is optimum in a tree and if the weather can stop its development during winter. So we decided to work on the <i>Bacillus subtilis</i> growth in function of the temperature during the year. Modeling bacterial growth in a tree section generates some difficulties, we need to know distance between two tree extremities (treetops and root) or the speed sap flow which can vary with temperatures during the day and seasons, cause of the type of sap (phloem, xylem). Furthermore a tree is not an homogeneous system, its roots, trunk and branch do not contain same amount of sap and wood. The average speed of the plane tree sap is 2.4m/h <b>[2]</b>, which means that in a day the sap will flow from one end to the other of a tree 30m. Tree is reduced to a bioreactor.<br />
</p><br />
<p class="texte"><br />
We make the following hypothesis:</p><br />
<ol class="list1"><br />
<li><br />
According to the publication of <b>Xianling Ji[1]</b>, after 6 months of <i>Bacillus subtilis</i> growth in a tree, bacteria cells reach a concentration of 10⁵ cells per gram of fresh plant. We assume that 10⁵ cells / g is the maximum concentration.<br />
</li><br />
<li><br />
The composition of the phloem is stable. There is no effect of depletion of the medium.<br />
</li><br />
<li><br />
Only temperature impact on bacterial growth.<br />
</li><br />
<li><br />
It is believed that there is no leakage of cells.<br />
</li><br />
</ol><br />
<br />
<br />
<p class="title2"><br />
Method<br />
</p><br />
<br />
<p class="texte"><br />
An assessment of the <i>Bacillus subtilis</i> growth in a similar sap, the birch sap <b>[3]</b> was performed in laboratory conditions with optimum growth medium for <i>Bacillus subtilis</i>. Thus, a growth rate μ opt. From this value we can extrapolate a growth curve as a function of temperature.<br />
For this we used to <b>cardinal temperature model [4]</b>: </p><br />
<br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/8/85/Formules_Rosso.png" alt="cardinal temperature model"></center><br />
<br />
<br />
<p class="texte"><br />
T: Temperature.</br><br />
µ_opt: optimal growth rate.</br><br />
µ: growth rate at T.</br><br />
T_max = max temperature supported by bacteria.</br><br />
T_min = min temperature supported by bacteria.</br><br />
T_opt = optimum temperature for the growth.</br></br><br />
<br />
Necessary parameters for this function are minimun temperature T_min and maximum temperature T_max, optimal temperature for the growth T_opt and optimal growth rate µ_opt.</br><br />
</br><br />
T_min: 10°C</br><br />
T_max: 52°C</br><br />
T_opt: 37°C</br><br />
µ_opt: 8.5968 cfu/d</br></br><br />
<br />
The optimal growth (µopt) rate is obtained experimentally with a similar birch sap environment.</br><br />
The growth rate is negative below 10°C ( growth test performed at 10 ° C and 4 ° C under similar conditions for the measurement of μ_opt), survival rate after 24h was 0.3 % at 10°C and null at 4°C.<br />
Conditions apply:</p><br />
<br />
<p class="texte"> <br />
If<span style="color:#FFFFFF; font-family:'Open Sans'; font-size:14px;">__</span>| T<= 4°C -> µ = -1</br><br />
<span style="color:#FFFFFF; font-family:'Open Sans'; font-size:14px;">____</span>| 4°C<T<= 10°C -> µ = -0.97</br><br />
<span style="color:#FFFFFF; font-family:'Open Sans'; font-size:14px;">____</span>| T > 10°C -> µ = f(T) with f(T) egal to cardinal temperature model.</p><br />
<br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/b/b1/Plot_growth_rate.png" alt="Figure1"></center><br />
<p class="legend">Fig 1: bacterial growth (µ) as a function of temperature</p><br />
<br />
<p class="texte"> A logistic model developed by <b>Hiroshi Fujikawa [5]</b> is used to study bacterial growth.</p><br />
<br />
<p class="legend">General logistics formulas</p><br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/c/c3/Form_general_fonction.png" alt="General logistics formulas"></center><br />
<br />
<br />
<p class="texte"><br />
In our cases, µ depending of the temperature. N corresponds to the bacterial population, Nmin and Nmax are two asymptotes.Parameter "m" is a curvature parameter; With a larger m, the curvature of the deceleration phase with the model is smaller. Parameter n is a parameter related to the period lag. With a larger n, the period of lag is shorter. Nmin is slightly lower than N0, when N is small, close to Nmin, as the initial state (N is equal to N0), Nmin / N is almost equal to 1 so the term ( 1 - ( Nmin / N) ) is less than 1, growth is very slow. If N decrease until reach Nmin the term (1-(Nmin/N)) is equal to 0 thus there are can not be any growth. Similarly when N is equal to Nmax the term (1- (N / Nmax ) ) is equal to 0 and the growth is blocked.</br><br />
<br />
To overcome this we labor under two conditions , positive growth and negative growth, so two equations.This led to the writing of this model:</p><br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/f/f8/Form_part.png" alt="model"></center><br />
<br />
<br />
<br />
<br />
<p class="texte"><br />
with n = 1 and m = 0.5</br></br><br />
<br />
Term (1-(Nmin/N)) is not taken into account when there is growth and (1-(N/Nmax)) is not taken into account when there is bacterial decay.</br><br />
Meteorological records of the Toulouse region of 2011-2013 are used to do averages daily temperatures. Thus we can determine <i>B.subtilis</i> growth during a year on Toulouse. This values are obtained for each day by the average on the hightest and the lowest temperature.<br />
</br><br />
<br />
The density of green wood plane is about 650kg / m³. The average diameter of the trunks of the trees in question is about 0.80m and 15m high. This represents a volume of 30 m³ . The weight of the trunk is therefore 19,604kg .<br />
Added to this weight the weight of branches, twigs, leaves about 25% and about 15% of roots (source).<br />
</br><br />
The average weight of a tree plane is 27,446kg where in inoculated 10mL of bacterial culture at 10⁹cfu/mL, ie 10^10 bacterial cells. This represents 3.64x10² cfu/g of fresh plant (N0).<br />
</p><br />
<br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/5/53/Bacterial_growth.png" alt="Figure2"></center><br />
<p class="legend">Fig 2: (<span style="color:#000000; font-family:'Open Sans'; font-size:14px;">black</span>) Bacillus Subtilis growth curve during one year. (<span style="color:#FF0040; font-family:'Open Sans'; font-size:14px;">red</span>) average temperature. (<span style="color:#0101DF; font-family:'Open Sans'; font-size:14px;">blue</span>) threshold at 10 °C.</p><br />
<br />
<p class="texte"><br />
In our model, growth starts only from 10°C, which happens between March and April. This period seems to be suitable to put the strain in the tree. From December the temperature decreased below 4°C, threshold below which bacteria die.<br />
</p><br />
<br />
<p class="title2"><br />
Discussion<br />
</p><br />
<br />
<p class="texte"><br />
In practice, temperature variations are certainly lower in tree than outside, especially if roots extend very deep. Composition of the tree sap must also intervene in the growth rate, nutrient content of sap is also temperature dependent. The effects of the decrease of the temperature in winter also involves a fall of the sap and this must also be involved in the disappearance of our strain in the tree. The period of <i>Bacillus subtilis</i> growth is certainly affected by the change in temperature, the rise of sap, its composition variations can consequently slow the growth rate.<br />
<br />
The modeling work is done with the programming language 'R' script attached.<br />
</p><br />
<br />
<p class="title2"><br />
References<br />
</p><br />
<br />
<p class="texte"><br />
<ul class="list2"><br />
<li><br />
[1] Xianling Ji, Guobing Lu, Yingping Gai, Chengchao Zheng & Zhimei Mu (2008) Biological control against bacterial wilt and colonization of mulberry by an endophytic Bacillus subtilis strain. FEMS Microbiol Ecol 65: 565–573<br />
</li><br />
<li><br />
[2] A. Garnier(1977) Transfert de sève brute dans le tronc des arbres aspects méthodologiques et physiologiques. Ann. Sci. Foresi. 34 (1): 17-45<br />
</li><br />
<li><br />
[3] Heikki Kallio , Tuija Teerinen , Seija Ahtonen , Meri Suihko , Reino R. Linko (1989) Composition and properties of birch syrup (Betula pubescens). J. Agric. Food Chem 37 (1): 51–54<br />
</li><br />
<li><br />
[4] L. Rosso, J. R. Lobry & J. P. Flandrois (1992) AN Unexpected Correlation between Cardinal Temperatures of Microbial Growth Highlighted by a New Model. J. theor. Biol. 162 : 447-463<br />
</li><br />
<li><br />
[5] Hiroshi Fujikawa (2010), Development of a New Logistic Model for Microbial Growth in Foods. Biocontrol of Science Vol 15: 75-80<br />
</li><br />
</ul><br />
<br />
<p class="title2"><br />
Annexe<br />
</p><br />
<br />
script<br />
tableau des temperatures.<br />
<br />
</p><br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Spreading" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Spreading<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;"><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Project/SpreadingTeam:Toulouse/Project/Spreading2014-10-13T12:46:37Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none; text-align: justify;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify;}<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify;}<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/5/59/Wind_and_leaves.png') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<div class="banniere-content"><br />
<h2>Spreading</h2><br />
<p>How to keep control on SubtiTree?</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Spreading</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<br />
<p class="texte">Our engineered bacterium has been designed to be inoculated in a tree and to cure fungal diseases. Understanding the environmental issues resulting from the use of a modified organism in the nature, our team worked on different aspects in order to ensure a safe use of SubtiTree. <br />
The first objective is to avoid the spreading of our smart bacterium outside the tree. In other words, the purpose is to ensure that once SubtiTree is in the tree, it is unable to live anywhere else. Another issue concerns the horizontal transfers of the genetic material between different bacteria. <br />
Taking into account these issues, we thought about three modules.<br />
</p><br />
<br />
<div id="Spreading"><br />
<center><img alt="schema" style="width:700px; z-index:2; " src="https://static.igem.org/mediawiki/2014/7/74/Spreading.png"></img></center><br />
<a class="Auxotro" HREF="#Auxotrophy"></a><br />
<a class="NSporing" HREF="#NonSporing"></a><br />
<a class="Tox" HREF="#Toxin"></a><br />
</div><br />
<br />
<p class="title1">Survival in the environment : proline auxotroph <i>B. subtilis</i></p><br />
<p class="texte">SubtiTree will live in sap tree, thus we use one endophyte <I>Bacillus subtilis</I> strain. In order to contain our bacteria in this area during a short period of time, we modified some of its survival characteristics. To make the bacterium dependant on the tree and to avoid its spreading in the environment, it should be preferable to use a strain of <i>B.subtilis</i> which is auxotroph to a particular amino acid. The bacterium should be unable to synthesize one essential amino acid, and should find it in its environment. The proline could be a good example since it is wide-spread in the phloem sap. It is the amino acid which is present in highest concentration in the phloem sap. If our bacterium is unable to synthesize the proline, it will be obliged to take it in its close environment, that is to say the phloem sap. <br />
Thus, if the bacterium is in the sap, it can grow normally without any deficiency since it uses the proline present in the sap ; but if it escapes from the tree and <i>a fortiori</i> from the sap, it will not be able to survive for a long time. Indeed, proline is found in low quantities in the ground. This system should guarantee that the bacterium develops only in the tree and not elsewhere in the surroundings of the tree.<br/> <br />
Auxotroph <i>B.sutbilis</i> strains already exist and are indexed in databases as BGSC (Bacillus Genetic Stock Center), therefore it is easy to find.</p><br />
<br />
<p class="title1">Preventing sporulation of <i>B. subtilis</i></p><br />
<p class="texte"><br />
It is known that endophyte bacteria must sporulate to survive to winter. In order to limit the spreading of our bacterium, we decided to limit its lifespan to only one season. The bacteria should be injected in spring, grow during the summer and finally should be inactivated in fall.<br\> <br />
<i>Bacillus subtilis</i> is a sporing bacterium : sporulation enable the microorganism to handle very harsh conditions and to spread tree to tree. Indeed, a spore is a very resistant form that is adapted for unfavorable conditions and for dispersal.<br/> <br />
To keep the control on the development of SubtiTree, our strain should therefore be non-sporing. We chose a <I>B.subtilis</I> strain without late genes of sporulation. Thus, after a season of treatment, the sape become less nutritious, the temperature is low and the engineered bacterium cannot survive the following winter.<br/><br />
In addition, deleting all the engineered bacterial community every year puts a brake on the evolution due to random mutation, thus it allows to keep control on the genetic constructions.<br />
</p><br />
<br />
<p class="texte">These two first characteristics of SubtiTree show that it is an annual bacterium, which can only grow in sap tree. By combinind them, they prevent any long term colonization of an ecological niche by SubtiTree against wild type bacteria. These prevent a long term effect.</p><br />
<br />
<br />
<p class="title1">Gene transfer : toxin-antitoxin system</p><br />
<br />
<p class="texte"> While we were trying to respond to bacterial spreading problem, we also wondered about horizontal gene transfer.The goal of this module is to prevent horizontal transfer between bacteria. Indeed, it is necessary to avoid any exchange of genetic material between wild type organisms and optimized organisms : it could be dangerous because of mutations, and considering ethics, it seems to be essential to avoid the spreading of synthetic genes.<br />
<br>Considering this issue, we thought about a system to avoid such transfers : a toxin-antitoxin module. It involves the addition of two genes to the bacterium : a gene encoding for a toxin (for example <i>tse2</i>) and a gene encoding for the antitoxin (<i>tsi1</i>), placing them in an opposite way on the genome. The large space between them prevents simultaneous transfers : if the optimised bacterium transfers the gene encoding for the toxin, the probability that the gene encoding for the antitoxin may be transferred simultaneously is really low since they are located far away from each other.<br/><br />
Therefore, if the host bacterium receives the gene encoding for the toxin, it will be unable to survive since it will not have the antitoxin. If it receives the antitoxin only, it will not be useful for the bacterium, and will not affect it.<br/><br />
To sum up, since a simultaneous transfer is dimly probable, the bacterium will either die because of the toxin or live while expressing the antitoxin. <br />
</p><br />
<br />
<p class="texte">Our synthetic genes are not the only problem in the design of SubtiTree. One of the side effects of our cloning method is the persistence of antibiotic resistance genes. This is incompatible with the introduction of SubtiTree in the environment. It is possible to delete this resistance in chromosome. To conclude, the spreading limitation shown previously makes the use of SubtiTree acceptable in the environment. <br />
</p><br />
<br />
<p class="title1">Using integrative plasmids</p><br />
<p class="texte"><br />
All our constructions are carried by integrative plasmids. Consequently, our different genetic modules would be integrated into the bacterium genome. The integration in the genome is more stable as the constructions are less likely to be transferred to other microorganisms. In addition to that, the expression of our genetic modules would not be dependant on a selective pressure based on an antibiotic resistance (as we can not inject antibiotics in the tree), allowing a high level of transcription in planta. <br />
</p><br />
<br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Fungicides" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Fungicides<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Modelling" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Modelling</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
<br />
<br />
<div class="clear"></div><br />
</div><br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/ModellingTeam:Toulouse/Modelling2014-10-12T15:53:12Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify;}<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify;}<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/d/de/Banniere.jpg') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
/*content*/<br />
<br />
.Title{<br />
text-shadow: 1px 3px 3px rgba(70, 156, 133, 0.75);<br />
color: #2CC23E;<br />
font-size: 50px;<br />
text-align: center;<br />
}<br />
<br />
.Sub_title{<br />
color: #2CC23E;<br />
font-size: 30px;<br />
}<br />
<br />
.Article {<br />
text-align: justify;<br />
text-indent:15px;<br />
}<br />
<br />
a.Link {<br />
font-size:10pt;<br />
color :#A4A4A4; <br />
text-decoration:none;<br />
}<br />
<br />
a.Link:hover{<br />
text-decoration:underline;}<br />
<br />
p.legend{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: center;}<br />
<br />
ol.list1{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px;}<br />
<br />
ul.list2{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px;}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Modelling</h2><br />
<p>To develop a predictive model</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Modelling</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<p class="texte"><br />
<br />
Modelling is a tool used to simplify and study systems. We can try to predict behaviour with bibliographic information or information obtained from experiment.</br><br />
<br />
Our project focuses on the development of our bacteria in tree. The way of bacterial growth in tree seems to be unknown, so we must infer bacillus subtilis behaviour.</p><br />
<br />
<p class="title1"><br />
Bacterial Growth<br />
</p><br />
<br />
<p class="title2"><br />
Objective<br />
</p><br />
<br />
<p class="texte"><br />
<i>Bacillus subtilis</i> is a trees endophyte strain, a study <b>[1]</b> showed that <i>Bacillus subtilis</i> could develop and colonize fully a tree and reach a concentration of 10⁵ cells per g of fresh plant. We need to know in which conditions the growth are optimum in a tree and if the weather can stop its development in winter. We work on the <i>Bacillus subtilis</i> growth in function of the temperature during year. Modeling bacterial growth in a tree section generate some difficulties, we need to know distance between two tree extremities (treetops and root) or the speed sap flow which can vary with temperature during the day and seasons, cause of the type of sap (phloem, xylem). Furthermore tree is not a homogeneous system, its roots, trunk and branch do not contain same amount of sap and wood. The average speed of the plane tree sap is 2.4m/h <b>[2]</b> that means that in a day the sap will flow from one end to the other of a tree 30m. Tree is reduced to a bioreactor.<br />
</p><br />
<p class="texte"><br />
We make the following hypothesis:</p><br />
<ol class="list1"><br />
<li><br />
According to the publication of <b>Xianling Ji[1]</b>, after 6 months of <i>Bacillus subtilis</i> growth in a tree bacteria cells reach a concentration of 10⁵ cells per gram of fresh plant. Assume that 10⁵ cells / g is the maximum concentration.<br />
</li><br />
<li><br />
The composition of the phloem is stable. There is no effect of depletion of the medium.<br />
</li><br />
<li><br />
Only temperature impact on bacterial growth.<br />
</li><br />
<li><br />
It is believed that there is no leakage of cells.<br />
</li><br />
</ol><br />
<br />
<br />
<p class="title2"><br />
Method<br />
</p><br />
<br />
<p class="texte"><br />
An assessment of the <i>Bacillus subtilis</i> growth in a similar sap, the birch sap <b>[3]</b> was performed in laboratory conditions with optimum growth medium for <i>Bacillus subtilis</i>. Thus, a growth rate μ opt. From this value we can extrapolate a growth curve as a function of temperature.<br />
For this we used to <b>cardinal temperature model [4]</b>: </p><br />
<br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/8/85/Formules_Rosso.png" alt="cardinal temperature model"></center><br />
<br />
<br />
<p class="texte"><br />
T: Temperature.</br><br />
µ_opt: optimal growth rate.</br><br />
µ: growth rate at T.</br><br />
T_max = max temperature supported by bacteria.</br><br />
T_min = min temperature supported by bacteria.</br><br />
T_opt = optimum temperature for the growth.</br></br><br />
<br />
Necessary parameter for this function is minimun temperature T_min and maximum temperature T_max, optimal temperature for the growth T_opt and the optimal growth rate µ_opt.</br><br />
</br><br />
T_min: 10°C</br><br />
T_max: 52°C</br><br />
T_opt: 37°C</br><br />
µ_opt: 8.5968 cfu/d</br></br><br />
<br />
The optimal growth (µopt) rate is obtained experimentally with a similar birch sap environment.</br><br />
The growth rate is negative below 10°C ( growth test performed at 10 ° C and 4 ° C under similar conditions for the measurement of μ_opt), survival rate after 24h was 0.3 % at 10°C and null at 4°C.<br />
Conditions apply:</p><br />
<br />
<p class="texte"> <br />
If<span style="color:#FFFFFF; font-family:'Open Sans'; font-size:14px;">__</span>| T<= 4°C -> µ = -1</br><br />
<span style="color:#FFFFFF; font-family:'Open Sans'; font-size:14px;">____</span>| 4°C<T<= 10°C -> µ = -0.97</br><br />
<span style="color:#FFFFFF; font-family:'Open Sans'; font-size:14px;">____</span>| T > 10°C -> µ = f(T) with f(T) egal to cardinal temperature model.</p><br />
<br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/b/b1/Plot_growth_rate.png" alt="Figure1"></center><br />
<p class="legend">Fig 1: bacterial growth (µ) as a function of temperature</p><br />
<br />
<p class="texte"> A logistic model developed by <b>Hiroshi Fujikawa [5]</b> is used to study bacterial growth.</p><br />
<br />
<p class="legend">General logistics formulas</p><br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/c/c3/Form_general_fonction.png" alt="General logistics formulas"></center><br />
<br />
<br />
<p class="texte"><br />
In our cases, µ depending of the temperature. N corresponds to the bacterial population, Nmin and Nmax are two asymptotes.Parameter "m" is a curvature parameter; With a larger m, the curvature of the deceleration phase with the model is smaller. Parameter n is a parameter related to the period lag. With a larger n, the period of lag is shorter. Nmin is slightly lower than N0, when N is small, close to Nmin, as the initial state (N is equal to N0), Nmin / N is almost equal to 1 so the term ( 1 - ( Nmin / N) ) is less than 1, growth is very slow. If N decrease until reach Nmin the term (1-(Nmin/N)) is equal to 0 thus there are can not be any growth. Similarly when N is equal to Nmax the term (1- (N / Nmax ) ) is equal to 0 and the growth is blocked.</br><br />
<br />
To overcome this we labor under two conditions , positive growth and negative growth, so two equations.This led to the writing of this model:</p><br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/f/f8/Form_part.png" alt="model"></center><br />
<br />
<br />
<br />
<br />
<p class="texte"><br />
with n = 1 and m = 0.5</br></br><br />
<br />
Term (1-(Nmin/N)) is not taken into account when there is growth and (1-(N/Nmax)) is not taken account when there is bacterial decay.</br><br />
Meteorological records of the Toulouse region of 2011-2013 are used to do averages daily temperatures. Thus we can determine <i>B.subtilis</i> growth during a year on Toulouse. This values are obtained for each day by the average on the hightest and the lowest temperature.<br />
</br><br />
<br />
The density of green wood plane is about 650kg / m³. The average diameter of the trunks of the trees in question is about 0.80m and 15m high. This represents a volume of 30 m³ . The weight of the trunk is therefore 19604kg .<br />
Added to this weight the weight of branches, twigs, leaves about 25 % and about 15% of roots (source).<br />
</br><br />
The average weight of a tree plane is 27446kg where in inoculated 10mL of bacterial culture at 10⁹cfu/mL, ie 10^10 bacterial cells. This represents 3.64x10² cfu/g of fresh plant (N0).<br />
</p><br />
<br />
<center><img style="" src="https://static.igem.org/mediawiki/2014/5/53/Bacterial_growth.png" alt="Figure2"></center><br />
<p class="legend">Fig 2: (<span style="color:#000000; font-family:'Open Sans'; font-size:14px;">black</span>) Bacillus Subtilis growth curve during one year. (<span style="color:#FF0040; font-family:'Open Sans'; font-size:14px;">red</span>) average temperature. (<span style="color:#0101DF; font-family:'Open Sans'; font-size:14px;">blue</span>) threshold at 10 °C.</p><br />
<br />
<p class="texte"><br />
In our model growth start only from 10°C this product between March and April. This period seems to be suitable to put the strain in the tree. From December the temperature down to be below 4°C, threshold below which bacterias dies.<br />
</p><br />
<br />
<p class="title2"><br />
Discussion<br />
</p><br />
<br />
<p class="texte"><br />
In practice, the temperature variations are certainly lower in tree than outside, especially if roots extend very deep. Composition of the tree sap must also intervene in the growth rate, nutrient content of sap is also temperature dependent. The effects of the decrease of the temperature in winter also involve a fall of the sap and this must also be involved in the disappearance of our strain in the tree. The period of <i>Bacillus subtilis</i> growth is certainly affected by the change in temperature, the rise of sap, its composition variations can consequently slow the growth rate.<br />
<br />
The modeling work is done with the programming language 'R' script attached.<br />
</p><br />
<br />
<p class="title2"><br />
References<br />
</p><br />
<br />
<p class="texte"><br />
<ul class="list2"><br />
<li><br />
[1] Xianling Ji, Guobing Lu, Yingping Gai, Chengchao Zheng & Zhimei Mu (2008) Biological control against bacterial wilt and colonization of mulberry by an endophytic Bacillus subtilis strain. FEMS Microbiol Ecol 65: 565–573<br />
</li><br />
<li><br />
[2] A. Garnier(1977) Transfert de sève brute dans le tronc des arbres aspects méthodologiques et physiologiques. Ann. Sci. Foresi. 34 (1): 17-45<br />
</li><br />
<li><br />
[3] Heikki Kallio , Tuija Teerinen , Seija Ahtonen , Meri Suihko , Reino R. Linko (1989) Composition and properties of birch syrup (Betula pubescens). J. Agric. Food Chem 37 (1): 51–54<br />
</li><br />
<li><br />
[4] L. Rosso, J. R. Lobry & J. P. Flandrois (1992) AN Unexpected Correlation between Cardinal Temperatures of Microbial Growth Highlighted by a New Model. J. theor. Biol. 162 : 447-463<br />
</li><br />
<li><br />
[5] Hiroshi Fujikawa (2010), Development of a New Logistic Model for Microbial Growth in Foods. Biocontrol of Science Vol 15: 75-80<br />
</li><br />
</ul><br />
<br />
<p class="title2"><br />
Annexe<br />
</p><br />
<br />
script<br />
tableau des temperatures.<br />
<br />
</p><br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Spreading" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Spreading<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;"><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Project/SpreadingTeam:Toulouse/Project/Spreading2014-10-12T15:02:54Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none; text-align: justify;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify;}<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify;}<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/5/59/Wind_and_leaves.png') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<div class="banniere-content"><br />
<h2>Spreading</h2><br />
<p>How to keep control on SubtiTree?</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Spreading</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<br />
<p class="texte">Our engineered bacterium has been designed to be inoculated in a tree and to cure fungal diseases. Understanding the environmental issues resulting from the use of a modified organism in the nature, our team worked on different aspects in order to ensure a safe use of SubtiTree. <br />
The first objective is to avoid the spreading of our smart bacterium outside the tree. In other words, the purpose is to ensure that once SubtiTree is in the tree, it is unable to live anywhere else. Another issue concerns the horizontal transfers of the genetic material between different bacteria. <br />
Taking into account these issues, we thought about three modules.<br />
</p><br />
<br />
<div id="Spreading"><br />
<center><img alt="schema" style="width:700px; z-index:2; " src="https://static.igem.org/mediawiki/2014/7/74/Spreading.png"></img></center><br />
<a class="Auxotro" HREF="#Auxotrophy"></a><br />
<a class="NSporing" HREF="#NonSporing"></a><br />
<a class="Tox" HREF="#Toxin"></a><br />
</div><br />
<br />
<br />
<p class="title1">Using a glutamin auxotroph <i>B. subtilis</i> strain</p><br />
<p class="texte"><br />
To make the bacterium dependant on the tree and to avoid its spreading in the environment, it should be preferable to use a strain of <i>B.subtilis</i> which is auxotroph to a particular amino acid. The bacterium should be unable to synthesize one essential amino acid, and should find it in its environment. The glutamine could be a good example since it is wide-spread in the phloem sap. It is the amino acid which is present in highest concentration in the phloem sap. If our bacterium is unable to synthesize the glutamine, it will be obliged to take it in its close environment, that is to say the phloem sap. <br />
Thus, if the bacterium is in the sap, it can grow normally without any deficiency since it uses the glutamine present in the sap ; but if it escapes from the tree and <i>a fortiori</i> from the sap, it will not be able to survive for a long time. Indeed, glutamine is found in low quantities in the ground. This system should guarantee that the bacterium develops only in the tree and not elsewhere in the surroundings of the tree.<br/> <br />
Auxotroph <i>B.sutbilis</i> strains already exist and are indexed in databases as BGSC (Bacillus Genetic Stock Center), therefore it is easy to find. <br />
</p><br />
<br />
<p class="title1">Preventing sporulation of <i>B. subtilis</i></p><br />
<p class="texte"><br />
In order to limit the spreading of our bacterium, we decided to limit its lifespan to only one season. The bacteria should be injected in spring, grow during the summer and finally should be inactivated in fall.<br\> <br />
<i>Bacillus subtilis</i> is a sporing bacterium : sporulation enable the microorganism to handle very harsh conditions and to spread tree to tree. Indeed, a spore is a very resistant form that is adapted for unfavorable conditions and for dispersal.<br/> <br />
To keep the control on the development of SubtiTree, our strain should therefore be non-sporing. Thus, after a season of treatment, the sape become less nutritious, the temperature is low and the engineered bacterium cannot survive the following winter.<br/><br />
In addition, deleting all the engineered bacterial community every year puts a brake on the evolution due to random mutation, thus it allows to keep control on the genetic constructions.<br />
</p><br />
<br />
<p class="title1">Using a toxin-antitoxin system</p><br />
<p class="texte"><br />
The goal of this module is to prevent horizontal transfer between bacteria. Indeed, it is necessary to avoid any exchange of genetic material between wild type organisms and optimized organisms : it could be dangerous because of mutations, and considering ethics, it seems to be essential to avoid the spreading of synthetic genes.<br/><br />
Considering this issue, we thought about a system to avoid such transfers : a toxin-antitoxin module. It involves the addition of two genes to the bacterium : a gene encoding for a toxin (for example <i>tse2</i>) and a gene encoding for the antitoxin (<i>tsi1</i>), placing them in an opposite way on the genome. The large space between them prevents simultaneous transfers : if the optimised bacterium transfers the gene encoding for the toxin, the probability that the gene encoding for the antitoxin may be transferred simultaneously is really low since they are located far away from each other.<br/><br />
Therefore, if the host bacterium receives the gene encoding for the toxin, it will be unable to survive since it will not have the antitoxin. If it receives the antitoxin only, it will not be useful for the bacterium, and will not affect it.<br/><br />
To sum up, since a simultaneous transfer is dimly probable, the bacterium will either die because of the toxin or live while expressing the antitoxin (useless). <br />
</p><br />
<br />
<p class="title1">Using integrative plasmids</p><br />
<p class="texte"><br />
All our constructions are carried by integrative plasmids. Consequently, our different genetic modules would be integrated into the bacterium genome. The integration in the genome is more stable as the constructions are less likely to be transferred to other microorganisms. In addition to that, the expression of our genetic modules would not be dependant on a selective pressure based on an antibiotic resistance (as we can not inject antibiotics in the tree), allowing a high level of transcription in planta. <br />
</p><br />
<br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Fungicides" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Fungicides<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Modelling" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Modelling</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
<br />
<br />
<div class="clear"></div><br />
</div><br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Project/SpreadingTeam:Toulouse/Project/Spreading2014-10-12T14:58:45Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none; text-align: justify;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify;}<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify;}<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/5/59/Wind_and_leaves.png') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<div class="banniere-content"><br />
<h2>Spreading</h2><br />
<p>How to keep control on SubtiTree?</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Spreading</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<br />
<p class="texte">Our engineered bacterium has been designed to be inoculated in a tree and to cure fungal diseases. Understanding the environmental issues resulting from the use of a modified organism in the nature, our team worked on different aspects in order to ensure a safe use of SubtiTree. <br />
The first objective is to avoid the spreading of our smart bacterium outside the tree. In other words, the purpose is to ensure that once SubtiTree is in the tree, it is unable to live anywhere else. Another issue concerns the horizontal transfers of the genetic material between different bacteria. <br />
Taking into account these issues, we thought about three modules.<br />
</p><br />
<br />
<div id="Spreading"><br />
<center><img alt="schema" style="width:700px; z-index:2; " src="https://static.igem.org/mediawiki/2014/7/74/Spreading.png"></img></center><br />
<a class="Auxotro" HREF="#Auxotrophy"></a><br />
<a class="NSporing" HREF="#NonSporing"></a><br />
<a class="Tox" HREF="#Toxin"></a><br />
</div><br />
<br />
<br />
<p class="title1">Using a glutamin auxotroph <i>B. subtilis</i> strain</p><br />
<p class="texte"><br />
To make the bacterium dependant on the tree and to avoid its spreading in the environment, it should be preferable to use a strain of <i>B.subtilis</i> which is auxotroph to a particular amino acid. The bacterium should be unable to synthesize one essential amino acid, and should find it in its environment. The glutamine could be a good example since it is wide-spread in the phloem sap. It is the amino acid which is present in highest concentration in the phloem sap. If our bacterium is unable to synthesize the glutamine, it will be obliged to take it in its close environment, that is to say the phloem sap. <br />
Thus, if the bacterium is in the sap, it can grow normally without any deficiency since it uses the glutamine present in the sap ; but if it escapes from the tree and <i>a fortiori</i> from the sap, it will not be able to survive for a long time. Indeed, glutamine is found in low quantities in the ground. This system should guarantee that the bacterium develops only in the tree and not elsewhere in the surroundings of the tree.<br/> <br />
Auxotroph <i>B.sutbilis</i> strains already exist and are indexed in databases as BGSC (Bacillus Genetic Stock Center), therefore it is easy to find. <br />
</p><br />
<br />
<p class="title1">Preventing sporulation of <i>B. subtilis</i></p><br />
<p class="texte"><br />
In order to limit the spreading of our bacterium, we decided to limit its lifespan to only one season. The bacteria should be injected in spring, grow during the summer and finally should be inactivated in fall.<br\> <br />
<i>Bacillus subtilis</i> is a sporing bacterium : sporulation enable the microorganism to handle very harsh conditions and to spread tree to tree. Indeed, a spore is a very resistant form that is adapted for unfavorable conditions and for dispersal.<br/> <br />
To keep the control on the development of SubtiTree, our strain should therefore be non-sporing. Thus, after a season of treatment, the sape become less nutritious, the temperature is low and the engineered bacterium cannot survive the following winter.<br/><br />
In addition, deleting all the engineered bacterial community every year puts a brake on the evolution due to random mutation, thus it allows to keep control on the genetic constructions.<br />
</p><br />
<br />
<p class="title1">Using a toxin-antitoxin system</p><br />
<p class="texte"><br />
The goal of this module is to prevent horizontal transfer between bacteria. Indeed, it is necessary to avoid any exchange of genetic material between wild type organisms and optimized organisms : it could be dangerous because of mutations, and considering ethics, it seems to be essential to avoid the spreading of synthetic genes.<br/><br />
Considering this issue, we thought about a system to avoid such transfers : a toxin-antitoxin module. It involves the addition of two genes to the bacterium : a gene encoding for a toxin (for example tse2) and a gene encoding for the antitoxin (tsi1), placing them in an opposite way on the genome. The large space between them will permit to avoid simultaneous transfers : if the optimised bacterium transfers the gene encoding for the toxin, the probability that the gene encoding for the antitoxin may be transferred simultaneously is really low since they are located far away from each other.<br/><br />
Therefore, if the host bacterium receives the gene encoding for the toxin, it will be unable to survive since it will not have the antitoxin. If it receives the antitoxin only, it will not be useful for the bacterium, and will not affect it.<br/><br />
To sum up, since a simultaneous transfer is dimly probable, the bacterium will either die because of the toxin or live while expressing the antitoxin (useless). <br />
</p><br />
<br />
<p class="title1">Using integrative plasmids</p><br />
<p class="texte"><br />
All our constructions should be carried by integrative plasmids. Consequently, our different genetic modules would be integrated into the bacterium genome. The integration in the genome is more stable as the constructions are less likely to be transferred to other microorganisms. In addition to that, the expression of our genetic modules would not be dependant on a selective pressure based on an antibiotic resistance (as we can not inject antibiotics in the tree), allowing a high level of transcription in planta. <br />
</p><br />
<br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Fungicides" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Fungicides<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Modelling" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Modelling</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
<br />
<br />
<div class="clear"></div><br />
</div><br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Project/SpreadingTeam:Toulouse/Project/Spreading2014-10-12T14:57:58Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none; text-align: justify;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify;}<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify;}<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/5/59/Wind_and_leaves.png') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<div class="banniere-content"><br />
<h2>Spreading</h2><br />
<p>How to keep control on SubtiTree?</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Spreading</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<br />
<p class="texte">Our engineered bacterium has been designed to be inoculated in a tree and to cure fungal diseases. Understanding the environmental issues resulting from the use of a modified organism in the nature, our team worked on different aspects in order to ensure a safe use of SubtiTree. <br />
The first objective is to avoid the spreading of our smart bacterium outside the tree. In other words, the purpose is to ensure that once SubtiTree is in the tree, it is unable to live anywhere else. Another issue concerns the horizontal transfers of the genetic material between different bacteria. <br />
Taking into account these issues, we thought about three modules.<br />
</p><br />
<br />
<div id="Spreading"><br />
<center><img alt="schema" style="width:700px; z-index:2; " src="https://static.igem.org/mediawiki/2014/7/74/Spreading.png"></img></center><br />
<a class="Auxotro" HREF="#Auxotrophy"></a><br />
<a class="NSporing" HREF="#NonSporing"></a><br />
<a class="Tox" HREF="#Toxin"></a><br />
</div><br />
<br />
<br />
<br />
<p class="title1">Using a glutamin auxotroph <i>B. subtilis</i> strain</p><br />
<p class="texte"><br />
To make the bacterium dependant on the tree and to avoid its spreading in the environment, it should be preferable to use a strain of <i>B.subtilis</i> which is auxotroph to a particular amino acid. The bacterium should be unable to synthesize one essential amino acid, and should find it in its environment. The glutamine could be a good example since it is wide-spread in the phloem sap. It is the amino acid which is present in highest concentration in the phloem sap. If our bacterium is unable to synthesize the glutamine, it will be obliged to take it in its close environment, that is to say the phloem sap. <br />
Thus, if the bacterium is in the sap, it can grow normally without any deficiency since it uses the glutamine present in the sap ; but if it escapes from the tree and <i>a fortiori</i> from the sap, it will not be able to survive for a long time. Indeed, glutamine is found in low quantities in the ground. This system should guarantee that the bacterium develops only in the tree and not elsewhere in the surroundings of the tree.<br/> <br />
Auxotroph <i>B.sutbilis</i> strains already exist and are indexed in databases as BGSC (Bacillus Genetic Stock Center), therefore it is easy to find. <br />
</p><br />
<br />
<p class="title1">Preventing sporulation of <i>B. subtilis</i></p><br />
<p class="texte"><br />
In order to limit the spreading of our bacterium, we decided to limit its lifespan to only one season. The bacteria should be injected in spring, grow during the summer and finally should be inactivated in fall.<br\> <br />
<i>Bacillus subtilis</i> is a sporing bacterium : sporulation enable the microorganism to handle very harsh conditions and to spread tree to tree. Indeed, a spore is a very resistant form that is adapted for unfavorable conditions and for dispersal.<br/> <br />
To keep the control on the development of SubtiTree, our strain should therefore be non-sporing. Thus, after a season of treatment, the sape become less nutritious, the temperature is low and the engineered bacterium cannot survive the following winter.<br/><br />
In addition, deleting all the engineered bacterial community every year puts a brake on the evolution due to random mutation, thus it allows to keep control on the genetic constructions.<br />
</p><br />
<br />
<p class="title1">Using a toxin-antitoxin system</p><br />
<p class="texte"><br />
The goal of this module is to prevent horizontal transfer between bacteria. Indeed, it is necessary to avoid any exchange of genetic material between wild type organisms and optimized organisms : it could be dangerous because of mutations, and considering ethics, it seems to be essential to avoid the spreading of synthetic genes.<br/><br />
Considering this issue, we thought about a system to avoid such transfers : a toxin-antitoxin module. It involves the addition of two genes to the bacterium : a gene encoding for a toxin (for example tse2) and a gene encoding for the antitoxin (tsi1), placing them in an opposite way on the genome. The large space between them will permit to avoid simultaneous transfers : if the optimised bacterium transfers the gene encoding for the toxin, the probability that the gene encoding for the antitoxin may be transferred simultaneously is really low since they are located far away from each other.<br/><br />
Therefore, if the host bacterium receives the gene encoding for the toxin, it will be unable to survive since it will not have the antitoxin. If it receives the antitoxin only, it will not be useful for the bacterium, and will not affect it.<br/><br />
To sum up, since a simultaneous transfer is dimly probable, the bacterium will either die because of the toxin or live while expressing the antitoxin (useless). <br />
</p><br />
<br />
<p class="title1">Using integrative plasmids</p><br />
<p class="texte"><br />
All our constructions should be carried by integrative plasmids. Consequently, our different genetic modules would be integrated into the bacterium genome. The integration in the genome is more stable as the constructions are less likely to be transferred to other microorganisms. In addition to that, the expression of our genetic modules would not be dependant on a selective pressure based on an antibiotic resistance (as we can not inject antibiotics in the tree), allowing a high level of transcription in planta. <br />
</p><br />
<br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Fungicides" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Fungicides<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Modelling" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Modelling</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
<br />
<br />
<div class="clear"></div><br />
</div><br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Project/SpreadingTeam:Toulouse/Project/Spreading2014-10-12T14:31:06Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none; text-align: justify;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify;}<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify;}<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/5/59/Wind_and_leaves.png') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<div class="banniere-content"><br />
<h2>Spreading</h2><br />
<p>How to keep control on SubtiTree?</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Spreading</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<br />
<p class="texte">Our engineered bacterium has been designed to be inoculated in a tree and to cure fungal diseases. Understanding the environmental issues resulting from the use of a modified organism in the nature, our team worked on different aspects in order to ensure a safe use of SubtiTree. <br />
The first objective is to avoid the spreading of our smart bacterium outside the tree. In other words, the purpose is to ensure that once SubtiTree is in the tree, it is unable to live anywhere else. Another issue concerns the horizontal transfers of the genetic material between different bacteria. <br />
Taking into account these issues, we thought about three modules.<br />
</p><br />
<br />
<div id="Spreading"><br />
<center><img alt="schema" style="width:700px; z-index:2; " src="https://static.igem.org/mediawiki/2014/7/74/Spreading.png"></img></center><br />
<a class="Auxotro" HREF="#Auxotrophy"></a><br />
<a class="NSporing" HREF="#NonSporing"></a><br />
<a class="Tox" HREF="#Toxin"></a><br />
</div><br />
<br />
<p class="title1">Using a glutamin auxotroph <i>B. subtilis</i> strain</p><br />
<p class="texte"><br />
To make the bacterium dependant on the tree and to avoid its spreading in the environment, it should be preferable to use a strain of <i>B.subtilis</i> which is auxotroph to a particular amino acid. The bacterium should be unable to synthesize one essential amino acid, and should find it in its environment. The glutamine could be a good example since it is wide-spread in the phloem sap. It is the amino acid which is present in highest concentration in the phloem sap. If our bacterium is unable to synthesize the glutamine, it will be obliged to take it in its close environment, that is to say the phloem sap. <br />
Thus, if the bacterium is in the sap, it can grow normally without any deficiency since it uses the glutamine present in the sap ; but if it escapes from the tree and <i>a fortiori</i> from the sap, it will not be able to survive for a long time. Indeed, glutamine is found in low quantities in the ground. This system should guarantee that the bacterium develops only in the tree and not elsewhere in the surroundings of the tree.<br/> <br />
Auxotroph <i>B.sutbilis</i> strains already exist and are indexed in databases as BGSC (Bacillus Genetic Stock Center), therefore it is easy to find. <br />
</p><br />
<br />
<p class="title1">Preventing sporulation of <i>B. subtilis</i></p><br />
<p class="texte"><br />
In order to limit the spreading of our bacterium, we decided to limit its lifespan to only one season. The bacteria should be injected in spring, grow during the summer and finally should be inactivated in fall.<br\> <br />
<i>Bacillus subtilis</i> is a sporing bacterium : sporulation enable the microorganism to handle very harsh conditions and to spread tree to tree. Indeed, a spore is a very resistant form that is adapted for unfavorable conditions and for dispersal.<br/> <br />
To keep the control on the development of SubtiTree, our strain should therefore be non-sporing. Thus, after a season of treatment, the sape become less nutritious, the temperature is low and the engineered bacterium cannot survive the following winter.<br/><br />
In addition, deleting all the engineered bacterial community every year puts a brake on the evolution due to random mutation = to keep control on the genetic constructions <br />
</p><br />
<br />
<p class="title1">Using a toxin-antitoxin system</p><br />
<p class="texte"><br />
The goal of this module is to prevent horizontal transfer between bacteria. Indeed, it is necessary to avoid any exchange of genetic material between wild type organisms and optimized organisms : it could be dangerous because of mutations, and considering ethics, it seems to be essential to avoid the spreading of synthetic genes.<br/><br />
Considering this issue, we thought about a system to avoid such transfers : a toxin-antitoxin module. It involves the addition of two genes to the bacterium : a gene encoding for a toxin (for example tse2) and a gene encoding for the antitoxin (tsi1), placing them in an opposite way on the genome. The large space between them will permit to avoid simultaneous transfers : if the optimised bacterium transfers the gene encoding for the toxin, the probability that the gene encoding for the antitoxin may be transferred simultaneously is really low since they are located far away from each other.<br/><br />
Therefore, if the host bacterium receives the gene encoding for the toxin, it will be unable to survive since it will not have the antitoxin. If it receives the antitoxin only, it will not be useful for the bacterium, and will not affect it.<br/><br />
To sum up, since a simultaneous transfer is dimly probable, the bacterium will either die because of the toxin or live while expressing the antitoxin (useless). <br />
</p><br />
<br />
<p class="title1">Using integrative plasmids</p><br />
<p class="texte"><br />
All our constructions should be carried by integrative plasmids. Consequently, our different genetic modules would be integrated into the bacterium genome. The integration in the genome is more stable as the constructions are less likely to be transferred to other microorganisms. In addition to that, the expression of our genetic modules would not be dependant on a selective pressure based on an antibiotic resistance (as we can not inject antibiotics in the tree), allowing a high level of transcription in planta. <br />
</p><br />
<br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Fungicides" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Fungicides<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Modelling" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Modelling</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
<br />
<br />
<div class="clear"></div><br />
</div><br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Project/SpreadingTeam:Toulouse/Project/Spreading2014-10-12T14:30:36Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none; text-align: justify;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify;}<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify;}<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/5/59/Wind_and_leaves.png') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<div class="banniere-content"><br />
<h2>Spreading</h2><br />
<p><br> How to keep control on SubtiTree?</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Spreading</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<br />
<p class="texte">Our engineered bacterium has been designed to be inoculated in a tree and to cure fungal diseases. Understanding the environmental issues resulting from the use of a modified organism in the nature, our team worked on different aspects in order to ensure a safe use of SubtiTree. <br />
The first objective is to avoid the spreading of our smart bacterium outside the tree. In other words, the purpose is to ensure that once SubtiTree is in the tree, it is unable to live anywhere else. Another issue concerns the horizontal transfers of the genetic material between different bacteria. <br />
Taking into account these issues, we thought about three modules.<br />
</p><br />
<br />
<div id="Spreading"><br />
<center><img alt="schema" style="width:700px; z-index:2; " src="https://static.igem.org/mediawiki/2014/7/74/Spreading.png"></img></center><br />
<a class="Auxotro" HREF="#Auxotrophy"></a><br />
<a class="NSporing" HREF="#NonSporing"></a><br />
<a class="Tox" HREF="#Toxin"></a><br />
</div><br />
<br />
<p class="title1">Using a glutamin auxotroph <i>B. subtilis</i> strain</p><br />
<p class="texte"><br />
To make the bacterium dependant on the tree and to avoid its spreading in the environment, it should be preferable to use a strain of <i>B.subtilis</i> which is auxotroph to a particular amino acid. The bacterium should be unable to synthesize one essential amino acid, and should find it in its environment. The glutamine could be a good example since it is wide-spread in the phloem sap. It is the amino acid which is present in highest concentration in the phloem sap. If our bacterium is unable to synthesize the glutamine, it will be obliged to take it in its close environment, that is to say the phloem sap. <br />
Thus, if the bacterium is in the sap, it can grow normally without any deficiency since it uses the glutamine present in the sap ; but if it escapes from the tree and <i>a fortiori</i> from the sap, it will not be able to survive for a long time. Indeed, glutamine is found in low quantities in the ground. This system should guarantee that the bacterium develops only in the tree and not elsewhere in the surroundings of the tree.<br/> <br />
Auxotroph <i>B.sutbilis</i> strains already exist and are indexed in databases as BGSC (Bacillus Genetic Stock Center), therefore it is easy to find. <br />
</p><br />
<br />
<p class="title1">Preventing sporulation of <i>B. subtilis</i></p><br />
<p class="texte"><br />
In order to limit the spreading of our bacterium, we decided to limit its lifespan to only one season. The bacteria should be injected in spring, grow during the summer and finally should be inactivated in fall.<br\> <br />
<i>Bacillus subtilis</i> is a sporing bacterium : sporulation enable the microorganism to handle very harsh conditions and to spread tree to tree. Indeed, a spore is a very resistant form that is adapted for unfavorable conditions and for dispersal.<br/> <br />
To keep the control on the development of SubtiTree, our strain should therefore be non-sporing. Thus, after a season of treatment, the sape become less nutritious, the temperature is low and the engineered bacterium cannot survive the following winter.<br/><br />
In addition, deleting all the engineered bacterial community every year puts a brake on the evolution due to random mutation = to keep control on the genetic constructions <br />
</p><br />
<br />
<p class="title1">Using a toxin-antitoxin system</p><br />
<p class="texte"><br />
The goal of this module is to prevent horizontal transfer between bacteria. Indeed, it is necessary to avoid any exchange of genetic material between wild type organisms and optimized organisms : it could be dangerous because of mutations, and considering ethics, it seems to be essential to avoid the spreading of synthetic genes.<br/><br />
Considering this issue, we thought about a system to avoid such transfers : a toxin-antitoxin module. It involves the addition of two genes to the bacterium : a gene encoding for a toxin (for example tse2) and a gene encoding for the antitoxin (tsi1), placing them in an opposite way on the genome. The large space between them will permit to avoid simultaneous transfers : if the optimised bacterium transfers the gene encoding for the toxin, the probability that the gene encoding for the antitoxin may be transferred simultaneously is really low since they are located far away from each other.<br/><br />
Therefore, if the host bacterium receives the gene encoding for the toxin, it will be unable to survive since it will not have the antitoxin. If it receives the antitoxin only, it will not be useful for the bacterium, and will not affect it.<br/><br />
To sum up, since a simultaneous transfer is dimly probable, the bacterium will either die because of the toxin or live while expressing the antitoxin (useless). <br />
</p><br />
<br />
<p class="title1">Using integrative plasmids</p><br />
<p class="texte"><br />
All our constructions should be carried by integrative plasmids. Consequently, our different genetic modules would be integrated into the bacterium genome. The integration in the genome is more stable as the constructions are less likely to be transferred to other microorganisms. In addition to that, the expression of our genetic modules would not be dependant on a selective pressure based on an antibiotic resistance (as we can not inject antibiotics in the tree), allowing a high level of transcription in planta. <br />
</p><br />
<br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Fungicides" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Fungicides<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Modelling" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Modelling</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
<br />
<br />
<div class="clear"></div><br />
</div><br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Project/SpreadingTeam:Toulouse/Project/Spreading2014-10-12T14:29:15Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none; text-align: justify;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify;}<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify;}<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/5/59/Wind_and_leaves.png') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<div class="banniere-content"><br />
<h2>Spreading</h2><br />
<p>How can SubtiTree respect the environment?<br> How do we keep control on SubtiTree?</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Spreading</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<br />
<p class="texte">Our engineered bacterium has been designed to be inoculated in a tree and to cure fungal diseases. Understanding the environmental issues resulting from the use of a modified organism in the nature, our team worked on different aspects in order to ensure a safe use of SubtiTree. <br />
The first objective is to avoid the spreading of our smart bacterium outside the tree. In other words, the purpose is to ensure that once SubtiTree is in the tree, it is unable to live anywhere else. Another issue concerns the horizontal transfers of the genetic material between different bacteria. <br />
Taking into account these issues, we thought about three modules.<br />
</p><br />
<br />
<div id="Spreading"><br />
<center><img alt="schema" style="width:700px; z-index:2; " src="https://static.igem.org/mediawiki/2014/7/74/Spreading.png"></img></center><br />
<a class="Auxotro" HREF="#Auxotrophy"></a><br />
<a class="NSporing" HREF="#NonSporing"></a><br />
<a class="Tox" HREF="#Toxin"></a><br />
</div><br />
<br />
<p class="title1">Using a glutamin auxotroph <i>B. subtilis</i> strain</p><br />
<p class="texte"><br />
To make the bacterium dependant on the tree and to avoid its spreading in the environment, it should be preferable to use a strain of <i>B.subtilis</i> which is auxotroph to a particular amino acid. The bacterium should be unable to synthesize one essential amino acid, and should find it in its environment. The glutamine could be a good example since it is wide-spread in the phloem sap. It is the amino acid which is present in highest concentration in the phloem sap. If our bacterium is unable to synthesize the glutamine, it will be obliged to take it in its close environment, that is to say the phloem sap. <br />
Thus, if the bacterium is in the sap, it can grow normally without any deficiency since it uses the glutamine present in the sap ; but if it escapes from the tree and <i>a fortiori</i> from the sap, it will not be able to survive for a long time. Indeed, glutamine is found in low quantities in the ground. This system should guarantee that the bacterium develops only in the tree and not elsewhere in the surroundings of the tree.<br/> <br />
Auxotroph <i>B.sutbilis</i> strains already exist and are indexed in databases as BGSC (Bacillus Genetic Stock Center), therefore it is easy to find. <br />
</p><br />
<br />
<p class="title1">Preventing sporulation of <i>B. subtilis</i></p><br />
<p class="texte"><br />
In order to limit the spreading of our bacterium, we decided to limit its lifespan to only one season. The bacteria should be injected in spring, grow during the summer and finally should be inactivated in fall.<br\> <br />
<i>Bacillus subtilis</i> is a sporing bacterium : sporulation enable the microorganism to handle very harsh conditions and to spread tree to tree. Indeed, a spore is a very resistant form that is adapted for unfavorable conditions and for dispersal.<br/> <br />
To keep the control on the development of SubtiTree, our strain should therefore be non-sporing. Thus, after a season of treatment, the sape become less nutritious, the temperature is low and the engineered bacterium cannot survive the following winter.<br/><br />
In addition, deleting all the engineered bacterial community every year puts a brake on the evolution due to random mutation = to keep control on the genetic constructions <br />
</p><br />
<br />
<p class="title1">Using a toxin-antitoxin system</p><br />
<p class="texte"><br />
The goal of this module is to prevent horizontal transfer between bacteria. Indeed, it is necessary to avoid any exchange of genetic material between wild type organisms and optimized organisms : it could be dangerous because of mutations, and considering ethics, it seems to be essential to avoid the spreading of synthetic genes.<br/><br />
Considering this issue, we thought about a system to avoid such transfers : a toxin-antitoxin module. It involves the addition of two genes to the bacterium : a gene encoding for a toxin (for example tse2) and a gene encoding for the antitoxin (tsi1), placing them in an opposite way on the genome. The large space between them will permit to avoid simultaneous transfers : if the optimised bacterium transfers the gene encoding for the toxin, the probability that the gene encoding for the antitoxin may be transferred simultaneously is really low since they are located far away from each other.<br/><br />
Therefore, if the host bacterium receives the gene encoding for the toxin, it will be unable to survive since it will not have the antitoxin. If it receives the antitoxin only, it will not be useful for the bacterium, and will not affect it.<br/><br />
To sum up, since a simultaneous transfer is dimly probable, the bacterium will either die because of the toxin or live while expressing the antitoxin (useless). <br />
</p><br />
<br />
<p class="title1">Using integrative plasmids</p><br />
<p class="texte"><br />
All our constructions should be carried by integrative plasmids. Consequently, our different genetic modules would be integrated into the bacterium genome. The integration in the genome is more stable as the constructions are less likely to be transferred to other microorganisms. In addition to that, the expression of our genetic modules would not be dependant on a selective pressure based on an antibiotic resistance (as we can not inject antibiotics in the tree), allowing a high level of transcription in planta. <br />
</p><br />
<br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Fungicides" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Fungicides<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Modelling" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Modelling</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
<br />
<br />
<div class="clear"></div><br />
</div><br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Project/FungicidesTeam:Toulouse/Project/Fungicides2014-10-12T14:20:33Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify;}<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify;}<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/3/38/Fungi.jpg') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Fungicides</h2><br />
<p>To eradicate fungal diseases</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Fungicides</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<!--Short description : à changer!!!--><br />
<br />
<img style="width:800px; " src="https://static.igem.org/mediawiki/2014/0/0c/Recap_fungicides.jpg"><br />
<br />
<p class="textesimple">The main objective of SubtiTree is to ensure the <b> destruction of the pathogenic fungi </b> inside the tree. In order to achieve this goal, we built a genetic module to produce three different peptides with antifungal activities. </p> <br><br />
<br />
<p class="textesimple"> Originated from plants, these peptides have different targets to maximize the lethality on <i>C. platani</i>. <br />
<p class="textesimple"><br />
- <b>D4E1</b> is a synthetic peptide analog to Cecropin B AMPs (antimicrobial peptides) made of 17 amino acids which has been shown to have an antifungal activity by complexing with a sterol present in the conidia’s wall of numerous fungi. </p><br />
<p class="textesimple"><br />
- <b>GAFP-1 </b>(<i>Gastrodia</i> Anti Fungal Protein 1), also known as gastrodianin, is a mannose and chitin binding lectin originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years. GAFP1 accumulates in nutritive corms where the fungal infection takes place, and <i>in vitro</i> assays demonstrated it can inhibit the growth of ascomycete and basidiomycete fungal plant pathogens.</p><br />
<br />
<p class="textesimple"><br />
- <b>EcAMP-1 </b>(<i>Echinochloa crus-galli </i> Anti Microbial Peptide) consists in 37 amino acids inhibiting hyphae elongation. EcAMP1 is the first example of AMP with a novel disulfide-stabilized-α helical hairpin fold. It is isolated from kernels of barnyard grass. EcAMP1 exhibits high activity against fungi of the genus Fusarium.</p><br />
<br />
</p><br />
<br><br />
<p class="title1" style="margin-top:30px;"><b>More information on this module </p></b> <br><br />
<p class="texte"><br />
We built different genetic constructions to test each fungicide separately and to test them all together on the same operon where the 3 genes coding for the antifungal peptides are placed under the control of a constitutive promoter in <i>Bacillus subtilis </i>: Pveg. </p><br />
<br />
<img style="width:930px; float:left; margin: 30px 0;" src="https://static.igem.org/mediawiki/parts/d/d0/Fungicideprod.jpg"> <br />
<br />
<p class="texte">EcAMP-1 was already present in the Registry, added by the Utah State 2013 iGEM team (<a ref="http://parts.igem.org/Part:BBa_K1162001"_blank"> BB_K1162001</a>) . We added D4E1 and GAFP-1 to the Registry of Standard Biological Parts (see parts). These new BioBricks were designed in order to be expressed and secreted with <i>Bacillus subtilis</i>. <br />
</p><br />
<br><br />
<p class="title1"><b>Secretion </b> </p><br />
<p class="texte">In order to export the peptides outside the bacteria, the coding sequence of D4E1 and GAFP-1 was flanked on the N-terminal end with a signal peptide (amyE signal peptide) followed by a pro peptide, cleaved during the secretion process. </p> <br><br />
<br />
<br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/2014/2/2e/Secretion.jpg"><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/2014/d/d7/Fongpep.jpg"><br />
<br><br />
<br />
<br><br />
<br />
</P> <br />
<p class="title1"><b>References</b> </p><br />
<br />
<p class="texte"><br />
- A.J De Lucca, J.M Bland, C. Grimm, T.J Jacks.<b> Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1 </b>. Canadian Journal of Microbiology. 1998, Vol. 44:514-520. <br><br />
Kanniah Rajasekaran, Kurt D. Stromberg, Jeffrey W. Cary, and Thomas E. Cleveland.<b> Broad-Spectrum Antimicrobial Activity in vitro of the Synthetic Peptide D4E1</b>. J. Agric. Food Chem. 2001, Vol. 49, 2799-2803.<br><br />
-M. Visser, D. Stephan, J.M. Jaynes and J.T. Burger.<b> A transient expression assay for the in planta efficacy screening of an antimicrobial peptide against grapevine bacterial pathogens</b>. Letters in Applied Microbiology. 2012, Vol. 54, 543–551.<br><br />
-K. D. Cox, D. R. Layne, R. Scorza, G Schnabel. <b>Gastrodia anti-fungal protein from the orchid Gastrodia elata confers disease resistance to root pathogens in transgenic tobacco</b>. Planta. 2006, Vol. 224:1373–1383<br><br />
-Xiaochen Wang, Guy Bauw, Els J.M. Van Damme, Willy J. Peumans, Zhang-Liang Chen, Marc Van Montagu and Willy Dillen. <b>Gastrodianin-like mannose-binding proteins: a novel class of plant proteins with antifungal properties</b>. The Plant Journal. 2001, Vol. 25(6), 651±661<br><br />
-Svetlana B. Nolde, Alexander A. Vassilevski, Eugene A. Rogozhin, Nikolay A. Barinov, Tamara A. Balashova, Olga V. Samsonova, Yuri V. Baranov, Alexey S. Arseniev and Eugene V. Grishin. <b>Disulfide-stabilized Helical Hairpin Structure and Activity of a Novel Antifungal Peptide EcAMP1 from Seeds of Barnyard Grass (Echinochloa crus-galli)</b>. The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153<br><br />
</p><br />
<br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Binding" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Binding<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Spreading" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Spreading</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Project/FungicidesTeam:Toulouse/Project/Fungicides2014-10-12T14:17:58Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify;}<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify;}<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/3/38/Fungi.jpg') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Fungicides</h2><br />
<p>To eradicate fungal diseases</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Fungicides</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<!--Short description : à changer!!!--><br />
<br />
<img style="width:800px; " src="https://static.igem.org/mediawiki/2014/0/0c/Recap_fungicides.jpg"><br />
<br />
<p class="textesimple">The main objective of SubtiTree is to ensure the <b> destruction of the pathogenic fungi </b> inside the tree. In order to achieve this goal, we built a genetic module to produce three different peptides with antifungal activities. </p> <br><br />
<br />
<p class="textesimple"> Originated from plants, these peptides have different targets to maximize the lethality on <i>C. platani</i>. <br />
<p class="textesimple"><br />
- <b>D4E1</b> is a synthetic peptide analog to Cecropin B AMPs (antimicrobial peptides) made of 17 amino acids which has been shown to have an antifungal activity by complexing with a sterol present in the conidia’s wall of numerous fungi. </p><br />
<p class="textesimple"><br />
- <b>GAFP-1 </b>(<i>Gastrodia</i> Anti Fungal Protein 1), also known as gastrodianin, is a mannose and chitin binding lectin originating from the Asiatic orchid Gastrodia elata, a traditional Chinese medicinal herb cultured for thousands of years. GAFP1 accumulates in nutritive corms where the fungal infection takes place, and in vitro assays demonstrated it can inhibit the growth of ascomycete and basidiomycete fungal plant pathogens.</p><br />
<br />
<p class="textesimple"><br />
- <b>EcAMP-1 </b>(<i>Echinochloa crus-galli </i> Anti Microbial Peptide) consists in 37 amino acids inhibiting hyphae elongation. EcAMP1 is the first example of AMP with a novel disulfide-stabilized-α helical hairpin fold. It is isolated from kernels of barnyard grass. EcAMP1 exhibits high activity against fungi of the genus Fusarium.</p><br />
<br />
</p><br />
<br><br />
<p class="title1" style="margin-top:30px;"><b>More information on this module </p></b> <br><br />
<p class="texte"><br />
We built different genetic constructions to test each fungicide separately and to test them all together on the same operon where the 3 genes coding for the antifungal peptides are placed under the control of a constitutive promoter in <i>Bacillus subtilis </i>: Pveg. </p><br />
<br />
<img style="width:930px; float:left; margin: 30px 0;" src="https://static.igem.org/mediawiki/parts/d/d0/Fungicideprod.jpg"> <br />
<br />
<p class="texte">EcAMP-1 was already present in the Registry, added by the Utah State 2013 iGEM team (<a ref="http://parts.igem.org/Part:BBa_K1162001"_blank"> BB_K1162001</a>) . We added D4E1 and GAFP-1 to the Registry of Standard Biological Parts (see parts). These new BioBricks were designed in order to be expressed and secreted with <i>Bacillus subtilis</i>. <br />
</p><br />
<br><br />
<p class="title1"><b>Secretion </b> </p><br />
<p class="texte">In order to export the peptides outside the bacteria, the coding sequence of D4E1 and GAFP-1 was flanked on the N-terminal end with a signal peptide (amyE signal peptide) followed by a pro peptide, cleaved during the secretion process. </p> <br><br />
<br />
<br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/2014/2/2e/Secretion.jpg"><br />
<img style="width:400px; " src="https://static.igem.org/mediawiki/2014/d/d7/Fongpep.jpg"><br />
<br><br />
<br />
<br><br />
<br />
</P> <br />
<p class="title1"><b>References</b> </p><br />
<br />
<p class="texte"><br />
- A.J De Lucca, J.M Bland, C. Grimm, T.J Jacks.<b> Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1 </b>. Canadian Journal of Microbiology. 1998, Vol. 44:514-520. <br><br />
Kanniah Rajasekaran, Kurt D. Stromberg, Jeffrey W. Cary, and Thomas E. Cleveland.<b> Broad-Spectrum Antimicrobial Activity in vitro of the Synthetic Peptide D4E1</b>. J. Agric. Food Chem. 2001, Vol. 49, 2799-2803.<br><br />
-M. Visser, D. Stephan, J.M. Jaynes and J.T. Burger.<b> A transient expression assay for the in planta efficacy screening of an antimicrobial peptide against grapevine bacterial pathogens</b>. Letters in Applied Microbiology. 2012, Vol. 54, 543–551.<br><br />
-K. D. Cox, D. R. Layne, R. Scorza, G Schnabel. <b>Gastrodia anti-fungal protein from the orchid Gastrodia elata confers disease resistance to root pathogens in transgenic tobacco</b>. Planta. 2006, Vol. 224:1373–1383<br><br />
-Xiaochen Wang, Guy Bauw, Els J.M. Van Damme, Willy J. Peumans, Zhang-Liang Chen, Marc Van Montagu and Willy Dillen. <b>Gastrodianin-like mannose-binding proteins: a novel class of plant proteins with antifungal properties</b>. The Plant Journal. 2001, Vol. 25(6), 651±661<br><br />
-Svetlana B. Nolde, Alexander A. Vassilevski, Eugene A. Rogozhin, Nikolay A. Barinov, Tamara A. Balashova, Olga V. Samsonova, Yuri V. Baranov, Alexey S. Arseniev and Eugene V. Grishin. <b>Disulfide-stabilized Helical Hairpin Structure and Activity of a Novel Antifungal Peptide EcAMP1 from Seeds of Barnyard Grass (Echinochloa crus-galli)</b>. The journal of Biological Chemistry. 2011, Vol. 286, 25145–25153<br><br />
</p><br />
<br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Binding" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Binding<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Spreading" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Spreading</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Project/SpreadingTeam:Toulouse/Project/Spreading2014-10-12T13:59:06Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none; text-align: justify;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title2simple{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 5px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify;}<br />
<br />
.textesimple{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify;}<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/5/59/Wind_and_leaves.png') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<div class="banniere-content"><br />
<h2>Spreading</h2><br />
<p>How can SubtiTree respect the environment?<br> How do we keep control on SubtiTree?</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Spreading</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<br />
<p class="texte">Our engineered bacterium has been designed to be inoculated in a tree and to cure fungal diseases. Understanding the environmental issues resulting from the use of a modified organism in the nature, our team worked on different aspects in order to ensure a safe use of SubtiTree. <br />
The first objective is to avoid the spreading of our smart bacterium outside the tree. In other words, the purpose is to ensure that once SubtiTree is in the tree, it is unable to live anywhere else. Another issue concerns the horizontal transfers of the genetic material between different bacteria. <br />
Taking into account these issues, we thought about three modules.<br />
</p><br />
<br />
<div id="Spreading"><br />
<center><img alt="schema" style="width:700px; z-index:2; " src="https://static.igem.org/mediawiki/2014/7/74/Spreading.png"></img></center><br />
<a class="Auxotro" HREF="#Auxotrophy"></a><br />
<a class="NSporing" HREF="#NonSporing"></a><br />
<a class="Tox" HREF="#Toxin"></a><br />
</div><br />
<br />
<p class="title1">Using a glutamin auxotroph <i>B. subtilis</i> strain</p><br />
<p class="texte"><br />
To make the bacterium dependant on the tree and to avoid its spreading in the environment, it should be preferable to use a strain of <i>B.subtilis</i> which is auxotroph to a particular amino acid. The bacterium should be unable to synthesize one essential amino acid, and should find it in its environment. The glutamine could be a good example since it is wide-spread in the phloem sap. It is the amino acid which is present in highest concentration in the phloem sap. If our bacterium is unable to synthesize the glutamine, it will be obliged to take it in its close environment, that is to say the phloem sap. <br />
Thus, if the bacterium is in the sap, it can grow normally without any deficiency since it uses the glutamine present in the sap ; but if it escapes from the tree and a fortiori from the sap, it will not be able to survive for a long time. Indeed, glutamine is found in very low quantities in the ground. This system should guarantee that the bacterium develops only in the tree and not elsewhere in the surroundings of the tree.<br/> <br />
Auxotroph <i>B.sutbilis</i> strains already exist and are indexed in databases as BGSC (Bacillus Genetic Stock Center), so it is easy to find. <br />
</p><br />
<br />
<p class="title1">Preventing sporulation of <i>B. subtilis</i></p><br />
<p class="texte"><br />
In order to limit the spreading of our bacterium, we decided to limit its lifespan to only one season. The bacteria should be injected in spring, grow during the summer and finally should be inactivated in fall.<br\> <br />
<i>Bacillus subtilis</i> is a sporing bacterium : sporulation enable the microorganism to handle very harsh conditions and to spread tree to tree. Indeed, a spore is a very resistant form that is adapted for unfavorable conditions and for dispersal.<br/> <br />
To keep the control on the development of SubtiTree, our strain should therefore be non-sporing. Thus, after a season of treatment, the sape become less nutritious, the temperature is low and the engineered bacterium cannot survive the following winter.<br/><br />
In addition, deleting all the engineered bacterial community every year puts a brake on the evolution due to random mutation = to keep control on the genetic constructions <br />
</p><br />
<br />
<p class="title1">Using a toxin-antitoxin system</p><br />
<p class="texte"><br />
The goal of this module is to prevent horizontal transfer between bacteria. Indeed, it is necessary to avoid any exchange of genetic material between wild type organisms and optimized organisms : it could be dangerous because of mutations, and considering ethics, it seems to be essential to avoid the spreading of synthetic genes.<br/><br />
Considering this issue, we thought about a system to avoid such transfers : a toxin-antitoxin module. It involves the addition of two genes to the bacterium : a gene encoding for a toxin (for example tse2) and a gene encoding for the antitoxin (tsi1), placing them in an opposite way on the genome. The large space between them will permit to avoid simultaneous transfers : if the optimised bacterium transfers the gene encoding for the toxin, the probability that the gene encoding for the antitoxin may be transferred simultaneously is really low since they are located far away from each other.<br/><br />
Therefore, if the host bacterium receives the gene encoding for the toxin, it will be unable to survive since it will not have the antitoxin. If it receives the antitoxin only, it will not be useful for the bacterium, and will not affect it.<br/><br />
To sum up, since a simultaneous transfer is dimly probable, the bacterium will either die because of the toxin or live while expressing the antitoxin (useless). <br />
</p><br />
<br />
<p class="title1">Using integrative plasmids</p><br />
<p class="texte"><br />
All our constructions should be carried by integrative plasmids. Consequently, our different genetic modules would be integrated into the bacterium genome. The integration in the genome is more stable as the constructions are less likely to be transferred to other microorganisms. In addition to that, the expression of our genetic modules would not be dependant on a selective pressure based on an antibiotic resistance (as we can not inject antibiotics in the tree), allowing a high level of transcription in planta. <br />
</p><br />
<br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Fungicides" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Fungicides<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Modelling" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Modelling</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
<br />
<br />
<div class="clear"></div><br />
</div><br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:ToulouseTeam:Toulouse2014-10-12T13:44:02Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : font Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:#20a8da; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify; }<br />
<br />
/* Removing wiki-like stuff */<br />
/****************************/<br />
#contentSub, #search-controls, .firstHeading, #-box, #catlinks, #p-logo {<br />
display:none;}<br />
<br />
/* Redesigning the topmenu */<br />
/***************************/<br />
body {<br />
: 0 0 0 0;<br />
padding: 0px;}<br />
#top-section {<br />
width: 965px;<br />
height: 0;<br />
margin: 0 auto ;<br />
padding: 0;<br />
border: none;}<br />
#menubar {<br />
font-size: 100%;<br />
top:-8px;}<br />
.left-menu:hover {<br />
background-color: transparent;}<br />
#menubar li a {<br />
background-color: transparent;}<br />
#menubar:hover {<br />
color: black;}<br />
#menubar li a {<br />
color: transparent;}<br />
#menubar:hover li a {<br />
color: black;}<br />
#menubar > ul > li:last-child {<br />
display:none;}<br />
<br />
/* Layout */<br />
/**********/<br />
.l-content{margin-top:40px;}<br />
<br />
.l-content img{<br />
float:left;<br />
padding-top: 60px;<br />
width:290px;<br />
}<br />
<br />
.l-content .text-presentation{<br />
float:right;<br />
margin-top: -35px;<br />
width:610px<br />
}<br />
<br />
.l-content .text-presentation h1{<br />
margin-left: -108px;<br />
border:none;<br />
}<br />
<br />
/* ------------------------------------ gallerie ----*/<br />
<br />
.gallerie-project{<br />
margin:0;<br />
padding 0;<br />
margin-top:40px;<br />
}<br />
<br />
.line-gallerie-project{<br />
background-color:#f0f0f0;<br />
height:310px;<br />
width:100%;<br />
}<br />
<br />
.part-little-projet{<br />
background-color:#2ecc71;<br />
display: block;<br />
position: relative;<br />
height:310px;<br />
width: 310px;<br />
float:left;<br />
}<br />
<br />
.part-little-projet:hover{<br />
background-color:#1bad59;<br />
}<br />
<br />
.title-part-projet{<br />
position: absolute;<br />
top:40px;<br />
left:33px;<br />
font-size:24px;<br />
color:white;<br />
}<br />
<br />
.part-large-projet{<br />
background-color:#f0f0f0;<br />
display: block;<br />
position: relative;<br />
height:310px;<br />
width: 650px;<br />
float:left;<br />
}<br />
<br />
.part-large-projet:hover{<br />
background-color:#dfdfdf;<br />
}<br />
<br />
<br />
/* ------------------------------------ aside ----------*/<br />
<br />
.wrapper-aside{<br />
margin: 0 auto;<br />
width:740px;<br />
}<br />
<br />
.sticker-aside{<br />
background-color: #fff;<br />
border: 1px solid #e7e7e7;<br />
float:left;<br />
height:359px;<br />
padding: 5px 5px;<br />
position:relative;<br />
text-align:center;<br />
width:220px;<br />
}<br />
<br />
.sticker-aside:after{<br />
content: 'wiki/images/b/bb/Template-igem2014-before-stickers.png';<br />
position: absolute;<br />
background: url('https://static.igem.org/mediawiki/2014/b/bb/Template-igem2014-before-stickers.png') no-repeat center;<br />
bottom: -32px;<br />
text-indent: -9999px;<br />
right: -13px;<br />
width: 260px;<br />
height: 32px;<br />
}<br />
<br />
.sticker-aside h2{<br />
border:none;<br />
color:#181818;<br />
font-size:20px;<br />
}<br />
<br />
.sticker-aside p{<br />
color:#888888;<br />
display: block;<br />
height: 40px;<br />
position: relative;<br />
bottom: 15px<br />
}<br />
<br />
.sticker-aside a{<br />
display: block;<br />
margin: 0 auto;<br />
top: 5px;<br />
position: relative;<br />
}<br />
<br />
<br />
<br />
</style><br />
<!-------------------------------- CONTENT ---------------------------------><br />
<br />
<div class="l-content"><br />
<br />
<div class="presentation-project"><br />
<img src="https://static.igem.org/mediawiki/2014/b/b6/Canal_du_Midi_-_plane_trees.jpeg" alt="Présentation igem toulouse" /><br />
<div class="text-presentation"><br />
<h1 class="title2" style="font-size:28px;"><br />
SubtiTree<br />
</h1><br />
<p class="texte"><br />
Fungal diseases lead to major economic losses all over the world. Some pathogens trigger trachemycosis by disturbing the vascular system of the plant. Canker is one of these infections especially affecting plane trees (Platanus sp.). Plane trees are widely present in Southern France and in particular along the famous Canal du Midi, participating to the site gorgeousness. Today, the only treatment consists in preventive tree-cutting which has a huge cost and implies significant ecological troubles. Facing this emergency, our team proposes an innovative solution originated from synthetic biology. <br />
</p><br />
<a class="button-home" href="https://2014.igem.org/Team:Toulouse/Project/project-context" style="border: 1px solid #282828;-webkit-border-radius: 5px;-moz-border-radius: 5px;border-radius: 5px; padding: 13px 25px 12px; color: #282828; text-decoration: none; font-size: 17px; background: none; display: block; width: 89px;">Learn more</a><br />
</div><br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
<div class="gallerie-project" style="margin-bottom:50px; margin-top:70px;"><br />
<br />
<div class="line-gallerie-project"><br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Overviews" class="part-little-projet"><br />
<div class="title-part-projet">Overwiew</div><br />
<div style="position:absolute; top:90px; left:34px; color:white; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px;">Let's save our trees with SubtiTree!</div><br />
<div style="border-bottom: 1px solid #fff; color:white; font-family:'Open Sans'; position:absolute; bottom:25px; right:22px; font-size:16px;">Read more</div><br />
</a><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Chemotaxis" class="part-large-projet"><br />
<div class="title-part-projet" style="color:#515553; ">Chemotaxis</div><br />
<div style="position:absolute; top:90px; left:50px; color:#515553; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px;">To target the pathogenic fungus</div><br />
<div style="border-bottom: 1px solid #515553; color:#515553; font-family:'Open Sans'; position:absolute; bottom:25px; left:50px; font-size:16px;">Read more</div><br />
</a><br />
</div><br />
<br />
<div class="line-gallerie-project"><br />
<a href="https://2014.igem.org/Team:Toulouse/Project/binding" class="part-large-projet"><br />
<div class="title-part-projet style="color:black; ">Binding</div><br />
<div style="position:absolute; top:90px; left:34px; color:#515553; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px">To be attached to the fungal pathogen wall</div><br />
<div style="border-bottom: 1px solid #515553; color:#515553; font-family:'Open Sans'; position:absolute; bottom:28px; right:28px; font-size:16px;">Read more</div><br />
</a><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Modelling" class="part-little-projet"><br />
<div class="title-part-projet">Modelling</div><br />
<div style="position:absolute; top:90px; left:34px; color:white; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px">To develop a predictive model</div><br />
<div style="border-bottom: 1px solid #fff; color:white; font-family:'Open Sans'; position:absolute; bottom:28px; right:22px; font-size:16px;">Read more</div><br />
</a><br />
</div><br />
<br />
<div class="line-gallerie-project"><br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Spreading" class="part-little-projet"><br />
<div class="title-part-projet">Spreading</div><br />
<div style="position:absolute; top:90px; left:34px; color:white; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px">How can SubtiTree respect the environment? How do we keep control on Subtitree?</div><br />
<div style="border-bottom: 1px solid #fff; color:white; font-family:'Open Sans'; position:absolute; bottom:25px; right:22px; font-size:16px;"">Read more</div><br />
</a><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Fungicides" class="part-large-projet"><br />
<div class="title-part-projet">Fungicides</div><br />
<div style="position:absolute; top:90px; left:50px; color:#515553; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px;">To eradicate fungal diseases</div><br />
<div style="border-bottom: 1px solid #515553; color:#515553; font-family:'Open Sans'; position:absolute; bottom:25px; right:22px; font-size:16px;">Read more</div><br />
</a><br />
</div><br />
<br />
</div><br />
<br />
</div><br />
<br />
</div><br />
<br />
</div><br />
<br />
<br />
<!-------------------------------- ASIDE ---------------------------------><br />
<br />
<div id="aside" style="width:100%; height:572px; background-color:#f9f9f9; border: 1px solid #ccc;"><br />
<br />
<div class="wrapper-aside"><br />
<br />
<h2 style="color:#033071; text-align:center; border:none; font-weight:bold; margin:30px 0px 50px 0px;">HUMAN PRACTICE</h2><br />
<br />
<div class="sticker-aside" style="margin-right:20px;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/67/Template-igem2014-HP-1.jpg" alt="image safety" /><br />
<h2>Safety</h2><br />
<p>Safety is not just a slogan, it is a way of life.</p><br />
<a href="https://2014.igem.org/Team:Toulouse/Safety"><img src="https://static.igem.org/mediawiki/2014/6/6f/Template-igem2014-iconeHP.png"/></a><br />
</div><br />
<br />
<div class="sticker-aside" style="margin-right:20px;"><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Ethics.jpg" alt="" /><br />
<h2>Ethics</h2><br />
<p>Ethics are more important than laws.</p><br />
<a href="https://2014.igem.org/Team:Toulouse/ethics"><img src="https://static.igem.org/mediawiki/2014/6/6f/Template-igem2014-iconeHP.png"/></a><br />
</div><br />
<br />
<div class="sticker-aside" style="position: relative;bottom:19px;"><br />
<img src="https://static.igem.org/mediawiki/2014/2/20/Bacteria_talk.png" alt="" style="margin-top:26px;" /><br />
<h2>Communication</h2><br />
<p>Good words are worth much, and cost little.</p><br />
<a href="https://2014.igem.org/Team:Toulouse/Communication"><img src="https://static.igem.org/mediawiki/2014/6/6f/Template-igem2014-iconeHP.png"/></a><br />
</div><br />
<br />
</div> <br />
</div><br />
<div class="clear"></div><br />
<!-------------------------------- SPONSOR ---------------------------------><br />
<div class="Sponsor" style="margin:0 auto; width:960px; padding:60px 0 70px;"><p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d5/Toulouse_sponsors.png" alt="Bandeau Sponsor"></p><br />
</div><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
</html><br />
<br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:ToulouseTeam:Toulouse2014-10-12T13:43:39Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : font Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:#20a8da; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify; }<br />
<br />
/* Removing wiki-like stuff */<br />
/****************************/<br />
#contentSub, #search-controls, .firstHeading, #-box, #catlinks, #p-logo {<br />
display:none;}<br />
<br />
/* Redesigning the topmenu */<br />
/***************************/<br />
body {<br />
: 0 0 0 0;<br />
padding: 0px;}<br />
#top-section {<br />
width: 965px;<br />
height: 0;<br />
margin: 0 auto ;<br />
padding: 0;<br />
border: none;}<br />
#menubar {<br />
font-size: 100%;<br />
top:-8px;}<br />
.left-menu:hover {<br />
background-color: transparent;}<br />
#menubar li a {<br />
background-color: transparent;}<br />
#menubar:hover {<br />
color: black;}<br />
#menubar li a {<br />
color: transparent;}<br />
#menubar:hover li a {<br />
color: black;}<br />
#menubar > ul > li:last-child {<br />
display:none;}<br />
<br />
/* Layout */<br />
/**********/<br />
.l-content{margin-top:40px;}<br />
<br />
.l-content img{<br />
float:left;<br />
padding-top: 60px;<br />
width:290px;<br />
}<br />
<br />
.l-content .text-presentation{<br />
float:right;<br />
margin-top: -35px;<br />
width:610px<br />
}<br />
<br />
.l-content .text-presentation h1{<br />
margin-left: -108px;<br />
border:none;<br />
}<br />
<br />
/* ------------------------------------ gallerie ----*/<br />
<br />
.gallerie-project{<br />
margin:0;<br />
padding 0;<br />
margin-top:40px;<br />
}<br />
<br />
.line-gallerie-project{<br />
background-color:#f0f0f0;<br />
height:310px;<br />
width:100%;<br />
}<br />
<br />
.part-little-projet{<br />
background-color:#2ecc71;<br />
display: block;<br />
position: relative;<br />
height:310px;<br />
width: 310px;<br />
float:left;<br />
}<br />
<br />
.part-little-projet:hover{<br />
background-color:#1bad59;<br />
}<br />
<br />
.title-part-projet{<br />
position: absolute;<br />
top:40px;<br />
left:33px;<br />
font-size:24px;<br />
color:white;<br />
}<br />
<br />
.part-large-projet{<br />
background-color:#f0f0f0;<br />
display: block;<br />
position: relative;<br />
height:310px;<br />
width: 650px;<br />
float:left;<br />
}<br />
<br />
.part-large-projet:hover{<br />
background-color:#dfdfdf;<br />
}<br />
<br />
<br />
/* ------------------------------------ aside ----------*/<br />
<br />
.wrapper-aside{<br />
margin: 0 auto;<br />
width:740px;<br />
}<br />
<br />
.sticker-aside{<br />
background-color: #fff;<br />
border: 1px solid #e7e7e7;<br />
float:left;<br />
height:359px;<br />
padding: 5px 5px;<br />
position:relative;<br />
text-align:center;<br />
width:220px;<br />
}<br />
<br />
.sticker-aside:after{<br />
content: 'wiki/images/b/bb/Template-igem2014-before-stickers.png';<br />
position: absolute;<br />
background: url('https://static.igem.org/mediawiki/2014/b/bb/Template-igem2014-before-stickers.png') no-repeat center;<br />
bottom: -32px;<br />
text-indent: -9999px;<br />
right: -13px;<br />
width: 260px;<br />
height: 32px;<br />
}<br />
<br />
.sticker-aside h2{<br />
border:none;<br />
color:#181818;<br />
font-size:20px;<br />
}<br />
<br />
.sticker-aside p{<br />
color:#888888;<br />
display: block;<br />
height: 40px;<br />
position: relative;<br />
bottom: 15px<br />
}<br />
<br />
.sticker-aside a{<br />
display: block;<br />
margin: 0 auto;<br />
top: 5px;<br />
position: relative;<br />
}<br />
<br />
<br />
<br />
</style><br />
<!-------------------------------- CONTENT ---------------------------------><br />
<br />
<div class="l-content"><br />
<br />
<div class="presentation-project"><br />
<img src="https://static.igem.org/mediawiki/2014/b/b6/Canal_du_Midi_-_plane_trees.jpeg" alt="Présentation igem toulouse" /><br />
<div class="text-presentation"><br />
<h1 class="title2" style="font-size:28px;"><br />
SubtiTree<br />
</h1><br />
<p class="texte"><br />
Fungal diseases lead to major economic losses all over the world. Some pathogens trigger trachemycosis by disturbing the vascular system of the plant. Canker is one of these infections especially affecting plane trees (Platanus sp.). Plane trees are widely present in Southern France and in particular along the famous Canal du Midi, participating to the site gorgeousness. Today, the only treatment consists in preventive tree-cutting which has a huge cost and implies significant ecological troubles. Facing this emergency, our team proposes an innovative solution originated from synthetic biology. <br />
</p><br />
<a class="button-home" href="https://2014.igem.org/Team:Toulouse/Project/project-context" style="border: 1px solid #282828;-webkit-border-radius: 5px;-moz-border-radius: 5px;border-radius: 5px; padding: 13px 25px 12px; color: #282828; text-decoration: none; font-size: 17px; background: none; display: block; width: 89px;">Learn more</a><br />
</div><br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
<div class="gallerie-project" style="margin-bottom:50px; margin-top:70px;"><br />
<br />
<div class="line-gallerie-project"><br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Overviews" class="part-little-projet"><br />
<div class="title-part-projet">Overwiew</div><br />
<div style="position:absolute; top:90px; left:34px; color:white; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px;">Let's save our trees with SubtiTree!</div><br />
<div style="border-bottom: 1px solid #fff; color:white; font-family:'Open Sans'; position:absolute; bottom:25px; right:22px; font-size:16px;">Read more</div><br />
</a><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Chemotaxis" class="part-large-projet"><br />
<div class="title-part-projet" style="color:#515553; ">Chemotaxis</div><br />
<div style="position:absolute; top:90px; left:50px; color:#515553; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px;">To target the pathogenic fungus</div><br />
<div style="border-bottom: 1px solid #515553; color:#515553; font-family:'Open Sans'; position:absolute; bottom:25px; left:50px; font-size:16px;">Read more</div><br />
</a><br />
</div><br />
<br />
<div class="line-gallerie-project"><br />
<a href="https://2014.igem.org/Team:Toulouse/Project/binding" class="part-large-projet"><br />
<div class="title-part-projet style="color:#515553; ">Binding</div><br />
<div style="position:absolute; top:90px; left:34px; color:#515553; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px">To be attached to the fungal pathogen wall</div><br />
<div style="border-bottom: 1px solid #515553; color:#515553; font-family:'Open Sans'; position:absolute; bottom:28px; right:28px; font-size:16px;">Read more</div><br />
</a><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Modelling" class="part-little-projet"><br />
<div class="title-part-projet">Modelling</div><br />
<div style="position:absolute; top:90px; left:34px; color:white; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px">To develop a predictive model</div><br />
<div style="border-bottom: 1px solid #fff; color:white; font-family:'Open Sans'; position:absolute; bottom:28px; right:22px; font-size:16px;">Read more</div><br />
</a><br />
</div><br />
<br />
<div class="line-gallerie-project"><br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Spreading" class="part-little-projet"><br />
<div class="title-part-projet">Spreading</div><br />
<div style="position:absolute; top:90px; left:34px; color:white; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px">How can SubtiTree respect the environment? How do we keep control on Subtitree?</div><br />
<div style="border-bottom: 1px solid #fff; color:white; font-family:'Open Sans'; position:absolute; bottom:25px; right:22px; font-size:16px;"">Read more</div><br />
</a><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Fungicides" class="part-large-projet"><br />
<div class="title-part-projet">Fungicides</div><br />
<div style="position:absolute; top:90px; left:50px; color:#515553; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px;">To eradicate fungal diseases</div><br />
<div style="border-bottom: 1px solid #515553; color:#515553; font-family:'Open Sans'; position:absolute; bottom:25px; right:22px; font-size:16px;">Read more</div><br />
</a><br />
</div><br />
<br />
</div><br />
<br />
</div><br />
<br />
</div><br />
<br />
</div><br />
<br />
<br />
<!-------------------------------- ASIDE ---------------------------------><br />
<br />
<div id="aside" style="width:100%; height:572px; background-color:#f9f9f9; border: 1px solid #ccc;"><br />
<br />
<div class="wrapper-aside"><br />
<br />
<h2 style="color:#033071; text-align:center; border:none; font-weight:bold; margin:30px 0px 50px 0px;">HUMAN PRACTICE</h2><br />
<br />
<div class="sticker-aside" style="margin-right:20px;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/67/Template-igem2014-HP-1.jpg" alt="image safety" /><br />
<h2>Safety</h2><br />
<p>Safety is not just a slogan, it is a way of life.</p><br />
<a href="https://2014.igem.org/Team:Toulouse/Safety"><img src="https://static.igem.org/mediawiki/2014/6/6f/Template-igem2014-iconeHP.png"/></a><br />
</div><br />
<br />
<div class="sticker-aside" style="margin-right:20px;"><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Ethics.jpg" alt="" /><br />
<h2>Ethics</h2><br />
<p>Ethics are more important than laws.</p><br />
<a href="https://2014.igem.org/Team:Toulouse/ethics"><img src="https://static.igem.org/mediawiki/2014/6/6f/Template-igem2014-iconeHP.png"/></a><br />
</div><br />
<br />
<div class="sticker-aside" style="position: relative;bottom:19px;"><br />
<img src="https://static.igem.org/mediawiki/2014/2/20/Bacteria_talk.png" alt="" style="margin-top:26px;" /><br />
<h2>Communication</h2><br />
<p>Good words are worth much, and cost little.</p><br />
<a href="https://2014.igem.org/Team:Toulouse/Communication"><img src="https://static.igem.org/mediawiki/2014/6/6f/Template-igem2014-iconeHP.png"/></a><br />
</div><br />
<br />
</div> <br />
</div><br />
<div class="clear"></div><br />
<!-------------------------------- SPONSOR ---------------------------------><br />
<div class="Sponsor" style="margin:0 auto; width:960px; padding:60px 0 70px;"><p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d5/Toulouse_sponsors.png" alt="Bandeau Sponsor"></p><br />
</div><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
</html><br />
<br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:ToulouseTeam:Toulouse2014-10-12T13:42:38Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : font Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:#20a8da; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 0 0; line-height:24px; text-align: justify; }<br />
<br />
/* Removing wiki-like stuff */<br />
/****************************/<br />
#contentSub, #search-controls, .firstHeading, #-box, #catlinks, #p-logo {<br />
display:none;}<br />
<br />
/* Redesigning the topmenu */<br />
/***************************/<br />
body {<br />
: 0 0 0 0;<br />
padding: 0px;}<br />
#top-section {<br />
width: 965px;<br />
height: 0;<br />
margin: 0 auto ;<br />
padding: 0;<br />
border: none;}<br />
#menubar {<br />
font-size: 100%;<br />
top:-8px;}<br />
.left-menu:hover {<br />
background-color: transparent;}<br />
#menubar li a {<br />
background-color: transparent;}<br />
#menubar:hover {<br />
color: black;}<br />
#menubar li a {<br />
color: transparent;}<br />
#menubar:hover li a {<br />
color: black;}<br />
#menubar > ul > li:last-child {<br />
display:none;}<br />
<br />
/* Layout */<br />
/**********/<br />
.l-content{margin-top:40px;}<br />
<br />
.l-content img{<br />
float:left;<br />
padding-top: 60px;<br />
width:290px;<br />
}<br />
<br />
.l-content .text-presentation{<br />
float:right;<br />
margin-top: -35px;<br />
width:610px<br />
}<br />
<br />
.l-content .text-presentation h1{<br />
margin-left: -108px;<br />
border:none;<br />
}<br />
<br />
/* ------------------------------------ gallerie ----*/<br />
<br />
.gallerie-project{<br />
margin:0;<br />
padding 0;<br />
margin-top:40px;<br />
}<br />
<br />
.line-gallerie-project{<br />
background-color:#f0f0f0;<br />
height:310px;<br />
width:100%;<br />
}<br />
<br />
.part-little-projet{<br />
background-color:#2ecc71;<br />
display: block;<br />
position: relative;<br />
height:310px;<br />
width: 310px;<br />
float:left;<br />
}<br />
<br />
.part-little-projet:hover{<br />
background-color:#1bad59;<br />
}<br />
<br />
.title-part-projet{<br />
position: absolute;<br />
top:40px;<br />
left:33px;<br />
font-size:24px;<br />
color:white;<br />
}<br />
<br />
.part-large-projet{<br />
background-color:#f0f0f0;<br />
display: block;<br />
position: relative;<br />
height:310px;<br />
width: 650px;<br />
float:left;<br />
}<br />
<br />
.part-large-projet:hover{<br />
background-color:#dfdfdf;<br />
}<br />
<br />
<br />
/* ------------------------------------ aside ----------*/<br />
<br />
.wrapper-aside{<br />
margin: 0 auto;<br />
width:740px;<br />
}<br />
<br />
.sticker-aside{<br />
background-color: #fff;<br />
border: 1px solid #e7e7e7;<br />
float:left;<br />
height:359px;<br />
padding: 5px 5px;<br />
position:relative;<br />
text-align:center;<br />
width:220px;<br />
}<br />
<br />
.sticker-aside:after{<br />
content: 'wiki/images/b/bb/Template-igem2014-before-stickers.png';<br />
position: absolute;<br />
background: url('https://static.igem.org/mediawiki/2014/b/bb/Template-igem2014-before-stickers.png') no-repeat center;<br />
bottom: -32px;<br />
text-indent: -9999px;<br />
right: -13px;<br />
width: 260px;<br />
height: 32px;<br />
}<br />
<br />
.sticker-aside h2{<br />
border:none;<br />
color:#181818;<br />
font-size:20px;<br />
}<br />
<br />
.sticker-aside p{<br />
color:#888888;<br />
display: block;<br />
height: 40px;<br />
position: relative;<br />
bottom: 15px<br />
}<br />
<br />
.sticker-aside a{<br />
display: block;<br />
margin: 0 auto;<br />
top: 5px;<br />
position: relative;<br />
}<br />
<br />
<br />
<br />
</style><br />
<!-------------------------------- CONTENT ---------------------------------><br />
<br />
<div class="l-content"><br />
<br />
<div class="presentation-project"><br />
<img src="https://static.igem.org/mediawiki/2014/b/b6/Canal_du_Midi_-_plane_trees.jpeg" alt="Présentation igem toulouse" /><br />
<div class="text-presentation"><br />
<h1 class="title2" style="font-size:28px;"><br />
SubtiTree<br />
</h1><br />
<p class="texte"><br />
Fungal diseases lead to major economic losses all over the world. Some pathogens trigger trachemycosis by disturbing the vascular system of the plant. Canker is one of these infections especially affecting plane trees (Platanus sp.). Plane trees are widely present in Southern France and in particular along the famous Canal du Midi, participating to the site gorgeousness. Today, the only treatment consists in preventive tree-cutting which has a huge cost and implies significant ecological troubles. Facing this emergency, our team proposes an innovative solution originated from synthetic biology. <br />
</p><br />
<a class="button-home" href="https://2014.igem.org/Team:Toulouse/Project/project-context" style="border: 1px solid #282828;-webkit-border-radius: 5px;-moz-border-radius: 5px;border-radius: 5px; padding: 13px 25px 12px; color: #282828; text-decoration: none; font-size: 17px; background: none; display: block; width: 89px;">Learn more</a><br />
</div><br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
<div class="gallerie-project" style="margin-bottom:50px; margin-top:70px;"><br />
<br />
<div class="line-gallerie-project"><br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Overviews" class="part-little-projet"><br />
<div class="title-part-projet">Overwiew</div><br />
<div style="position:absolute; top:90px; left:34px; color:white; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px;">Let's save our trees with SubtiTree!</div><br />
<div style="border-bottom: 1px solid #fff; color:white; font-family:'Open Sans'; position:absolute; bottom:25px; right:22px; font-size:16px;">Read more</div><br />
</a><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Chemotaxis" class="part-large-projet"><br />
<div class="title-part-projet" style="color:#515553; ">Chemotaxis</div><br />
<div style="position:absolute; top:90px; left:50px; color:#515553; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px;">To target the pathogenic fungus</div><br />
<div style="border-bottom: 1px solid #515553; color:#515553; font-family:'Open Sans'; position:absolute; bottom:25px; left:50px; font-size:16px;">Read more</div><br />
</a><br />
</div><br />
<br />
<div class="line-gallerie-project"><br />
<a href="https://2014.igem.org/Team:Toulouse/Project/binding" class="part-large-projet"><br />
<div class="title-part-projet style="color:black; ">Binding</div><br />
<div style="position:absolute; top:90px; left:34px; color:#515553; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px">To be attached to the fungal pathogen wall</div><br />
<div style="border-bottom: 1px solid #515553; color:#515553; font-family:'Open Sans'; position:absolute; bottom:28px; right:28px; font-size:16px;">Read more</div><br />
</a><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Modelling" class="part-little-projet"><br />
<div class="title-part-projet">Modelling</div><br />
<div style="position:absolute; top:90px; left:34px; color:white; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px">To develop a predictive model</div><br />
<div style="border-bottom: 1px solid #fff; color:white; font-family:'Open Sans'; position:absolute; bottom:28px; right:22px; font-size:16px;">Read more</div><br />
</a><br />
</div><br />
<br />
<div class="line-gallerie-project"><br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Spreading" class="part-little-projet"><br />
<div class="title-part-projet">Spreading</div><br />
<div style="position:absolute; top:90px; left:34px; color:white; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px">How can SubtiTree respect the environment? How do we keep control on Subtitree?</div><br />
<div style="border-bottom: 1px solid #fff; color:white; font-family:'Open Sans'; position:absolute; bottom:25px; right:22px; font-size:16px;"">Read more</div><br />
</a><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Fungicides" class="part-large-projet"><br />
<div class="title-part-projet">Fungicides</div><br />
<div style="position:absolute; top:90px; left:50px; color:#515553; font-family:'Open Sans'; font-size:16px; width:200px; line-height:24px;">To eradicate fungal diseases</div><br />
<div style="border-bottom: 1px solid #515553; color:#515553; font-family:'Open Sans'; position:absolute; bottom:25px; right:22px; font-size:16px;">Read more</div><br />
</a><br />
</div><br />
<br />
</div><br />
<br />
</div><br />
<br />
</div><br />
<br />
</div><br />
<br />
<br />
<!-------------------------------- ASIDE ---------------------------------><br />
<br />
<div id="aside" style="width:100%; height:572px; background-color:#f9f9f9; border: 1px solid #ccc;"><br />
<br />
<div class="wrapper-aside"><br />
<br />
<h2 style="color:#033071; text-align:center; border:none; font-weight:bold; margin:30px 0px 50px 0px;">HUMAN PRACTICE</h2><br />
<br />
<div class="sticker-aside" style="margin-right:20px;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/67/Template-igem2014-HP-1.jpg" alt="image safety" /><br />
<h2>Safety</h2><br />
<p>Safety is not just a slogan, it is a way of life.</p><br />
<a href="https://2014.igem.org/Team:Toulouse/Safety"><img src="https://static.igem.org/mediawiki/2014/6/6f/Template-igem2014-iconeHP.png"/></a><br />
</div><br />
<br />
<div class="sticker-aside" style="margin-right:20px;"><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Ethics.jpg" alt="" /><br />
<h2>Ethics</h2><br />
<p>Ethics are more important than laws.</p><br />
<a href="https://2014.igem.org/Team:Toulouse/ethics"><img src="https://static.igem.org/mediawiki/2014/6/6f/Template-igem2014-iconeHP.png"/></a><br />
</div><br />
<br />
<div class="sticker-aside" style="position: relative;bottom:19px;"><br />
<img src="https://static.igem.org/mediawiki/2014/2/20/Bacteria_talk.png" alt="" style="margin-top:26px;" /><br />
<h2>Communication</h2><br />
<p>Good words are worth much, and cost little.</p><br />
<a href="https://2014.igem.org/Team:Toulouse/Communication"><img src="https://static.igem.org/mediawiki/2014/6/6f/Template-igem2014-iconeHP.png"/></a><br />
</div><br />
<br />
</div> <br />
</div><br />
<div class="clear"></div><br />
<!-------------------------------- SPONSOR ---------------------------------><br />
<div class="Sponsor" style="margin:0 auto; width:960px; padding:60px 0 70px;"><p align="center"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d5/Toulouse_sponsors.png" alt="Bandeau Sponsor"></p><br />
</div><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
</html><br />
<br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Project/ChemotaxisTeam:Toulouse/Project/Chemotaxis2014-10-12T13:36:40Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:#20a8da; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 50px 0; line-height:24px; text-align: justify; }<br />
<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/a/aa/Chimio.jpg') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Chemotaxis</h2><br />
<p>To target the pathogenic fungus</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Chemotaxis</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<!--Short description : à changer!!!--><br />
<img style="width:420px; " src="https://static.igem.org/mediawiki/parts/e/e9/Recap_chemotax.jpg"><br />
<br />
<p class="title1"> </p><br />
<br />
<p class="texte">Coming soon!</p><br />
<br />
<p class="title1"> </p><br />
<br />
<p class="texte"> </p><br />
<br />
<p class="title1"> </p><br />
<br />
<p class="texte"></p> <br />
<br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Overviews" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Overview<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/binding" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Binding</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Project/project-contextTeam:Toulouse/Project/project-context2014-10-12T13:27:31Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 35px 0; line-height:24px; text-align: justify; }<br />
<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/2/2a/Contexte.jpg') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Project context</h2><br />
<p>A threatened heritage</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Context project</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<!--Short description : à changer!!!--><br />
<br />
<p class="texte">One of Europe Wonders is without a doubt the Toulouse Canal du Midi. Situated in the heart of our city and going through Southern France, our lab is only 5 minutes walk from the Canal du Midi. Everyone in the team could enjoy the quietness and loveliness of this charming place. Unfortunately, this heritage is threatened: a phytopathogenic fugus, <i>Ceratocystis platani</i>, is devastating the trees lining the Canal.<br />
</p><br />
<br />
<p class="texte">The situation is alarming: today the only solution is preventive tree cutting to stem the fungal epidemic. The consequences are disastrous. Not only for the beauty of the landscapes but it has also huge environmental, social and economic costs. Today, 42.000 plane trees are threatened, knowing that cutting and planting a new resistant tree is about €4,000.<br />
</p><br />
<br />
<p class="title1">The Canal du midi</p> <br />
<p class="texte">This ingenious masterpiece respects the environment in which it is harmoniously integrated since 1681 by Pierre-Paul Riquet.</br><br />
Characterized as a summit level canal, it culminates at 189 meters of altitude and permits the achievement of water through the sea.</br><br />
In December 7th, 1996, the UNESCO registers Le Canal du Midi on the list of world’s legacy.</br><br />
The canal du midi is today primarily used for tourism, recreation and housing. Busier than the Seine (Paris), it accounts for one fifth of French river tourism and 80 % tourists are foreigners. Boaters seeking for tranquility, quietness and a unique environment mainly navigate over it.<br />
</p><br />
<br />
<p class="title1">Canker Stain</p> <br />
<p class="texte">The first plane trees bordering the channel appeared lately after its creation in 1776. Resolving the erosion of riverbanks and the evaporation of the water, the plane imposes itself as a dominant and emblematic marrow. The wooded legacy is estimated to about 42.000 trees, it contributes to the landscape and aesthetic.<br />
The canker stain is a disease caused by <i>Ceratocystis platani</i>, a microscopic fungus that exclusively attacks Plane Trees.</br><br />
Probably introduced in France by infected wooden munitions cases coming from United States of America in 1945, the fungus introduces itself inside the sain tree’s heart, blocks the sap flow and kills it in only 2 to 5 years.<br />
On the regulatory side, the July 31th 2000 modified ministerial order classes <i>Ceratocystis</i> as a harmful organism for plants. This order leads to a mandatory fight against the fungus.<br />
</p><br />
<br />
<p class="title1">Fighting the plane trees pathogen</p> <br />
<p class="texte">Regarding this imminent threat, some solutions emerged.<br />
</br>First of all, chemical fungicides were used. However, the community realized it was impossible to eradicate the whole fungus. <br />
</br>Therefore, the French Institut National de Recherche Agronomique (INRA) created a new type of plane trees named Platanor which were resistant to the fungus infection. Unfortunately, this extreme measure could not save the already contaminated trees nearby the Canal du Midi.<br />
</br>Today, the only option remains in the tree-cutting of the contaminated plane trees. Nevertheless, this previous solution is not optimal because of the high cost and the major ecological, touristic and social impact for our heritage.<br />
</p><br />
<br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Home<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Overviews" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Overview</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Project/project-contextTeam:Toulouse/Project/project-context2014-10-12T13:26:33Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 35px 0; line-height:24px; text-align: justify; }<br />
<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/2/2a/Contexte.jpg') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Project context</h2><br />
<p>A threatened heritage</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Context project</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<!--Short description : à changer!!!--><br />
<br />
<p class="texte">One of Europe Wonders is without a doubt the Toulouse Canal du Midi. Situated in the heart of our city and going through Southern France, our lab is only 5 minutes walk from the Canal du Midi. Everyone in the team could enjoy the quietness and loveliness of this charming place. Unfortunately, this heritage is threatened: a phytopathogenic fugus, <i>Ceratocystis platani</i>, is devastating the trees lining the Canal.<br />
</p><br />
<br />
<p class="texte">The situation is alarming: today the only solution is preventive tree cutting to stem the fungal epidemic. The consequences are disastrous. Not only for the beauty of the landscapes but it has also huge environmental, social and economic costs. Today, 42.000 plane trees are threatened, knowing that cutting and planting a new resistant tree is about €4,000.<br />
</p><br />
<br />
<p class="title1">The Canal du midi</p> <br />
<p class="texte">This ingenious masterpiece respects the environment in which it is harmoniously integrated since 1681 by Pierre-Paul Riquet.</br><br />
Characterized as a summit level canal, it culminates at 189 meters of altitude and permits the achievement of water through the sea.</br><br />
In December 7th, 1996, the UNESCO registers Le Canal du Midi on the list of world’s legacy.</br><br />
The canal du midi is today primarily used for tourism, recreation and housing. Busier than the Seine (Paris), it accounts for one fifth of French river tourism and 80 % tourists are foreigners. Boaters seeking for tranquility, quietness and a unique environment mainly navigate over it.<br />
</p><br />
<br />
<p class="title1">Canker Stain</p> <br />
<p class="texte">The first plane trees bordering the channel appeared lately after its creation in 1776. Resolving the erosion of riverbanks and the evaporation of the water, the plane imposes itself as a dominant and emblematic marrow. The wooded legacy is estimated to about 42.000 trees, it contributes to the landscape and aesthetic.<br />
The canker stain is a disease caused by Ceratocystis platani, a microscopic fungus that exclusively attacks Plane Trees.</br><br />
Probably introduced in France by infected wooden munitions cases coming from United States of America in 1945, the fungus introduces itself inside the sain tree’s heart, blocks the sap flow and kills it in only 2 to 5 years.<br />
On the regulatory side, the July 31th 2000 modified ministerial order classes Ceratocystis as a harmful organism for plants. This order leads to a mandatory fight against the fungus.<br />
</p><br />
<br />
<p class="title1">Fighting the plane trees pathogen</p> <br />
<p class="texte">Regarding this imminent threat, some solutions emerged.<br />
</br>First of all, chemical fungicides were used. However, the community realized it was impossible to eradicate the whole fungus. <br />
</br>Therefore, the French Institut National de Recherche Agronomique (INRA) created a new type of plane trees named Platanor which were resistant to the fungus infection. Unfortunately, this extreme measure could not save the already contaminated trees nearby the Canal du Midi.<br />
</br>Today, the only option remains in the tree-cutting of the contaminated plane trees. Nevertheless, this previous solution is not optimal because of the high cost and the major ecological, touristic and social impact for our heritage.<br />
</p><br />
<br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Home<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Overviews" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Overview</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineauhttp://2014.igem.org/Team:Toulouse/Project/project-contextTeam:Toulouse/Project/project-context2014-10-12T13:25:36Z<p>FannyPineau: </p>
<hr />
<div>{{:Team:Toulouse/template/header-base}}<br />
<br />
<html><br />
<br />
<!--/* open Sans : fonnt Google*/--><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:400,600' rel='stylesheet' type='text/css'><br />
<br />
<link href='http://fonts.googleapis.com/css?family=Open+Sans:800' rel='stylesheet' type='text/css'><br />
<br />
<!--/* CSS contenu*/--><br />
<br />
<style type="text/css"><br />
<br />
.title1{color:green; font-family:'Open Sans'; font-weight:600; font-size:24px; margin:0 0 33px 0; border:none;}<br />
<br />
.title2{color:#5a6060; font-family:'Open Sans'; font-weight:600; font-size:18px; margin:0 0 30px 0; border:none;}<br />
<br />
.title3{color:#7f8c8c; font-family:'Open Sans'; font-weight:400; font-size:16px; margin:0 0 20px 0; border:none;}<br />
<br />
.texte{color:#5a6060; font-family:'Open Sans'; font-size:14px; margin:0 0 35px 0; line-height:24px; text-align: justify; }<br />
<br />
<br />
.banniere{<br />
background: url('https://static.igem.org/mediawiki/2014/2/2a/Contexte.jpg') no-repeat center;<br />
-webkit-background-size: cover; /* pour Chrome et Safari */<br />
-moz-background-size: cover; /* pour Firefox */<br />
-o-background-size: cover; /* pour Opera */<br />
background-size: cover;<br />
height:315px;<br />
margin: 30px 0 0;<br />
width:100%;<br />
}<br />
<br />
.banniere-content{<br />
background-color: rgba(46,204,113, 0.6);<br />
padding:28px 28px 0;<br />
position:absolute;<br />
bottom:0;<br />
right: 0;<br />
height:92px;<br />
width:392px;<br />
color:white<br />
}<br />
<br />
.banniere-content h2{<br />
color:white;<br />
font-size:24px<br />
}<br />
<br />
.banniere-content p{<br />
color:white;<br />
font-size:16px<br />
}<br />
<br />
</style><br />
<body><br />
<br />
<div class="banniere"><br />
<div class="banniere-wrapper" style="width:960px; height:100%; margin: 0 auto; position:relative;"><br />
<br />
<br />
<div class="banniere-content"><br />
<h2>Project context</h2><br />
<p>A threatened heritage</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="fils-ariane" style="width:100%; height:60px; background:#ededed;"><br />
<p style="margin:0 auto; color:#696969; width:960px; padding-top:20px; font-size:16px;"> Project&nbsp;&nbsp;&nbsp;>&nbsp;&nbsp;&nbsp;Context project</p> <br />
</div><br />
<br />
<br />
<div id="innercontenthome"><br />
<div class="centering" style="padding-top: 85px; padding-bottom:40px;"><br />
<br />
<!--Short description : à changer!!!--><br />
<br />
<p class="texte">One of Europe Wonders is without a doubt the Toulouse Canal du Midi. Situated in the heart of our city and going through Southern France, our lab is only 5 minutes walk from the Canal du Midi. Everyone in the team could enjoy the quietness and loveliness of this charming place. Unfortunately, this heritage is threatened: a phytopathogenic fugus, <i>Ceratocystis platani</i>, is devastating the trees lining the Canal.<br />
</p><br />
<br />
<p class="texte">The situation is alarming: today the only solution is preventive tree cutting to stem the fungal epidemic. The consequences are disastrous. Not only for the beauty of the landscapes but it has also huge environmental, social and economic costs. Today, 42.000 plane trees are threatened, knowing that cutting and planting a new resistant tree is about €4,000.<br />
</p><br />
<br />
<p class="title1">The Canal du midi</p> <br />
<p class="texte">This ingenious masterpiece respects the environment in which it is harmoniously integrated since 1681 by Pierre-Paul Riquet.</br><br />
Characterized as a summit level canal, it culminates at 189 meters of altitude and permits the achievement of water through the sea.</br><br />
In December 7th, 1996, the UNESCO registers Le Canal du Midi on the list of world’s legacy.</br><br />
The canal du midi is today primarily used for tourism, recreation and housing. Busier than the Seine (Paris), it accounts for one fifth of French river tourism and 80 % tourists are foreigners. Boaters seeking for tranquility, quietness and a unique environment mainly navigate over it.<br />
</p><br />
<br />
<p class="title1">Canker Stain</p> <br />
<p class="texte">The first plane trees bordering the channel appeared lately after its creation in 1776. Resolving the erosion of riverbanks and the evaporation of the water, the plane imposes itself as a dominant and emblematic marrow. The wooded legacy is estimated to about 42.000 trees, it contributes to the landscape and aesthetic.<br />
The canker stain is a disease caused by ''Ceratocystis platani'', a microscopic fungus that exclusively attacks Plane Trees.</br><br />
Probably introduced in France by infected wooden munitions cases coming from United States of America in 1945, the fungus introduces itself inside the sain tree’s heart, blocks the sap flow and kills it in only 2 to 5 years.<br />
On the regulatory side, the July 31th 2000 modified ministerial order classes ''Ceratocystis'' as a harmful organism for plants. This order leads to a mandatory fight against the fungus.<br />
</p><br />
<br />
<p class="title1">Fighting the plane trees pathogen</p> <br />
<p class="texte">Regarding this imminent threat, some solutions emerged.<br />
</br>First of all, chemical fungicides were used. However, the community realized it was impossible to eradicate the whole fungus. <br />
</br>Therefore, the French Institut National de Recherche Agronomique (INRA) created a new type of plane trees named Platanor which were resistant to the fungus infection. Unfortunately, this extreme measure could not save the already contaminated trees nearby the Canal du Midi.<br />
</br>Today, the only option remains in the tree-cutting of the contaminated plane trees. Nevertheless, this previous solution is not optimal because of the high cost and the major ecological, touristic and social impact for our heritage.<br />
</p><br />
<br />
<br />
<!-- Navigation section --> <br />
<br />
<div class="page-nav" style="border-top:1px solid #cccccc; padding-top:40px; margin-top:40px;"><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse" class="page-nav-right" style="width:447px; float:left; display:block;text-decoration:none; color:#666; font-size:18px;">Home<br />
<img src="https://static.igem.org/mediawiki/2014/2/26/Template-igem2014-img-arrowleft.png" style="display:block; padding-top:10px;"/><br />
</a> <br />
<br />
<div class="page-nav-center" style="width:65px; float:left;"><br />
<img src="https://static.igem.org/mediawiki/2014/6/66/Template-igem2014-logo-nav.png" alt="logo" /><br />
</div><br />
<br />
<a href="https://2014.igem.org/Team:Toulouse/Project/Overviews" class="page-nav-left" style="width:447px; float:right; display:block; text-align:right; text-decoration:none;<br />
color:#666; font-size:18px;">Overview</br><br />
<img src="https://static.igem.org/mediawiki/2014/e/ea/Template-igem2014-img-arrowright.png" style="display:block; float:right; padding-top:10px; " /><br />
</a><br />
<br />
</div><br />
<br />
<div class="clear"></div><br />
<br />
</div><br />
</div><br />
</body><br />
</html><br />
<br />
<!-------------------------------- FOOTER ---------------------------------><br />
{{:Team:Toulouse/template/footer}}</div>FannyPineau