http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=500&target=Lujemomo&year=&month=
2012.igem.org - User contributions [en]
2024-03-29T05:25:58Z
From 2012.igem.org
MediaWiki 1.16.0
http://2012.igem.org/Team:Valencia_Biocampus/Attributions
Team:Valencia Biocampus/Attributions
2013-06-15T13:33:41Z
<p>Lujemomo: </p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
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<br />
<center><br />
<div id="Titulos"><br />
<h2>Attributions</h2><br />
<br><br />
<br />
<div id="PorDefecto"><br />
<br />
== '''Students''' ==<br />
<br><br />
Talking Life has been performed by twelve students, organized in sub-teams. The work has personally been done by us, and distributed as follows:<br />
<br />
<br />
<br />
-<b>Mariano Collantes:</b> Wiki, Talk, Human Practices<br> <br />
-<b>Maria Marin:</b> Bacteria, Talk, Human Practices<br><br />
-<b>Lucia Gómez:</b> Yeast, Wiki<br><br />
-<b>Alba Iglesias:</b> Bacteria, Cheater characterization<br><br />
-<b>Pedro Dorado:</b> Bacteria, Human Practices, Cheater characterization<br><br />
-<b>Lucas Morales:</b> Yeast, Modelling, Wiki<br><br />
-<b>Tamara Carrillo:</b> Wiki, Modeling, Voice interphase<br><br />
-<b>José Joaquin Alcaina:</b> Wiki, modeling, Fluorimeter construction<br><br />
-<b>José Luis Racero:</b> Wiki, Modeling, Fluorimeter construction<br><br />
-<b>Guillermo Zafrilla:</b> Bacteria, Yeast<br><br />
-<b>Luisa Martínez:</b> Yeast, Human Practices<br><br />
-<b>Lamberto Torralba:</b> Bacteria, Human Practices<br><br />
<br><br><br />
<br />
== '''Advisors and supervisors''' ==<br />
<br><br />
-Bacteria. Lab work supervised by <b>Cristina Vilanova</b> and <b>Manuel Porcar</b>.<br><br />
-Yeast. Supervised by <b>Marta Tortajada</b>.<br><br />
-Modelling and hardware. Mainly supervised by <b>Jesús Picó</b>.<br><br />
-Voice interphase. Supervised by <b>Miguel Lozano</b>.<br><br />
-Human Practices. Supervised by <b>Ana Delgado</b>.<br><br />
-Talking Life (movie). Script made by the students of Human Practices as listed above. Professional help (Artefactando) was required for recording and edition. Actor Alfred Picó kindly worked at no cost for this project.<br><br />
<br><br></div>
Lujemomo
http://2012.igem.org/File:Rsz_wbbanner.jpg
File:Rsz wbbanner.jpg
2013-06-15T09:59:40Z
<p>Lujemomo: </p>
<hr />
<div></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/estiloc3po
Team:Valencia Biocampus/estiloc3po
2012-09-26T23:30:18Z
<p>Lujemomo: </p>
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Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/estiloc3po
Team:Valencia Biocampus/estiloc3po
2012-09-26T23:29:58Z
<p>Lujemomo: </p>
<hr />
<div><html><br />
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display:none;<br />
}<br />
<br />
<br />
<br />
<br />
/*background:transparent url("https://static.igem.org/mediawiki/2010/0/0c/Transpa.png") center top fixed repeat-y;*/<br />
/* El que estaba antes 26/10 https://static.igem.org/mediawiki/2010/8/86/Transpa30.png */<br />
<br />
#content{<br />
background:transparent url("https://static.igem.org/mediawiki/2010/f/f4/Transpa50.png") center top fixed repeat-y;<br />
padding: 0px;<br />
margin:0 auto;<br />
border: none; <br />
}<br />
<br />
<br />
/*Now to set heading and body fonts*/<br />
<br />
body {<br />
background: #000000 url(https://static.igem.org/mediawiki/2012/f/f9/Fondo_gris.jpeg) no-repeat top center fixed; <br />
/* background:#121212;*/<br />
font-family:Arial, Helvetica, sans-serif;<br />
font-size:8px; /* tamaño de fuente*/<br />
color:#fff;<br />
overflow-x:hidden; <br />
}<br />
<br />
#Titulos{<br />
// text-align: left;<br />
//font-family: Fantasy; /*Kautiva, Verdana, Geneva, sans-serif;*/<br />
//font-size: 30px;<br />
//font-weight: bold;<br />
//color: #000000;<br />
<br />
margin-top: 68px;<br />
background: none;<br />
width: 740px;<br />
padding: 10px;<br />
display: block;<br />
text-align: center;<br />
font-size: 18px;<br />
<br />
}<br />
<br />
#Titulos h2 {<br />
text-align: left;<br />
}<br />
<br />
/* To make the images borders transparents!!! */<br />
div.thumbinner {background-color:transparent;}<br />
div.thumb {border-color: transparent;}<br />
div.thumb {background-color: transparent;}<br />
h1 { color: #000000 }<br />
h2 { color: #000000 }<br />
h3 { color: #000000 }<br />
h4 { color: #000000 }<br />
<br />
#HomeCenter {<br />
width: 660px;<br />
height: auto;<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 14px;<br />
text-align: justify;<br />
margin-top: 8px;<br />
margin-bottom: 20px; <br />
margin-left: 12px; <br />
margin-right: 12px; <br />
padding: 0;<br />
}<br />
<br />
<br />
#HomeRight {<br />
width: 270px;<br />
height: auto;<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
}<br />
#HomeRight2 {<br />
width: 370px;<br />
height: auto;<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
}<br />
<br />
#IzqSup {<br />
width: 360px; <br />
height:auto; /* altura del fondo */<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: justify;<br />
margin-top: 0px;<br />
margin-bottom: 0px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 0;<br />
}<br />
<br />
#DerSup {<br />
width: 360px; <br />
height: auto; /* altura del fondo */<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
margin-top: 8px;<br />
margin-bottom: 30px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 30;<br />
} <br />
#DerSup2 {<br />
width: 250px; <br />
height: auto; /* altura del fondo */<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
margin-top: 8px;<br />
margin-bottom: 5px; <br />
margin-left: 12px; /* cuando espacio izquierdo dejo */<br />
margin-right: 8px; <br />
padding: 8;<br />
} <br />
<br />
<br />
#Inf {<br />
width: 710px; <br />
height: auto<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: justify;<br />
margin-top: 650px;<br />
margin-bottom: 10px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 0;<br />
}<br />
<br />
#PorDefecto {<br />
width: auto; <br />
height: auto<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: justify;<br />
margin-top: -30px; // para que el texto suba<br />
margin-bottom: 20px; <br />
margin-left: 12px; /* cuando espacio izquierdo dejo */<br />
margin-right: 12px; <br />
padding: 0;<br />
}<br />
<br />
#PosBocadillo {<br />
width: 360px; <br />
height: auto; /* altura del fondo */<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
margin-top: 8px;<br />
margin-bottom: 30px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 30;<br />
} <br />
//// <br />
#Contact { <br />
position: relative;<br />
text-align: left;<br />
}<br />
#Contact h5 {<br />
color: #D0D9E1;<br />
font-family: Georgia;<br />
font-size: 140px;<br />
letter-spacing: -6px;<br />
margin: 0;<br />
opacity: .9;<br />
padding: 0;<br />
-moz-transform: skew(20deg);<br />
-o-transform: skew(20deg);<br />
-webkit-transform: skew(20deg);<br />
}<br />
<br />
#Contact h6 {<br />
color: #4682B4;<br />
font-family: Verdana;<br />
font-size: 60px;<br />
<br />
left: 120px;<br />
letter-spacing: 20px;<br />
<br />
margin: 0;<br />
padding: 0;<br />
position: absolute;<br />
<br />
top: 20px;<br />
}<br />
<br />
<br />
////<br />
<!--<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
%<br />
% ESTILO 2: PARA LOS SPONSORS<br />
%<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
#sponsors {<br />
background: none;<br />
width: 740px;<br />
padding: 10px;<br />
display: block;<br />
text-align: center;<br />
}<br />
<br />
/* imagen de sponsor: separacion vertical respecto a la horizontal inferior */<br />
#sponsors img {<br />
padding: 15px;<br />
}<br />
<br />
/* encabezado: alineacion*/<br />
#sponsors h2 {<br />
text-align: left;<br />
}<br />
<br />
/* tamaños de las imagenes */<br />
a.tam1 img{<br />
height: 120px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam2 img{<br />
height: 100px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam3 img{<br />
height: 90px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam4 img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tamLogo img{<br />
height: 70px;<br />
width: 360px;<br />
float: left;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.meneillos img{<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
/* al pasar el ratón */<br />
a.tam1:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam2:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam3:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam4 img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tamLogo img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
<br />
a.meneillos:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
<br />
</style><br />
<br />
</html><br />
<br />
<!--<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
%<br />
% ESTILO 2: GALERIA DE EQUIPO: STUDENTS, INSTRUCTORS,...<br />
%<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
<br />
<html><br />
<style><br />
body, ul, li, h1, h2, h3{<br />
margin:-7; /* grosor del menú superior de edición */<br />
padding:-7; /* separación vertical del título con el borde superior */<br />
z-index:1;<br />
}<br />
<br />
.ps_overlay{<br />
z-index:1;<br />
background:#111;<br />
width:100%;<br />
height:100%;<br />
position:fixed;<br />
top:0px;<br />
left:0px;<br />
opacity:0.5;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=80);<br />
}<br />
/* Image container style */<br />
.ps_container{<br />
width:480px;<br />
height:350px;<br />
position:absolute;<br />
top:50%;<br />
margin-top:-175px;<br />
left:50%;<br />
margin-left:-240px;<br />
z-index:1;<br />
}<br />
.ps_container img{<br />
border:10px solid #fff;<br />
position:absolute;<br />
top:50%;<br />
left:50%;<br />
-moz-box-shadow:1px 1px 10px #000;<br />
-webkit-box-shadow:1px 1px 10px #000;<br />
box-shadow:1px 1px 10px #000;<br />
}<br />
<br />
/* Next photo button for preview mode */<br />
a.ps_next_photo{<br />
position:absolute;<br />
top:50%;<br />
left:50%;<br />
width:56px;<br />
height:56px;<br />
margin:-28px 0 0 -28px;<br />
z-index:200;<br />
cursor:pointer;<br />
background:#000 url(https://static.igem.org/mediawiki/2012/7/7f/Next_photoValencia.png) no-repeat 50% 50%;<br />
opacity:0.6;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=60); <br />
-moz-border-radius:10px;<br />
-webkit-border-radius:10px;<br />
border-radius:10px;<br />
}<br />
a.ps_next_photo:hover,<br />
a.ps_close:hover{<br />
opacity:0.8;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=80); <br />
}<br />
/* Thumbnail slider style */<br />
<br />
/* posicion espacial de todo el slider en conjunto*/<br />
.ps_slider{<br />
width:845px;<br />
height:700px; /* cuantos pixeles verticales ocupa el fondo del slier*/<br />
position:relative;<br />
margin:40px auto 0px auto;<br />
}<br />
.ps_slider a.next,<br />
.ps_slider a.prev{<br />
position:absolute;<br />
background-color:#000;<br />
background-position:center center;<br />
background-repeat:no-repeat;<br />
border:1px solid #232323;<br />
width:20px;<br />
height:20px;<br />
top:50%;<br />
margin-top:-10px;<br />
opacity:0.6;<br />
-moz-border-radius:5px;<br />
-webkit-border-radius:5px;<br />
border-radius:5px;<br />
cursor:pointer;<br />
outline:none;<br />
}<br />
<br />
.ps_slider a.prev:hover,<br />
.ps_slider a.next:hover{<br />
border:1px solid #333;<br />
opacity:0.9;<br />
}<br />
<br />
.ps_slider a.disabled,<br />
<br />
<br />
.ps_slider a.disabled:hover{<br />
opacity:0.4;<br />
border:1px solid #111;<br />
cursor:default;<br />
}<br />
.ps_slider a.prev{<br />
left:-30px;<br />
background-image:url(https://static.igem.org/mediawiki/2012/1/12/PrevValencia.png);<br />
}<br />
.ps_slider a.next{<br />
right:-30px;<br />
background-image:url(https://static.igem.org/mediawiki/2012/3/37/NextValencia.png);<br />
}<br />
<br />
/* cuadrado gris claro de cada slider*/<br />
.ps_slider .ps_album{<br />
width:140px;<br />
height:660px; /* altura*/<br />
padding:10px;<br />
background-color:#333;<br />
border:1px solid #444;<br />
position:absolute;<br />
top:0px;<br />
text-align:center;<br />
/*cursor:pointer;*/ /* QUITAMOS EL PUNTERO DE MOMENTO */<br />
-moz-box-shadow:1px 1px 4px #000;<br />
-webkit-box-shadow:1px 1px 4px #000;<br />
box-shadow:1px 1px 4px #000;<br />
-webkit-box-reflect:<br />
below 5px <br />
-webkit-gradient(<br />
linear, <br />
left top, <br />
left bottom, <br />
from(transparent), <br />
color-stop(0.6, transparent), <br />
to(rgb(18, 18, 18))<br />
);<br />
}<br />
/*LUZ DE CADA SLIDER, DE MOMENTO COMENTADO*/<br />
/*<br />
.ps_slider .ps_album:hover{<br />
background-color:#383838;<br />
}*/<br />
<br />
/* imagen de cada slider */<br />
.ps_slider .ps_album img{<br />
height:90px;<br />
border:1px solid #444;<br />
-moz-box-shadow:1px 1px 4px #000;<br />
-webkit-box-shadow:1px 1px 4px #000;<br />
box-shadow:1px 1px 4px #000;<br />
}<br />
<br />
/*cuadrado gris oscuro de cada slider*/<br />
.ps_slider .ps_album .ps_desc{<br />
display:block;<br />
color:#666;<br />
background:#111 url(https://static.igem.org/mediawiki/2012/7/7d/OverlayValencia.png) no-repeat bottom right;<br />
height:550px;<br />
margin-top:10px;<br />
text-align:left;<br />
line-height:20px;<br />
overflow:hidden;<br />
text-overflow:ellipsis;<br />
border:1px solid #393939;<br />
-moz-box-shadow:0px 0px 2px #000 inset;<br />
-webkit-box-shadow:0px 0px 2px #000 inset;<br />
box-shadow:0px 0px 2px #000 inset;<br />
}<br />
/*LUZ DE CADA SLIDER, DE MOMENTO COMENTADO*/<br />
/*<br />
.ps_slider .ps_album:hover .ps_desc{<br />
background-image:none;<br />
}*/<br />
.ps_slider .ps_album .ps_desc span{<br />
display:block;<br />
margin:0px 10px 10px 10px;<br />
border-top:1px solid #333;<br />
padding-top:5px;<br />
}<br />
.ps_slider .ps_album .ps_desc h2{<br />
margin:10px 10px 0px 10px;<br />
text-align:left;<br />
padding-bottom:5px;<br />
font-weight:normal;<br />
color:#ddd;<br />
text-shadow:0px 0px 1px #fff;<br />
border-bottom:1px solid #000;<br />
}<br />
.ps_slider .loading{<br />
background:#121212 url(https://static.igem.org/mediawiki/2012/5/57/LoadingValencia.gif) no-repeat 50% 50%;<br />
position:absolute;<br />
top:0px;<br />
left:0px;<br />
width:100%;<br />
height:100%;<br />
opacity:0.7;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=70);<br />
}<br />
<br />
span.reference{<br />
position:fixed;<br />
top:10px;<br />
right:10px;<br />
font-size:9px;<br />
}<br />
<br />
span.reference a{<br />
color:#aaa;<br />
text-decoration:none;<br />
text-transform:uppercase;<br />
margin-left:10px;<br />
}<br />
<br />
span.reference a:hover{<br />
color:#ddd;<br />
}<br />
<br />
h1.title{<br />
text-indent:-9000px;<br />
/*background:transparent url(https://static.igem.org/mediawiki/2012/2/22/StudentMembers.png) no-repeat top left;*/<br />
width:640px;<br />
height:52px;<br />
position:absolute;<br />
top:15px;<br />
left:15px;<br />
} <br />
<br />
</style><br />
</html></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/estiloc3po
Team:Valencia Biocampus/estiloc3po
2012-09-26T23:29:23Z
<p>Lujemomo: </p>
<hr />
<div><html><br />
<br />
<style type="text/css"><br />
<br />
#globalWrapper { background-color: transparent; border: none; margin: 0; padding: 0;}<br />
<br />
.firstHeading { /**/<br />
height:0px;<br />
visibility: hidden;<br />
}<br />
<br />
#menubar { <br />
background-color: #696969; /* left menu bar color*/<br />
}<br />
#menubar ul li a { <br />
color: #000000; } /* menu text color*/<br />
.right-menu li a { <br />
color: #A5C5EE; <br />
background-color: #FFFFFF; /* right menu bar color*/<br />
}<br />
#p-logo { height 0px; overflow:hidden; display: none;<br />
}<br />
#top-section { <br />
background-position: center center; <br />
background-repeat: no-repeat; <br />
background-color: #FFFFFF; /* top header background color*/<br />
border-width:0px;<br />
height:0px; /* top header height (determines how low content will start to appear)*/<br />
}<br />
#siteSub {<br />
display:none;<br />
}<br />
#search-controls { /* gets rid of the search bar (it was ugly) */<br />
display:none;<br />
margin-top:0px;<br />
}<br />
#contentSub {<br />
display:none;<br />
}<br />
<br />
<br />
<br />
<br />
/*background:transparent url("https://static.igem.org/mediawiki/2010/0/0c/Transpa.png") center top fixed repeat-y;*/<br />
/* El que estaba antes 26/10 https://static.igem.org/mediawiki/2010/8/86/Transpa30.png */<br />
<br />
#content{<br />
background:transparent url("https://static.igem.org/mediawiki/2010/f/f4/Transpa50.png") center top fixed repeat-y;<br />
padding: 0px;<br />
margin:0 auto;<br />
border: none; <br />
}<br />
<br />
<br />
/*Now to set heading and body fonts*/<br />
<br />
body {<br />
background: #000000 url(https://static.igem.org/mediawiki/2012/f/f9/Fondo_gris.jpeg) no-repeat top center fixed; <br />
/* background:#121212;*/<br />
font-family:Arial, Helvetica, sans-serif;<br />
font-size:8px; /* tamaño de fuente*/<br />
color:#fff;<br />
overflow-x:hidden; <br />
}<br />
<br />
#Titulos{<br />
// text-align: left;<br />
//font-family: Fantasy; /*Kautiva, Verdana, Geneva, sans-serif;*/<br />
//font-size: 30px;<br />
//font-weight: bold;<br />
//color: #000000;<br />
<br />
margin-top: 68px;<br />
background: none;<br />
width: 740px;<br />
padding: 10px;<br />
display: block;<br />
text-align: center;<br />
font-size: 18px;<br />
<br />
}<br />
<br />
#Titulos h2 {<br />
text-align: left;<br />
}<br />
<br />
/* To make the images borders transparents!!! */<br />
div.thumbinner {background-color:transparent;}<br />
div.thumb {border-color: transparent;}<br />
div.thumb {background-color: transparent;}<br />
h1 { color: #000000 }<br />
h2 { color: #000000 }<br />
h3 { color: #000000 }<br />
h4 { color: #000000 }<br />
<br />
#HomeCenter {<br />
width: 660px;<br />
height: auto;<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 14px;<br />
text-align: justify;<br />
margin-top: 8px;<br />
margin-bottom: 20px; <br />
margin-left: 12px; <br />
margin-right: 12px; <br />
padding: 0;<br />
}<br />
<br />
<br />
#HomeRight {<br />
width: 270px;<br />
height: auto;<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
}<br />
#HomeRight2 {<br />
width: 370px;<br />
height: auto;<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
}<br />
<br />
#IzqSup {<br />
width: 360px; <br />
height:auto; /* altura del fondo */<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: justify;<br />
margin-top: 0px;<br />
margin-bottom: 0px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 0;<br />
}<br />
<br />
#DerSup {<br />
width: 360px; <br />
height: auto; /* altura del fondo */<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
margin-top: 8px;<br />
margin-bottom: 30px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 30;<br />
} <br />
#DerSup2 {<br />
width: 250px; <br />
height: auto; /* altura del fondo */<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
margin-top: 8px;<br />
margin-bottom: 5px; <br />
margin-left: 12px; /* cuando espacio izquierdo dejo */<br />
margin-right: 8px; <br />
padding: 8;<br />
} <br />
<br />
<br />
#Inf {<br />
width: 710px; <br />
height: auto<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: justify;<br />
margin-top: 650px;<br />
margin-bottom: 10px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 0;<br />
}<br />
<br />
#PorDefecto {<br />
width: auto; <br />
height: auto<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: justify;<br />
margin-top: -30px; // para que el texto suba<br />
margin-bottom: 20px; <br />
margin-left: 12px; /* cuando espacio izquierdo dejo */<br />
margin-right: 12px; <br />
padding: 0;<br />
}<br />
<br />
#PosBocadillo {<br />
width: 360px; <br />
height: auto; /* altura del fondo */<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
margin-top: 8px;<br />
margin-bottom: 30px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 30;<br />
} <br />
//// <br />
#Contact { <br />
position: relative;<br />
text-align: left;<br />
}<br />
#Contact h5 {<br />
color: #D0D9E1;<br />
font-family: Georgia;<br />
font-size: 140px;<br />
letter-spacing: -6px;<br />
margin: 0;<br />
opacity: .9;<br />
padding: 0;<br />
-moz-transform: skew(20deg);<br />
-o-transform: skew(20deg);<br />
-webkit-transform: skew(20deg);<br />
}<br />
<br />
#Contact h6 {<br />
color: #4682B4;<br />
font-family: Verdana;<br />
font-size: 60px;<br />
<br />
left: 120px;<br />
letter-spacing: 20px;<br />
<br />
margin: 0;<br />
padding: 0;<br />
position: absolute;<br />
<br />
top: 20px;<br />
}<br />
<br />
<br />
////<br />
<!--<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
%<br />
% ESTILO 2: PARA LOS SPONSORS<br />
%<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
#sponsors {<br />
background: none;<br />
width: 740px;<br />
padding: 10px;<br />
display: block;<br />
text-align: center;<br />
}<br />
<br />
/* imagen de sponsor: separacion vertical respecto a la horizontal inferior */<br />
#sponsors img {<br />
padding: 15px;<br />
}<br />
<br />
/* encabezado: alineacion*/<br />
#sponsors h2 {<br />
text-align: left;<br />
}<br />
<br />
/* tamaños de las imagenes */<br />
a.tam1 img{<br />
height: 120px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam2 img{<br />
height: 100px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam3 img{<br />
height: 90px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam4 img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tamLogo img{<br />
height: 70px;<br />
width: 360px;<br />
float: left;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.meneillos img{<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
/* al pasar el ratón */<br />
a.tam1:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam2:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam3:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam4 img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tamLogo img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
<br />
a.meneillos:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
<br />
</style><br />
<br />
</html><br />
<br />
<!--<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
%<br />
% ESTILO 2: GALERIA DE EQUIPO: STUDENTS, INSTRUCTORS,...<br />
%<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
<br />
<html><br />
<style><br />
body, ul, li, h1, h2, h3{<br />
margin:-7; /* grosor del menú superior de edición */<br />
padding:-7; /* separación vertical del título con el borde superior */<br />
z-index:1;<br />
}<br />
<br />
.ps_overlay{<br />
z-index:1;<br />
background:#111;<br />
width:100%;<br />
height:100%;<br />
position:fixed;<br />
top:0px;<br />
left:0px;<br />
opacity:0.5;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=80);<br />
}<br />
/* Image container style */<br />
.ps_container{<br />
width:480px;<br />
height:350px;<br />
position:absolute;<br />
top:50%;<br />
margin-top:-175px;<br />
left:50%;<br />
margin-left:-240px;<br />
z-index:1;<br />
}<br />
.ps_container img{<br />
border:10px solid #fff;<br />
position:absolute;<br />
top:50%;<br />
left:50%;<br />
-moz-box-shadow:1px 1px 10px #000;<br />
-webkit-box-shadow:1px 1px 10px #000;<br />
box-shadow:1px 1px 10px #000;<br />
}<br />
<br />
/* Next photo button for preview mode */<br />
a.ps_next_photo{<br />
position:absolute;<br />
top:50%;<br />
left:50%;<br />
width:56px;<br />
height:56px;<br />
margin:-28px 0 0 -28px;<br />
z-index:200;<br />
cursor:pointer;<br />
background:#000 url(https://static.igem.org/mediawiki/2012/7/7f/Next_photoValencia.png) no-repeat 50% 50%;<br />
opacity:0.6;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=60); <br />
-moz-border-radius:10px;<br />
-webkit-border-radius:10px;<br />
border-radius:10px;<br />
}<br />
a.ps_next_photo:hover,<br />
a.ps_close:hover{<br />
opacity:0.8;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=80); <br />
}<br />
/* Thumbnail slider style */<br />
<br />
/* posicion espacial de todo el slider en conjunto*/<br />
.ps_slider{<br />
width:845px;<br />
height:700px; /* cuantos pixeles verticales ocupa el fondo del slier*/<br />
position:relative;<br />
margin:40px auto 0px auto;<br />
}<br />
.ps_slider a.next,<br />
.ps_slider a.prev{<br />
position:absolute;<br />
background-color:#000;<br />
background-position:center center;<br />
background-repeat:no-repeat;<br />
border:1px solid #232323;<br />
width:20px;<br />
height:20px;<br />
top:50%;<br />
margin-top:-10px;<br />
opacity:0.6;<br />
-moz-border-radius:5px;<br />
-webkit-border-radius:5px;<br />
border-radius:5px;<br />
cursor:pointer;<br />
outline:none;<br />
}<br />
<br />
.ps_slider a.prev:hover,<br />
.ps_slider a.next:hover{<br />
border:1px solid #333;<br />
opacity:0.9;<br />
}<br />
<br />
.ps_slider a.disabled,<br />
<br />
<br />
.ps_slider a.disabled:hover{<br />
opacity:0.4;<br />
border:1px solid #111;<br />
cursor:default;<br />
}<br />
.ps_slider a.prev{<br />
left:-30px;<br />
background-image:url(https://static.igem.org/mediawiki/2012/1/12/PrevValencia.png);<br />
}<br />
.ps_slider a.next{<br />
right:-30px;<br />
background-image:url(https://static.igem.org/mediawiki/2012/3/37/NextValencia.png);<br />
}<br />
<br />
/* cuadrado gris claro de cada slider*/<br />
.ps_slider .ps_album{<br />
width:140px;<br />
height:660px; /* altura*/<br />
padding:10px;<br />
background-color:#333;<br />
border:1px solid #444;<br />
position:absolute;<br />
top:0px;<br />
text-align:center;<br />
/*cursor:pointer;*/ /* QUITAMOS EL PUNTERO DE MOMENTO */<br />
-moz-box-shadow:1px 1px 4px #000;<br />
-webkit-box-shadow:1px 1px 4px #000;<br />
box-shadow:1px 1px 4px #000;<br />
-webkit-box-reflect:<br />
below 5px <br />
-webkit-gradient(<br />
linear, <br />
left top, <br />
left bottom, <br />
from(transparent), <br />
color-stop(0.6, transparent), <br />
to(rgb(18, 18, 18))<br />
);<br />
}<br />
/*LUZ DE CADA SLIDER, DE MOMENTO COMENTADO*/<br />
/*<br />
.ps_slider .ps_album:hover{<br />
background-color:#383838;<br />
}*/<br />
<br />
/* imagen de cada slider */<br />
.ps_slider .ps_album img{<br />
height:90px;<br />
border:1px solid #444;<br />
-moz-box-shadow:1px 1px 4px #000;<br />
-webkit-box-shadow:1px 1px 4px #000;<br />
box-shadow:1px 1px 4px #000;<br />
}<br />
<br />
/*cuadrado gris oscuro de cada slider*/<br />
.ps_slider .ps_album .ps_desc{<br />
display:block;<br />
color:#666;<br />
background:#111 url(https://static.igem.org/mediawiki/2012/7/7d/OverlayValencia.png) no-repeat bottom right;<br />
height:550px;<br />
margin-top:10px;<br />
text-align:left;<br />
line-height:20px;<br />
overflow:hidden;<br />
text-overflow:ellipsis;<br />
border:1px solid #393939;<br />
-moz-box-shadow:0px 0px 2px #000 inset;<br />
-webkit-box-shadow:0px 0px 2px #000 inset;<br />
box-shadow:0px 0px 2px #000 inset;<br />
}<br />
/*LUZ DE CADA SLIDER, DE MOMENTO COMENTADO*/<br />
/*<br />
.ps_slider .ps_album:hover .ps_desc{<br />
background-image:none;<br />
}*/<br />
.ps_slider .ps_album .ps_desc span{<br />
display:block;<br />
margin:0px 10px 10px 10px;<br />
border-top:1px solid #333;<br />
padding-top:5px;<br />
}<br />
.ps_slider .ps_album .ps_desc h2{<br />
margin:10px 10px 0px 10px;<br />
text-align:left;<br />
padding-bottom:5px;<br />
font-weight:normal;<br />
color:#ddd;<br />
text-shadow:0px 0px 1px #fff;<br />
border-bottom:1px solid #000;<br />
}<br />
.ps_slider .loading{<br />
background:#121212 url(https://static.igem.org/mediawiki/2012/5/57/LoadingValencia.gif) no-repeat 50% 50%;<br />
position:absolute;<br />
top:0px;<br />
left:0px;<br />
width:100%;<br />
height:100%;<br />
opacity:0.7;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=70);<br />
}<br />
<br />
span.reference{<br />
position:fixed;<br />
top:10px;<br />
right:10px;<br />
font-size:9px;<br />
}<br />
<br />
span.reference a{<br />
color:#aaa;<br />
text-decoration:none;<br />
text-transform:uppercase;<br />
margin-left:10px;<br />
}<br />
<br />
span.reference a:hover{<br />
color:#ddd;<br />
}<br />
<br />
h1.title{<br />
text-indent:-9000px;<br />
/*background:transparent url(https://static.igem.org/mediawiki/2012/2/22/StudentMembers.png) no-repeat top left;*/<br />
width:640px;<br />
height:52px;<br />
position:absolute;<br />
top:15px;<br />
left:15px;<br />
} <br />
<br />
</style><br />
<br />
TOCBG {<br />
background-color":#9F9F9F";<br />
}<br />
<br />
</html></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/estiloc3po
Team:Valencia Biocampus/estiloc3po
2012-09-26T23:26:58Z
<p>Lujemomo: </p>
<hr />
<div><html><br />
<br />
<style type="text/css"><br />
<br />
#globalWrapper { background-color: transparent; border: none; margin: 0; padding: 0;}<br />
<br />
.firstHeading { /**/<br />
height:0px;<br />
visibility: hidden;<br />
}<br />
<br />
#menubar { <br />
background-color: #696969; /* left menu bar color*/<br />
}<br />
#menubar ul li a { <br />
color: #000000; } /* menu text color*/<br />
.right-menu li a { <br />
color: #A5C5EE; <br />
background-color: #FFFFFF; /* right menu bar color*/<br />
}<br />
#p-logo { height 0px; overflow:hidden; display: none;<br />
}<br />
#top-section { <br />
background-position: center center; <br />
background-repeat: no-repeat; <br />
background-color: #FFFFFF; /* top header background color*/<br />
border-width:0px;<br />
height:0px; /* top header height (determines how low content will start to appear)*/<br />
}<br />
#siteSub {<br />
display:none;<br />
}<br />
#search-controls { /* gets rid of the search bar (it was ugly) */<br />
display:none;<br />
margin-top:0px;<br />
}<br />
#contentSub {<br />
display:none;<br />
}<br />
<br />
<br />
<br />
<br />
/*background:transparent url("https://static.igem.org/mediawiki/2010/0/0c/Transpa.png") center top fixed repeat-y;*/<br />
/* El que estaba antes 26/10 https://static.igem.org/mediawiki/2010/8/86/Transpa30.png */<br />
<br />
#content{<br />
background:transparent url("https://static.igem.org/mediawiki/2010/f/f4/Transpa50.png") center top fixed repeat-y;<br />
padding: 0px;<br />
margin:0 auto;<br />
border: none; <br />
}<br />
<br />
<br />
/*Now to set heading and body fonts*/<br />
<br />
body {<br />
background: #000000 url(https://static.igem.org/mediawiki/2012/f/f9/Fondo_gris.jpeg) no-repeat top center fixed; <br />
/* background:#121212;*/<br />
font-family:Arial, Helvetica, sans-serif;<br />
font-size:8px; /* tamaño de fuente*/<br />
color:#fff;<br />
overflow-x:hidden; <br />
}<br />
<br />
#Titulos{<br />
// text-align: left;<br />
//font-family: Fantasy; /*Kautiva, Verdana, Geneva, sans-serif;*/<br />
//font-size: 30px;<br />
//font-weight: bold;<br />
//color: #000000;<br />
<br />
margin-top: 68px;<br />
background: none;<br />
width: 740px;<br />
padding: 10px;<br />
display: block;<br />
text-align: center;<br />
font-size: 18px;<br />
<br />
}<br />
<br />
#Titulos h2 {<br />
text-align: left;<br />
}<br />
<br />
/* To make the images borders transparents!!! */<br />
div.thumbinner {background-color:transparent;}<br />
div.thumb {border-color: transparent;}<br />
div.thumb {background-color: transparent;}<br />
h1 { color: #000000 }<br />
h2 { color: #000000 }<br />
h3 { color: #000000 }<br />
h4 { color: #000000 }<br />
<br />
#HomeCenter {<br />
width: 660px;<br />
height: auto;<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 14px;<br />
text-align: justify;<br />
margin-top: 8px;<br />
margin-bottom: 20px; <br />
margin-left: 12px; <br />
margin-right: 12px; <br />
padding: 0;<br />
}<br />
<br />
<br />
#HomeRight {<br />
width: 270px;<br />
height: auto;<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
}<br />
#HomeRight2 {<br />
width: 370px;<br />
height: auto;<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
}<br />
<br />
#IzqSup {<br />
width: 360px; <br />
height:auto; /* altura del fondo */<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: justify;<br />
margin-top: 0px;<br />
margin-bottom: 0px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 0;<br />
}<br />
<br />
#DerSup {<br />
width: 360px; <br />
height: auto; /* altura del fondo */<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
margin-top: 8px;<br />
margin-bottom: 30px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 30;<br />
} <br />
#DerSup2 {<br />
width: 250px; <br />
height: auto; /* altura del fondo */<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
margin-top: 8px;<br />
margin-bottom: 5px; <br />
margin-left: 12px; /* cuando espacio izquierdo dejo */<br />
margin-right: 8px; <br />
padding: 8;<br />
} <br />
<br />
<br />
#Inf {<br />
width: 710px; <br />
height: auto<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: justify;<br />
margin-top: 650px;<br />
margin-bottom: 10px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 0;<br />
}<br />
<br />
#PorDefecto {<br />
width: auto; <br />
height: auto<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: justify;<br />
margin-top: -30px; // para que el texto suba<br />
margin-bottom: 20px; <br />
margin-left: 12px; /* cuando espacio izquierdo dejo */<br />
margin-right: 12px; <br />
padding: 0;<br />
}<br />
<br />
#PosBocadillo {<br />
width: 360px; <br />
height: auto; /* altura del fondo */<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
margin-top: 8px;<br />
margin-bottom: 30px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 30;<br />
} <br />
//// <br />
#Contact { <br />
position: relative;<br />
text-align: left;<br />
}<br />
#Contact h5 {<br />
color: #D0D9E1;<br />
font-family: Georgia;<br />
font-size: 140px;<br />
letter-spacing: -6px;<br />
margin: 0;<br />
opacity: .9;<br />
padding: 0;<br />
-moz-transform: skew(20deg);<br />
-o-transform: skew(20deg);<br />
-webkit-transform: skew(20deg);<br />
}<br />
<br />
#Contact h6 {<br />
color: #4682B4;<br />
font-family: Verdana;<br />
font-size: 60px;<br />
<br />
left: 120px;<br />
letter-spacing: 20px;<br />
<br />
margin: 0;<br />
padding: 0;<br />
position: absolute;<br />
<br />
top: 20px;<br />
}<br />
<br />
<br />
////<br />
<!--<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
%<br />
% ESTILO 2: PARA LOS SPONSORS<br />
%<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
#sponsors {<br />
background: none;<br />
width: 740px;<br />
padding: 10px;<br />
display: block;<br />
text-align: center;<br />
}<br />
<br />
/* imagen de sponsor: separacion vertical respecto a la horizontal inferior */<br />
#sponsors img {<br />
padding: 15px;<br />
}<br />
<br />
/* encabezado: alineacion*/<br />
#sponsors h2 {<br />
text-align: left;<br />
}<br />
<br />
/* tamaños de las imagenes */<br />
a.tam1 img{<br />
height: 120px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam2 img{<br />
height: 100px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam3 img{<br />
height: 90px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam4 img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tamLogo img{<br />
height: 70px;<br />
width: 360px;<br />
float: left;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.meneillos img{<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
/* al pasar el ratón */<br />
a.tam1:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam2:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam3:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam4 img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tamLogo img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
<br />
a.meneillos:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
<br />
</style><br />
<br />
</html><br />
<br />
<!--<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
%<br />
% ESTILO 2: GALERIA DE EQUIPO: STUDENTS, INSTRUCTORS,...<br />
%<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
<br />
<html><br />
<style><br />
body, ul, li, h1, h2, h3{<br />
margin:-7; /* grosor del menú superior de edición */<br />
padding:-7; /* separación vertical del título con el borde superior */<br />
z-index:1;<br />
}<br />
<br />
.ps_overlay{<br />
z-index:1;<br />
background:#111;<br />
width:100%;<br />
height:100%;<br />
position:fixed;<br />
top:0px;<br />
left:0px;<br />
opacity:0.5;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=80);<br />
}<br />
/* Image container style */<br />
.ps_container{<br />
width:480px;<br />
height:350px;<br />
position:absolute;<br />
top:50%;<br />
margin-top:-175px;<br />
left:50%;<br />
margin-left:-240px;<br />
z-index:1;<br />
}<br />
.ps_container img{<br />
border:10px solid #fff;<br />
position:absolute;<br />
top:50%;<br />
left:50%;<br />
-moz-box-shadow:1px 1px 10px #000;<br />
-webkit-box-shadow:1px 1px 10px #000;<br />
box-shadow:1px 1px 10px #000;<br />
}<br />
<br />
/* Next photo button for preview mode */<br />
a.ps_next_photo{<br />
position:absolute;<br />
top:50%;<br />
left:50%;<br />
width:56px;<br />
height:56px;<br />
margin:-28px 0 0 -28px;<br />
z-index:200;<br />
cursor:pointer;<br />
background:#000 url(https://static.igem.org/mediawiki/2012/7/7f/Next_photoValencia.png) no-repeat 50% 50%;<br />
opacity:0.6;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=60); <br />
-moz-border-radius:10px;<br />
-webkit-border-radius:10px;<br />
border-radius:10px;<br />
}<br />
a.ps_next_photo:hover,<br />
a.ps_close:hover{<br />
opacity:0.8;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=80); <br />
}<br />
/* Thumbnail slider style */<br />
<br />
/* posicion espacial de todo el slider en conjunto*/<br />
.ps_slider{<br />
width:845px;<br />
height:700px; /* cuantos pixeles verticales ocupa el fondo del slier*/<br />
position:relative;<br />
margin:40px auto 0px auto;<br />
}<br />
.ps_slider a.next,<br />
.ps_slider a.prev{<br />
position:absolute;<br />
background-color:#000;<br />
background-position:center center;<br />
background-repeat:no-repeat;<br />
border:1px solid #232323;<br />
width:20px;<br />
height:20px;<br />
top:50%;<br />
margin-top:-10px;<br />
opacity:0.6;<br />
-moz-border-radius:5px;<br />
-webkit-border-radius:5px;<br />
border-radius:5px;<br />
cursor:pointer;<br />
outline:none;<br />
}<br />
<br />
.ps_slider a.prev:hover,<br />
.ps_slider a.next:hover{<br />
border:1px solid #333;<br />
opacity:0.9;<br />
}<br />
<br />
.ps_slider a.disabled,<br />
<br />
<br />
.ps_slider a.disabled:hover{<br />
opacity:0.4;<br />
border:1px solid #111;<br />
cursor:default;<br />
}<br />
.ps_slider a.prev{<br />
left:-30px;<br />
background-image:url(https://static.igem.org/mediawiki/2012/1/12/PrevValencia.png);<br />
}<br />
.ps_slider a.next{<br />
right:-30px;<br />
background-image:url(https://static.igem.org/mediawiki/2012/3/37/NextValencia.png);<br />
}<br />
<br />
/* cuadrado gris claro de cada slider*/<br />
.ps_slider .ps_album{<br />
width:140px;<br />
height:660px; /* altura*/<br />
padding:10px;<br />
background-color:#333;<br />
border:1px solid #444;<br />
position:absolute;<br />
top:0px;<br />
text-align:center;<br />
/*cursor:pointer;*/ /* QUITAMOS EL PUNTERO DE MOMENTO */<br />
-moz-box-shadow:1px 1px 4px #000;<br />
-webkit-box-shadow:1px 1px 4px #000;<br />
box-shadow:1px 1px 4px #000;<br />
-webkit-box-reflect:<br />
below 5px <br />
-webkit-gradient(<br />
linear, <br />
left top, <br />
left bottom, <br />
from(transparent), <br />
color-stop(0.6, transparent), <br />
to(rgb(18, 18, 18))<br />
);<br />
}<br />
/*LUZ DE CADA SLIDER, DE MOMENTO COMENTADO*/<br />
/*<br />
.ps_slider .ps_album:hover{<br />
background-color:#383838;<br />
}*/<br />
<br />
/* imagen de cada slider */<br />
.ps_slider .ps_album img{<br />
height:90px;<br />
border:1px solid #444;<br />
-moz-box-shadow:1px 1px 4px #000;<br />
-webkit-box-shadow:1px 1px 4px #000;<br />
box-shadow:1px 1px 4px #000;<br />
}<br />
<br />
/*cuadrado gris oscuro de cada slider*/<br />
.ps_slider .ps_album .ps_desc{<br />
display:block;<br />
color:#666;<br />
background:#111 url(https://static.igem.org/mediawiki/2012/7/7d/OverlayValencia.png) no-repeat bottom right;<br />
height:550px;<br />
margin-top:10px;<br />
text-align:left;<br />
line-height:20px;<br />
overflow:hidden;<br />
text-overflow:ellipsis;<br />
border:1px solid #393939;<br />
-moz-box-shadow:0px 0px 2px #000 inset;<br />
-webkit-box-shadow:0px 0px 2px #000 inset;<br />
box-shadow:0px 0px 2px #000 inset;<br />
}<br />
/*LUZ DE CADA SLIDER, DE MOMENTO COMENTADO*/<br />
/*<br />
.ps_slider .ps_album:hover .ps_desc{<br />
background-image:none;<br />
}*/<br />
.ps_slider .ps_album .ps_desc span{<br />
display:block;<br />
margin:0px 10px 10px 10px;<br />
border-top:1px solid #333;<br />
padding-top:5px;<br />
}<br />
.ps_slider .ps_album .ps_desc h2{<br />
margin:10px 10px 0px 10px;<br />
text-align:left;<br />
padding-bottom:5px;<br />
font-weight:normal;<br />
color:#ddd;<br />
text-shadow:0px 0px 1px #fff;<br />
border-bottom:1px solid #000;<br />
}<br />
.ps_slider .loading{<br />
background:#121212 url(https://static.igem.org/mediawiki/2012/5/57/LoadingValencia.gif) no-repeat 50% 50%;<br />
position:absolute;<br />
top:0px;<br />
left:0px;<br />
width:100%;<br />
height:100%;<br />
opacity:0.7;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=70);<br />
}<br />
<br />
span.reference{<br />
position:fixed;<br />
top:10px;<br />
right:10px;<br />
font-size:9px;<br />
}<br />
<br />
span.reference a{<br />
color:#aaa;<br />
text-decoration:none;<br />
text-transform:uppercase;<br />
margin-left:10px;<br />
}<br />
<br />
span.reference a:hover{<br />
color:#ddd;<br />
}<br />
<br />
h1.title{<br />
text-indent:-9000px;<br />
/*background:transparent url(https://static.igem.org/mediawiki/2012/2/22/StudentMembers.png) no-repeat top left;*/<br />
width:640px;<br />
height:52px;<br />
position:absolute;<br />
top:15px;<br />
left:15px;<br />
} <br />
<br />
</style><br />
TOCBG {<br />
background-color:#9F9F9F;<br />
}<br />
</html></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/estiloc3po
Team:Valencia Biocampus/estiloc3po
2012-09-26T23:25:13Z
<p>Lujemomo: </p>
<hr />
<div><html><br />
<br />
<style type="text/css"><br />
<br />
#globalWrapper { background-color: transparent; border: none; margin: 0; padding: 0;}<br />
<br />
.firstHeading { /**/<br />
height:0px;<br />
visibility: hidden;<br />
}<br />
<br />
#menubar { <br />
background-color: #696969; /* left menu bar color*/<br />
}<br />
#menubar ul li a { <br />
color: #000000; } /* menu text color*/<br />
.right-menu li a { <br />
color: #A5C5EE; <br />
background-color: #FFFFFF; /* right menu bar color*/<br />
}<br />
#p-logo { height 0px; overflow:hidden; display: none;<br />
}<br />
#top-section { <br />
background-position: center center; <br />
background-repeat: no-repeat; <br />
background-color: #FFFFFF; /* top header background color*/<br />
border-width:0px;<br />
height:0px; /* top header height (determines how low content will start to appear)*/<br />
}<br />
#siteSub {<br />
display:none;<br />
}<br />
#search-controls { /* gets rid of the search bar (it was ugly) */<br />
display:none;<br />
margin-top:0px;<br />
}<br />
#contentSub {<br />
display:none;<br />
}<br />
<br />
<br />
<br />
<br />
/*background:transparent url("https://static.igem.org/mediawiki/2010/0/0c/Transpa.png") center top fixed repeat-y;*/<br />
/* El que estaba antes 26/10 https://static.igem.org/mediawiki/2010/8/86/Transpa30.png */<br />
<br />
#content{<br />
background:transparent url("https://static.igem.org/mediawiki/2010/f/f4/Transpa50.png") center top fixed repeat-y;<br />
padding: 0px;<br />
margin:0 auto;<br />
border: none; <br />
}<br />
<br />
<br />
/*Now to set heading and body fonts*/<br />
<br />
body {<br />
background: #000000 url(https://static.igem.org/mediawiki/2012/f/f9/Fondo_gris.jpeg) no-repeat top center fixed; <br />
/* background:#121212;*/<br />
font-family:Arial, Helvetica, sans-serif;<br />
font-size:8px; /* tamaño de fuente*/<br />
color:#fff;<br />
overflow-x:hidden; <br />
}<br />
<br />
#Titulos{<br />
// text-align: left;<br />
//font-family: Fantasy; /*Kautiva, Verdana, Geneva, sans-serif;*/<br />
//font-size: 30px;<br />
//font-weight: bold;<br />
//color: #000000;<br />
<br />
margin-top: 68px;<br />
background: none;<br />
width: 740px;<br />
padding: 10px;<br />
display: block;<br />
text-align: center;<br />
font-size: 18px;<br />
<br />
}<br />
<br />
#Titulos h2 {<br />
text-align: left;<br />
}<br />
<br />
/* To make the images borders transparents!!! */<br />
div.thumbinner {background-color:transparent;}<br />
div.thumb {border-color: transparent;}<br />
div.thumb {background-color: transparent;}<br />
h1 { color: #000000 }<br />
h2 { color: #000000 }<br />
h3 { color: #000000 }<br />
h4 { color: #000000 }<br />
<br />
#HomeCenter {<br />
width: 660px;<br />
height: auto;<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 14px;<br />
text-align: justify;<br />
margin-top: 8px;<br />
margin-bottom: 20px; <br />
margin-left: 12px; <br />
margin-right: 12px; <br />
padding: 0;<br />
}<br />
<br />
<br />
#HomeRight {<br />
width: 270px;<br />
height: auto;<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
}<br />
#HomeRight2 {<br />
width: 370px;<br />
height: auto;<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
}<br />
<br />
#IzqSup {<br />
width: 360px; <br />
height:auto; /* altura del fondo */<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: justify;<br />
margin-top: 0px;<br />
margin-bottom: 0px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 0;<br />
}<br />
<br />
#DerSup {<br />
width: 360px; <br />
height: auto; /* altura del fondo */<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
margin-top: 8px;<br />
margin-bottom: 30px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 30;<br />
} <br />
#DerSup2 {<br />
width: 250px; <br />
height: auto; /* altura del fondo */<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
margin-top: 8px;<br />
margin-bottom: 5px; <br />
margin-left: 12px; /* cuando espacio izquierdo dejo */<br />
margin-right: 8px; <br />
padding: 8;<br />
} <br />
<br />
#TOCBG {<br />
background-color:#9F9F9F;<br />
}<br />
<br />
#Inf {<br />
width: 710px; <br />
height: auto<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: justify;<br />
margin-top: 650px;<br />
margin-bottom: 10px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 0;<br />
}<br />
<br />
#PorDefecto {<br />
width: auto; <br />
height: auto<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: justify;<br />
margin-top: -30px; // para que el texto suba<br />
margin-bottom: 20px; <br />
margin-left: 12px; /* cuando espacio izquierdo dejo */<br />
margin-right: 12px; <br />
padding: 0;<br />
}<br />
<br />
#PosBocadillo {<br />
width: 360px; <br />
height: auto; /* altura del fondo */<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
margin-top: 8px;<br />
margin-bottom: 30px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 30;<br />
} <br />
//// <br />
#Contact { <br />
position: relative;<br />
text-align: left;<br />
}<br />
#Contact h5 {<br />
color: #D0D9E1;<br />
font-family: Georgia;<br />
font-size: 140px;<br />
letter-spacing: -6px;<br />
margin: 0;<br />
opacity: .9;<br />
padding: 0;<br />
-moz-transform: skew(20deg);<br />
-o-transform: skew(20deg);<br />
-webkit-transform: skew(20deg);<br />
}<br />
<br />
#Contact h6 {<br />
color: #4682B4;<br />
font-family: Verdana;<br />
font-size: 60px;<br />
<br />
left: 120px;<br />
letter-spacing: 20px;<br />
<br />
margin: 0;<br />
padding: 0;<br />
position: absolute;<br />
<br />
top: 20px;<br />
}<br />
<br />
<br />
////<br />
<!--<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
%<br />
% ESTILO 2: PARA LOS SPONSORS<br />
%<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
#sponsors {<br />
background: none;<br />
width: 740px;<br />
padding: 10px;<br />
display: block;<br />
text-align: center;<br />
}<br />
<br />
/* imagen de sponsor: separacion vertical respecto a la horizontal inferior */<br />
#sponsors img {<br />
padding: 15px;<br />
}<br />
<br />
/* encabezado: alineacion*/<br />
#sponsors h2 {<br />
text-align: left;<br />
}<br />
<br />
/* tamaños de las imagenes */<br />
a.tam1 img{<br />
height: 120px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam2 img{<br />
height: 100px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam3 img{<br />
height: 90px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam4 img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tamLogo img{<br />
height: 70px;<br />
width: 360px;<br />
float: left;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.meneillos img{<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
/* al pasar el ratón */<br />
a.tam1:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam2:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam3:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam4 img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tamLogo img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
<br />
a.meneillos:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
<br />
</style><br />
<br />
</html><br />
<br />
<!--<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
%<br />
% ESTILO 2: GALERIA DE EQUIPO: STUDENTS, INSTRUCTORS,...<br />
%<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
<br />
<html><br />
<style><br />
body, ul, li, h1, h2, h3{<br />
margin:-7; /* grosor del menú superior de edición */<br />
padding:-7; /* separación vertical del título con el borde superior */<br />
z-index:1;<br />
}<br />
<br />
.ps_overlay{<br />
z-index:1;<br />
background:#111;<br />
width:100%;<br />
height:100%;<br />
position:fixed;<br />
top:0px;<br />
left:0px;<br />
opacity:0.5;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=80);<br />
}<br />
/* Image container style */<br />
.ps_container{<br />
width:480px;<br />
height:350px;<br />
position:absolute;<br />
top:50%;<br />
margin-top:-175px;<br />
left:50%;<br />
margin-left:-240px;<br />
z-index:1;<br />
}<br />
.ps_container img{<br />
border:10px solid #fff;<br />
position:absolute;<br />
top:50%;<br />
left:50%;<br />
-moz-box-shadow:1px 1px 10px #000;<br />
-webkit-box-shadow:1px 1px 10px #000;<br />
box-shadow:1px 1px 10px #000;<br />
}<br />
<br />
/* Next photo button for preview mode */<br />
a.ps_next_photo{<br />
position:absolute;<br />
top:50%;<br />
left:50%;<br />
width:56px;<br />
height:56px;<br />
margin:-28px 0 0 -28px;<br />
z-index:200;<br />
cursor:pointer;<br />
background:#000 url(https://static.igem.org/mediawiki/2012/7/7f/Next_photoValencia.png) no-repeat 50% 50%;<br />
opacity:0.6;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=60); <br />
-moz-border-radius:10px;<br />
-webkit-border-radius:10px;<br />
border-radius:10px;<br />
}<br />
a.ps_next_photo:hover,<br />
a.ps_close:hover{<br />
opacity:0.8;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=80); <br />
}<br />
/* Thumbnail slider style */<br />
<br />
/* posicion espacial de todo el slider en conjunto*/<br />
.ps_slider{<br />
width:845px;<br />
height:700px; /* cuantos pixeles verticales ocupa el fondo del slier*/<br />
position:relative;<br />
margin:40px auto 0px auto;<br />
}<br />
.ps_slider a.next,<br />
.ps_slider a.prev{<br />
position:absolute;<br />
background-color:#000;<br />
background-position:center center;<br />
background-repeat:no-repeat;<br />
border:1px solid #232323;<br />
width:20px;<br />
height:20px;<br />
top:50%;<br />
margin-top:-10px;<br />
opacity:0.6;<br />
-moz-border-radius:5px;<br />
-webkit-border-radius:5px;<br />
border-radius:5px;<br />
cursor:pointer;<br />
outline:none;<br />
}<br />
<br />
.ps_slider a.prev:hover,<br />
.ps_slider a.next:hover{<br />
border:1px solid #333;<br />
opacity:0.9;<br />
}<br />
<br />
.ps_slider a.disabled,<br />
<br />
<br />
.ps_slider a.disabled:hover{<br />
opacity:0.4;<br />
border:1px solid #111;<br />
cursor:default;<br />
}<br />
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Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/Protocols
Team:Valencia Biocampus/Protocols
2012-09-26T23:24:56Z
<p>Lujemomo: </p>
<hr />
<div><br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estilo}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
<br />
<br><br />
<br />
<ol><p style="font-size:x-large;">Protocols</p><br />
------------<br />
</ol><br />
<ol><br />
__TOC__<br />
<br />
<br />
= '''General Protocols''' =<br />
<ol><br />
<br />
== '''Transformation protocols''' ==<br />
<br />
=== '''Heat Shock Protocol for bacteria transformation''' ===<br />
<html><br />
<ol><br />
<br />
<li>Take competent E.coli cells from –80°C freezer.<br />
<li>Turn on water bath to 42°C.<br />
<li>Put 100 ul of competent cells in an Eppendorf tube.<br />
<li>Keep tubes on ice.<br />
<li>Add 50 ng of circular DNA into E.coli cells. Incubate on ice for 10 minutes to thaw competent cells.<br />
<li>Put tube(s) with DNA and E.coli into water bath at 42°C for 45 seconds.<br />
<li>Put tubes back on ice for 2 minutes to reduce damage to the E.coli cells.<br />
<li>Add 1 ml of <a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#SOC_broth_for_bacteria">SOC Broth</a> (with no antibiotic added). Incubate tubes for 1 hour at 37°C.<br />
<li>Spread about 100 ul of the resulting culture on LB plates (with Ampicillin added). Grow overnight.<br />
<li>Pick colonies about 12-16 hours later.<br />
<br />
</ol><br />
</html><br />
<br />
=== '''Yeast transformation''' ===<br />
<br />
<html><br />
<ol><br />
<br />
<li>Prepare a 2 ml preculture in the selection medium of the strain to be transformed.<br />
<li>Inoculate 20 ml of YPD2% for each transformation, in order to get an OD600 = 1 next day.<br />
<ol></html>[[File:Asffsfasasf.png]]<html><br />
<br>Where n is the number of divisions (generation time: 1´5 h for S. cerevisiae).</ol><br />
From now, it is not necessary to work below sterility conditions.<br />
Once reached the OD600 = 1:<br />
<li>Centrifuge at 3000 rpm 5 min.<br />
<li>Wash with sterile water.<br />
<li>Resuspend in 30 ml of LISORB.<br />
<li>Shake at ambient temperature for 30 min.<br />
<li>Centrifuge at 3000 rpm 5 min and resuspend in 1 ml of LISORB. Transfer to an eppendorf tube.<br />
<li>Centrifuge at 3000 rpm 5 min.<br />
<li>Resuspend in 100 μl of LISORB for each transformation. Transfer 100 μL aliquots in different tubes for each transformation.<br />
<li>Add 7 μl of salmon sperm DNA + 1 μl of transforming DNA.<br />
<li>Incubate 10 min at ambient temperature.<br />
<li>Add 260 μl of 40%PEG/LiAc/TE. Mix well.<br />
<li>Incubate 1 h at 30°C.<br />
<li>Add 43 μl of DMSO and give a thermal shock of 5 minutes at 42°C.<br />
<li>Centrifuge at 3000 rpm 5 min.<br />
<li>Wash with 1 ml of sterile water.<br />
<li>Centrifuge at 3000 rpm 5 min.<br />
<li>Resuspend in 0´5 ml of water and plaque:<br />
<ol>-50 μl<br />
<br>-Rest (centrifuge and decant leaving 50-100 μl).</ol><br />
<br />
<br />
</ol><br />
</html><br />
<br />
== '''DNA extraction and purification protocols''' ==<br />
<br />
=== '''Mini-prep''' ===<br />
<br />
<html><br />
<ol><br />
<br />
<li>Different cultures (each one with a different construction), which are growing in a selective media (LB + Ampicillin), get centrifuged at 4500g 5 min.<br />
<li>Supernatant is removed.<br />
<li>The cells can be washed (x2) with a saline solution (PBS) in order to remove impurities.<br />
<li>The pellet is resuspended in 250 μL of Resuspension Solution (RNase A added to it previously. This solution is kept at 4ºC). Important: resuspend it completely.<br />
<li>Transfer the suspension to an eppendorf tube.<br />
<li>Add 250 μL of Lysis Solution.<br />
<li>Mix it inverting the tube 4-6 times <b>(DO NOT VORTEX!)</b> until solution gets viscous and slightly clear. <br />
<ol><b>Important: Do not incubate more than 5 min.</b></ol><br />
<li>Add 350 μL of Neutralization Solution.<br />
<li>Mix it inverting the tube 4-6 times. Incubate in ice for 15-30 min.<br />
<ol>Now if it was necessary, the process could stop here keeping the eppendorf tube in ice.</ol><br />
<li>Centrifuge 10’ (max. rpm) in order to pellet cell debris and chromosomal DNA.<br />
<li>Transfer the supernatant (≈ 800 μL) to the spin column (pipetting to avoid carrying impurities).<br />
<ol><b>Important: DO NOT TRANSFER THE PRECIPITATE!</ol></b><br />
<li>Centrifuge 1’.<br />
<li>Flow-though liquid is removed.<br />
<li>Add 500 μL of Wash Solution (Solution stock has to be perfectly closed, it contains ethanol!).<br />
<li>Centrifuge ≈ 1’.<br />
<li>Flow-though liquid is removed.<br />
<li>14, 15, 16 steps are repeated.<br />
<li>Centrifuge 1’ in order to eliminate residual Wash Solution.<br />
<li>The spin column is transferred into an eppendorf tube (the collection tube is eliminated).<br />
<li>Add 50 μL of Elution Buffer to the center of spin column membrane and let it 5’ getting soaked (it increases the efficiency of process).<br />
<ol><b>Important: DO NOT CONTACT THE COLUMN MEMBRANE WITH THE PIPETTE TIP!</ol></b><br />
<li>Centrifuge ≈ 2’.<br />
<li>To increase the efficiency (≈ 20%) we can get the flow-though liquid and repeat the steps previously described (20 and 21).<br />
<li>The column is discarded and the solution which contains the purified plasmid can be stored in cold.<br />
<br />
</ol><br />
</html><br />
<br />
==='''Protocol for Gel Extraction'''===<br />
<html><br />
<ol><br />
<br />
<li>Cut bands of interest from the agarose gel.<br />
<li>Add 300 uL of Solution L1 for each 100 mg of gel.<br />
<li>Incubate at 50ºC for 15 minutes.<br />
<li>Centrifugate in a 2 ml column at 12000xg for 1 minute.<br />
<li>Re-insert the spin column into the resaver tube and add 500 uL of Buffer L2.<br />
<li>Centrifugate 12000xg for 1 minute.<br />
<li>Discard the flow-through.<br />
<li>Centrifuge 12000xg for 1 minute. <br />
<li>Place the spin column into a new 1.5 mL microfuge tube.<br />
<li>Add 50 uL of mQ water. <br />
<li>Centrifuge 12000xg for 2 minutes.<br />
<br />
</ol><br />
</html><br />
<br />
== '''DNA digestion and ligation protocols''' ==<br />
<br />
<br />
=== '''Digestion Protocol For Plasmid Backbone Using EcoRI and PstI''' ===<br />
<br />
<html><br />
<ol><br />
<table border="1" style="background:transparent"><br />
<tr><br />
<td>DNA linearized plasmid Backbone (25 ng/uL)</td><br />
<td> </td><br />
<td>8 uL </td><br />
</tr><br />
<tr><br />
<td>PstI</td><br />
<td> </td><br />
<td>1 uL </td><br />
</tr><br />
<tr><br />
<td>EcoRI</td><br />
<td> </td><br />
<td>1 uL </td><br />
</tr><br />
<tr><br />
<td>Buffer 10x must be a common buffer for <br />
<br>EcoRI and PstI (e.g. buffer H in Roche system)</td><br />
<td> </td><br />
<td>2.5 uL</td><br />
</tr><br />
<tr><br />
<td>mQ Water</td><br />
<td> </td><br />
<td>x uL</td><br />
</tr><br />
<tr><br />
<td><b>TOTAL</b></td><br />
<td> </td><br />
<td>25 uL</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<br />
Mix by pipetting when both enzymes have been added. Avoid vortexing. Enzymes are kept in cooler or ice throughout all experiments. <br />
<ol><br />
<li>The digestion mixture is kept for 3 hours at 37 ° C<br />
<br />
<li>The mixture is kept for 20 minutes at 80ºC<br />
<br />
</ol><br />
</ol><br />
</html><br />
<br />
=== '''Digestion Protocol For Plasmid pUC57 + Construction Using EcoRI and PstI''' ===<br />
<br />
<html><br />
<ol><br />
<table border="1" style="background:transparent"><br />
<tr><br />
<td>Plasmid DNA</td><br />
<td> </td><br />
<td>96 uL </td><br />
</tr><br />
<tr><br />
<td>PstI</td><br />
<td> </td><br />
<td>2 uL + 2 uL</td><br />
</tr><br />
<tr><br />
<td>EcoRI</td><br />
<td> </td><br />
<td>2 uL + 2 uL</td><br />
</tr><br />
<tr><br />
<td>Buffer 10x must be a common buffer for <br />
<br>EcoRI and PstI (e.g. buffer H in Roche system)</td><br />
<td> </td><br />
<td>12 uL</td><br />
</tr><br />
<tr><br />
<td>mQ Water</td><br />
<td> </td><br />
<td>4 uL</td><br />
</tr><br />
<tr><br />
<td><b>TOTAL</b></td><br />
<td> </td><br />
<td>120 uL</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<br />
The volume used is so high because after digestion we were going to purify the different inserts from an agarose gel. <br>In order to optimize the digestion reaction we follow these steps <br />
<ol><br />
<li>2 uL of EcoRI is added and incubated for 1 hour.<br />
<li>2 uL of EcoRI is added and incubated for 1 hour.<br />
<li>2 uL of PstI is added and incubated for 1 hour.<br />
<li>2 uL of PstI is added and incubated for 1 hour.<br />
<br />
</ol><br />
</ol><br />
</html><br />
<br />
=== '''Ligation''' ===<br />
<br />
<html><br />
<ol><br />
<table border="1" style="background:transparent"><br />
<tr><br />
<td>Plasmid DNA*</td><br />
<td> </td><br />
<td>X uL </td><br />
</tr><br />
<tr><br />
<td>Insert DNA*</td><br />
<td> </td><br />
<td>Y uL </td><br />
</tr><br />
<tr><br />
<td>10X ligase buffer</td><br />
<td> </td><br />
<td>1 uL </td><br />
</tr><br />
<tr><br />
<td>T4 ligase</td><br />
<td> </td><br />
<td>1 uL </td><br />
</tr><br />
<tr><br />
<td>mQ Water</td><br />
<td> </td><br />
<td>8-(X+Y) uL</td><br />
</tr><br />
<tr><br />
<td><b>TOTAL</b></td><br />
<td> </td><br />
<td>10 uL</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<br />
<b>OBSERVATION:</b> The ratio that has to exist between the number of molecules of plasmid DNA and insert is 1:3 (the volumes depends on the concentration of DNAp and the insert). <br />
</ol><br />
</html><br />
<br />
=== '''Colony PCR''' ===<br />
<br />
<html><br />
<ol><br />
<br />
<li>Each colony is taken from the petri dish and <br />
<ol><ol type="a"><br />
<li>resuspended in 15 μl of mQ water in a eppendorf if we are working with bacteria.<br />
<li>resuspended in 15 μl of NaOH 20 mM in a eppendorf if we are working with yeast.<br />
</ol></ol><br />
<li>Incubate for 15 minutes at room temperature.<br />
<li>The PCR mix is prepared as shown:<br />
<ol> 2 ul of yeast DNA solution<br />
<br> 5 ul of 10X PCR buffer<br />
<br> 4 ul of dNTPs 2.5 mM<br />
<br> 2 ul of A oligo<br />
<br> 2 ul of B oligo<br />
<br> 31 ul of water<br />
</ol><br />
<li>Once mixed, 4 ul of 10X TAQ polymerase solution is added.<br />
<li>The PCR reaction program is the next one:<br />
<ol> 94ºC 3 minutes<br />
<br> 30 cycles of:<br />
<ol> 94ºC 1 minutes<br />
<br> 45ºC 1 minutes 30 seconds<br />
<br> 72ºC 2 minutes<br />
</ol><br />
72ºC 10 minutes<br />
<br> 4ºC Hold<br />
</ol><br />
<br />
</ol><br />
</html><br />
<br />
== '''Biobricks protocols''' ==<br />
<br />
<html><br />
<ol><br />
<br />
<li><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Digestion_Protocol_For_Plasmid_Backbone_Using_EcoRI_and_PstI"> Digestion Protocol For Plasmid Backbone Using EcoRI and PstI</a><br />
<li><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Digestion_Protocol_For_Plasmid_pUC57_.2B_Construction_Using_EcoRI_and_PstI"> Digestion Protocol For Plasmid pUC57 + Construction Using EcoRI and PstI </a><br />
<li><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Heat_Shock_Protocol_for_bacteria_transformation"> Transformation Protocol Using Heat Shock </a><br />
<li><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Ligation"> Ligation Protocol </a><br />
<li><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep"> Mini-preps. Purification protocol. </a><br />
<li><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Protocol_for_Gel_Extraction"> Protocol for Gel Extraction </a><br />
<br />
<br />
</ol><br />
</html><br />
<br />
</ol><br />
= '''Media and solutions protocols''' =<br />
<ol><br />
<br />
=== '''LB broth for bacteria''' ===<br />
<br />
<html><br />
<ol><br />
<br />
<table style="background:transparent"><br />
<tr><br />
<td><b>Bacterial peptone</b></td><br />
<td> </td><br />
<td>1% (p/v)</td><br />
</tr><br />
<tr><br />
<td><b>Yeast extract</b></td><br />
<td> </td><br />
<td>0.5% (p/v)</td><br />
</tr><br />
<tr><br />
<td><b>NaCl</b></td><br />
<td> </td><br />
<td>1% (p/v)</td><br />
</tr><br />
</table><br />
In order to obtain solid LB, add 15 g/L of bacteriologic agar.<br />
<br />
<br />
</html><br />
<br />
==== '''LBA broth for bacteria''' ====<br />
<br />
<html><br />
<ol><br />
<br />
Add 1 mL of ampicilin 100mg/mL (of mQ water) in 1L of <a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#LB_broth_for_bacteria">LB Broth</a><br />
<br />
</ol><br />
</html><br />
<br />
==== '''LB + Chloramphenicol broth for bacteria''' ====<br />
<br />
<html><br />
<ol><br />
<br />
Add 1 mL of Chloramphenicol 34mg/mL (of pure ethanol) in 1L of <a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#LB_broth_for_bacteria">LB Broth</a><br />
<br />
</ol><br />
</ol><br />
</html><br />
<br />
=== '''SOC broth for bacteria''' ===<br />
<br />
<html><br />
<ol><br />
<table style="background:transparent"><br />
<tr><br />
<td><b>Bacterial triptone</b></td><br />
<td> </td><br />
<td>4g</td><br />
</tr><br />
<tr><br />
<td><b>Yeast extract</b></td><br />
<td> </td><br />
<td>1g</td><br />
</tr><br />
<tr><br />
<td><b>NaCl 5M</b></td><br />
<td> </td><br />
<td>0.4 mL</td><br />
</tr><br />
<tr><br />
<td><b>KCl 3M</b></td><br />
<td> </td><br />
<td>0.167 mL</td><br />
</tr><br />
<tr><br />
<td><b>MgSO4 </b></td><br />
<td> </td><br />
<td>2.465 g</td><br />
</tr><br />
<tr><br />
<td><b>MgCl2 </b></td><br />
<td> </td><br />
<td>2.033 mL</td><br />
</tr><br />
<tr><br />
<td><b>Glucose</b></td><br />
<td> </td><br />
<td>3.603 g</td><br />
</tr><br />
<tr><br />
<td><b>Distiled water</b></td><br />
<td> </td><br />
<td>220 mL</td><br />
</tr><br />
</table><br />
Sterilization by filtration<br />
<br />
</ol><br />
</html><br />
<br />
=== '''YP broth for yeast''' ===<br />
<br />
<html><br />
<ol><br />
<br />
<table style="background:transparent"><br />
<tr><br />
<td><b>Bacterial peptone</b></td><br />
<td> </td><br />
<td>2% (p/v)</td><br />
</tr><br />
<tr><br />
<td><b>Yeast extract</b></td><br />
<td> </td><br />
<td>1% (p/v)</td><br />
</tr><br />
</table><br />
In order to obtain solid YP, add 2% of agar before sterilization.<br />
<br />
<br />
</html><br />
<br />
==== '''YPD broth for yeast''' ====<br />
<br />
<html><br />
<ol><br />
<br />
Add x% (p/v) of dextrose or glucose (sterilized by filtration) in <a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#YP_broth_for_yeast">YP Broth</a> after sterilization.<br />
<br />
</ol><br />
</html><br />
<br />
==== '''YPRE broth for yeast''' ====<br />
<br />
<html><br />
<ol><br />
<br />
Add 2% (p/v) of raffinose and 2% (v/v) of ethanol 100% in <a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#YP_broth_for_yeast">YP Broth</a> after sterilization.<br />
<br />
</ol><br />
</ol><br />
</html><br />
<br />
=== '''SD broth for yeast''' ===<br />
<br />
<html><br />
<ol><br />
<br />
<table style="background:transparent"><br />
<tr><br />
<td><b>Yeast nitrogen base</b> <br />
<br> w/o aminoacids and w/ amonium persulfate</td><br />
<td> &nbsp; &nbsp; &nbsp;</td><br />
<td>0.67% (p/v)</td><br />
</tr><br />
<tr><br />
<td><b>Glucose</b></td><br />
<td> </td><br />
<td>2% (p/v)</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<b>Aminoacids</b> (leucine, metionine, histidine) and <b>nucleotides</b> (uracil) are added after sterilization in a final concentration of 20 mg/mL.<br />
<br> In order to obtain solid SD, add 2% (p/v) of agar before sterilization.<br><br />
<br />
</ol><br />
</html><br />
<br />
=== '''LISORB solution for yeast transformation''' ===<br />
<html><br />
<b>For 100 mL</b><br />
<br><br />
<ol><br />
<table style="background:transparent"><br />
<tr><br />
<td><b>LiAc 1M</b></td><br />
<td> </td><br />
<td>10 mL</td><br />
</tr><br />
<tr><br />
<td><b>Sorbitol 2.4M</b></td><br />
<td> </td><br />
<td>41.6 mL</td><br />
</tr><br />
<tr><br />
<td><b>TE 100X</b></td><br />
<td> </td><br />
<td>1 mL</td><br />
</tr><br />
</table><br />
Bring to 100 ml of distilled H2O<br />
<br />
</ol><br />
</html><br />
<br />
=== '''40%PEG/LiAc/TE solution for yeast transformation''' ===<br />
<html><br />
<b>For 20 mL</b><br />
<br><br />
<ol><br />
<table style="background:transparent"><br />
<tr><br />
<td><b>LiAc 1M</b></td><br />
<td> </td><br />
<td>2 mL</td><br />
</tr><br />
<tr><br />
<td><b>PEG 3500 50%</b></td><br />
<td> </td><br />
<td>16 mL</td><br />
</tr><br />
<tr><br />
<td><b>TE 100X</b></td><br />
<td> </td><br />
<td>0.2 mL</td><br />
</tr><br />
</table><br />
Bring to 20 ml of distilled H2O<br />
</ol><br />
</html><br />
</ol><br />
<br />
= '''Yeast Induction protocol''' =<br />
<br />
<html><br />
<ol><br />
<br />
<li>Colonies are picked from petri dish and suspended in suplemented SD media.<br />
<li>Incubate overnight<br />
<li>Resuspend in YPD8%<br />
<li>Incubate overnight to 5 OD.<br />
<li>Resuspend in YPRE<br />
<li>Incubate for 8 to 24 hours<br />
<li>Add a final concentration of 0.3 mM of H2O2<br />
<li>Measure OD and fluorescence intensity.<br />
<br />
</ol></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/estiloc3po
Team:Valencia Biocampus/estiloc3po
2012-09-26T23:24:52Z
<p>Lujemomo: </p>
<hr />
<div><html><br />
<br />
<style type="text/css"><br />
<br />
#globalWrapper { background-color: transparent; border: none; margin: 0; padding: 0;}<br />
<br />
.firstHeading { /**/<br />
height:0px;<br />
visibility: hidden;<br />
}<br />
<br />
#menubar { <br />
background-color: #696969; /* left menu bar color*/<br />
}<br />
#menubar ul li a { <br />
color: #000000; } /* menu text color*/<br />
.right-menu li a { <br />
color: #A5C5EE; <br />
background-color: #FFFFFF; /* right menu bar color*/<br />
}<br />
#p-logo { height 0px; overflow:hidden; display: none;<br />
}<br />
#top-section { <br />
background-position: center center; <br />
background-repeat: no-repeat; <br />
background-color: #FFFFFF; /* top header background color*/<br />
border-width:0px;<br />
height:0px; /* top header height (determines how low content will start to appear)*/<br />
}<br />
#siteSub {<br />
display:none;<br />
}<br />
#search-controls { /* gets rid of the search bar (it was ugly) */<br />
display:none;<br />
margin-top:0px;<br />
}<br />
#contentSub {<br />
display:none;<br />
}<br />
<br />
<br />
<br />
<br />
/*background:transparent url("https://static.igem.org/mediawiki/2010/0/0c/Transpa.png") center top fixed repeat-y;*/<br />
/* El que estaba antes 26/10 https://static.igem.org/mediawiki/2010/8/86/Transpa30.png */<br />
<br />
#content{<br />
background:transparent url("https://static.igem.org/mediawiki/2010/f/f4/Transpa50.png") center top fixed repeat-y;<br />
padding: 0px;<br />
margin:0 auto;<br />
border: none; <br />
}<br />
<br />
<br />
/*Now to set heading and body fonts*/<br />
<br />
body {<br />
background: #000000 url(https://static.igem.org/mediawiki/2012/f/f9/Fondo_gris.jpeg) no-repeat top center fixed; <br />
/* background:#121212;*/<br />
font-family:Arial, Helvetica, sans-serif;<br />
font-size:8px; /* tamaño de fuente*/<br />
color:#fff;<br />
overflow-x:hidden; <br />
}<br />
<br />
#Titulos{<br />
// text-align: left;<br />
//font-family: Fantasy; /*Kautiva, Verdana, Geneva, sans-serif;*/<br />
//font-size: 30px;<br />
//font-weight: bold;<br />
//color: #000000;<br />
<br />
margin-top: 68px;<br />
background: none;<br />
width: 740px;<br />
padding: 10px;<br />
display: block;<br />
text-align: center;<br />
font-size: 18px;<br />
<br />
}<br />
<br />
#Titulos h2 {<br />
text-align: left;<br />
}<br />
<br />
/* To make the images borders transparents!!! */<br />
div.thumbinner {background-color:transparent;}<br />
div.thumb {border-color: transparent;}<br />
div.thumb {background-color: transparent;}<br />
h1 { color: #000000 }<br />
h2 { color: #000000 }<br />
h3 { color: #000000 }<br />
h4 { color: #000000 }<br />
<br />
#HomeCenter {<br />
width: 660px;<br />
height: auto;<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 14px;<br />
text-align: justify;<br />
margin-top: 8px;<br />
margin-bottom: 20px; <br />
margin-left: 12px; <br />
margin-right: 12px; <br />
padding: 0;<br />
}<br />
<br />
<br />
#HomeRight {<br />
width: 270px;<br />
height: auto;<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
}<br />
#HomeRight2 {<br />
width: 370px;<br />
height: auto;<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
}<br />
<br />
#IzqSup {<br />
width: 360px; <br />
height:auto; /* altura del fondo */<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: justify;<br />
margin-top: 0px;<br />
margin-bottom: 0px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 0;<br />
}<br />
<br />
#DerSup {<br />
width: 360px; <br />
height: auto; /* altura del fondo */<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
margin-top: 8px;<br />
margin-bottom: 30px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 30;<br />
} <br />
#DerSup2 {<br />
width: 250px; <br />
height: auto; /* altura del fondo */<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
margin-top: 8px;<br />
margin-bottom: 5px; <br />
margin-left: 12px; /* cuando espacio izquierdo dejo */<br />
margin-right: 8px; <br />
padding: 8;<br />
} <br />
#TOCBG{<br />
background-color:#9F9F9F;<br />
}<br />
<br />
#Inf {<br />
width: 710px; <br />
height: auto<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: justify;<br />
margin-top: 650px;<br />
margin-bottom: 10px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 0;<br />
}<br />
<br />
#PorDefecto {<br />
width: auto; <br />
height: auto<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: justify;<br />
margin-top: -30px; // para que el texto suba<br />
margin-bottom: 20px; <br />
margin-left: 12px; /* cuando espacio izquierdo dejo */<br />
margin-right: 12px; <br />
padding: 0;<br />
}<br />
<br />
#PosBocadillo {<br />
width: 360px; <br />
height: auto; /* altura del fondo */<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
margin-top: 8px;<br />
margin-bottom: 30px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 30;<br />
} <br />
//// <br />
#Contact { <br />
position: relative;<br />
text-align: left;<br />
}<br />
#Contact h5 {<br />
color: #D0D9E1;<br />
font-family: Georgia;<br />
font-size: 140px;<br />
letter-spacing: -6px;<br />
margin: 0;<br />
opacity: .9;<br />
padding: 0;<br />
-moz-transform: skew(20deg);<br />
-o-transform: skew(20deg);<br />
-webkit-transform: skew(20deg);<br />
}<br />
<br />
#Contact h6 {<br />
color: #4682B4;<br />
font-family: Verdana;<br />
font-size: 60px;<br />
<br />
left: 120px;<br />
letter-spacing: 20px;<br />
<br />
margin: 0;<br />
padding: 0;<br />
position: absolute;<br />
<br />
top: 20px;<br />
}<br />
<br />
<br />
////<br />
<!--<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
%<br />
% ESTILO 2: PARA LOS SPONSORS<br />
%<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
#sponsors {<br />
background: none;<br />
width: 740px;<br />
padding: 10px;<br />
display: block;<br />
text-align: center;<br />
}<br />
<br />
/* imagen de sponsor: separacion vertical respecto a la horizontal inferior */<br />
#sponsors img {<br />
padding: 15px;<br />
}<br />
<br />
/* encabezado: alineacion*/<br />
#sponsors h2 {<br />
text-align: left;<br />
}<br />
<br />
/* tamaños de las imagenes */<br />
a.tam1 img{<br />
height: 120px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam2 img{<br />
height: 100px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam3 img{<br />
height: 90px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam4 img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tamLogo img{<br />
height: 70px;<br />
width: 360px;<br />
float: left;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.meneillos img{<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
/* al pasar el ratón */<br />
a.tam1:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam2:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam3:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam4 img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tamLogo img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
<br />
a.meneillos:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
<br />
</style><br />
<br />
</html><br />
<br />
<!--<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
%<br />
% ESTILO 2: GALERIA DE EQUIPO: STUDENTS, INSTRUCTORS,...<br />
%<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
<br />
<html><br />
<style><br />
body, ul, li, h1, h2, h3{<br />
margin:-7; /* grosor del menú superior de edición */<br />
padding:-7; /* separación vertical del título con el borde superior */<br />
z-index:1;<br />
}<br />
<br />
.ps_overlay{<br />
z-index:1;<br />
background:#111;<br />
width:100%;<br />
height:100%;<br />
position:fixed;<br />
top:0px;<br />
left:0px;<br />
opacity:0.5;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=80);<br />
}<br />
/* Image container style */<br />
.ps_container{<br />
width:480px;<br />
height:350px;<br />
position:absolute;<br />
top:50%;<br />
margin-top:-175px;<br />
left:50%;<br />
margin-left:-240px;<br />
z-index:1;<br />
}<br />
.ps_container img{<br />
border:10px solid #fff;<br />
position:absolute;<br />
top:50%;<br />
left:50%;<br />
-moz-box-shadow:1px 1px 10px #000;<br />
-webkit-box-shadow:1px 1px 10px #000;<br />
box-shadow:1px 1px 10px #000;<br />
}<br />
<br />
/* Next photo button for preview mode */<br />
a.ps_next_photo{<br />
position:absolute;<br />
top:50%;<br />
left:50%;<br />
width:56px;<br />
height:56px;<br />
margin:-28px 0 0 -28px;<br />
z-index:200;<br />
cursor:pointer;<br />
background:#000 url(https://static.igem.org/mediawiki/2012/7/7f/Next_photoValencia.png) no-repeat 50% 50%;<br />
opacity:0.6;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=60); <br />
-moz-border-radius:10px;<br />
-webkit-border-radius:10px;<br />
border-radius:10px;<br />
}<br />
a.ps_next_photo:hover,<br />
a.ps_close:hover{<br />
opacity:0.8;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=80); <br />
}<br />
/* Thumbnail slider style */<br />
<br />
/* posicion espacial de todo el slider en conjunto*/<br />
.ps_slider{<br />
width:845px;<br />
height:700px; /* cuantos pixeles verticales ocupa el fondo del slier*/<br />
position:relative;<br />
margin:40px auto 0px auto;<br />
}<br />
.ps_slider a.next,<br />
.ps_slider a.prev{<br />
position:absolute;<br />
background-color:#000;<br />
background-position:center center;<br />
background-repeat:no-repeat;<br />
border:1px solid #232323;<br />
width:20px;<br />
height:20px;<br />
top:50%;<br />
margin-top:-10px;<br />
opacity:0.6;<br />
-moz-border-radius:5px;<br />
-webkit-border-radius:5px;<br />
border-radius:5px;<br />
cursor:pointer;<br />
outline:none;<br />
}<br />
<br />
.ps_slider a.prev:hover,<br />
.ps_slider a.next:hover{<br />
border:1px solid #333;<br />
opacity:0.9;<br />
}<br />
<br />
.ps_slider a.disabled,<br />
<br />
<br />
.ps_slider a.disabled:hover{<br />
opacity:0.4;<br />
border:1px solid #111;<br />
cursor:default;<br />
}<br />
.ps_slider a.prev{<br />
left:-30px;<br />
background-image:url(https://static.igem.org/mediawiki/2012/1/12/PrevValencia.png);<br />
}<br />
.ps_slider a.next{<br />
right:-30px;<br />
background-image:url(https://static.igem.org/mediawiki/2012/3/37/NextValencia.png);<br />
}<br />
<br />
/* cuadrado gris claro de cada slider*/<br />
.ps_slider .ps_album{<br />
width:140px;<br />
height:660px; /* altura*/<br />
padding:10px;<br />
background-color:#333;<br />
border:1px solid #444;<br />
position:absolute;<br />
top:0px;<br />
text-align:center;<br />
/*cursor:pointer;*/ /* QUITAMOS EL PUNTERO DE MOMENTO */<br />
-moz-box-shadow:1px 1px 4px #000;<br />
-webkit-box-shadow:1px 1px 4px #000;<br />
box-shadow:1px 1px 4px #000;<br />
-webkit-box-reflect:<br />
below 5px <br />
-webkit-gradient(<br />
linear, <br />
left top, <br />
left bottom, <br />
from(transparent), <br />
color-stop(0.6, transparent), <br />
to(rgb(18, 18, 18))<br />
);<br />
}<br />
/*LUZ DE CADA SLIDER, DE MOMENTO COMENTADO*/<br />
/*<br />
.ps_slider .ps_album:hover{<br />
background-color:#383838;<br />
}*/<br />
<br />
/* imagen de cada slider */<br />
.ps_slider .ps_album img{<br />
height:90px;<br />
border:1px solid #444;<br />
-moz-box-shadow:1px 1px 4px #000;<br />
-webkit-box-shadow:1px 1px 4px #000;<br />
box-shadow:1px 1px 4px #000;<br />
}<br />
<br />
/*cuadrado gris oscuro de cada slider*/<br />
.ps_slider .ps_album .ps_desc{<br />
display:block;<br />
color:#666;<br />
background:#111 url(https://static.igem.org/mediawiki/2012/7/7d/OverlayValencia.png) no-repeat bottom right;<br />
height:550px;<br />
margin-top:10px;<br />
text-align:left;<br />
line-height:20px;<br />
overflow:hidden;<br />
text-overflow:ellipsis;<br />
border:1px solid #393939;<br />
-moz-box-shadow:0px 0px 2px #000 inset;<br />
-webkit-box-shadow:0px 0px 2px #000 inset;<br />
box-shadow:0px 0px 2px #000 inset;<br />
}<br />
/*LUZ DE CADA SLIDER, DE MOMENTO COMENTADO*/<br />
/*<br />
.ps_slider .ps_album:hover .ps_desc{<br />
background-image:none;<br />
}*/<br />
.ps_slider .ps_album .ps_desc span{<br />
display:block;<br />
margin:0px 10px 10px 10px;<br />
border-top:1px solid #333;<br />
padding-top:5px;<br />
}<br />
.ps_slider .ps_album .ps_desc h2{<br />
margin:10px 10px 0px 10px;<br />
text-align:left;<br />
padding-bottom:5px;<br />
font-weight:normal;<br />
color:#ddd;<br />
text-shadow:0px 0px 1px #fff;<br />
border-bottom:1px solid #000;<br />
}<br />
.ps_slider .loading{<br />
background:#121212 url(https://static.igem.org/mediawiki/2012/5/57/LoadingValencia.gif) no-repeat 50% 50%;<br />
position:absolute;<br />
top:0px;<br />
left:0px;<br />
width:100%;<br />
height:100%;<br />
opacity:0.7;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=70);<br />
}<br />
<br />
span.reference{<br />
position:fixed;<br />
top:10px;<br />
right:10px;<br />
font-size:9px;<br />
}<br />
<br />
span.reference a{<br />
color:#aaa;<br />
text-decoration:none;<br />
text-transform:uppercase;<br />
margin-left:10px;<br />
}<br />
<br />
span.reference a:hover{<br />
color:#ddd;<br />
}<br />
<br />
h1.title{<br />
text-indent:-9000px;<br />
/*background:transparent url(https://static.igem.org/mediawiki/2012/2/22/StudentMembers.png) no-repeat top left;*/<br />
width:640px;<br />
height:52px;<br />
position:absolute;<br />
top:15px;<br />
left:15px;<br />
} <br />
<br />
</style><br />
</html></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/estiloc3po
Team:Valencia Biocampus/estiloc3po
2012-09-26T23:24:37Z
<p>Lujemomo: </p>
<hr />
<div><html><br />
<br />
<style type="text/css"><br />
<br />
#globalWrapper { background-color: transparent; border: none; margin: 0; padding: 0;}<br />
<br />
.firstHeading { /**/<br />
height:0px;<br />
visibility: hidden;<br />
}<br />
<br />
#menubar { <br />
background-color: #696969; /* left menu bar color*/<br />
}<br />
#menubar ul li a { <br />
color: #000000; } /* menu text color*/<br />
.right-menu li a { <br />
color: #A5C5EE; <br />
background-color: #FFFFFF; /* right menu bar color*/<br />
}<br />
#p-logo { height 0px; overflow:hidden; display: none;<br />
}<br />
#top-section { <br />
background-position: center center; <br />
background-repeat: no-repeat; <br />
background-color: #FFFFFF; /* top header background color*/<br />
border-width:0px;<br />
height:0px; /* top header height (determines how low content will start to appear)*/<br />
}<br />
#siteSub {<br />
display:none;<br />
}<br />
#search-controls { /* gets rid of the search bar (it was ugly) */<br />
display:none;<br />
margin-top:0px;<br />
}<br />
#contentSub {<br />
display:none;<br />
}<br />
<br />
<br />
<br />
<br />
/*background:transparent url("https://static.igem.org/mediawiki/2010/0/0c/Transpa.png") center top fixed repeat-y;*/<br />
/* El que estaba antes 26/10 https://static.igem.org/mediawiki/2010/8/86/Transpa30.png */<br />
<br />
#content{<br />
background:transparent url("https://static.igem.org/mediawiki/2010/f/f4/Transpa50.png") center top fixed repeat-y;<br />
padding: 0px;<br />
margin:0 auto;<br />
border: none; <br />
}<br />
<br />
<br />
/*Now to set heading and body fonts*/<br />
<br />
body {<br />
background: #000000 url(https://static.igem.org/mediawiki/2012/f/f9/Fondo_gris.jpeg) no-repeat top center fixed; <br />
/* background:#121212;*/<br />
font-family:Arial, Helvetica, sans-serif;<br />
font-size:8px; /* tamaño de fuente*/<br />
color:#fff;<br />
overflow-x:hidden; <br />
}<br />
<br />
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Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/Yeast
Team:Valencia Biocampus/Yeast
2012-09-26T23:15:03Z
<p>Lujemomo: /* INDUSTRIAL APPLICATIONS */</p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
<br />
<center><br />
<br><br><br />
<ol><br />
<div id="Titulos"><br />
<h2>Yeast Subteam</h2><br />
<br />
<br />
<div id="PorDefecto"><br />
=== '''THE IDEA''' ===<br />
<br><br />
Our aim in this part of the project is to detect when the yeast starts fermenting. At the end of the project we will be able to “ask” the yeast if there is still any glucose in the medium or not through the addition of H2O2. Furthermore, we will be able to know for how long the media has been running out of glucose.<br />
In conclusion, this project allows us to know how much time has elapsed since the fermentation began.<br />
<br />
To do this, we are going to use two gene constructions:<br><br><br />
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<br><br><br />
<br />
• '''The ADH2 promoter is fused to the YAP1 protein coding sequence.''' The YAP1 protein is a yeast transcription factor regulator of H2O2 adaptive response. It is stored in the cytoplasm in normal conditions and, in the presence of H2O2, it is transported to the nucleus where it acts as a transcription factor. The ADH2 promoter is activated in the absence of glucose.<br />
<br />
Thus, complete disappearance of glucose triggers the production of YAP1 in the cytoplasm, and YAP1 concentration increases if the lack of glucose continues. Notice that we are working with a delta-yap1 mutant strain as a recipient of our construct.<br />
<br />
• '''The TRR promoter is fused to the GFP (Green Fluorescence Protein) coding sequence.''' The green fluorescent protein can be detected by fluorescent emission. The tiorredoxin reductase promoter is activated by two transcriptional factors, YAP1 and SKN7 in the oxidative form. Both of them can only bind to the promoter if H2O2 has been previously added to the culture medium.<br />
<br><br />
<br><br><br />
<br />
=== '''MOLECULAR MECHANISMS''' ===<br />
<br />
<br><br />
Click on each plasmid to learn how our constructions work!<br />
<br><br />
<div style="text-align: center;"><br />
<table align="center" border="0.01" bordercolor="#9F9F9F" style="background-color:#9F9F9F"><br />
<tr><br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#Yeast"><br />
<img src="https://static.igem.org/mediawiki/2012/f/fe/Yeast_fermentative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#OXIDATIVE_STRESS_RESPONSE"><br />
<img src="https://static.igem.org/mediawiki/2012/9/9e/Yeast_oxidative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
</tr><br />
</table><br />
</div><br />
<br />
<br><br />
Each construction was carried in different plasmids for their expression in yeast:<br />
<br><br />
'''YEplac181 carried the Fermentative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4893/">click here</a></html>.<br />
<br><br />
'''YEp352 carried the Oxidative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4867/">click here</a></html>.<br />
<br><br><br><br />
<br />
=== '''INDUSTRIAL APPLICATIONS''' ===<br />
<html><br />
<br><br />
<div id="DerSup2"><br />
<br />
</html>[[Image:Aplicacionesindustriales.jpg|250px|right]]<br />
</div><br />
The main industrial application of our talking yeast is to know the moment at which fermentation started. This is possible because we can calculate the time elapsed since the medium ran out of glucose according to the fluorescence intensity. Taking this into account, we could ask the yeast, through the addition of hydrogen peroxide: <b>“How long have you been fermenting?”</b> And the cells would answer telling us how long it has been since all the glucose was consumed. One possible answer could be <b>“I have been fermenting for 1 hour”</b>. <br />
<br><br><br />
There is also a second possible application, but it needs a more detailed explanation:<br />
<br><br><br />
There are several factors affecting yeast ethanol production during alcoholic fermentation. <i>Saccharomyces cerevisiae</i> seems to have adapted all along its evolution to optimize its growing rate in environments that are rich in easily assimilable nutrients, such as sugars and amino acids. <br />
<br><br><br />
There are two important characteristics responsible of <i>S. cerevisiae</i> adaptation to this particular niche. One of them is its capability of metabolizing glucose and fructose through both the respiration and the fermentation pathways. The second one is its ability to grow in aerobic and anaerobic conditions. All of this makes this species exhibit some metabolic peculiarities, such as the Pasteur and the Crabtree effects. The Crabtree effect, described in <i>S. cerevisiae</i> and in a few other yeast species, must be taken into account when ethanol production is being studied or modified during fermentation. This effect causes that, even under high concentration of oxygen and with a relatively low amount of glucose, a considerable fraction of the consumed sugar is used to produce ethanol through the fermentative pathway. This is why in every industrial process which requires yeast growth (such as starter yeast production) it is necessary to provide the optimum amount of glucose that permits its direct consumption through the respiratory pathway (or the other way round if fermentation is desirable, such in bioethanol production). <br />
<br><br><br />
We present here one way of monitoring the fate of glucose in the medium. By adding a small amount of hydrogen peroxide as a chemical input, we plan to ask our culture: <b>“Is there any glucose left?”</b>, and the culture would answer -according to the amount of glucose already present in the medium- what concentration would be necessary to optimize either yeast growth or fermentation through the Crabtree effect. In this way, the Crabtree effect could be monitored (perhaps even online) by measuring fluorescence and used to regulate the amount of glucose to be fed. <br />
<br><br />
<br><br />
<br />
=== '''EXPERIMENTAL OUTLINE''' ===<br />
<br />
- We ordered the DNA constructions: pADH2-YAP1 protein and pTRR-GFP protein that were cloned in plasmid pUC57 with a bacterial origin of replication. <br />
<br><br />
- We obtain the Yeplac181 and Yep352 yeast vectors by our laboratory.<br />
<br><br />
- We carried out four transformations of <i>E. coli</i> strain DH5, one for each plasmid DNA (the two constructions and the two vectors), in order to amplify them. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Heat_Shock_Protocol_for_bacteria_transformation"> Transformation Protocol Using Heat Shock </a></html>.<br />
<br><br />
- We obtained several <i>E. coli</i> transformants in four plates and took some colonies of each DNA construction (from both constructions and both vectors) and cultured them in liquid medium over night at 37ºC in shaking flasks. <br />
<br><br />
- A day after, we extracted the plasmid DNA. See the Mini-preps protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- We obtained the purified constructions (both in pUC57 plasmid) and the vectors for yeast (YEplac181 and YEp352) also purified. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Protocol_for_Gel_Extraction"> Protocol for Gel Extraction </a></html><br />
<br><br />
- We digested the four DNAs with restriction enzymes EcoRI and PstI in order to obtain compatible ends. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Digestion_Protocol_For_Plasmid_pUC57_.2B_Construction_Using_EcoRI_and_PstI"> Digestion Protocol </a></html>.<br />
<br><br />
- We ligated the pTRR-GFP construction with the Yep352 vector and the ADH2-YAP1 construction with the Yeplac181 vector. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Ligation">ligation Protocol</a></html>.<br />
<br><br />
- The day after that we transformed <i>E. coli</i> with the results of the ligation in order to amplify the final constructions (pTRR-GFP/Yep352 and pADH2-YAP1/YEplac181). We located the recombinant constructs using X-Gal and white/blue selection. <br />
<br><br />
- We took some of these white colonies and cultured them in liquid medium overnight at 37ºC in shaking flasks. <br />
<br><br />
- The day after we extracted the plasmid DNA. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- After this, we checked the final purified recombinant constructs by electrophoresis, restriction digest and DNA capilar sequencing.<br />
<br><br />
- We introduced the first of the DNA recombinant plasmids in the yeast. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- We selected the transformants by growth in solid and liquid mineral medium attending to the auxotrophic markers and checked the presence of the construction by PCR. See the protocol <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Colony_PCR">here</a></html>.<br />
<br><br />
- We used the transformed yeast obtained at that moment and transformed it with the second recombinant construct. See the yeast transformation protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- Once again we identified the transformants by growth in selective medium and after that we used a PCR protocol to check the presence of both constructions.<br />
<br><br />
- In order to detect fluorescence, we carried out a protocol to induce the expression of GFP. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_Induction_protocol"> Yeast induction protocol </a></html>.<br />
<br><br />
<br />
</div></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/Yeast
Team:Valencia Biocampus/Yeast
2012-09-26T23:14:47Z
<p>Lujemomo: /* INDUSTRIAL APPLICATIONS */</p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
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<div id="Titulos"><br />
<h2>Yeast Subteam</h2><br />
<br />
<br />
<div id="PorDefecto"><br />
=== '''THE IDEA''' ===<br />
<br><br />
Our aim in this part of the project is to detect when the yeast starts fermenting. At the end of the project we will be able to “ask” the yeast if there is still any glucose in the medium or not through the addition of H2O2. Furthermore, we will be able to know for how long the media has been running out of glucose.<br />
In conclusion, this project allows us to know how much time has elapsed since the fermentation began.<br />
<br />
To do this, we are going to use two gene constructions:<br><br><br />
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<br><br><br />
<br />
• '''The ADH2 promoter is fused to the YAP1 protein coding sequence.''' The YAP1 protein is a yeast transcription factor regulator of H2O2 adaptive response. It is stored in the cytoplasm in normal conditions and, in the presence of H2O2, it is transported to the nucleus where it acts as a transcription factor. The ADH2 promoter is activated in the absence of glucose.<br />
<br />
Thus, complete disappearance of glucose triggers the production of YAP1 in the cytoplasm, and YAP1 concentration increases if the lack of glucose continues. Notice that we are working with a delta-yap1 mutant strain as a recipient of our construct.<br />
<br />
• '''The TRR promoter is fused to the GFP (Green Fluorescence Protein) coding sequence.''' The green fluorescent protein can be detected by fluorescent emission. The tiorredoxin reductase promoter is activated by two transcriptional factors, YAP1 and SKN7 in the oxidative form. Both of them can only bind to the promoter if H2O2 has been previously added to the culture medium.<br />
<br><br />
<br><br><br />
<br />
=== '''MOLECULAR MECHANISMS''' ===<br />
<br />
<br><br />
Click on each plasmid to learn how our constructions work!<br />
<br><br />
<div style="text-align: center;"><br />
<table align="center" border="0.01" bordercolor="#9F9F9F" style="background-color:#9F9F9F"><br />
<tr><br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#Yeast"><br />
<img src="https://static.igem.org/mediawiki/2012/f/fe/Yeast_fermentative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#OXIDATIVE_STRESS_RESPONSE"><br />
<img src="https://static.igem.org/mediawiki/2012/9/9e/Yeast_oxidative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
</tr><br />
</table><br />
</div><br />
<br />
<br><br />
Each construction was carried in different plasmids for their expression in yeast:<br />
<br><br />
'''YEplac181 carried the Fermentative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4893/">click here</a></html>.<br />
<br><br />
'''YEp352 carried the Oxidative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4867/">click here</a></html>.<br />
<br><br><br><br />
<br />
=== '''INDUSTRIAL APPLICATIONS''' ===<br />
<html><br />
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<div id="DerSup2"><br />
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</html>[[Image:Aplicacionesindustriales.jpg|250px|right]]<br />
</div><br />
The main industrial application of our talking yeast is to know the moment at which fermentation started. This is possible because we can calculate the time elapsed since the medium ran out of glucose according to the fluorescence intensity. Taking this into account, we could ask the yeast, through the addition of hydrogen peroxide: <b>“How long have you been fermenting?”</b> And the cells would answer telling us how long it has been since all the glucose was consumed. One possible answer could be <b>“I have been fermenting for 1 hour”</b>. <br />
<br><br><br />
There is also a second possible application, but it needs a more detailed explanation:<br />
<br><br><br />
There are several factors affecting yeast ethanol production during alcoholic fermentation. <i>Saccharomyces cerevisiae</i> seems to have adapted all along its evolution to optimize its growing rate in environments that are rich in easily assimilable nutrients, such as sugars and amino acids. <br />
<br><br><br />
There are two important characteristics responsible of <i>S. cerevisiae</i> adaptation to this particular niche. One of them is its capability of metabolizing glucose and fructose through both the respiration and the fermentation pathways. The second one is its ability to grow in aerobic and anaerobic conditions. All of this makes this species exhibit some metabolic peculiarities, such as the Pasteur and the Crabtree effects. The Crabtree effect, described in <i>S. cerevisiae</i> and in a few other yeast species, must be taken into account when ethanol production is being studied or modified during fermentation. This effect causes that, even under high concentration of oxygen and with a relatively low amount of glucose, a considerable fraction of the consumed sugar is used to produce ethanol through the fermentative pathway. This is why in every industrial process which requires yeast growth (such as starter yeast production) it is necessary to provide the optimum amount of glucose that permits its direct consumption through the respiratory pathway (or the other way round if fermentation is desirable, such in bioethanol production). <br />
<br><br><br />
We present here one way of monitoring the fate of glucose in the medium. By adding a small amount of hydrogen peroxide as a chemical input, we plan to ask our culture: <b>“Is there any glucose left?”</b>, and the culture would answer -according to the amount of glucose already present in the medium- what concentration would be necessary to optimize either yeast growth or fermentation through the Crabtree effect. In this way, the Crabtree effect could be monitored (perhaps even online) by measuring fluorescence and used to regulate the amount of glucose to be fed. <br />
<br><br />
<br><br><br />
</html><br />
<br />
=== '''EXPERIMENTAL OUTLINE''' ===<br />
<br />
- We ordered the DNA constructions: pADH2-YAP1 protein and pTRR-GFP protein that were cloned in plasmid pUC57 with a bacterial origin of replication. <br />
<br><br />
- We obtain the Yeplac181 and Yep352 yeast vectors by our laboratory.<br />
<br><br />
- We carried out four transformations of <i>E. coli</i> strain DH5, one for each plasmid DNA (the two constructions and the two vectors), in order to amplify them. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Heat_Shock_Protocol_for_bacteria_transformation"> Transformation Protocol Using Heat Shock </a></html>.<br />
<br><br />
- We obtained several <i>E. coli</i> transformants in four plates and took some colonies of each DNA construction (from both constructions and both vectors) and cultured them in liquid medium over night at 37ºC in shaking flasks. <br />
<br><br />
- A day after, we extracted the plasmid DNA. See the Mini-preps protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- We obtained the purified constructions (both in pUC57 plasmid) and the vectors for yeast (YEplac181 and YEp352) also purified. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Protocol_for_Gel_Extraction"> Protocol for Gel Extraction </a></html><br />
<br><br />
- We digested the four DNAs with restriction enzymes EcoRI and PstI in order to obtain compatible ends. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Digestion_Protocol_For_Plasmid_pUC57_.2B_Construction_Using_EcoRI_and_PstI"> Digestion Protocol </a></html>.<br />
<br><br />
- We ligated the pTRR-GFP construction with the Yep352 vector and the ADH2-YAP1 construction with the Yeplac181 vector. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Ligation">ligation Protocol</a></html>.<br />
<br><br />
- The day after that we transformed <i>E. coli</i> with the results of the ligation in order to amplify the final constructions (pTRR-GFP/Yep352 and pADH2-YAP1/YEplac181). We located the recombinant constructs using X-Gal and white/blue selection. <br />
<br><br />
- We took some of these white colonies and cultured them in liquid medium overnight at 37ºC in shaking flasks. <br />
<br><br />
- The day after we extracted the plasmid DNA. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- After this, we checked the final purified recombinant constructs by electrophoresis, restriction digest and DNA capilar sequencing.<br />
<br><br />
- We introduced the first of the DNA recombinant plasmids in the yeast. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- We selected the transformants by growth in solid and liquid mineral medium attending to the auxotrophic markers and checked the presence of the construction by PCR. See the protocol <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Colony_PCR">here</a></html>.<br />
<br><br />
- We used the transformed yeast obtained at that moment and transformed it with the second recombinant construct. See the yeast transformation protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- Once again we identified the transformants by growth in selective medium and after that we used a PCR protocol to check the presence of both constructions.<br />
<br><br />
- In order to detect fluorescence, we carried out a protocol to induce the expression of GFP. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_Induction_protocol"> Yeast induction protocol </a></html>.<br />
<br><br />
<br />
</div></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/estiloc3po
Team:Valencia Biocampus/estiloc3po
2012-09-26T23:13:36Z
<p>Lujemomo: </p>
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Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/Yeast
Team:Valencia Biocampus/Yeast
2012-09-26T23:13:03Z
<p>Lujemomo: /* INDUSTRIAL APPLICATIONS */</p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
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<center><br />
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<ol><br />
<div id="Titulos"><br />
<h2>Yeast Subteam</h2><br />
<br />
<br />
<div id="PorDefecto"><br />
=== '''THE IDEA''' ===<br />
<br><br />
Our aim in this part of the project is to detect when the yeast starts fermenting. At the end of the project we will be able to “ask” the yeast if there is still any glucose in the medium or not through the addition of H2O2. Furthermore, we will be able to know for how long the media has been running out of glucose.<br />
In conclusion, this project allows us to know how much time has elapsed since the fermentation began.<br />
<br />
To do this, we are going to use two gene constructions:<br><br><br />
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<br><br><br />
<br />
• '''The ADH2 promoter is fused to the YAP1 protein coding sequence.''' The YAP1 protein is a yeast transcription factor regulator of H2O2 adaptive response. It is stored in the cytoplasm in normal conditions and, in the presence of H2O2, it is transported to the nucleus where it acts as a transcription factor. The ADH2 promoter is activated in the absence of glucose.<br />
<br />
Thus, complete disappearance of glucose triggers the production of YAP1 in the cytoplasm, and YAP1 concentration increases if the lack of glucose continues. Notice that we are working with a delta-yap1 mutant strain as a recipient of our construct.<br />
<br />
• '''The TRR promoter is fused to the GFP (Green Fluorescence Protein) coding sequence.''' The green fluorescent protein can be detected by fluorescent emission. The tiorredoxin reductase promoter is activated by two transcriptional factors, YAP1 and SKN7 in the oxidative form. Both of them can only bind to the promoter if H2O2 has been previously added to the culture medium.<br />
<br><br />
<br><br><br />
<br />
=== '''MOLECULAR MECHANISMS''' ===<br />
<br />
<br><br />
Click on each plasmid to learn how our constructions work!<br />
<br><br />
<div style="text-align: center;"><br />
<table align="center" border="0.01" bordercolor="#9F9F9F" style="background-color:#9F9F9F"><br />
<tr><br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#Yeast"><br />
<img src="https://static.igem.org/mediawiki/2012/f/fe/Yeast_fermentative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#OXIDATIVE_STRESS_RESPONSE"><br />
<img src="https://static.igem.org/mediawiki/2012/9/9e/Yeast_oxidative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
</tr><br />
</table><br />
</div><br />
<br />
<br><br />
Each construction was carried in different plasmids for their expression in yeast:<br />
<br><br />
'''YEplac181 carried the Fermentative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4893/">click here</a></html>.<br />
<br><br />
'''YEp352 carried the Oxidative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4867/">click here</a></html>.<br />
<br><br><br><br />
<br />
=== '''INDUSTRIAL APPLICATIONS''' ===<br />
<html><br />
<br><br />
<div id="DerSup2"><br />
<br />
</html>[[Image:Aplicacionesindustriales.jpg|250px|right]]<html><br />
</div><br />
The main industrial application of our talking yeast is to know the moment at which fermentation started. This is possible because we can calculate the time elapsed since the medium ran out of glucose according to the fluorescence intensity. Taking this into account, we could ask the yeast, through the addition of hydrogen peroxide: <b>“How long have you been fermenting?”</b> And the cells would answer telling us how long it has been since all the glucose was consumed. One possible answer could be <b>“I have been fermenting for 1 hour”</b>. <br />
<br><br><br />
There is also a second possible application, but it needs a more detailed explanation:<br />
<br><br><br />
There are several factors affecting yeast ethanol production during alcoholic fermentation. <i>Saccharomyces cerevisiae</i> seems to have adapted all along its evolution to optimize its growing rate in environments that are rich in easily assimilable nutrients, such as sugars and amino acids. <br />
<br><br><br />
There are two important characteristics responsible of <i>S. cerevisiae</i> adaptation to this particular niche. One of them is its capability of metabolizing glucose and fructose through both the respiration and the fermentation pathways. The second one is its ability to grow in aerobic and anaerobic conditions. All of this makes this species exhibit some metabolic peculiarities, such as the Pasteur and the Crabtree effects. The Crabtree effect, described in <i>S. cerevisiae</i> and in a few other yeast species, must be taken into account when ethanol production is being studied or modified during fermentation. This effect causes that, even under high concentration of oxygen and with a relatively low amount of glucose, a considerable fraction of the consumed sugar is used to produce ethanol through the fermentative pathway. This is why in every industrial process which requires yeast growth (such as starter yeast production) it is necessary to provide the optimum amount of glucose that permits its direct consumption through the respiratory pathway (or the other way round if fermentation is desirable, such in bioethanol production). <br />
<br><br><br />
We present here one way of monitoring the fate of glucose in the medium. By adding a small amount of hydrogen peroxide as a chemical input, we plan to ask our culture: <b>“Is there any glucose left?”</b>, and the culture would answer -according to the amount of glucose already present in the medium- what concentration would be necessary to optimize either yeast growth or fermentation through the Crabtree effect. In this way, the Crabtree effect could be monitored (perhaps even online) by measuring fluorescence and used to regulate the amount of glucose to be fed. <br />
<br><br />
<br><br><br />
</html><br />
<br />
=== '''EXPERIMENTAL OUTLINE''' ===<br />
<br />
- We ordered the DNA constructions: pADH2-YAP1 protein and pTRR-GFP protein that were cloned in plasmid pUC57 with a bacterial origin of replication. <br />
<br><br />
- We obtain the Yeplac181 and Yep352 yeast vectors by our laboratory.<br />
<br><br />
- We carried out four transformations of <i>E. coli</i> strain DH5, one for each plasmid DNA (the two constructions and the two vectors), in order to amplify them. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Heat_Shock_Protocol_for_bacteria_transformation"> Transformation Protocol Using Heat Shock </a></html>.<br />
<br><br />
- We obtained several <i>E. coli</i> transformants in four plates and took some colonies of each DNA construction (from both constructions and both vectors) and cultured them in liquid medium over night at 37ºC in shaking flasks. <br />
<br><br />
- A day after, we extracted the plasmid DNA. See the Mini-preps protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- We obtained the purified constructions (both in pUC57 plasmid) and the vectors for yeast (YEplac181 and YEp352) also purified. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Protocol_for_Gel_Extraction"> Protocol for Gel Extraction </a></html><br />
<br><br />
- We digested the four DNAs with restriction enzymes EcoRI and PstI in order to obtain compatible ends. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Digestion_Protocol_For_Plasmid_pUC57_.2B_Construction_Using_EcoRI_and_PstI"> Digestion Protocol </a></html>.<br />
<br><br />
- We ligated the pTRR-GFP construction with the Yep352 vector and the ADH2-YAP1 construction with the Yeplac181 vector. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Ligation">ligation Protocol</a></html>.<br />
<br><br />
- The day after that we transformed <i>E. coli</i> with the results of the ligation in order to amplify the final constructions (pTRR-GFP/Yep352 and pADH2-YAP1/YEplac181). We located the recombinant constructs using X-Gal and white/blue selection. <br />
<br><br />
- We took some of these white colonies and cultured them in liquid medium overnight at 37ºC in shaking flasks. <br />
<br><br />
- The day after we extracted the plasmid DNA. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- After this, we checked the final purified recombinant constructs by electrophoresis, restriction digest and DNA capilar sequencing.<br />
<br><br />
- We introduced the first of the DNA recombinant plasmids in the yeast. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- We selected the transformants by growth in solid and liquid mineral medium attending to the auxotrophic markers and checked the presence of the construction by PCR. See the protocol <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Colony_PCR">here</a></html>.<br />
<br><br />
- We used the transformed yeast obtained at that moment and transformed it with the second recombinant construct. See the yeast transformation protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- Once again we identified the transformants by growth in selective medium and after that we used a PCR protocol to check the presence of both constructions.<br />
<br><br />
- In order to detect fluorescence, we carried out a protocol to induce the expression of GFP. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_Induction_protocol"> Yeast induction protocol </a></html>.<br />
<br><br />
<br />
</div></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/estiloc3po
Team:Valencia Biocampus/estiloc3po
2012-09-26T23:12:57Z
<p>Lujemomo: </p>
<hr />
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margin-bottom: 10px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 0;<br />
}<br />
<br />
#PorDefecto {<br />
width: auto; <br />
height: auto<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: justify;<br />
margin-top: -30px; // para que el texto suba<br />
margin-bottom: 20px; <br />
margin-left: 12px; /* cuando espacio izquierdo dejo */<br />
margin-right: 12px; <br />
padding: 0;<br />
}<br />
<br />
#PosBocadillo {<br />
width: 360px; <br />
height: auto; /* altura del fondo */<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
margin-top: 8px;<br />
margin-bottom: 30px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 30;<br />
} <br />
//// <br />
#Contact { <br />
position: relative;<br />
text-align: left;<br />
}<br />
#Contact h5 {<br />
color: #D0D9E1;<br />
font-family: Georgia;<br />
font-size: 140px;<br />
letter-spacing: -6px;<br />
margin: 0;<br />
opacity: .9;<br />
padding: 0;<br />
-moz-transform: skew(20deg);<br />
-o-transform: skew(20deg);<br />
-webkit-transform: skew(20deg);<br />
}<br />
<br />
#Contact h6 {<br />
color: #4682B4;<br />
font-family: Verdana;<br />
font-size: 60px;<br />
<br />
left: 120px;<br />
letter-spacing: 20px;<br />
<br />
margin: 0;<br />
padding: 0;<br />
position: absolute;<br />
<br />
top: 20px;<br />
}<br />
<br />
<br />
////<br />
<!--<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
%<br />
% ESTILO 2: PARA LOS SPONSORS<br />
%<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
#sponsors {<br />
background: none;<br />
width: 740px;<br />
padding: 10px;<br />
display: block;<br />
text-align: center;<br />
}<br />
<br />
/* imagen de sponsor: separacion vertical respecto a la horizontal inferior */<br />
#sponsors img {<br />
padding: 15px;<br />
}<br />
<br />
/* encabezado: alineacion*/<br />
#sponsors h2 {<br />
text-align: left;<br />
}<br />
<br />
/* tamaños de las imagenes */<br />
a.tam1 img{<br />
height: 120px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam2 img{<br />
height: 100px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam3 img{<br />
height: 90px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam4 img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tamLogo img{<br />
height: 70px;<br />
width: 360px;<br />
float: left;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.meneillos img{<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
/* al pasar el ratón */<br />
a.tam1:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam2:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam3:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam4 img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tamLogo img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
<br />
a.meneillos:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
<br />
</style><br />
<br />
</html><br />
<br />
<!--<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
%<br />
% ESTILO 2: GALERIA DE EQUIPO: STUDENTS, INSTRUCTORS,...<br />
%<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
<br />
<html><br />
<style><br />
body, ul, li, h1, h2, h3{<br />
margin:-7; /* grosor del menú superior de edición */<br />
padding:-7; /* separación vertical del título con el borde superior */<br />
z-index:1;<br />
}<br />
<br />
.ps_overlay{<br />
z-index:1;<br />
background:#111;<br />
width:100%;<br />
height:100%;<br />
position:fixed;<br />
top:0px;<br />
left:0px;<br />
opacity:0.5;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=80);<br />
}<br />
/* Image container style */<br />
.ps_container{<br />
width:480px;<br />
height:350px;<br />
position:absolute;<br />
top:50%;<br />
margin-top:-175px;<br />
left:50%;<br />
margin-left:-240px;<br />
z-index:1;<br />
}<br />
.ps_container img{<br />
border:10px solid #fff;<br />
position:absolute;<br />
top:50%;<br />
left:50%;<br />
-moz-box-shadow:1px 1px 10px #000;<br />
-webkit-box-shadow:1px 1px 10px #000;<br />
box-shadow:1px 1px 10px #000;<br />
}<br />
<br />
/* Next photo button for preview mode */<br />
a.ps_next_photo{<br />
position:absolute;<br />
top:50%;<br />
left:50%;<br />
width:56px;<br />
height:56px;<br />
margin:-28px 0 0 -28px;<br />
z-index:200;<br />
cursor:pointer;<br />
background:#000 url(https://static.igem.org/mediawiki/2012/7/7f/Next_photoValencia.png) no-repeat 50% 50%;<br />
opacity:0.6;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=60); <br />
-moz-border-radius:10px;<br />
-webkit-border-radius:10px;<br />
border-radius:10px;<br />
}<br />
a.ps_next_photo:hover,<br />
a.ps_close:hover{<br />
opacity:0.8;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=80); <br />
}<br />
/* Thumbnail slider style */<br />
<br />
/* posicion espacial de todo el slider en conjunto*/<br />
.ps_slider{<br />
width:845px;<br />
height:700px; /* cuantos pixeles verticales ocupa el fondo del slier*/<br />
position:relative;<br />
margin:40px auto 0px auto;<br />
}<br />
.ps_slider a.next,<br />
.ps_slider a.prev{<br />
position:absolute;<br />
background-color:#000;<br />
background-position:center center;<br />
background-repeat:no-repeat;<br />
border:1px solid #232323;<br />
width:20px;<br />
height:20px;<br />
top:50%;<br />
margin-top:-10px;<br />
opacity:0.6;<br />
-moz-border-radius:5px;<br />
-webkit-border-radius:5px;<br />
border-radius:5px;<br />
cursor:pointer;<br />
outline:none;<br />
}<br />
<br />
.ps_slider a.prev:hover,<br />
.ps_slider a.next:hover{<br />
border:1px solid #333;<br />
opacity:0.9;<br />
}<br />
<br />
.ps_slider a.disabled,<br />
<br />
<br />
.ps_slider a.disabled:hover{<br />
opacity:0.4;<br />
border:1px solid #111;<br />
cursor:default;<br />
}<br />
.ps_slider a.prev{<br />
left:-30px;<br />
background-image:url(https://static.igem.org/mediawiki/2012/1/12/PrevValencia.png);<br />
}<br />
.ps_slider a.next{<br />
right:-30px;<br />
background-image:url(https://static.igem.org/mediawiki/2012/3/37/NextValencia.png);<br />
}<br />
<br />
/* cuadrado gris claro de cada slider*/<br />
.ps_slider .ps_album{<br />
width:140px;<br />
height:660px; /* altura*/<br />
padding:10px;<br />
background-color:#333;<br />
border:1px solid #444;<br />
position:absolute;<br />
top:0px;<br />
text-align:center;<br />
/*cursor:pointer;*/ /* QUITAMOS EL PUNTERO DE MOMENTO */<br />
-moz-box-shadow:1px 1px 4px #000;<br />
-webkit-box-shadow:1px 1px 4px #000;<br />
box-shadow:1px 1px 4px #000;<br />
-webkit-box-reflect:<br />
below 5px <br />
-webkit-gradient(<br />
linear, <br />
left top, <br />
left bottom, <br />
from(transparent), <br />
color-stop(0.6, transparent), <br />
to(rgb(18, 18, 18))<br />
);<br />
}<br />
/*LUZ DE CADA SLIDER, DE MOMENTO COMENTADO*/<br />
/*<br />
.ps_slider .ps_album:hover{<br />
background-color:#383838;<br />
}*/<br />
<br />
/* imagen de cada slider */<br />
.ps_slider .ps_album img{<br />
height:90px;<br />
border:1px solid #444;<br />
-moz-box-shadow:1px 1px 4px #000;<br />
-webkit-box-shadow:1px 1px 4px #000;<br />
box-shadow:1px 1px 4px #000;<br />
}<br />
<br />
/*cuadrado gris oscuro de cada slider*/<br />
.ps_slider .ps_album .ps_desc{<br />
display:block;<br />
color:#666;<br />
background:#111 url(https://static.igem.org/mediawiki/2012/7/7d/OverlayValencia.png) no-repeat bottom right;<br />
height:550px;<br />
margin-top:10px;<br />
text-align:left;<br />
line-height:20px;<br />
overflow:hidden;<br />
text-overflow:ellipsis;<br />
border:1px solid #393939;<br />
-moz-box-shadow:0px 0px 2px #000 inset;<br />
-webkit-box-shadow:0px 0px 2px #000 inset;<br />
box-shadow:0px 0px 2px #000 inset;<br />
}<br />
/*LUZ DE CADA SLIDER, DE MOMENTO COMENTADO*/<br />
/*<br />
.ps_slider .ps_album:hover .ps_desc{<br />
background-image:none;<br />
}*/<br />
.ps_slider .ps_album .ps_desc span{<br />
display:block;<br />
margin:0px 10px 10px 10px;<br />
border-top:1px solid #333;<br />
padding-top:5px;<br />
}<br />
.ps_slider .ps_album .ps_desc h2{<br />
margin:10px 10px 0px 10px;<br />
text-align:left;<br />
padding-bottom:5px;<br />
font-weight:normal;<br />
color:#ddd;<br />
text-shadow:0px 0px 1px #fff;<br />
border-bottom:1px solid #000;<br />
}<br />
.ps_slider .loading{<br />
background:#121212 url(https://static.igem.org/mediawiki/2012/5/57/LoadingValencia.gif) no-repeat 50% 50%;<br />
position:absolute;<br />
top:0px;<br />
left:0px;<br />
width:100%;<br />
height:100%;<br />
opacity:0.7;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=70);<br />
}<br />
<br />
span.reference{<br />
position:fixed;<br />
top:10px;<br />
right:10px;<br />
font-size:9px;<br />
}<br />
<br />
span.reference a{<br />
color:#aaa;<br />
text-decoration:none;<br />
text-transform:uppercase;<br />
margin-left:10px;<br />
}<br />
<br />
span.reference a:hover{<br />
color:#ddd;<br />
}<br />
<br />
h1.title{<br />
text-indent:-9000px;<br />
/*background:transparent url(https://static.igem.org/mediawiki/2012/2/22/StudentMembers.png) no-repeat top left;*/<br />
width:640px;<br />
height:52px;<br />
position:absolute;<br />
top:15px;<br />
left:15px;<br />
} <br />
<br />
</style><br />
</html></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/estiloc3po
Team:Valencia Biocampus/estiloc3po
2012-09-26T23:11:36Z
<p>Lujemomo: </p>
<hr />
<div><html><br />
<br />
<style type="text/css"><br />
<br />
#globalWrapper { background-color: transparent; border: none; margin: 0; padding: 0;}<br />
<br />
.firstHeading { /**/<br />
height:0px;<br />
visibility: hidden;<br />
}<br />
<br />
#menubar { <br />
background-color: #696969; /* left menu bar color*/<br />
}<br />
#menubar ul li a { <br />
color: #000000; } /* menu text color*/<br />
.right-menu li a { <br />
color: #A5C5EE; <br />
background-color: #FFFFFF; /* right menu bar color*/<br />
}<br />
#p-logo { height 0px; overflow:hidden; display: none;<br />
}<br />
#top-section { <br />
background-position: center center; <br />
background-repeat: no-repeat; <br />
background-color: #FFFFFF; /* top header background color*/<br />
border-width:0px;<br />
height:0px; /* top header height (determines how low content will start to appear)*/<br />
}<br />
#siteSub {<br />
display:none;<br />
}<br />
#search-controls { /* gets rid of the search bar (it was ugly) */<br />
display:none;<br />
margin-top:0px;<br />
}<br />
#contentSub {<br />
display:none;<br />
}<br />
<br />
<br />
<br />
<br />
/*background:transparent url("https://static.igem.org/mediawiki/2010/0/0c/Transpa.png") center top fixed repeat-y;*/<br />
/* El que estaba antes 26/10 https://static.igem.org/mediawiki/2010/8/86/Transpa30.png */<br />
<br />
#content{<br />
background:transparent url("https://static.igem.org/mediawiki/2010/f/f4/Transpa50.png") center top fixed repeat-y;<br />
padding: 0px;<br />
margin:0 auto;<br />
border: none; <br />
}<br />
<br />
<br />
/*Now to set heading and body fonts*/<br />
<br />
body {<br />
background: #000000 url(https://static.igem.org/mediawiki/2012/f/f9/Fondo_gris.jpeg) no-repeat top center fixed; <br />
/* background:#121212;*/<br />
font-family:Arial, Helvetica, sans-serif;<br />
font-size:8px; /* tamaño de fuente*/<br />
color:#fff;<br />
overflow-x:hidden; <br />
}<br />
<br />
#Titulos{<br />
// text-align: left;<br />
//font-family: Fantasy; /*Kautiva, Verdana, Geneva, sans-serif;*/<br />
//font-size: 30px;<br />
//font-weight: bold;<br />
//color: #000000;<br />
<br />
margin-top: 68px;<br />
background: none;<br />
width: 740px;<br />
padding: 10px;<br />
display: block;<br />
text-align: center;<br />
font-size: 18px;<br />
<br />
}<br />
<br />
#Titulos h2 {<br />
text-align: left;<br />
}<br />
<br />
/* To make the images borders transparents!!! */<br />
div.thumbinner {background-color:transparent;}<br />
div.thumb {border-color: transparent;}<br />
div.thumb {background-color: transparent;}<br />
h1 { color: #000000 }<br />
h2 { color: #000000 }<br />
h3 { color: #000000 }<br />
h4 { color: #000000 }<br />
<br />
#HomeCenter {<br />
width: 660px;<br />
height: auto;<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 14px;<br />
text-align: justify;<br />
margin-top: 8px;<br />
margin-bottom: 20px; <br />
margin-left: 12px; <br />
margin-right: 12px; <br />
padding: 0;<br />
}<br />
<br />
<br />
#HomeRight {<br />
width: 270px;<br />
height: auto;<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
}<br />
#HomeRight2 {<br />
width: 370px;<br />
height: auto;<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
}<br />
<br />
#IzqSup {<br />
width: 360px; <br />
height:auto; /* altura del fondo */<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: justify;<br />
margin-top: 0px;<br />
margin-bottom: 0px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 0;<br />
}<br />
<br />
#DerSup {<br />
width: 360px; <br />
height: auto; /* altura del fondo */<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
margin-top: 8px;<br />
margin-bottom: 30px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 30;<br />
} <br />
#DerSup2 {<br />
width: 200px; <br />
height: auto; /* altura del fondo */<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
margin-top: 8px;<br />
margin-bottom: 8px; <br />
margin-left: 8px; /* cuando espacio izquierdo dejo */<br />
margin-right: 8px; <br />
padding: 8;<br />
} <br />
<br />
#Inf {<br />
width: 710px; <br />
height: auto<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: justify;<br />
margin-top: 650px;<br />
margin-bottom: 10px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 0;<br />
}<br />
<br />
#PorDefecto {<br />
width: auto; <br />
height: auto<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: justify;<br />
margin-top: -30px; // para que el texto suba<br />
margin-bottom: 20px; <br />
margin-left: 12px; /* cuando espacio izquierdo dejo */<br />
margin-right: 12px; <br />
padding: 0;<br />
}<br />
<br />
#PosBocadillo {<br />
width: 360px; <br />
height: auto; /* altura del fondo */<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
margin-top: 8px;<br />
margin-bottom: 30px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 30;<br />
} <br />
//// <br />
#Contact { <br />
position: relative;<br />
text-align: left;<br />
}<br />
#Contact h5 {<br />
color: #D0D9E1;<br />
font-family: Georgia;<br />
font-size: 140px;<br />
letter-spacing: -6px;<br />
margin: 0;<br />
opacity: .9;<br />
padding: 0;<br />
-moz-transform: skew(20deg);<br />
-o-transform: skew(20deg);<br />
-webkit-transform: skew(20deg);<br />
}<br />
<br />
#Contact h6 {<br />
color: #4682B4;<br />
font-family: Verdana;<br />
font-size: 60px;<br />
<br />
left: 120px;<br />
letter-spacing: 20px;<br />
<br />
margin: 0;<br />
padding: 0;<br />
position: absolute;<br />
<br />
top: 20px;<br />
}<br />
<br />
<br />
////<br />
<!--<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
%<br />
% ESTILO 2: PARA LOS SPONSORS<br />
%<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
#sponsors {<br />
background: none;<br />
width: 740px;<br />
padding: 10px;<br />
display: block;<br />
text-align: center;<br />
}<br />
<br />
/* imagen de sponsor: separacion vertical respecto a la horizontal inferior */<br />
#sponsors img {<br />
padding: 15px;<br />
}<br />
<br />
/* encabezado: alineacion*/<br />
#sponsors h2 {<br />
text-align: left;<br />
}<br />
<br />
/* tamaños de las imagenes */<br />
a.tam1 img{<br />
height: 120px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam2 img{<br />
height: 100px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam3 img{<br />
height: 90px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam4 img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tamLogo img{<br />
height: 70px;<br />
width: 360px;<br />
float: left;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.meneillos img{<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
/* al pasar el ratón */<br />
a.tam1:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam2:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam3:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam4 img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tamLogo img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
<br />
a.meneillos:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
<br />
</style><br />
<br />
</html><br />
<br />
<!--<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
%<br />
% ESTILO 2: GALERIA DE EQUIPO: STUDENTS, INSTRUCTORS,...<br />
%<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
<br />
<html><br />
<style><br />
body, ul, li, h1, h2, h3{<br />
margin:-7; /* grosor del menú superior de edición */<br />
padding:-7; /* separación vertical del título con el borde superior */<br />
z-index:1;<br />
}<br />
<br />
.ps_overlay{<br />
z-index:1;<br />
background:#111;<br />
width:100%;<br />
height:100%;<br />
position:fixed;<br />
top:0px;<br />
left:0px;<br />
opacity:0.5;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=80);<br />
}<br />
/* Image container style */<br />
.ps_container{<br />
width:480px;<br />
height:350px;<br />
position:absolute;<br />
top:50%;<br />
margin-top:-175px;<br />
left:50%;<br />
margin-left:-240px;<br />
z-index:1;<br />
}<br />
.ps_container img{<br />
border:10px solid #fff;<br />
position:absolute;<br />
top:50%;<br />
left:50%;<br />
-moz-box-shadow:1px 1px 10px #000;<br />
-webkit-box-shadow:1px 1px 10px #000;<br />
box-shadow:1px 1px 10px #000;<br />
}<br />
<br />
/* Next photo button for preview mode */<br />
a.ps_next_photo{<br />
position:absolute;<br />
top:50%;<br />
left:50%;<br />
width:56px;<br />
height:56px;<br />
margin:-28px 0 0 -28px;<br />
z-index:200;<br />
cursor:pointer;<br />
background:#000 url(https://static.igem.org/mediawiki/2012/7/7f/Next_photoValencia.png) no-repeat 50% 50%;<br />
opacity:0.6;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=60); <br />
-moz-border-radius:10px;<br />
-webkit-border-radius:10px;<br />
border-radius:10px;<br />
}<br />
a.ps_next_photo:hover,<br />
a.ps_close:hover{<br />
opacity:0.8;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=80); <br />
}<br />
/* Thumbnail slider style */<br />
<br />
/* posicion espacial de todo el slider en conjunto*/<br />
.ps_slider{<br />
width:845px;<br />
height:700px; /* cuantos pixeles verticales ocupa el fondo del slier*/<br />
position:relative;<br />
margin:40px auto 0px auto;<br />
}<br />
.ps_slider a.next,<br />
.ps_slider a.prev{<br />
position:absolute;<br />
background-color:#000;<br />
background-position:center center;<br />
background-repeat:no-repeat;<br />
border:1px solid #232323;<br />
width:20px;<br />
height:20px;<br />
top:50%;<br />
margin-top:-10px;<br />
opacity:0.6;<br />
-moz-border-radius:5px;<br />
-webkit-border-radius:5px;<br />
border-radius:5px;<br />
cursor:pointer;<br />
outline:none;<br />
}<br />
<br />
.ps_slider a.prev:hover,<br />
.ps_slider a.next:hover{<br />
border:1px solid #333;<br />
opacity:0.9;<br />
}<br />
<br />
.ps_slider a.disabled,<br />
<br />
<br />
.ps_slider a.disabled:hover{<br />
opacity:0.4;<br />
border:1px solid #111;<br />
cursor:default;<br />
}<br />
.ps_slider a.prev{<br />
left:-30px;<br />
background-image:url(https://static.igem.org/mediawiki/2012/1/12/PrevValencia.png);<br />
}<br />
.ps_slider a.next{<br />
right:-30px;<br />
background-image:url(https://static.igem.org/mediawiki/2012/3/37/NextValencia.png);<br />
}<br />
<br />
/* cuadrado gris claro de cada slider*/<br />
.ps_slider .ps_album{<br />
width:140px;<br />
height:660px; /* altura*/<br />
padding:10px;<br />
background-color:#333;<br />
border:1px solid #444;<br />
position:absolute;<br />
top:0px;<br />
text-align:center;<br />
/*cursor:pointer;*/ /* QUITAMOS EL PUNTERO DE MOMENTO */<br />
-moz-box-shadow:1px 1px 4px #000;<br />
-webkit-box-shadow:1px 1px 4px #000;<br />
box-shadow:1px 1px 4px #000;<br />
-webkit-box-reflect:<br />
below 5px <br />
-webkit-gradient(<br />
linear, <br />
left top, <br />
left bottom, <br />
from(transparent), <br />
color-stop(0.6, transparent), <br />
to(rgb(18, 18, 18))<br />
);<br />
}<br />
/*LUZ DE CADA SLIDER, DE MOMENTO COMENTADO*/<br />
/*<br />
.ps_slider .ps_album:hover{<br />
background-color:#383838;<br />
}*/<br />
<br />
/* imagen de cada slider */<br />
.ps_slider .ps_album img{<br />
height:90px;<br />
border:1px solid #444;<br />
-moz-box-shadow:1px 1px 4px #000;<br />
-webkit-box-shadow:1px 1px 4px #000;<br />
box-shadow:1px 1px 4px #000;<br />
}<br />
<br />
/*cuadrado gris oscuro de cada slider*/<br />
.ps_slider .ps_album .ps_desc{<br />
display:block;<br />
color:#666;<br />
background:#111 url(https://static.igem.org/mediawiki/2012/7/7d/OverlayValencia.png) no-repeat bottom right;<br />
height:550px;<br />
margin-top:10px;<br />
text-align:left;<br />
line-height:20px;<br />
overflow:hidden;<br />
text-overflow:ellipsis;<br />
border:1px solid #393939;<br />
-moz-box-shadow:0px 0px 2px #000 inset;<br />
-webkit-box-shadow:0px 0px 2px #000 inset;<br />
box-shadow:0px 0px 2px #000 inset;<br />
}<br />
/*LUZ DE CADA SLIDER, DE MOMENTO COMENTADO*/<br />
/*<br />
.ps_slider .ps_album:hover .ps_desc{<br />
background-image:none;<br />
}*/<br />
.ps_slider .ps_album .ps_desc span{<br />
display:block;<br />
margin:0px 10px 10px 10px;<br />
border-top:1px solid #333;<br />
padding-top:5px;<br />
}<br />
.ps_slider .ps_album .ps_desc h2{<br />
margin:10px 10px 0px 10px;<br />
text-align:left;<br />
padding-bottom:5px;<br />
font-weight:normal;<br />
color:#ddd;<br />
text-shadow:0px 0px 1px #fff;<br />
border-bottom:1px solid #000;<br />
}<br />
.ps_slider .loading{<br />
background:#121212 url(https://static.igem.org/mediawiki/2012/5/57/LoadingValencia.gif) no-repeat 50% 50%;<br />
position:absolute;<br />
top:0px;<br />
left:0px;<br />
width:100%;<br />
height:100%;<br />
opacity:0.7;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=70);<br />
}<br />
<br />
span.reference{<br />
position:fixed;<br />
top:10px;<br />
right:10px;<br />
font-size:9px;<br />
}<br />
<br />
span.reference a{<br />
color:#aaa;<br />
text-decoration:none;<br />
text-transform:uppercase;<br />
margin-left:10px;<br />
}<br />
<br />
span.reference a:hover{<br />
color:#ddd;<br />
}<br />
<br />
h1.title{<br />
text-indent:-9000px;<br />
/*background:transparent url(https://static.igem.org/mediawiki/2012/2/22/StudentMembers.png) no-repeat top left;*/<br />
width:640px;<br />
height:52px;<br />
position:absolute;<br />
top:15px;<br />
left:15px;<br />
} <br />
<br />
</style><br />
</html></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/estiloc3po
Team:Valencia Biocampus/estiloc3po
2012-09-26T23:11:13Z
<p>Lujemomo: </p>
<hr />
<div><html><br />
<br />
<style type="text/css"><br />
<br />
#globalWrapper { background-color: transparent; border: none; margin: 0; padding: 0;}<br />
<br />
.firstHeading { /**/<br />
height:0px;<br />
visibility: hidden;<br />
}<br />
<br />
#menubar { <br />
background-color: #696969; /* left menu bar color*/<br />
}<br />
#menubar ul li a { <br />
color: #000000; } /* menu text color*/<br />
.right-menu li a { <br />
color: #A5C5EE; <br />
background-color: #FFFFFF; /* right menu bar color*/<br />
}<br />
#p-logo { height 0px; overflow:hidden; display: none;<br />
}<br />
#top-section { <br />
background-position: center center; <br />
background-repeat: no-repeat; <br />
background-color: #FFFFFF; /* top header background color*/<br />
border-width:0px;<br />
height:0px; /* top header height (determines how low content will start to appear)*/<br />
}<br />
#siteSub {<br />
display:none;<br />
}<br />
#search-controls { /* gets rid of the search bar (it was ugly) */<br />
display:none;<br />
margin-top:0px;<br />
}<br />
#contentSub {<br />
display:none;<br />
}<br />
<br />
<br />
<br />
<br />
/*background:transparent url("https://static.igem.org/mediawiki/2010/0/0c/Transpa.png") center top fixed repeat-y;*/<br />
/* El que estaba antes 26/10 https://static.igem.org/mediawiki/2010/8/86/Transpa30.png */<br />
<br />
#content{<br />
background:transparent url("https://static.igem.org/mediawiki/2010/f/f4/Transpa50.png") center top fixed repeat-y;<br />
padding: 0px;<br />
margin:0 auto;<br />
border: none; <br />
}<br />
<br />
<br />
/*Now to set heading and body fonts*/<br />
<br />
body {<br />
background: #000000 url(https://static.igem.org/mediawiki/2012/f/f9/Fondo_gris.jpeg) no-repeat top center fixed; <br />
/* background:#121212;*/<br />
font-family:Arial, Helvetica, sans-serif;<br />
font-size:8px; /* tamaño de fuente*/<br />
color:#fff;<br />
overflow-x:hidden; <br />
}<br />
<br />
#Titulos{<br />
// text-align: left;<br />
//font-family: Fantasy; /*Kautiva, Verdana, Geneva, sans-serif;*/<br />
//font-size: 30px;<br />
//font-weight: bold;<br />
//color: #000000;<br />
<br />
margin-top: 68px;<br />
background: none;<br />
width: 740px;<br />
padding: 10px;<br />
display: block;<br />
text-align: center;<br />
font-size: 18px;<br />
<br />
}<br />
<br />
#Titulos h2 {<br />
text-align: left;<br />
}<br />
<br />
/* To make the images borders transparents!!! */<br />
div.thumbinner {background-color:transparent;}<br />
div.thumb {border-color: transparent;}<br />
div.thumb {background-color: transparent;}<br />
h1 { color: #000000 }<br />
h2 { color: #000000 }<br />
h3 { color: #000000 }<br />
h4 { color: #000000 }<br />
<br />
#HomeCenter {<br />
width: 660px;<br />
height: auto;<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 14px;<br />
text-align: justify;<br />
margin-top: 8px;<br />
margin-bottom: 20px; <br />
margin-left: 12px; <br />
margin-right: 12px; <br />
padding: 0;<br />
}<br />
<br />
<br />
#HomeRight {<br />
width: 270px;<br />
height: auto;<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
}<br />
#HomeRight2 {<br />
width: 370px;<br />
height: auto;<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
}<br />
<br />
#IzqSup {<br />
width: 360px; <br />
height:auto; /* altura del fondo */<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: justify;<br />
margin-top: 0px;<br />
margin-bottom: 0px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 0;<br />
}<br />
<br />
#DerSup {<br />
width: 360px; <br />
height: auto; /* altura del fondo */<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
margin-top: 8px;<br />
margin-bottom: 30px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 30;<br />
} <br />
#DerSup2 {<br />
width: 200px; <br />
height: auto; /* altura del fondo */<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
margin-top: 8px;<br />
margin-bottom: 8px; <br />
margin-left: 8px; /* cuando espacio izquierdo dejo */<br />
margin-right: 8px; <br />
padding: 30;<br />
} <br />
<br />
#Inf {<br />
width: 710px; <br />
height: auto<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: justify;<br />
margin-top: 650px;<br />
margin-bottom: 10px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 0;<br />
}<br />
<br />
#PorDefecto {<br />
width: auto; <br />
height: auto<br />
float: left;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: justify;<br />
margin-top: -30px; // para que el texto suba<br />
margin-bottom: 20px; <br />
margin-left: 12px; /* cuando espacio izquierdo dejo */<br />
margin-right: 12px; <br />
padding: 0;<br />
}<br />
<br />
#PosBocadillo {<br />
width: 360px; <br />
height: auto; /* altura del fondo */<br />
float: right;<br />
font-family: Verdana, Verdana, Geneva, sans-serif;<br />
font-size: 12px;<br />
text-align: left;<br />
margin-top: 8px;<br />
margin-bottom: 30px; <br />
margin-left: 30px; /* cuando espacio izquierdo dejo */<br />
margin-right: 30px; <br />
padding: 30;<br />
} <br />
//// <br />
#Contact { <br />
position: relative;<br />
text-align: left;<br />
}<br />
#Contact h5 {<br />
color: #D0D9E1;<br />
font-family: Georgia;<br />
font-size: 140px;<br />
letter-spacing: -6px;<br />
margin: 0;<br />
opacity: .9;<br />
padding: 0;<br />
-moz-transform: skew(20deg);<br />
-o-transform: skew(20deg);<br />
-webkit-transform: skew(20deg);<br />
}<br />
<br />
#Contact h6 {<br />
color: #4682B4;<br />
font-family: Verdana;<br />
font-size: 60px;<br />
<br />
left: 120px;<br />
letter-spacing: 20px;<br />
<br />
margin: 0;<br />
padding: 0;<br />
position: absolute;<br />
<br />
top: 20px;<br />
}<br />
<br />
<br />
////<br />
<!--<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
%<br />
% ESTILO 2: PARA LOS SPONSORS<br />
%<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
#sponsors {<br />
background: none;<br />
width: 740px;<br />
padding: 10px;<br />
display: block;<br />
text-align: center;<br />
}<br />
<br />
/* imagen de sponsor: separacion vertical respecto a la horizontal inferior */<br />
#sponsors img {<br />
padding: 15px;<br />
}<br />
<br />
/* encabezado: alineacion*/<br />
#sponsors h2 {<br />
text-align: left;<br />
}<br />
<br />
/* tamaños de las imagenes */<br />
a.tam1 img{<br />
height: 120px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam2 img{<br />
height: 100px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam3 img{<br />
height: 90px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tam4 img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tamLogo img{<br />
height: 70px;<br />
width: 360px;<br />
float: left;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.meneillos img{<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
/* al pasar el ratón */<br />
a.tam1:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam2:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam3:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
a.tam4 img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
a.tamLogo img{<br />
height: 70px;<br />
opacity: .50;<br />
filter: alpha(opacity=50);<br />
filter: "alpha(opacity=50)";<br />
}<br />
<br />
<br />
a.meneillos:hover img{<br />
opacity:1;<br />
filter:alpha(opacity=100);<br />
filter: "alpha(opacity=100)";<br />
} <br />
<br />
<br />
</style><br />
<br />
</html><br />
<br />
<!--<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
%<br />
% ESTILO 2: GALERIA DE EQUIPO: STUDENTS, INSTRUCTORS,...<br />
%<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
<br />
<html><br />
<style><br />
body, ul, li, h1, h2, h3{<br />
margin:-7; /* grosor del menú superior de edición */<br />
padding:-7; /* separación vertical del título con el borde superior */<br />
z-index:1;<br />
}<br />
<br />
.ps_overlay{<br />
z-index:1;<br />
background:#111;<br />
width:100%;<br />
height:100%;<br />
position:fixed;<br />
top:0px;<br />
left:0px;<br />
opacity:0.5;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=80);<br />
}<br />
/* Image container style */<br />
.ps_container{<br />
width:480px;<br />
height:350px;<br />
position:absolute;<br />
top:50%;<br />
margin-top:-175px;<br />
left:50%;<br />
margin-left:-240px;<br />
z-index:1;<br />
}<br />
.ps_container img{<br />
border:10px solid #fff;<br />
position:absolute;<br />
top:50%;<br />
left:50%;<br />
-moz-box-shadow:1px 1px 10px #000;<br />
-webkit-box-shadow:1px 1px 10px #000;<br />
box-shadow:1px 1px 10px #000;<br />
}<br />
<br />
/* Next photo button for preview mode */<br />
a.ps_next_photo{<br />
position:absolute;<br />
top:50%;<br />
left:50%;<br />
width:56px;<br />
height:56px;<br />
margin:-28px 0 0 -28px;<br />
z-index:200;<br />
cursor:pointer;<br />
background:#000 url(https://static.igem.org/mediawiki/2012/7/7f/Next_photoValencia.png) no-repeat 50% 50%;<br />
opacity:0.6;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=60); <br />
-moz-border-radius:10px;<br />
-webkit-border-radius:10px;<br />
border-radius:10px;<br />
}<br />
a.ps_next_photo:hover,<br />
a.ps_close:hover{<br />
opacity:0.8;<br />
filter:progid:DXImageTransform.Microsoft.Alpha(opacity=80); <br />
}<br />
/* Thumbnail slider style */<br />
<br />
/* posicion espacial de todo el slider en conjunto*/<br />
.ps_slider{<br />
width:845px;<br />
height:700px; /* cuantos pixeles verticales ocupa el fondo del slier*/<br />
position:relative;<br />
margin:40px auto 0px auto;<br />
}<br />
.ps_slider a.next,<br />
.ps_slider a.prev{<br />
position:absolute;<br />
background-color:#000;<br />
background-position:center center;<br />
background-repeat:no-repeat;<br />
border:1px solid #232323;<br />
width:20px;<br />
height:20px;<br />
top:50%;<br />
margin-top:-10px;<br />
opacity:0.6;<br />
-moz-border-radius:5px;<br />
-webkit-border-radius:5px;<br />
border-radius:5px;<br />
cursor:pointer;<br />
outline:none;<br />
}<br />
<br />
.ps_slider a.prev:hover,<br />
.ps_slider a.next:hover{<br />
border:1px solid #333;<br />
opacity:0.9;<br />
}<br />
<br />
.ps_slider a.disabled,<br />
<br />
<br />
.ps_slider a.disabled:hover{<br />
opacity:0.4;<br />
border:1px solid #111;<br />
cursor:default;<br />
}<br />
.ps_slider a.prev{<br />
left:-30px;<br />
background-image:url(https://static.igem.org/mediawiki/2012/1/12/PrevValencia.png);<br />
}<br />
.ps_slider a.next{<br />
right:-30px;<br />
background-image:url(https://static.igem.org/mediawiki/2012/3/37/NextValencia.png);<br />
}<br />
<br />
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Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/Yeast
Team:Valencia Biocampus/Yeast
2012-09-26T23:10:45Z
<p>Lujemomo: </p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
<br />
<center><br />
<br><br><br />
<ol><br />
<div id="Titulos"><br />
<h2>Yeast Subteam</h2><br />
<br />
<br />
<div id="PorDefecto"><br />
=== '''THE IDEA''' ===<br />
<br><br />
Our aim in this part of the project is to detect when the yeast starts fermenting. At the end of the project we will be able to “ask” the yeast if there is still any glucose in the medium or not through the addition of H2O2. Furthermore, we will be able to know for how long the media has been running out of glucose.<br />
In conclusion, this project allows us to know how much time has elapsed since the fermentation began.<br />
<br />
To do this, we are going to use two gene constructions:<br><br><br />
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<br><br><br />
<br />
• '''The ADH2 promoter is fused to the YAP1 protein coding sequence.''' The YAP1 protein is a yeast transcription factor regulator of H2O2 adaptive response. It is stored in the cytoplasm in normal conditions and, in the presence of H2O2, it is transported to the nucleus where it acts as a transcription factor. The ADH2 promoter is activated in the absence of glucose.<br />
<br />
Thus, complete disappearance of glucose triggers the production of YAP1 in the cytoplasm, and YAP1 concentration increases if the lack of glucose continues. Notice that we are working with a delta-yap1 mutant strain as a recipient of our construct.<br />
<br />
• '''The TRR promoter is fused to the GFP (Green Fluorescence Protein) coding sequence.''' The green fluorescent protein can be detected by fluorescent emission. The tiorredoxin reductase promoter is activated by two transcriptional factors, YAP1 and SKN7 in the oxidative form. Both of them can only bind to the promoter if H2O2 has been previously added to the culture medium.<br />
<br><br />
<br><br><br />
<br />
=== '''MOLECULAR MECHANISMS''' ===<br />
<br />
<br><br />
Click on each plasmid to learn how our constructions work!<br />
<br><br />
<div style="text-align: center;"><br />
<table align="center" border="0.01" bordercolor="#9F9F9F" style="background-color:#9F9F9F"><br />
<tr><br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#Yeast"><br />
<img src="https://static.igem.org/mediawiki/2012/f/fe/Yeast_fermentative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#OXIDATIVE_STRESS_RESPONSE"><br />
<img src="https://static.igem.org/mediawiki/2012/9/9e/Yeast_oxidative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
</tr><br />
</table><br />
</div><br />
<br />
<br><br />
Each construction was carried in different plasmids for their expression in yeast:<br />
<br><br />
'''YEplac181 carried the Fermentative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4893/">click here</a></html>.<br />
<br><br />
'''YEp352 carried the Oxidative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4867/">click here</a></html>.<br />
<br><br><br><br />
<br />
=== '''INDUSTRIAL APPLICATIONS''' ===<br />
<html><br />
<br><br />
<div id="DerSup2"><br />
<br />
</html>[[Image:Aplicacionesindustriales.jpg|200px|right]]<html><br />
</div><br />
The main industrial application of our talking yeast is to know the moment at which fermentation started. This is possible because we can calculate the time elapsed since the medium ran out of glucose according to the fluorescence intensity. Taking this into account, we could ask the yeast, through the addition of hydrogen peroxide: <b>“How long have you been fermenting?”</b> And the cells would answer telling us how long it has been since all the glucose was consumed. One possible answer could be <b>“I have been fermenting for 1 hour”</b>. <br />
<br><br><br />
There is also a second possible application, but it needs a more detailed explanation:<br />
<br><br><br />
There are several factors affecting yeast ethanol production during alcoholic fermentation. <i>Saccharomyces cerevisiae</i> seems to have adapted all along its evolution to optimize its growing rate in environments that are rich in easily assimilable nutrients, such as sugars and amino acids. <br />
<br><br><br />
There are two important characteristics responsible of <i>S. cerevisiae</i> adaptation to this particular niche. One of them is its capability of metabolizing glucose and fructose through both the respiration and the fermentation pathways. The second one is its ability to grow in aerobic and anaerobic conditions. All of this makes this species exhibit some metabolic peculiarities, such as the Pasteur and the Crabtree effects. The Crabtree effect, described in <i>S. cerevisiae</i> and in a few other yeast species, must be taken into account when ethanol production is being studied or modified during fermentation. This effect causes that, even under high concentration of oxygen and with a relatively low amount of glucose, a considerable fraction of the consumed sugar is used to produce ethanol through the fermentative pathway. This is why in every industrial process which requires yeast growth (such as starter yeast production) it is necessary to provide the optimum amount of glucose that permits its direct consumption through the respiratory pathway (or the other way round if fermentation is desirable, such in bioethanol production). <br />
<br><br><br />
We present here one way of monitoring the fate of glucose in the medium. By adding a small amount of hydrogen peroxide as a chemical input, we plan to ask our culture: <b>“Is there any glucose left?”</b>, and the culture would answer -according to the amount of glucose already present in the medium- what concentration would be necessary to optimize either yeast growth or fermentation through the Crabtree effect. In this way, the Crabtree effect could be monitored (perhaps even online) by measuring fluorescence and used to regulate the amount of glucose to be fed. <br />
<br><br />
<br><br><br />
</html><br />
<br />
=== '''EXPERIMENTAL OUTLINE''' ===<br />
<br />
- We ordered the DNA constructions: pADH2-YAP1 protein and pTRR-GFP protein that were cloned in plasmid pUC57 with a bacterial origin of replication. <br />
<br><br />
- We obtain the Yeplac181 and Yep352 yeast vectors by our laboratory.<br />
<br><br />
- We carried out four transformations of <i>E. coli</i> strain DH5, one for each plasmid DNA (the two constructions and the two vectors), in order to amplify them. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Heat_Shock_Protocol_for_bacteria_transformation"> Transformation Protocol Using Heat Shock </a></html>.<br />
<br><br />
- We obtained several <i>E. coli</i> transformants in four plates and took some colonies of each DNA construction (from both constructions and both vectors) and cultured them in liquid medium over night at 37ºC in shaking flasks. <br />
<br><br />
- A day after, we extracted the plasmid DNA. See the Mini-preps protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- We obtained the purified constructions (both in pUC57 plasmid) and the vectors for yeast (YEplac181 and YEp352) also purified. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Protocol_for_Gel_Extraction"> Protocol for Gel Extraction </a></html><br />
<br><br />
- We digested the four DNAs with restriction enzymes EcoRI and PstI in order to obtain compatible ends. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Digestion_Protocol_For_Plasmid_pUC57_.2B_Construction_Using_EcoRI_and_PstI"> Digestion Protocol </a></html>.<br />
<br><br />
- We ligated the pTRR-GFP construction with the Yep352 vector and the ADH2-YAP1 construction with the Yeplac181 vector. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Ligation">ligation Protocol</a></html>.<br />
<br><br />
- The day after that we transformed <i>E. coli</i> with the results of the ligation in order to amplify the final constructions (pTRR-GFP/Yep352 and pADH2-YAP1/YEplac181). We located the recombinant constructs using X-Gal and white/blue selection. <br />
<br><br />
- We took some of these white colonies and cultured them in liquid medium overnight at 37ºC in shaking flasks. <br />
<br><br />
- The day after we extracted the plasmid DNA. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- After this, we checked the final purified recombinant constructs by electrophoresis, restriction digest and DNA capilar sequencing.<br />
<br><br />
- We introduced the first of the DNA recombinant plasmids in the yeast. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- We selected the transformants by growth in solid and liquid mineral medium attending to the auxotrophic markers and checked the presence of the construction by PCR. See the protocol <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Colony_PCR">here</a></html>.<br />
<br><br />
- We used the transformed yeast obtained at that moment and transformed it with the second recombinant construct. See the yeast transformation protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- Once again we identified the transformants by growth in selective medium and after that we used a PCR protocol to check the presence of both constructions.<br />
<br><br />
- In order to detect fluorescence, we carried out a protocol to induce the expression of GFP. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_Induction_protocol"> Yeast induction protocol </a></html>.<br />
<br><br />
<br />
</div></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/estiloc3po
Team:Valencia Biocampus/estiloc3po
2012-09-26T23:10:43Z
<p>Lujemomo: </p>
<hr />
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////<br />
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%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
%<br />
% ESTILO 2: PARA LOS SPONSORS<br />
%<br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
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Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/Yeast
Team:Valencia Biocampus/Yeast
2012-09-26T23:09:33Z
<p>Lujemomo: /* INDUSTRIAL APPLICATIONS */</p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
<br />
<center><br />
<br><br><br />
<ol><br />
<div id="Titulos"><br />
<h2>Yeast Subteam</h2><br />
<br />
<br />
<div id="PorDefecto"><br />
=== '''THE IDEA''' ===<br />
<br><br />
Our aim in this part of the project is to detect when the yeast starts fermenting. At the end of the project we will be able to “ask” the yeast if there is still any glucose in the medium or not through the addition of H2O2. Furthermore, we will be able to know for how long the media has been running out of glucose.<br />
In conclusion, this project allows us to know how much time has elapsed since the fermentation began.<br />
<br />
To do this, we are going to use two gene constructions:<br><br><br />
<html><br />
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<param name=wmode value="transparent"><br />
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</embed><br />
</object> <br />
</html><br />
<br><br><br />
<br />
• '''The ADH2 promoter is fused to the YAP1 protein coding sequence.''' The YAP1 protein is a yeast transcription factor regulator of H2O2 adaptive response. It is stored in the cytoplasm in normal conditions and, in the presence of H2O2, it is transported to the nucleus where it acts as a transcription factor. The ADH2 promoter is activated in the absence of glucose.<br />
<br />
Thus, complete disappearance of glucose triggers the production of YAP1 in the cytoplasm, and YAP1 concentration increases if the lack of glucose continues. Notice that we are working with a delta-yap1 mutant strain as a recipient of our construct.<br />
<br />
• '''The TRR promoter is fused to the GFP (Green Fluorescence Protein) coding sequence.''' The green fluorescent protein can be detected by fluorescent emission. The tiorredoxin reductase promoter is activated by two transcriptional factors, YAP1 and SKN7 in the oxidative form. Both of them can only bind to the promoter if H2O2 has been previously added to the culture medium.<br />
<br><br />
<br><br><br />
<br />
=== '''MOLECULAR MECHANISMS''' ===<br />
<br />
<br><br />
Click on each plasmid to learn how our constructions work!<br />
<br><br />
<div style="text-align: center;"><br />
<table align="center" border="0.01" bordercolor="#9F9F9F" style="background-color:#9F9F9F"><br />
<tr><br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#Yeast"><br />
<img src="https://static.igem.org/mediawiki/2012/f/fe/Yeast_fermentative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#OXIDATIVE_STRESS_RESPONSE"><br />
<img src="https://static.igem.org/mediawiki/2012/9/9e/Yeast_oxidative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
</tr><br />
</table><br />
</div><br />
<br />
<br><br />
Each construction was carried in different plasmids for their expression in yeast:<br />
<br><br />
'''YEplac181 carried the Fermentative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4893/">click here</a></html>.<br />
<br><br />
'''YEp352 carried the Oxidative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4867/">click here</a></html>.<br />
<br><br><br><br />
<br />
=== '''INDUSTRIAL APPLICATIONS''' ===<br />
<html><br />
<br><br />
<div id="DerSup"><br />
<br />
</html>[[Image:Aplicacionesindustriales.jpg|200px|right]]<html><br />
</div><br />
The main industrial application of our talking yeast is to know the moment at which fermentation started. This is possible because we can calculate the time elapsed since the medium ran out of glucose according to the fluorescence intensity. Taking this into account, we could ask the yeast, through the addition of hydrogen peroxide: <b>“How long have you been fermenting?”</b> And the cells would answer telling us how long it has been since all the glucose was consumed. One possible answer could be <b>“I have been fermenting for 1 hour”</b>. <br />
<br><br><br />
There is also a second possible application, but it needs a more detailed explanation:<br />
<br><br><br />
There are several factors affecting yeast ethanol production during alcoholic fermentation. <i>Saccharomyces cerevisiae</i> seems to have adapted all along its evolution to optimize its growing rate in environments that are rich in easily assimilable nutrients, such as sugars and amino acids. <br />
<br><br><br />
There are two important characteristics responsible of <i>S. cerevisiae</i> adaptation to this particular niche. One of them is its capability of metabolizing glucose and fructose through both the respiration and the fermentation pathways. The second one is its ability to grow in aerobic and anaerobic conditions. All of this makes this species exhibit some metabolic peculiarities, such as the Pasteur and the Crabtree effects. The Crabtree effect, described in <i>S. cerevisiae</i> and in a few other yeast species, must be taken into account when ethanol production is being studied or modified during fermentation. This effect causes that, even under high concentration of oxygen and with a relatively low amount of glucose, a considerable fraction of the consumed sugar is used to produce ethanol through the fermentative pathway. This is why in every industrial process which requires yeast growth (such as starter yeast production) it is necessary to provide the optimum amount of glucose that permits its direct consumption through the respiratory pathway (or the other way round if fermentation is desirable, such in bioethanol production). <br />
<br><br><br />
We present here one way of monitoring the fate of glucose in the medium. By adding a small amount of hydrogen peroxide as a chemical input, we plan to ask our culture: <b>“Is there any glucose left?”</b>, and the culture would answer -according to the amount of glucose already present in the medium- what concentration would be necessary to optimize either yeast growth or fermentation through the Crabtree effect. In this way, the Crabtree effect could be monitored (perhaps even online) by measuring fluorescence and used to regulate the amount of glucose to be fed. <br />
<br><br />
<br><br><br />
</html><br />
<br />
=== '''EXPERIMENTAL OUTLINE''' ===<br />
<br />
- We ordered the DNA constructions: pADH2-YAP1 protein and pTRR-GFP protein that were cloned in plasmid pUC57 with a bacterial origin of replication. <br />
<br><br />
- We obtain the Yeplac181 and Yep352 yeast vectors by our laboratory.<br />
<br><br />
- We carried out four transformations of <i>E. coli</i> strain DH5, one for each plasmid DNA (the two constructions and the two vectors), in order to amplify them. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Heat_Shock_Protocol_for_bacteria_transformation"> Transformation Protocol Using Heat Shock </a></html>.<br />
<br><br />
- We obtained several <i>E. coli</i> transformants in four plates and took some colonies of each DNA construction (from both constructions and both vectors) and cultured them in liquid medium over night at 37ºC in shaking flasks. <br />
<br><br />
- A day after, we extracted the plasmid DNA. See the Mini-preps protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- We obtained the purified constructions (both in pUC57 plasmid) and the vectors for yeast (YEplac181 and YEp352) also purified. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Protocol_for_Gel_Extraction"> Protocol for Gel Extraction </a></html><br />
<br><br />
- We digested the four DNAs with restriction enzymes EcoRI and PstI in order to obtain compatible ends. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Digestion_Protocol_For_Plasmid_pUC57_.2B_Construction_Using_EcoRI_and_PstI"> Digestion Protocol </a></html>.<br />
<br><br />
- We ligated the pTRR-GFP construction with the Yep352 vector and the ADH2-YAP1 construction with the Yeplac181 vector. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Ligation">ligation Protocol</a></html>.<br />
<br><br />
- The day after that we transformed <i>E. coli</i> with the results of the ligation in order to amplify the final constructions (pTRR-GFP/Yep352 and pADH2-YAP1/YEplac181). We located the recombinant constructs using X-Gal and white/blue selection. <br />
<br><br />
- We took some of these white colonies and cultured them in liquid medium overnight at 37ºC in shaking flasks. <br />
<br><br />
- The day after we extracted the plasmid DNA. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- After this, we checked the final purified recombinant constructs by electrophoresis, restriction digest and DNA capilar sequencing.<br />
<br><br />
- We introduced the first of the DNA recombinant plasmids in the yeast. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- We selected the transformants by growth in solid and liquid mineral medium attending to the auxotrophic markers and checked the presence of the construction by PCR. See the protocol <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Colony_PCR">here</a></html>.<br />
<br><br />
- We used the transformed yeast obtained at that moment and transformed it with the second recombinant construct. See the yeast transformation protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- Once again we identified the transformants by growth in selective medium and after that we used a PCR protocol to check the presence of both constructions.<br />
<br><br />
- In order to detect fluorescence, we carried out a protocol to induce the expression of GFP. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_Induction_protocol"> Yeast induction protocol </a></html>.<br />
<br><br />
<br />
</div></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/Yeast
Team:Valencia Biocampus/Yeast
2012-09-26T23:09:11Z
<p>Lujemomo: /* INDUSTRIAL APPLICATIONS */</p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
<br />
<center><br />
<br><br><br />
<ol><br />
<div id="Titulos"><br />
<h2>Yeast Subteam</h2><br />
<br />
<br />
<div id="PorDefecto"><br />
=== '''THE IDEA''' ===<br />
<br><br />
Our aim in this part of the project is to detect when the yeast starts fermenting. At the end of the project we will be able to “ask” the yeast if there is still any glucose in the medium or not through the addition of H2O2. Furthermore, we will be able to know for how long the media has been running out of glucose.<br />
In conclusion, this project allows us to know how much time has elapsed since the fermentation began.<br />
<br />
To do this, we are going to use two gene constructions:<br><br><br />
<html><br />
<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=4,0,2,0" width="800" height="600" align="middle"><br />
<param name=wmode value="transparent"><br />
<param name=movie value="https://static.igem.org/mediawiki/2012/5/52/Yeast_Outline_VLC.swf"><br />
<param name=quality value=high><br />
<embed src="https://static.igem.org/mediawiki/2012/5/52/Yeast_Outline_VLC.swf" wmode=transparent quality=high pluginspage="http://www.macromedia.com/shockwave/download/index.cgi?P1_Prod_Version=ShockwaveFlash" type="application/x-shockwave-flash" width="800" height="600" align="middle"><br />
</embed><br />
</object> <br />
</html><br />
<br><br><br />
<br />
• '''The ADH2 promoter is fused to the YAP1 protein coding sequence.''' The YAP1 protein is a yeast transcription factor regulator of H2O2 adaptive response. It is stored in the cytoplasm in normal conditions and, in the presence of H2O2, it is transported to the nucleus where it acts as a transcription factor. The ADH2 promoter is activated in the absence of glucose.<br />
<br />
Thus, complete disappearance of glucose triggers the production of YAP1 in the cytoplasm, and YAP1 concentration increases if the lack of glucose continues. Notice that we are working with a delta-yap1 mutant strain as a recipient of our construct.<br />
<br />
• '''The TRR promoter is fused to the GFP (Green Fluorescence Protein) coding sequence.''' The green fluorescent protein can be detected by fluorescent emission. The tiorredoxin reductase promoter is activated by two transcriptional factors, YAP1 and SKN7 in the oxidative form. Both of them can only bind to the promoter if H2O2 has been previously added to the culture medium.<br />
<br><br />
<br><br><br />
<br />
=== '''MOLECULAR MECHANISMS''' ===<br />
<br />
<br><br />
Click on each plasmid to learn how our constructions work!<br />
<br><br />
<div style="text-align: center;"><br />
<table align="center" border="0.01" bordercolor="#9F9F9F" style="background-color:#9F9F9F"><br />
<tr><br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#Yeast"><br />
<img src="https://static.igem.org/mediawiki/2012/f/fe/Yeast_fermentative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#OXIDATIVE_STRESS_RESPONSE"><br />
<img src="https://static.igem.org/mediawiki/2012/9/9e/Yeast_oxidative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
</tr><br />
</table><br />
</div><br />
<br />
<br><br />
Each construction was carried in different plasmids for their expression in yeast:<br />
<br><br />
'''YEplac181 carried the Fermentative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4893/">click here</a></html>.<br />
<br><br />
'''YEp352 carried the Oxidative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4867/">click here</a></html>.<br />
<br><br><br><br />
<br />
=== '''INDUSTRIAL APPLICATIONS''' ===<br />
<html><br />
<br><br />
<div id="DerSup"><br />
<br />
</html>[[Image:Aplicacionesindustriales.jpg|200px|left]]<html><br />
</div><br />
The main industrial application of our talking yeast is to know the moment at which fermentation started. This is possible because we can calculate the time elapsed since the medium ran out of glucose according to the fluorescence intensity. Taking this into account, we could ask the yeast, through the addition of hydrogen peroxide: <b>“How long have you been fermenting?”</b> And the cells would answer telling us how long it has been since all the glucose was consumed. One possible answer could be <b>“I have been fermenting for 1 hour”</b>. <br />
<br><br><br />
There is also a second possible application, but it needs a more detailed explanation:<br />
<br><br><br />
There are several factors affecting yeast ethanol production during alcoholic fermentation. <i>Saccharomyces cerevisiae</i> seems to have adapted all along its evolution to optimize its growing rate in environments that are rich in easily assimilable nutrients, such as sugars and amino acids. <br />
<br><br><br />
There are two important characteristics responsible of <i>S. cerevisiae</i> adaptation to this particular niche. One of them is its capability of metabolizing glucose and fructose through both the respiration and the fermentation pathways. The second one is its ability to grow in aerobic and anaerobic conditions. All of this makes this species exhibit some metabolic peculiarities, such as the Pasteur and the Crabtree effects. The Crabtree effect, described in <i>S. cerevisiae</i> and in a few other yeast species, must be taken into account when ethanol production is being studied or modified during fermentation. This effect causes that, even under high concentration of oxygen and with a relatively low amount of glucose, a considerable fraction of the consumed sugar is used to produce ethanol through the fermentative pathway. This is why in every industrial process which requires yeast growth (such as starter yeast production) it is necessary to provide the optimum amount of glucose that permits its direct consumption through the respiratory pathway (or the other way round if fermentation is desirable, such in bioethanol production). <br />
<br><br><br />
We present here one way of monitoring the fate of glucose in the medium. By adding a small amount of hydrogen peroxide as a chemical input, we plan to ask our culture: <b>“Is there any glucose left?”</b>, and the culture would answer -according to the amount of glucose already present in the medium- what concentration would be necessary to optimize either yeast growth or fermentation through the Crabtree effect. In this way, the Crabtree effect could be monitored (perhaps even online) by measuring fluorescence and used to regulate the amount of glucose to be fed. <br />
<br><br />
<br><br><br />
</html><br />
<br />
=== '''EXPERIMENTAL OUTLINE''' ===<br />
<br />
- We ordered the DNA constructions: pADH2-YAP1 protein and pTRR-GFP protein that were cloned in plasmid pUC57 with a bacterial origin of replication. <br />
<br><br />
- We obtain the Yeplac181 and Yep352 yeast vectors by our laboratory.<br />
<br><br />
- We carried out four transformations of <i>E. coli</i> strain DH5, one for each plasmid DNA (the two constructions and the two vectors), in order to amplify them. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Heat_Shock_Protocol_for_bacteria_transformation"> Transformation Protocol Using Heat Shock </a></html>.<br />
<br><br />
- We obtained several <i>E. coli</i> transformants in four plates and took some colonies of each DNA construction (from both constructions and both vectors) and cultured them in liquid medium over night at 37ºC in shaking flasks. <br />
<br><br />
- A day after, we extracted the plasmid DNA. See the Mini-preps protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- We obtained the purified constructions (both in pUC57 plasmid) and the vectors for yeast (YEplac181 and YEp352) also purified. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Protocol_for_Gel_Extraction"> Protocol for Gel Extraction </a></html><br />
<br><br />
- We digested the four DNAs with restriction enzymes EcoRI and PstI in order to obtain compatible ends. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Digestion_Protocol_For_Plasmid_pUC57_.2B_Construction_Using_EcoRI_and_PstI"> Digestion Protocol </a></html>.<br />
<br><br />
- We ligated the pTRR-GFP construction with the Yep352 vector and the ADH2-YAP1 construction with the Yeplac181 vector. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Ligation">ligation Protocol</a></html>.<br />
<br><br />
- The day after that we transformed <i>E. coli</i> with the results of the ligation in order to amplify the final constructions (pTRR-GFP/Yep352 and pADH2-YAP1/YEplac181). We located the recombinant constructs using X-Gal and white/blue selection. <br />
<br><br />
- We took some of these white colonies and cultured them in liquid medium overnight at 37ºC in shaking flasks. <br />
<br><br />
- The day after we extracted the plasmid DNA. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- After this, we checked the final purified recombinant constructs by electrophoresis, restriction digest and DNA capilar sequencing.<br />
<br><br />
- We introduced the first of the DNA recombinant plasmids in the yeast. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- We selected the transformants by growth in solid and liquid mineral medium attending to the auxotrophic markers and checked the presence of the construction by PCR. See the protocol <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Colony_PCR">here</a></html>.<br />
<br><br />
- We used the transformed yeast obtained at that moment and transformed it with the second recombinant construct. See the yeast transformation protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- Once again we identified the transformants by growth in selective medium and after that we used a PCR protocol to check the presence of both constructions.<br />
<br><br />
- In order to detect fluorescence, we carried out a protocol to induce the expression of GFP. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_Induction_protocol"> Yeast induction protocol </a></html>.<br />
<br><br />
<br />
</div></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/Yeast
Team:Valencia Biocampus/Yeast
2012-09-26T23:08:50Z
<p>Lujemomo: /* INDUSTRIAL APPLICATIONS */</p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
<br />
<center><br />
<br><br><br />
<ol><br />
<div id="Titulos"><br />
<h2>Yeast Subteam</h2><br />
<br />
<br />
<div id="PorDefecto"><br />
=== '''THE IDEA''' ===<br />
<br><br />
Our aim in this part of the project is to detect when the yeast starts fermenting. At the end of the project we will be able to “ask” the yeast if there is still any glucose in the medium or not through the addition of H2O2. Furthermore, we will be able to know for how long the media has been running out of glucose.<br />
In conclusion, this project allows us to know how much time has elapsed since the fermentation began.<br />
<br />
To do this, we are going to use two gene constructions:<br><br><br />
<html><br />
<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=4,0,2,0" width="800" height="600" align="middle"><br />
<param name=wmode value="transparent"><br />
<param name=movie value="https://static.igem.org/mediawiki/2012/5/52/Yeast_Outline_VLC.swf"><br />
<param name=quality value=high><br />
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</embed><br />
</object> <br />
</html><br />
<br><br><br />
<br />
• '''The ADH2 promoter is fused to the YAP1 protein coding sequence.''' The YAP1 protein is a yeast transcription factor regulator of H2O2 adaptive response. It is stored in the cytoplasm in normal conditions and, in the presence of H2O2, it is transported to the nucleus where it acts as a transcription factor. The ADH2 promoter is activated in the absence of glucose.<br />
<br />
Thus, complete disappearance of glucose triggers the production of YAP1 in the cytoplasm, and YAP1 concentration increases if the lack of glucose continues. Notice that we are working with a delta-yap1 mutant strain as a recipient of our construct.<br />
<br />
• '''The TRR promoter is fused to the GFP (Green Fluorescence Protein) coding sequence.''' The green fluorescent protein can be detected by fluorescent emission. The tiorredoxin reductase promoter is activated by two transcriptional factors, YAP1 and SKN7 in the oxidative form. Both of them can only bind to the promoter if H2O2 has been previously added to the culture medium.<br />
<br><br />
<br><br><br />
<br />
=== '''MOLECULAR MECHANISMS''' ===<br />
<br />
<br><br />
Click on each plasmid to learn how our constructions work!<br />
<br><br />
<div style="text-align: center;"><br />
<table align="center" border="0.01" bordercolor="#9F9F9F" style="background-color:#9F9F9F"><br />
<tr><br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#Yeast"><br />
<img src="https://static.igem.org/mediawiki/2012/f/fe/Yeast_fermentative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#OXIDATIVE_STRESS_RESPONSE"><br />
<img src="https://static.igem.org/mediawiki/2012/9/9e/Yeast_oxidative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
</tr><br />
</table><br />
</div><br />
<br />
<br><br />
Each construction was carried in different plasmids for their expression in yeast:<br />
<br><br />
'''YEplac181 carried the Fermentative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4893/">click here</a></html>.<br />
<br><br />
'''YEp352 carried the Oxidative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4867/">click here</a></html>.<br />
<br><br><br><br />
<br />
=== '''INDUSTRIAL APPLICATIONS''' ===<br />
<html><br />
<br><br />
<div id="DerSup"><br />
<br />
</html>[[Image:Aplicacionesindustriales.jpg|360px|left]]<html><br />
</div><br />
The main industrial application of our talking yeast is to know the moment at which fermentation started. This is possible because we can calculate the time elapsed since the medium ran out of glucose according to the fluorescence intensity. Taking this into account, we could ask the yeast, through the addition of hydrogen peroxide: <b>“How long have you been fermenting?”</b> And the cells would answer telling us how long it has been since all the glucose was consumed. One possible answer could be <b>“I have been fermenting for 1 hour”</b>. <br />
<br><br><br />
There is also a second possible application, but it needs a more detailed explanation:<br />
<br><br><br />
There are several factors affecting yeast ethanol production during alcoholic fermentation. <i>Saccharomyces cerevisiae</i> seems to have adapted all along its evolution to optimize its growing rate in environments that are rich in easily assimilable nutrients, such as sugars and amino acids. <br />
<br><br><br />
There are two important characteristics responsible of <i>S. cerevisiae</i> adaptation to this particular niche. One of them is its capability of metabolizing glucose and fructose through both the respiration and the fermentation pathways. The second one is its ability to grow in aerobic and anaerobic conditions. All of this makes this species exhibit some metabolic peculiarities, such as the Pasteur and the Crabtree effects. The Crabtree effect, described in <i>S. cerevisiae</i> and in a few other yeast species, must be taken into account when ethanol production is being studied or modified during fermentation. This effect causes that, even under high concentration of oxygen and with a relatively low amount of glucose, a considerable fraction of the consumed sugar is used to produce ethanol through the fermentative pathway. This is why in every industrial process which requires yeast growth (such as starter yeast production) it is necessary to provide the optimum amount of glucose that permits its direct consumption through the respiratory pathway (or the other way round if fermentation is desirable, such in bioethanol production). <br />
<br><br><br />
We present here one way of monitoring the fate of glucose in the medium. By adding a small amount of hydrogen peroxide as a chemical input, we plan to ask our culture: <b>“Is there any glucose left?”</b>, and the culture would answer -according to the amount of glucose already present in the medium- what concentration would be necessary to optimize either yeast growth or fermentation through the Crabtree effect. In this way, the Crabtree effect could be monitored (perhaps even online) by measuring fluorescence and used to regulate the amount of glucose to be fed. <br />
<br><br />
<br><br><br />
</html><br />
<br />
=== '''EXPERIMENTAL OUTLINE''' ===<br />
<br />
- We ordered the DNA constructions: pADH2-YAP1 protein and pTRR-GFP protein that were cloned in plasmid pUC57 with a bacterial origin of replication. <br />
<br><br />
- We obtain the Yeplac181 and Yep352 yeast vectors by our laboratory.<br />
<br><br />
- We carried out four transformations of <i>E. coli</i> strain DH5, one for each plasmid DNA (the two constructions and the two vectors), in order to amplify them. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Heat_Shock_Protocol_for_bacteria_transformation"> Transformation Protocol Using Heat Shock </a></html>.<br />
<br><br />
- We obtained several <i>E. coli</i> transformants in four plates and took some colonies of each DNA construction (from both constructions and both vectors) and cultured them in liquid medium over night at 37ºC in shaking flasks. <br />
<br><br />
- A day after, we extracted the plasmid DNA. See the Mini-preps protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- We obtained the purified constructions (both in pUC57 plasmid) and the vectors for yeast (YEplac181 and YEp352) also purified. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Protocol_for_Gel_Extraction"> Protocol for Gel Extraction </a></html><br />
<br><br />
- We digested the four DNAs with restriction enzymes EcoRI and PstI in order to obtain compatible ends. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Digestion_Protocol_For_Plasmid_pUC57_.2B_Construction_Using_EcoRI_and_PstI"> Digestion Protocol </a></html>.<br />
<br><br />
- We ligated the pTRR-GFP construction with the Yep352 vector and the ADH2-YAP1 construction with the Yeplac181 vector. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Ligation">ligation Protocol</a></html>.<br />
<br><br />
- The day after that we transformed <i>E. coli</i> with the results of the ligation in order to amplify the final constructions (pTRR-GFP/Yep352 and pADH2-YAP1/YEplac181). We located the recombinant constructs using X-Gal and white/blue selection. <br />
<br><br />
- We took some of these white colonies and cultured them in liquid medium overnight at 37ºC in shaking flasks. <br />
<br><br />
- The day after we extracted the plasmid DNA. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- After this, we checked the final purified recombinant constructs by electrophoresis, restriction digest and DNA capilar sequencing.<br />
<br><br />
- We introduced the first of the DNA recombinant plasmids in the yeast. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- We selected the transformants by growth in solid and liquid mineral medium attending to the auxotrophic markers and checked the presence of the construction by PCR. See the protocol <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Colony_PCR">here</a></html>.<br />
<br><br />
- We used the transformed yeast obtained at that moment and transformed it with the second recombinant construct. See the yeast transformation protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- Once again we identified the transformants by growth in selective medium and after that we used a PCR protocol to check the presence of both constructions.<br />
<br><br />
- In order to detect fluorescence, we carried out a protocol to induce the expression of GFP. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_Induction_protocol"> Yeast induction protocol </a></html>.<br />
<br><br />
<br />
</div></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/Yeast
Team:Valencia Biocampus/Yeast
2012-09-26T23:08:19Z
<p>Lujemomo: /* INDUSTRIAL APPLICATIONS */</p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
<br />
<center><br />
<br><br><br />
<ol><br />
<div id="Titulos"><br />
<h2>Yeast Subteam</h2><br />
<br />
<br />
<div id="PorDefecto"><br />
=== '''THE IDEA''' ===<br />
<br><br />
Our aim in this part of the project is to detect when the yeast starts fermenting. At the end of the project we will be able to “ask” the yeast if there is still any glucose in the medium or not through the addition of H2O2. Furthermore, we will be able to know for how long the media has been running out of glucose.<br />
In conclusion, this project allows us to know how much time has elapsed since the fermentation began.<br />
<br />
To do this, we are going to use two gene constructions:<br><br><br />
<html><br />
<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=4,0,2,0" width="800" height="600" align="middle"><br />
<param name=wmode value="transparent"><br />
<param name=movie value="https://static.igem.org/mediawiki/2012/5/52/Yeast_Outline_VLC.swf"><br />
<param name=quality value=high><br />
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</embed><br />
</object> <br />
</html><br />
<br><br><br />
<br />
• '''The ADH2 promoter is fused to the YAP1 protein coding sequence.''' The YAP1 protein is a yeast transcription factor regulator of H2O2 adaptive response. It is stored in the cytoplasm in normal conditions and, in the presence of H2O2, it is transported to the nucleus where it acts as a transcription factor. The ADH2 promoter is activated in the absence of glucose.<br />
<br />
Thus, complete disappearance of glucose triggers the production of YAP1 in the cytoplasm, and YAP1 concentration increases if the lack of glucose continues. Notice that we are working with a delta-yap1 mutant strain as a recipient of our construct.<br />
<br />
• '''The TRR promoter is fused to the GFP (Green Fluorescence Protein) coding sequence.''' The green fluorescent protein can be detected by fluorescent emission. The tiorredoxin reductase promoter is activated by two transcriptional factors, YAP1 and SKN7 in the oxidative form. Both of them can only bind to the promoter if H2O2 has been previously added to the culture medium.<br />
<br><br />
<br><br><br />
<br />
=== '''MOLECULAR MECHANISMS''' ===<br />
<br />
<br><br />
Click on each plasmid to learn how our constructions work!<br />
<br><br />
<div style="text-align: center;"><br />
<table align="center" border="0.01" bordercolor="#9F9F9F" style="background-color:#9F9F9F"><br />
<tr><br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#Yeast"><br />
<img src="https://static.igem.org/mediawiki/2012/f/fe/Yeast_fermentative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#OXIDATIVE_STRESS_RESPONSE"><br />
<img src="https://static.igem.org/mediawiki/2012/9/9e/Yeast_oxidative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
</tr><br />
</table><br />
</div><br />
<br />
<br><br />
Each construction was carried in different plasmids for their expression in yeast:<br />
<br><br />
'''YEplac181 carried the Fermentative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4893/">click here</a></html>.<br />
<br><br />
'''YEp352 carried the Oxidative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4867/">click here</a></html>.<br />
<br><br><br><br />
<br />
=== '''INDUSTRIAL APPLICATIONS''' ===<br />
<html><br />
<br><br />
<div id="DerSup"><br />
<br />
</html>[[Image:Aplicacionesindustriales.jpg.JPG|360px|left]]<html><br />
</div><br />
The main industrial application of our talking yeast is to know the moment at which fermentation started. This is possible because we can calculate the time elapsed since the medium ran out of glucose according to the fluorescence intensity. Taking this into account, we could ask the yeast, through the addition of hydrogen peroxide: <b>“How long have you been fermenting?”</b> And the cells would answer telling us how long it has been since all the glucose was consumed. One possible answer could be <b>“I have been fermenting for 1 hour”</b>. <br />
<br><br><br />
There is also a second possible application, but it needs a more detailed explanation:<br />
<br><br><br />
There are several factors affecting yeast ethanol production during alcoholic fermentation. <i>Saccharomyces cerevisiae</i> seems to have adapted all along its evolution to optimize its growing rate in environments that are rich in easily assimilable nutrients, such as sugars and amino acids. <br />
<br><br><br />
There are two important characteristics responsible of <i>S. cerevisiae</i> adaptation to this particular niche. One of them is its capability of metabolizing glucose and fructose through both the respiration and the fermentation pathways. The second one is its ability to grow in aerobic and anaerobic conditions. All of this makes this species exhibit some metabolic peculiarities, such as the Pasteur and the Crabtree effects. The Crabtree effect, described in <i>S. cerevisiae</i> and in a few other yeast species, must be taken into account when ethanol production is being studied or modified during fermentation. This effect causes that, even under high concentration of oxygen and with a relatively low amount of glucose, a considerable fraction of the consumed sugar is used to produce ethanol through the fermentative pathway. This is why in every industrial process which requires yeast growth (such as starter yeast production) it is necessary to provide the optimum amount of glucose that permits its direct consumption through the respiratory pathway (or the other way round if fermentation is desirable, such in bioethanol production). <br />
<br><br><br />
We present here one way of monitoring the fate of glucose in the medium. By adding a small amount of hydrogen peroxide as a chemical input, we plan to ask our culture: <b>“Is there any glucose left?”</b>, and the culture would answer -according to the amount of glucose already present in the medium- what concentration would be necessary to optimize either yeast growth or fermentation through the Crabtree effect. In this way, the Crabtree effect could be monitored (perhaps even online) by measuring fluorescence and used to regulate the amount of glucose to be fed. <br />
<br><br />
<br><br><br />
</html><br />
<br />
=== '''EXPERIMENTAL OUTLINE''' ===<br />
<br />
- We ordered the DNA constructions: pADH2-YAP1 protein and pTRR-GFP protein that were cloned in plasmid pUC57 with a bacterial origin of replication. <br />
<br><br />
- We obtain the Yeplac181 and Yep352 yeast vectors by our laboratory.<br />
<br><br />
- We carried out four transformations of <i>E. coli</i> strain DH5, one for each plasmid DNA (the two constructions and the two vectors), in order to amplify them. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Heat_Shock_Protocol_for_bacteria_transformation"> Transformation Protocol Using Heat Shock </a></html>.<br />
<br><br />
- We obtained several <i>E. coli</i> transformants in four plates and took some colonies of each DNA construction (from both constructions and both vectors) and cultured them in liquid medium over night at 37ºC in shaking flasks. <br />
<br><br />
- A day after, we extracted the plasmid DNA. See the Mini-preps protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- We obtained the purified constructions (both in pUC57 plasmid) and the vectors for yeast (YEplac181 and YEp352) also purified. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Protocol_for_Gel_Extraction"> Protocol for Gel Extraction </a></html><br />
<br><br />
- We digested the four DNAs with restriction enzymes EcoRI and PstI in order to obtain compatible ends. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Digestion_Protocol_For_Plasmid_pUC57_.2B_Construction_Using_EcoRI_and_PstI"> Digestion Protocol </a></html>.<br />
<br><br />
- We ligated the pTRR-GFP construction with the Yep352 vector and the ADH2-YAP1 construction with the Yeplac181 vector. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Ligation">ligation Protocol</a></html>.<br />
<br><br />
- The day after that we transformed <i>E. coli</i> with the results of the ligation in order to amplify the final constructions (pTRR-GFP/Yep352 and pADH2-YAP1/YEplac181). We located the recombinant constructs using X-Gal and white/blue selection. <br />
<br><br />
- We took some of these white colonies and cultured them in liquid medium overnight at 37ºC in shaking flasks. <br />
<br><br />
- The day after we extracted the plasmid DNA. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- After this, we checked the final purified recombinant constructs by electrophoresis, restriction digest and DNA capilar sequencing.<br />
<br><br />
- We introduced the first of the DNA recombinant plasmids in the yeast. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- We selected the transformants by growth in solid and liquid mineral medium attending to the auxotrophic markers and checked the presence of the construction by PCR. See the protocol <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Colony_PCR">here</a></html>.<br />
<br><br />
- We used the transformed yeast obtained at that moment and transformed it with the second recombinant construct. See the yeast transformation protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- Once again we identified the transformants by growth in selective medium and after that we used a PCR protocol to check the presence of both constructions.<br />
<br><br />
- In order to detect fluorescence, we carried out a protocol to induce the expression of GFP. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_Induction_protocol"> Yeast induction protocol </a></html>.<br />
<br><br />
<br />
</div></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/Yeast
Team:Valencia Biocampus/Yeast
2012-09-26T22:56:36Z
<p>Lujemomo: /* EXPERIMENTAL OUTLINE */</p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
<br />
<center><br />
<br><br><br />
<ol><br />
<div id="Titulos"><br />
<h2>Yeast Subteam</h2><br />
<br />
<br />
<div id="PorDefecto"><br />
=== '''THE IDEA''' ===<br />
<br><br />
Our aim in this part of the project is to detect when the yeast starts fermenting. At the end of the project we will be able to “ask” the yeast if there is still any glucose in the medium or not through the addition of H2O2. Furthermore, we will be able to know for how long the media has been running out of glucose.<br />
In conclusion, this project allows us to know how much time has elapsed since the fermentation began.<br />
<br />
To do this, we are going to use two gene constructions:<br><br><br />
<html><br />
<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=4,0,2,0" width="800" height="600" align="middle"><br />
<param name=wmode value="transparent"><br />
<param name=movie value="https://static.igem.org/mediawiki/2012/5/52/Yeast_Outline_VLC.swf"><br />
<param name=quality value=high><br />
<embed src="https://static.igem.org/mediawiki/2012/5/52/Yeast_Outline_VLC.swf" wmode=transparent quality=high pluginspage="http://www.macromedia.com/shockwave/download/index.cgi?P1_Prod_Version=ShockwaveFlash" type="application/x-shockwave-flash" width="800" height="600" align="middle"><br />
</embed><br />
</object> <br />
</html><br />
<br><br><br />
<br />
• '''The ADH2 promoter is fused to the YAP1 protein coding sequence.''' The YAP1 protein is a yeast transcription factor regulator of H2O2 adaptive response. It is stored in the cytoplasm in normal conditions and, in the presence of H2O2, it is transported to the nucleus where it acts as a transcription factor. The ADH2 promoter is activated in the absence of glucose.<br />
<br />
Thus, complete disappearance of glucose triggers the production of YAP1 in the cytoplasm, and YAP1 concentration increases if the lack of glucose continues. Notice that we are working with a delta-yap1 mutant strain as a recipient of our construct.<br />
<br />
• '''The TRR promoter is fused to the GFP (Green Fluorescence Protein) coding sequence.''' The green fluorescent protein can be detected by fluorescent emission. The tiorredoxin reductase promoter is activated by two transcriptional factors, YAP1 and SKN7 in the oxidative form. Both of them can only bind to the promoter if H2O2 has been previously added to the culture medium.<br />
<br><br />
<br><br><br />
<br />
=== '''MOLECULAR MECHANISMS''' ===<br />
<br />
<br><br />
Click on each plasmid to learn how our constructions work!<br />
<br><br />
<div style="text-align: center;"><br />
<table align="center" border="0.01" bordercolor="#9F9F9F" style="background-color:#9F9F9F"><br />
<tr><br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#Yeast"><br />
<img src="https://static.igem.org/mediawiki/2012/f/fe/Yeast_fermentative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#OXIDATIVE_STRESS_RESPONSE"><br />
<img src="https://static.igem.org/mediawiki/2012/9/9e/Yeast_oxidative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
</tr><br />
</table><br />
</div><br />
<br />
<br><br />
Each construction was carried in different plasmids for their expression in yeast:<br />
<br><br />
'''YEplac181 carried the Fermentative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4893/">click here</a></html>.<br />
<br><br />
'''YEp352 carried the Oxidative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4867/">click here</a></html>.<br />
<br><br><br><br />
<br />
=== '''INDUSTRIAL APPLICATIONS''' ===<br />
<html><br />
<br><br />
The main industrial application of our talking yeast is to know the moment at which fermentation started. This is possible because we can calculate the time elapsed since the medium ran out of glucose according to the fluorescence intensity. Taking this into account, we could ask the yeast, through the addition of hydrogen peroxide: <b>“How long have you been fermenting?”</b> And the cells would answer telling us how long it has been since all the glucose was consumed. One possible answer could be <b>“I have been fermenting for 1 hour”</b>. <br />
<br><br><br />
There is also a second possible application, but it needs a more detailed explanation:<br />
<br><br><br />
There are several factors affecting yeast ethanol production during alcoholic fermentation. <i>Saccharomyces cerevisiae</i> seems to have adapted all along its evolution to optimize its growing rate in environments that are rich in easily assimilable nutrients, such as sugars and amino acids. <br />
<br><br><br />
There are two important characteristics responsible of <i>S. cerevisiae</i> adaptation to this particular niche. One of them is its capability of metabolizing glucose and fructose through both the respiration and the fermentation pathways. The second one is its ability to grow in aerobic and anaerobic conditions. All of this makes this species exhibit some metabolic peculiarities, such as the Pasteur and the Crabtree effects. The Crabtree effect, described in <i>S. cerevisiae</i> and in a few other yeast species, must be taken into account when ethanol production is being studied or modified during fermentation. This effect causes that, even under high concentration of oxygen and with a relatively low amount of glucose, a considerable fraction of the consumed sugar is used to produce ethanol through the fermentative pathway. This is why in every industrial process which requires yeast growth (such as starter yeast production) it is necessary to provide the optimum amount of glucose that permits its direct consumption through the respiratory pathway (or the other way round if fermentation is desirable, such in bioethanol production). <br />
<br><br><br />
We present here one way of monitoring the fate of glucose in the medium. By adding a small amount of hydrogen peroxide as a chemical input, we plan to ask our culture: <b>“Is there any glucose left?”</b>, and the culture would answer -according to the amount of glucose already present in the medium- what concentration would be necessary to optimize either yeast growth or fermentation through the Crabtree effect. In this way, the Crabtree effect could be monitored (perhaps even online) by measuring fluorescence and used to regulate the amount of glucose to be fed. <br />
<br><br />
<br><br><br />
</html><br />
<br />
=== '''EXPERIMENTAL OUTLINE''' ===<br />
<br />
- We ordered the DNA constructions: pADH2-YAP1 protein and pTRR-GFP protein that were cloned in plasmid pUC57 with a bacterial origin of replication. <br />
<br><br />
- We obtain the Yeplac181 and Yep352 yeast vectors by our laboratory.<br />
<br><br />
- We carried out four transformations of <i>E. coli</i> strain DH5, one for each plasmid DNA (the two constructions and the two vectors), in order to amplify them. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Heat_Shock_Protocol_for_bacteria_transformation"> Transformation Protocol Using Heat Shock </a></html>.<br />
<br><br />
- We obtained several <i>E. coli</i> transformants in four plates and took some colonies of each DNA construction (from both constructions and both vectors) and cultured them in liquid medium over night at 37ºC in shaking flasks. <br />
<br><br />
- A day after, we extracted the plasmid DNA. See the Mini-preps protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- We obtained the purified constructions (both in pUC57 plasmid) and the vectors for yeast (YEplac181 and YEp352) also purified. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Protocol_for_Gel_Extraction"> Protocol for Gel Extraction </a></html><br />
<br><br />
- We digested the four DNAs with restriction enzymes EcoRI and PstI in order to obtain compatible ends. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Digestion_Protocol_For_Plasmid_pUC57_.2B_Construction_Using_EcoRI_and_PstI"> Digestion Protocol </a></html>.<br />
<br><br />
- We ligated the pTRR-GFP construction with the Yep352 vector and the ADH2-YAP1 construction with the Yeplac181 vector. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Ligation">ligation Protocol</a></html>.<br />
<br><br />
- The day after that we transformed <i>E. coli</i> with the results of the ligation in order to amplify the final constructions (pTRR-GFP/Yep352 and pADH2-YAP1/YEplac181). We located the recombinant constructs using X-Gal and white/blue selection. <br />
<br><br />
- We took some of these white colonies and cultured them in liquid medium overnight at 37ºC in shaking flasks. <br />
<br><br />
- The day after we extracted the plasmid DNA. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- After this, we checked the final purified recombinant constructs by electrophoresis, restriction digest and DNA capilar sequencing.<br />
<br><br />
- We introduced the first of the DNA recombinant plasmids in the yeast. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- We selected the transformants by growth in solid and liquid mineral medium attending to the auxotrophic markers and checked the presence of the construction by PCR. See the protocol <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Colony_PCR">here</a></html>.<br />
<br><br />
- We used the transformed yeast obtained at that moment and transformed it with the second recombinant construct. See the yeast transformation protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- Once again we identified the transformants by growth in selective medium and after that we used a PCR protocol to check the presence of both constructions.<br />
<br><br />
- In order to detect fluorescence, we carried out a protocol to induce the expression of GFP. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_Induction_protocol"> Yeast induction protocol </a></html>.<br />
<br><br />
<br />
</div></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/Yeast
Team:Valencia Biocampus/Yeast
2012-09-26T22:53:36Z
<p>Lujemomo: /* EXPERIMENTAL OUTLINE */</p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
<br />
<center><br />
<br><br><br />
<ol><br />
<div id="Titulos"><br />
<h2>Yeast Subteam</h2><br />
<br />
<br />
<div id="PorDefecto"><br />
=== '''THE IDEA''' ===<br />
<br><br />
Our aim in this part of the project is to detect when the yeast starts fermenting. At the end of the project we will be able to “ask” the yeast if there is still any glucose in the medium or not through the addition of H2O2. Furthermore, we will be able to know for how long the media has been running out of glucose.<br />
In conclusion, this project allows us to know how much time has elapsed since the fermentation began.<br />
<br />
To do this, we are going to use two gene constructions:<br><br><br />
<html><br />
<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=4,0,2,0" width="800" height="600" align="middle"><br />
<param name=wmode value="transparent"><br />
<param name=movie value="https://static.igem.org/mediawiki/2012/5/52/Yeast_Outline_VLC.swf"><br />
<param name=quality value=high><br />
<embed src="https://static.igem.org/mediawiki/2012/5/52/Yeast_Outline_VLC.swf" wmode=transparent quality=high pluginspage="http://www.macromedia.com/shockwave/download/index.cgi?P1_Prod_Version=ShockwaveFlash" type="application/x-shockwave-flash" width="800" height="600" align="middle"><br />
</embed><br />
</object> <br />
</html><br />
<br><br><br />
<br />
• '''The ADH2 promoter is fused to the YAP1 protein coding sequence.''' The YAP1 protein is a yeast transcription factor regulator of H2O2 adaptive response. It is stored in the cytoplasm in normal conditions and, in the presence of H2O2, it is transported to the nucleus where it acts as a transcription factor. The ADH2 promoter is activated in the absence of glucose.<br />
<br />
Thus, complete disappearance of glucose triggers the production of YAP1 in the cytoplasm, and YAP1 concentration increases if the lack of glucose continues. Notice that we are working with a delta-yap1 mutant strain as a recipient of our construct.<br />
<br />
• '''The TRR promoter is fused to the GFP (Green Fluorescence Protein) coding sequence.''' The green fluorescent protein can be detected by fluorescent emission. The tiorredoxin reductase promoter is activated by two transcriptional factors, YAP1 and SKN7 in the oxidative form. Both of them can only bind to the promoter if H2O2 has been previously added to the culture medium.<br />
<br><br />
<br><br><br />
<br />
=== '''MOLECULAR MECHANISMS''' ===<br />
<br />
<br><br />
Click on each plasmid to learn how our constructions work!<br />
<br><br />
<div style="text-align: center;"><br />
<table align="center" border="0.01" bordercolor="#9F9F9F" style="background-color:#9F9F9F"><br />
<tr><br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#Yeast"><br />
<img src="https://static.igem.org/mediawiki/2012/f/fe/Yeast_fermentative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#OXIDATIVE_STRESS_RESPONSE"><br />
<img src="https://static.igem.org/mediawiki/2012/9/9e/Yeast_oxidative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
</tr><br />
</table><br />
</div><br />
<br />
<br><br />
Each construction was carried in different plasmids for their expression in yeast:<br />
<br><br />
'''YEplac181 carried the Fermentative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4893/">click here</a></html>.<br />
<br><br />
'''YEp352 carried the Oxidative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4867/">click here</a></html>.<br />
<br><br><br><br />
<br />
=== '''INDUSTRIAL APPLICATIONS''' ===<br />
<html><br />
<br><br />
The main industrial application of our talking yeast is to know the moment at which fermentation started. This is possible because we can calculate the time elapsed since the medium ran out of glucose according to the fluorescence intensity. Taking this into account, we could ask the yeast, through the addition of hydrogen peroxide: <b>“How long have you been fermenting?”</b> And the cells would answer telling us how long it has been since all the glucose was consumed. One possible answer could be <b>“I have been fermenting for 1 hour”</b>. <br />
<br><br><br />
There is also a second possible application, but it needs a more detailed explanation:<br />
<br><br><br />
There are several factors affecting yeast ethanol production during alcoholic fermentation. <i>Saccharomyces cerevisiae</i> seems to have adapted all along its evolution to optimize its growing rate in environments that are rich in easily assimilable nutrients, such as sugars and amino acids. <br />
<br><br><br />
There are two important characteristics responsible of <i>S. cerevisiae</i> adaptation to this particular niche. One of them is its capability of metabolizing glucose and fructose through both the respiration and the fermentation pathways. The second one is its ability to grow in aerobic and anaerobic conditions. All of this makes this species exhibit some metabolic peculiarities, such as the Pasteur and the Crabtree effects. The Crabtree effect, described in <i>S. cerevisiae</i> and in a few other yeast species, must be taken into account when ethanol production is being studied or modified during fermentation. This effect causes that, even under high concentration of oxygen and with a relatively low amount of glucose, a considerable fraction of the consumed sugar is used to produce ethanol through the fermentative pathway. This is why in every industrial process which requires yeast growth (such as starter yeast production) it is necessary to provide the optimum amount of glucose that permits its direct consumption through the respiratory pathway (or the other way round if fermentation is desirable, such in bioethanol production). <br />
<br><br><br />
We present here one way of monitoring the fate of glucose in the medium. By adding a small amount of hydrogen peroxide as a chemical input, we plan to ask our culture: <b>“Is there any glucose left?”</b>, and the culture would answer -according to the amount of glucose already present in the medium- what concentration would be necessary to optimize either yeast growth or fermentation through the Crabtree effect. In this way, the Crabtree effect could be monitored (perhaps even online) by measuring fluorescence and used to regulate the amount of glucose to be fed. <br />
<br><br />
<br><br><br />
</html><br />
<br />
=== '''EXPERIMENTAL OUTLINE''' ===<br />
<br />
- We ordered the DNA constructions: pADH2-YAP1 protein and pTRR-GFP protein that were cloned in plasmid pUC57 with a bacterial origin of replication. <br />
<br><br />
- We obtain the Yeplac181 and Yep352 yeast vectors by our laboratory.<br />
<br><br />
- We carried out four transformations of <i>E. coli</i> strain DH5, one for each plasmid DNA (the two constructions and the two vectors), in order to amplify them. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Heat_Shock_Protocol_for_bacteria_transformation"> Transformation Protocol Using Heat Shock </a></html>.<br />
<br><br />
- We obtained several <i>E. coli</i> transformants in four plates and took some colonies of each DNA construction (from both constructions and both vectors) and cultured them in liquid medium over night at 37ºC in shaking flasks. <br />
<br><br />
- A day after, we extracted the plasmid DNA. See the Mini-preps protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- We obtained the purified constructions (both in pUC57 plasmid) and the vectors for yeast (YEplac181 and YEp352) also purified. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Protocol_for_Gel_Extraction"> Protocol for Gel Extraction </a></html><br />
<br><br />
- We digested the four DNAs with restriction enzymes EcoRI and PstI in order to obtain compatible ends. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Digestion_Protocol_For_Plasmid_pUC57_.2B_Construction_Using_EcoRI_and_PstI"> Digestion Protocol </a></html>.<br />
<br><br />
- We ligated the pTRR-GFP construction with the Yep352 vector and the ADH2-YAP1 construction with the Yeplac181 vector. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Ligation">ligation Protocol</a></html>.<br />
<br><br />
- The day after that we transformed <i>E. coli</i> with the results of the ligation in order to amplify the final constructions (pTRR-GFP/Yep352 and pADH2-YAP1/YEplac181). We located the recombinant constructs using X-Gal and white/blue selection. <br />
<br><br />
- We took some of these white colonies and cultured them in liquid medium overnight at 37ºC in shaking flasks. <br />
<br><br />
- The day after we extracted the plasmid DNA. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- After this, we checked the final purified recombinant constructs by electrophoresis, restriction digest and DNA capilar sequencing.<br />
<br><br />
- We introduced the first of the DNA recombinant plasmids in the yeast. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- We selected the transformants by growth in solid and liquid mineral medium attending to the auxotrophic markers and checked the presence of the construction by PCR. See the protocol here.<br />
<br><br />
- We used the transformed yeast obtained at that moment and transformed it with the second recombinant construct. See the yeast transformation protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- Once again we identified the transformants by growth in selective medium and after that we used a PCR protocol to check the presence of both constructions.<br />
<br><br />
- In order to detect fluorescence, we carried out a protocol to induce the expression of GFP. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_Induction_protocol"> Yeast induction protocol </a></html>.<br />
<br><br />
<br />
</div></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/Yeast
Team:Valencia Biocampus/Yeast
2012-09-26T22:49:14Z
<p>Lujemomo: /* MOLECULAR MECHANISMS */</p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
<br />
<center><br />
<br><br><br />
<ol><br />
<div id="Titulos"><br />
<h2>Yeast Subteam</h2><br />
<br />
<br />
<div id="PorDefecto"><br />
=== '''THE IDEA''' ===<br />
<br><br />
Our aim in this part of the project is to detect when the yeast starts fermenting. At the end of the project we will be able to “ask” the yeast if there is still any glucose in the medium or not through the addition of H2O2. Furthermore, we will be able to know for how long the media has been running out of glucose.<br />
In conclusion, this project allows us to know how much time has elapsed since the fermentation began.<br />
<br />
To do this, we are going to use two gene constructions:<br><br><br />
<html><br />
<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=4,0,2,0" width="800" height="600" align="middle"><br />
<param name=wmode value="transparent"><br />
<param name=movie value="https://static.igem.org/mediawiki/2012/5/52/Yeast_Outline_VLC.swf"><br />
<param name=quality value=high><br />
<embed src="https://static.igem.org/mediawiki/2012/5/52/Yeast_Outline_VLC.swf" wmode=transparent quality=high pluginspage="http://www.macromedia.com/shockwave/download/index.cgi?P1_Prod_Version=ShockwaveFlash" type="application/x-shockwave-flash" width="800" height="600" align="middle"><br />
</embed><br />
</object> <br />
</html><br />
<br><br><br />
<br />
• '''The ADH2 promoter is fused to the YAP1 protein coding sequence.''' The YAP1 protein is a yeast transcription factor regulator of H2O2 adaptive response. It is stored in the cytoplasm in normal conditions and, in the presence of H2O2, it is transported to the nucleus where it acts as a transcription factor. The ADH2 promoter is activated in the absence of glucose.<br />
<br />
Thus, complete disappearance of glucose triggers the production of YAP1 in the cytoplasm, and YAP1 concentration increases if the lack of glucose continues. Notice that we are working with a delta-yap1 mutant strain as a recipient of our construct.<br />
<br />
• '''The TRR promoter is fused to the GFP (Green Fluorescence Protein) coding sequence.''' The green fluorescent protein can be detected by fluorescent emission. The tiorredoxin reductase promoter is activated by two transcriptional factors, YAP1 and SKN7 in the oxidative form. Both of them can only bind to the promoter if H2O2 has been previously added to the culture medium.<br />
<br><br />
<br><br><br />
<br />
=== '''MOLECULAR MECHANISMS''' ===<br />
<br />
<br><br />
Click on each plasmid to learn how our constructions work!<br />
<br><br />
<div style="text-align: center;"><br />
<table align="center" border="0.01" bordercolor="#9F9F9F" style="background-color:#9F9F9F"><br />
<tr><br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#Yeast"><br />
<img src="https://static.igem.org/mediawiki/2012/f/fe/Yeast_fermentative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#OXIDATIVE_STRESS_RESPONSE"><br />
<img src="https://static.igem.org/mediawiki/2012/9/9e/Yeast_oxidative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
</tr><br />
</table><br />
</div><br />
<br />
<br><br />
Each construction was carried in different plasmids for their expression in yeast:<br />
<br><br />
'''YEplac181 carried the Fermentative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4893/">click here</a></html>.<br />
<br><br />
'''YEp352 carried the Oxidative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4867/">click here</a></html>.<br />
<br><br><br><br />
<br />
=== '''INDUSTRIAL APPLICATIONS''' ===<br />
<html><br />
<br><br />
The main industrial application of our talking yeast is to know the moment at which fermentation started. This is possible because we can calculate the time elapsed since the medium ran out of glucose according to the fluorescence intensity. Taking this into account, we could ask the yeast, through the addition of hydrogen peroxide: <b>“How long have you been fermenting?”</b> And the cells would answer telling us how long it has been since all the glucose was consumed. One possible answer could be <b>“I have been fermenting for 1 hour”</b>. <br />
<br><br><br />
There is also a second possible application, but it needs a more detailed explanation:<br />
<br><br><br />
There are several factors affecting yeast ethanol production during alcoholic fermentation. <i>Saccharomyces cerevisiae</i> seems to have adapted all along its evolution to optimize its growing rate in environments that are rich in easily assimilable nutrients, such as sugars and amino acids. <br />
<br><br><br />
There are two important characteristics responsible of <i>S. cerevisiae</i> adaptation to this particular niche. One of them is its capability of metabolizing glucose and fructose through both the respiration and the fermentation pathways. The second one is its ability to grow in aerobic and anaerobic conditions. All of this makes this species exhibit some metabolic peculiarities, such as the Pasteur and the Crabtree effects. The Crabtree effect, described in <i>S. cerevisiae</i> and in a few other yeast species, must be taken into account when ethanol production is being studied or modified during fermentation. This effect causes that, even under high concentration of oxygen and with a relatively low amount of glucose, a considerable fraction of the consumed sugar is used to produce ethanol through the fermentative pathway. This is why in every industrial process which requires yeast growth (such as starter yeast production) it is necessary to provide the optimum amount of glucose that permits its direct consumption through the respiratory pathway (or the other way round if fermentation is desirable, such in bioethanol production). <br />
<br><br><br />
We present here one way of monitoring the fate of glucose in the medium. By adding a small amount of hydrogen peroxide as a chemical input, we plan to ask our culture: <b>“Is there any glucose left?”</b>, and the culture would answer -according to the amount of glucose already present in the medium- what concentration would be necessary to optimize either yeast growth or fermentation through the Crabtree effect. In this way, the Crabtree effect could be monitored (perhaps even online) by measuring fluorescence and used to regulate the amount of glucose to be fed. <br />
<br><br />
<br><br><br />
</html><br />
<br />
=== '''EXPERIMENTAL OUTLINE''' ===<br />
<br />
- We ordered the DNA constructions: pADH2-YAP1 protein and pTRR-GFP protein that were cloned in plasmid pUC57 with a bacterial origin of replication. <br />
<br><br />
- We were supplied the Yeplac181 and Yep352 yeast vectors by our laboratory.<br />
<br><br />
- We carried out four transformations of E. coli strain DH5, one for each plasmid DNA (the two constructions and the two vectors), in order to amplify them. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Heat_Shock_Protocol_for_bacteria_transformation"> Transformation Protocol Using Heat Shock </a></html>.<br />
<br><br />
- We obtained several transformants of E. coli in four plates and took some colonies of each DNA (from both constructions and both vectors) and cultured them in liquid medium over night at 37ºC in shaking flasks. <br />
<br><br />
- A day after, we extracted the plasmid DNA. See the Mini-preps protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- We obtained the purified constructions (both in pUC57 plasmid) and the vectors for yeast (YEplac181 and YEp352) also purified. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Protocol_for_Gel_Extraction"> Protocol for Gel Extraction </a></html><br />
<br><br />
- We digested the four DNAs with restriction enzymes EcoRI and PstI in order to obtain compatible ends. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Digestion_Protocol_For_Plasmid_pUC57_.2B_Construction_Using_EcoRI_and_PstI"> Digestion Protocol </a></html>.<br />
<br><br />
- We ligated the pTRR-GFP construction with the Yep352 vector and the ADH2-YAP1 construction with the Yeplac181 vector. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Ligation">ligation Protocol</a></html>.<br />
<br><br />
- The day after that we transformed E. coli with the results of the ligation in order to amplify the final constructions (pTRR-GFP/Yep352 and pADH2-YAP1/YEplac181). We located the recombinant constructs using X-Gal and white/blue selection. <br />
<br><br />
- We took some of these white colonies and cultured them in liquid medium overnight at 37ºC in shaking flasks. <br />
<br><br />
- The day after we extracted the plasmid DNA. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- After this, we checked the final purified recombinant constructs by electrophoresis, restriction digest and DNA capilar sequencing.<br />
<br><br />
- We introduced the first of the DNA recombinant plasmids in the yeast. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- We selected the transformants by growth in solid and liquid mineral medium attending to the auxotrophic markers and checked the presence of the construction by PCR. See the protocol here.<br />
<br><br />
- We used the transformed yeast obtained at that moment and transformed it with the second recombinant construct. See the yeast transformation protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- Once again we identified the transformants by growth in selective medium and after that we used a PCR protocol to check the presence of both constructions.<br />
<br><br />
- In order to detect fluorescence, we carried out a protocol to induce the expression of GFP. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_Induction_protocol"> Yeast induction protocol </a></html>.<br />
<br><br />
<br />
</div></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/Yeast
Team:Valencia Biocampus/Yeast
2012-09-26T22:48:23Z
<p>Lujemomo: /* INDUSTRIAL APPLICATIONS */</p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
<br />
<center><br />
<br><br><br />
<ol><br />
<div id="Titulos"><br />
<h2>Yeast Subteam</h2><br />
<br />
<br />
<div id="PorDefecto"><br />
=== '''THE IDEA''' ===<br />
<br><br />
Our aim in this part of the project is to detect when the yeast starts fermenting. At the end of the project we will be able to “ask” the yeast if there is still any glucose in the medium or not through the addition of H2O2. Furthermore, we will be able to know for how long the media has been running out of glucose.<br />
In conclusion, this project allows us to know how much time has elapsed since the fermentation began.<br />
<br />
To do this, we are going to use two gene constructions:<br><br><br />
<html><br />
<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=4,0,2,0" width="800" height="600" align="middle"><br />
<param name=wmode value="transparent"><br />
<param name=movie value="https://static.igem.org/mediawiki/2012/5/52/Yeast_Outline_VLC.swf"><br />
<param name=quality value=high><br />
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</embed><br />
</object> <br />
</html><br />
<br><br><br />
<br />
• '''The ADH2 promoter is fused to the YAP1 protein coding sequence.''' The YAP1 protein is a yeast transcription factor regulator of H2O2 adaptive response. It is stored in the cytoplasm in normal conditions and, in the presence of H2O2, it is transported to the nucleus where it acts as a transcription factor. The ADH2 promoter is activated in the absence of glucose.<br />
<br />
Thus, complete disappearance of glucose triggers the production of YAP1 in the cytoplasm, and YAP1 concentration increases if the lack of glucose continues. Notice that we are working with a delta-yap1 mutant strain as a recipient of our construct.<br />
<br />
• '''The TRR promoter is fused to the GFP (Green Fluorescence Protein) coding sequence.''' The green fluorescent protein can be detected by fluorescent emission. The tiorredoxin reductase promoter is activated by two transcriptional factors, YAP1 and SKN7 in the oxidative form. Both of them can only bind to the promoter if H2O2 has been previously added to the culture medium.<br />
<br><br />
<br><br><br />
<br />
=== '''MOLECULAR MECHANISMS''' ===<br />
<br />
<br><br />
Click on each plasmid to learn how our constructions work!<br />
<br><br />
<div style="text-align: center;"><br />
<table align="center" border="0.01" bordercolor="#9F9F9F" style="background-color:#9F9F9F"><br />
<tr><br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#Yeast"><br />
<img src="https://static.igem.org/mediawiki/2012/f/fe/Yeast_fermentative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#OXIDATIVE_STRESS_RESPONSE"><br />
<img src="https://static.igem.org/mediawiki/2012/9/9e/Yeast_oxidative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
</tr><br />
</table><br />
</div><br />
<br />
<br><br />
Each construction was carried in diferent plasmids for their expression in yeast:<br />
<br><br />
'''YEplac181 carried Fermentative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4893/">click here</a></html>.<br />
<br><br />
'''YEp352 carried Oxidative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4867/">click here</a></html>.<br />
<br><br><br><br />
<br />
=== '''INDUSTRIAL APPLICATIONS''' ===<br />
<html><br />
<br><br />
The main industrial application of our talking yeast is to know the moment at which fermentation started. This is possible because we can calculate the time elapsed since the medium ran out of glucose according to the fluorescence intensity. Taking this into account, we could ask the yeast, through the addition of hydrogen peroxide: <b>“How long have you been fermenting?”</b> And the cells would answer telling us how long it has been since all the glucose was consumed. One possible answer could be <b>“I have been fermenting for 1 hour”</b>. <br />
<br><br><br />
There is also a second possible application, but it needs a more detailed explanation:<br />
<br><br><br />
There are several factors affecting yeast ethanol production during alcoholic fermentation. <i>Saccharomyces cerevisiae</i> seems to have adapted all along its evolution to optimize its growing rate in environments that are rich in easily assimilable nutrients, such as sugars and amino acids. <br />
<br><br><br />
There are two important characteristics responsible of <i>S. cerevisiae</i> adaptation to this particular niche. One of them is its capability of metabolizing glucose and fructose through both the respiration and the fermentation pathways. The second one is its ability to grow in aerobic and anaerobic conditions. All of this makes this species exhibit some metabolic peculiarities, such as the Pasteur and the Crabtree effects. The Crabtree effect, described in <i>S. cerevisiae</i> and in a few other yeast species, must be taken into account when ethanol production is being studied or modified during fermentation. This effect causes that, even under high concentration of oxygen and with a relatively low amount of glucose, a considerable fraction of the consumed sugar is used to produce ethanol through the fermentative pathway. This is why in every industrial process which requires yeast growth (such as starter yeast production) it is necessary to provide the optimum amount of glucose that permits its direct consumption through the respiratory pathway (or the other way round if fermentation is desirable, such in bioethanol production). <br />
<br><br><br />
We present here one way of monitoring the fate of glucose in the medium. By adding a small amount of hydrogen peroxide as a chemical input, we plan to ask our culture: <b>“Is there any glucose left?”</b>, and the culture would answer -according to the amount of glucose already present in the medium- what concentration would be necessary to optimize either yeast growth or fermentation through the Crabtree effect. In this way, the Crabtree effect could be monitored (perhaps even online) by measuring fluorescence and used to regulate the amount of glucose to be fed. <br />
<br><br />
<br><br><br />
</html><br />
<br />
=== '''EXPERIMENTAL OUTLINE''' ===<br />
<br />
- We ordered the DNA constructions: pADH2-YAP1 protein and pTRR-GFP protein that were cloned in plasmid pUC57 with a bacterial origin of replication. <br />
<br><br />
- We were supplied the Yeplac181 and Yep352 yeast vectors by our laboratory.<br />
<br><br />
- We carried out four transformations of E. coli strain DH5, one for each plasmid DNA (the two constructions and the two vectors), in order to amplify them. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Heat_Shock_Protocol_for_bacteria_transformation"> Transformation Protocol Using Heat Shock </a></html>.<br />
<br><br />
- We obtained several transformants of E. coli in four plates and took some colonies of each DNA (from both constructions and both vectors) and cultured them in liquid medium over night at 37ºC in shaking flasks. <br />
<br><br />
- A day after, we extracted the plasmid DNA. See the Mini-preps protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- We obtained the purified constructions (both in pUC57 plasmid) and the vectors for yeast (YEplac181 and YEp352) also purified. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Protocol_for_Gel_Extraction"> Protocol for Gel Extraction </a></html><br />
<br><br />
- We digested the four DNAs with restriction enzymes EcoRI and PstI in order to obtain compatible ends. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Digestion_Protocol_For_Plasmid_pUC57_.2B_Construction_Using_EcoRI_and_PstI"> Digestion Protocol </a></html>.<br />
<br><br />
- We ligated the pTRR-GFP construction with the Yep352 vector and the ADH2-YAP1 construction with the Yeplac181 vector. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Ligation">ligation Protocol</a></html>.<br />
<br><br />
- The day after that we transformed E. coli with the results of the ligation in order to amplify the final constructions (pTRR-GFP/Yep352 and pADH2-YAP1/YEplac181). We located the recombinant constructs using X-Gal and white/blue selection. <br />
<br><br />
- We took some of these white colonies and cultured them in liquid medium overnight at 37ºC in shaking flasks. <br />
<br><br />
- The day after we extracted the plasmid DNA. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- After this, we checked the final purified recombinant constructs by electrophoresis, restriction digest and DNA capilar sequencing.<br />
<br><br />
- We introduced the first of the DNA recombinant plasmids in the yeast. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- We selected the transformants by growth in solid and liquid mineral medium attending to the auxotrophic markers and checked the presence of the construction by PCR. See the protocol here.<br />
<br><br />
- We used the transformed yeast obtained at that moment and transformed it with the second recombinant construct. See the yeast transformation protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- Once again we identified the transformants by growth in selective medium and after that we used a PCR protocol to check the presence of both constructions.<br />
<br><br />
- In order to detect fluorescence, we carried out a protocol to induce the expression of GFP. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_Induction_protocol"> Yeast induction protocol </a></html>.<br />
<br><br />
<br />
</div></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/Yeast
Team:Valencia Biocampus/Yeast
2012-09-26T22:45:54Z
<p>Lujemomo: /* THE IDEA */</p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
<br />
<center><br />
<br><br><br />
<ol><br />
<div id="Titulos"><br />
<h2>Yeast Subteam</h2><br />
<br />
<br />
<div id="PorDefecto"><br />
=== '''THE IDEA''' ===<br />
<br><br />
Our aim in this part of the project is to detect when the yeast starts fermenting. At the end of the project we will be able to “ask” the yeast if there is still any glucose in the medium or not through the addition of H2O2. Furthermore, we will be able to know for how long the media has been running out of glucose.<br />
In conclusion, this project allows us to know how much time has elapsed since the fermentation began.<br />
<br />
To do this, we are going to use two gene constructions:<br><br><br />
<html><br />
<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=4,0,2,0" width="800" height="600" align="middle"><br />
<param name=wmode value="transparent"><br />
<param name=movie value="https://static.igem.org/mediawiki/2012/5/52/Yeast_Outline_VLC.swf"><br />
<param name=quality value=high><br />
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</embed><br />
</object> <br />
</html><br />
<br><br><br />
<br />
• '''The ADH2 promoter is fused to the YAP1 protein coding sequence.''' The YAP1 protein is a yeast transcription factor regulator of H2O2 adaptive response. It is stored in the cytoplasm in normal conditions and, in the presence of H2O2, it is transported to the nucleus where it acts as a transcription factor. The ADH2 promoter is activated in the absence of glucose.<br />
<br />
Thus, complete disappearance of glucose triggers the production of YAP1 in the cytoplasm, and YAP1 concentration increases if the lack of glucose continues. Notice that we are working with a delta-yap1 mutant strain as a recipient of our construct.<br />
<br />
• '''The TRR promoter is fused to the GFP (Green Fluorescence Protein) coding sequence.''' The green fluorescent protein can be detected by fluorescent emission. The tiorredoxin reductase promoter is activated by two transcriptional factors, YAP1 and SKN7 in the oxidative form. Both of them can only bind to the promoter if H2O2 has been previously added to the culture medium.<br />
<br><br />
<br><br><br />
<br />
=== '''MOLECULAR MECHANISMS''' ===<br />
<br />
<br><br />
Click on each plasmid to learn how our constructions work!<br />
<br><br />
<div style="text-align: center;"><br />
<table align="center" border="0.01" bordercolor="#9F9F9F" style="background-color:#9F9F9F"><br />
<tr><br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#Yeast"><br />
<img src="https://static.igem.org/mediawiki/2012/f/fe/Yeast_fermentative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#OXIDATIVE_STRESS_RESPONSE"><br />
<img src="https://static.igem.org/mediawiki/2012/9/9e/Yeast_oxidative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
</tr><br />
</table><br />
</div><br />
<br />
<br><br />
Each construction was carried in diferent plasmids for their expression in yeast:<br />
<br><br />
'''YEplac181 carried Fermentative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4893/">click here</a></html>.<br />
<br><br />
'''YEp352 carried Oxidative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4867/">click here</a></html>.<br />
<br><br><br><br />
<br />
=== '''INDUSTRIAL APPLICATIONS''' ===<br />
<html><br />
<br><br />
The main industrial application of our talking yeast is to know the moment at which fermentation started. This is possible because we can calculate the time passed since the medium ran out of glucose according to the fluorescence intensity. Taking this into account, we could ask the yeast, through the addition of hydrogen peroxide: <b>“How long have you been fermenting?”</b> And the cells would answer telling us how long it has been since all the glucose was consumed. One possible answer could be <b>“I have been fermenting for 1 hour”</b>. <br />
<br><br><br />
There is also a second possible application, but it needs a more detailed explanation:<br />
<br><br><br />
There are several factors affecting yeast ethanol production during alcoholic fermentation. <i>Saccharomyces cerevisiae</i> seems to have adapted all along its evolution to optimize its growing rate in environments that are rich in easily assimilable nutrients, such as sugars and amino acids. <br />
<br><br><br />
There are two important characteristics responsible of <i>S. cerevisiae</i> adaptation to this particular niche. One of them is its capability of metabolizing glucose and fructose through both the respiration and the fermentation pathways. The second one is its ability to grow in aerobic and anaerobic conditions. All of this makes this species exhibit some metabolic peculiarities, such as the Pasteur and the Crabtree effects. The Crabtree effect, described in <i>S. cerevisiae</i> and in a few other yeast species, must be taken into account when ethanol production is being studied or modified during fermentation. This effect causes that, even under high concentration of oxygen and with a relatively low amount of glucose, a considerable fraction of the consumed sugar is used to produce ethanol through the fermentative pathway. This is why in every industrial process which requires yeast growth (such as starter yeast production) it is necessary to provide the optimum amount of glucose that permits its direct consumption through the respiratory pathway (or the other way round if fermentation is desirable, such in bioethanol production). <br />
<br><br><br />
We present here one way of monitoring the fate of glucose in the medium. By adding a small amount of hydrogen peroxide as a chemical input, we plan to ask our culture: <b>“Is there any glucose left?”</b>, and the culture would answer -according to the amount of glucose already present in the medium- what concentration would be necessary to optimize either yeast growth or fermentation through the Crabtree effect. In this way, the Crabtree effect could be monitored (perhaps even online) by measuring fluorescence and used to regulate the amount of glucose to be fed. <br />
<br><br />
<br><br><br />
</html><br />
<br />
=== '''EXPERIMENTAL OUTLINE''' ===<br />
<br />
- We ordered the DNA constructions: pADH2-YAP1 protein and pTRR-GFP protein that were cloned in plasmid pUC57 with a bacterial origin of replication. <br />
<br><br />
- We were supplied the Yeplac181 and Yep352 yeast vectors by our laboratory.<br />
<br><br />
- We carried out four transformations of E. coli strain DH5, one for each plasmid DNA (the two constructions and the two vectors), in order to amplify them. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Heat_Shock_Protocol_for_bacteria_transformation"> Transformation Protocol Using Heat Shock </a></html>.<br />
<br><br />
- We obtained several transformants of E. coli in four plates and took some colonies of each DNA (from both constructions and both vectors) and cultured them in liquid medium over night at 37ºC in shaking flasks. <br />
<br><br />
- A day after, we extracted the plasmid DNA. See the Mini-preps protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- We obtained the purified constructions (both in pUC57 plasmid) and the vectors for yeast (YEplac181 and YEp352) also purified. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Protocol_for_Gel_Extraction"> Protocol for Gel Extraction </a></html><br />
<br><br />
- We digested the four DNAs with restriction enzymes EcoRI and PstI in order to obtain compatible ends. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Digestion_Protocol_For_Plasmid_pUC57_.2B_Construction_Using_EcoRI_and_PstI"> Digestion Protocol </a></html>.<br />
<br><br />
- We ligated the pTRR-GFP construction with the Yep352 vector and the ADH2-YAP1 construction with the Yeplac181 vector. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Ligation">ligation Protocol</a></html>.<br />
<br><br />
- The day after that we transformed E. coli with the results of the ligation in order to amplify the final constructions (pTRR-GFP/Yep352 and pADH2-YAP1/YEplac181). We located the recombinant constructs using X-Gal and white/blue selection. <br />
<br><br />
- We took some of these white colonies and cultured them in liquid medium overnight at 37ºC in shaking flasks. <br />
<br><br />
- The day after we extracted the plasmid DNA. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- After this, we checked the final purified recombinant constructs by electrophoresis, restriction digest and DNA capilar sequencing.<br />
<br><br />
- We introduced the first of the DNA recombinant plasmids in the yeast. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- We selected the transformants by growth in solid and liquid mineral medium attending to the auxotrophic markers and checked the presence of the construction by PCR. See the protocol here.<br />
<br><br />
- We used the transformed yeast obtained at that moment and transformed it with the second recombinant construct. See the yeast transformation protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- Once again we identified the transformants by growth in selective medium and after that we used a PCR protocol to check the presence of both constructions.<br />
<br><br />
- In order to detect fluorescence, we carried out a protocol to induce the expression of GFP. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_Induction_protocol"> Yeast induction protocol </a></html>.<br />
<br><br />
<br />
</div></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/talking
Team:Valencia Biocampus/talking
2012-09-26T21:20:18Z
<p>Lujemomo: </p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
<br />
<ol><br />
<br><br />
<center><br />
<div id="Titulos"><br />
<h2>Talking Interfaces</h2><br />
<!-- ya cambiaré esto de sitio pero no lo borréis please!--><br />
<br><br />
<br />
<div id="PorDefecto"><br />
=== '''THE PROCESS''' ===<br />
<br><br />
<br />
The main objective of our project is to accomplish a verbal communication with our microorganisms.<br />
To do that, we need to establish the following process:<br />
<br><br />
<html><br />
<br />
<!-- FLASH--><br />
<br />
</html><br />
<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/0/07/ProcesoEn2.png" height="450" width="780"><br />
</html><br />
<br />
<ol><br />
<li>The input used is a voice signal (question), which will be collected by our voice recognizer.<br />
<li>The voice recognizer identifies the question and, through the program in charge of establishing the communication, its corresponding identifier is written in the <br />
assigned port of the arduino.<br />
<li>The software of the arduino reads the written identifier and, according to it, the corresponding port is selected, indicating the flourimeter which wavelength has <br />
to be emitted on the culture. There are four possible questions (q), and each of them is associated to a different wavelength. <br />
<li>The fluorimeter emits light (Bioinput), exciting the compound through optic filters.<br />
<li>Due to the excitation produced, the compound emits fluorescence (BioOutput), which is measured by the fluorimeter with a sensor.<br />
<li>This fluorescence corresponds to one of the four possible answers (r: response).<br />
<li>The program of the arduino identifies the answer and writes its identifier in the corresponding port.<br />
<li>The communication program reads the identifier of the answer from the port.<br />
<li>"Espeak" emits the answer via a voice signal (Output).<br />
</ol><br />
<br><br><br />
You can see an explicative animation of this process here:<br><br><br />
<html><br />
<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=4,0,2,0" width="550" height="300" align="middle"><br />
<param name=wmode value="transparent"><br />
<param name=movie value="https://static.igem.org/mediawiki/2012/0/06/Animacion_ordenador.swf"><br />
<param name=quality value=high><br />
<embed src="https://static.igem.org/mediawiki/2012/0/06/Animacion_ordenador.swf" wmode=transparent quality=high pluginspage="http://www.macromedia.com/shockwave/download/index.cgi?P1_Prod_Version=ShockwaveFlash" type="application/x-shockwave-flash" width="550" height="300" align="middle"><br />
</embed><br />
</object> <br />
</html><br />
<br><br><br><br />
<!--<br />
<html><br />
<table style="background-color:transparent"><br />
<tr><br />
<td width ="400" align="center"><img src="https://static.igem.org/mediawiki/2012/7/78/ZG36O.jpeg" width="392" height="246" BORDER=0></td><br />
<td width ="400" align="center">Bla bla</td><br />
</tr><br />
</table><br />
</html><br />
--><br />
<br />
<br><br />
In the following sections we analyse in detail the main elements used in the process: <br />
<ul> <br />
<li><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/talking#Voice_recognizer"><b>Voice recognizer</b></a></html><br />
<li><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/talking#Arduino"><b>Arduino</b></a></html><br />
<li><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/talking#Fluorimeter"><b>Fluorimeter</b></a></html><br />
</ul><br />
<br><br />
<br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
=== '''Voice recognizer''' ===<br />
<br />
<html><br />
<center><br />
<br><br />
<iframe width="420" height="315" src="http://www.youtube.com/embed/nhM99FoQYSg" frameborder="0" allowfullscreen></iframe><br />
<br><br />
</center><br />
</html><br />
<br />
Julius is a continuous speech real-time recongizer engine. It is based on Markov's interpretation of hidden models. It's opensource and distributed with a BSD licence. Its main platfomorm is Linux and other Unix systems, but it also works in Windows. It has been developed as a part of a free software kit for research in large-vocabulary continuous speech recognition (LVCSR) from 1977, and the Kyoto University of Japan has continued the work from 1999 to 2003.<br />
<!--<br />
Julius es un motor de reconocimiento de habla continua en tiempo real basado en la interpretación de Modelos Ocultos de Markov. Es de código abierto y se distribuye con licencia BSD. Su principal plataforma es Linux y otros sistemas Unix, también funciona en Windows. Ha sido desarrollado como parte de un kit de software libre para investigación en Reconocimiento de habla continua de amplio vocabulario (LVCSR) desde 1977 y el trabajo ha sido continuado por la Kyoto University de Japón de 1999 hasta el 2003.--><br />
<br />
In order to use Julius, it is necessary to create a language model and an acoustic model. Julius adopts the acoustic models and the pronunctiation dictionaries from the HTK software, which is not opensource, but can be used and downloaded for its use and posterior generation of acoustic models. <br />
<!-- <br />
Para usar Julius es necesario crear un modelo de lenguaje y un modelo acústico. Julius adopta los modelos acústicos y los diccionarios de pronunciación del software HTK, que a diferencia de Julius no es opensource, pero que puede ser usado y descargado para su uso y posterior generación de los modelos acústicos. <br />
--><br />
<br><br><br />
<b> Acoustic model </b><br />
----------------------------------<br />
An acoustic model is a file which contains an statistical representation of each of the different sounds that form a word (phoneme). Julius allows voice recognition through continuous dictation or by using a previously introduced grammar. However, the use of continuous dictation carries a problem. It requires an acoustic model trained with lots of voice files. As the amount of sound files containing voices and different texts increases, the ratio of good hits of the acoustic model will improve. This implies several variations: the pronunciation of the person training the model, the dialect used, etc. Nowadays, Julius doesn't count with any model good enough for continuous dictation in English.<br />
<br />
Due to the different problems presented by this kind of recognition, we chose the second option. We designed a grammar using the Voxforce acoustic model based in Markov's Hidden Models.<br />
To do this, we need the following file: <br><br><br />
-file .dict:a list of all the words that we want our culture to recognize and its corresponding decomposition into phonemes.<br />
<br />
<!--<br />
Un modelo acústico es un fichero que contiene una representación estadística de cada uno de los diferentes sonidos que conforman una palabra (fonemas). Julius permite reconocer voz mediante dictado continuo o mediante el uso de una gramática previamente introducida. Sin embargo el uso del dictado continuo conlleva un problema. Para ello es necesario un modelo acústico entrenado con una gran cantidad de ficheros de voz. A mayor número de ficheros de sonido conteniendo voces y textos diferentes mayor ratio de acierto tendrá el modelo acústico. Esto conllevo muchas variaciones: la pronunciación de la persona que entrena el modelo, el dialecto utilizado,... Actualmente, Julius no dispone de un modelo en Inglés lo suficientemente bueno para dictado continuo.<br />
<br />
Debido los diferentes problemas que conlleva este tipo de reconocimiento, nosotros optamos por la segunda opción. Diseñamos una gramática empleando el modelo acústico de Voxforce, basado en modelos ocultos de Markov.<br />
Para ello es necesario el siguiente fichero: <br><br><br />
- fichero.dict: lista de todas las palabras que queremos que reconozca nuestro cultivo y su correspondiente descomposición en fonemas.<br />
--><br />
<br />
<ul><br />
<li><b>Acoustic Analysis</b><br><br />
Acoustic models take the acoustic properties of the input signal. They acquire a group of vectors of certain characteristics that will later be compared with a group of <br />
patterns that represent symbols of a phonetic alphabet and return the symbols which resembles them the most. This is the basis of the mathematical probabilistic <br />
process called Hidden Markov Model. The acoustic analysis is based on the extraction of a vector similar to the input acoustic signal with the purpose of applying the <br />
theory of pattern recognition.<br />
<br />
This vector is a parametric representation of the acoustic signal, containing the most relevant information and storing as compressed as possible.<br />
In order to obtain a good group of vectors, the signal is pre-processed, reducing background noise and correlation.<br />
<br><br><br />
<br />
<li><b>HMM, Hidden Markov Model</b><br><br />
Hidden Markov Models (HMMs) provide a simple and effective framework<br />
for modelling time-varying spectral vector sequences. As a consequence,<br />
almost all present day large vocabulary continuous speech<br />
recognition systems are based on HMMs.<br><br><br />
<br />
The core of all speech recognition systems consists of a set<br />
of statistical models representing the various sounds of the language to<br />
be recognised. Since speech has temporal structure and can be encoded<br />
as a sequence of spectral vectors spanning the audio frequency range,<br />
the hidden Markov model (HMM) provides a natural framework for<br />
constructing such models.<br><br><br />
<br />
Basically, the input audio waveform from<br />
a microphone is converted into a sequence of fixed size acoustic vectors<br />
Y[1:T] = y1, . . . ,yT in a process called feature extraction. The decoder<br />
then attempts to find the sequence of words w'[1:L] = w1,...,wL which<br />
is most likely to have generated Y , i.e. the decoder tries to find.<br><br><br />
<br />
w' = argmax{P(w|Y)} for all the words w considered<br><br><br />
<br />
operating this (Bayes rule) the likelihood p(Y|w) is determined by the <br />
acoustic model and the prior P(w) is determined by the language model.<br />
<br />
<br><br><br />
</ul><br />
<br />
<!--<br />
<ul><br />
<li>Análisis acústico: <br><br />
Los modelos acústicos toman las propiedades acústicas de la señal de entrada, obtienen un conjunto de vectores de características que después compararán con un <br />
conjunto de patrones que representan los símbolos de un alfabeto fonético y devuelve los símbolos que más se parecen. En esto se basa el proceso matemático <br />
probabilístico llamado Modelo Oculto de Markov. El análisis acústico se basa en la extracción de un vector de características de la señal acústica de entrada con el fin <br />
de aplicar la teoría de reconocimiento de patrones.<br />
<br />
Éste vector es una representación paramétrica de la señal acústica, conteniendo la información más importante y almacenándola de la forma más compacta posible. <br />
Con el fin de extraer un buen conjunto de vectores, la señal es preprocesada reduciendo el ruido y la correlación.<br />
<br><br><br />
<br />
<li><b>Modelos Ocultos de Markov (HMM, Hidden Markov Model):</b> <br><br />
Hidden Markov Models (HMMs) provide a simple and effective framework<br />
for modelling time-varying spectral vector sequences. As a consequence,<br />
almost all present day large vocabulary continuous speech<br />
recognition systems are based on HMMs.<br><br><br />
<br />
The core of all speech recognition systems consists of a set<br />
of statistical models representing the various sounds of the language to<br />
be recognised. Since speech has temporal structure and can be encoded<br />
as a sequence of spectral vectors spanning the audio frequency range,<br />
the hidden Markov model (HMM) provides a natural framework for<br />
constructing such models.<br><br><br />
<br />
Basically, the input audio waveform from<br />
a microphone is converted into a sequence of fixed size acoustic vectors<br />
Y[1:T] = y1, . . . ,yT in a process called feature extraction. The decoder<br />
then attempts to find the sequence of words w'[1:L] = w1,...,wL which<br />
is most likely to have generated Y , i.e. the decoder tries to find.<br><br><br />
<br />
w' = argmax{P(w|Y)} for all the words w considered<br><br><br />
<br />
operating this (Bayes rule) the likelihood p(Y|w) is determined by the <br />
acoustic model and the prior P(w) is determined by the language model.<br />
<br><br><br />
</ul><br />
--><br />
<br />
<b>Language model</b><br />
---------------------------------<br />
Language model refers to the grammar on which the recognizer will work and specify the phrases that it will be able to identify.<br />
<br />
In Julius, recognition grammar is composed by two separate files:<br><br><br />
<br />
- .voca: List of words that the grammar contains. <br><br />
- .grammar: specifies the grammar of the language to be recognised.<br><br><br />
<br />
Both files must be converted to .dfa and to .dict using the grammar compilator "mkdfa.pl"<br />
The .dfa file generated represents a finite automat. The .dict archive contains the dictionary of words in Julius format.<br />
<br />
<br />
<!--<br />
El modelo de lenguaje hace referencia a la gramática sobre la que el reconocedor va a trabajar y especificará las frases que podrá identificar.<br />
En Julius, la gramática de reconocimiento se compone por dos archivos separados: <br><br><br />
- fichero . voca: listado de las palabras que contendrá la gramática. <br><br />
- fichero .grammar: Se especifica la gramática del lenguaje a reconocer. <br><br><br />
Ambos archivos deben ser convertidos a .dfa y a .dict utilizando el compilador de gramáticas "mkdfa.pl"<br />
El archivo generado .dfa representa un autómata finito y el archivo .dict contiene el diccionario de palabras en el formato de Julius.<br />
--><br />
<br />
<br><br />
<html><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/5/59/Grammar.png" height="230" width="280"><br />
</center><br />
</html><br />
<br><br><br />
<br />
Once the acoustic language and the language model have been defined, all we need is the implementation of the main program in Python.<br />
<br />
<!--<br />
Una vez hemos definido el modelo acústico y el modelo de lenguaje, únicamente falta la implementación del programa principal en Phyton.<br />
En él ejecutamos Julius y a través del reconocimiento de voz identificamos la pregunta realizada al cultivo.<br />
--><br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
=== '''Arduino''' ===<br />
<br />
<br><br />
<br />
[[Image:arduino.png|700 px||center]]<br />
<br />
<br />
<br />
The Arduino Uno is a microcontroller board based on the ATmega328 (datasheet). It has 14 digital input/output pins (of which 6 can be used as PWM outputs), 6 analog inputs, a 16 MHz crystal oscillator, a USB connection, a power jack, an ICSP header, and a reset button. It contains everything needed to support the microcontroller; simply connect it to a computer with a USB cable or power it with a AC-to-DC adapter or battery to get started.<br />
<br />
The Uno differs from all preceding boards in that it does not use the FTDI USB-to-serial driver chip. Instead, it features the Atmega16U2 (Atmega8U2 up to version R2) programmed as a USB-to-serial converter.<br />
<br />
Revision 2 of the Uno board has a resistor pulling the 8U2 HWB line to ground, making it easier to put into DFU mode.<br />
<br />
Revision 3 of the board has the following new features:<br />
<ol><br />
<li>1.0 pinout: added SDA and SCL pins that are near to the AREF pin and two other new pins placed near to the RESET pin, the IOREF that allow the shields to adapt to the voltage provided from the board. In future, shields will be compatible both with the board that use the AVR, which operate with 5V and with the Arduino Due that operate with 3.3V. The second one is a not connected pin, that is reserved for future purposes.<br />
<br />
<li> Stronger RESET circuit.<br />
<li> Atmega 16U2 replace the 8U2.<br />
</ol><br />
<br />
"Uno" means one in Italian and is named to mark the upcoming release of Arduino 1.0. The Uno and version 1.0 will be the reference versions of Arduino, moving forward. The Uno is the latest in a series of USB Arduino boards, and the reference model for the Arduino platform; for a comparison with previous versions, see the index of Arduino boards.<br />
<br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
=== '''Fluorimeter''' ===<br />
<br><br />
A fluorimeter is an electronic device that can read the light and transform it in an electrical signal. In our project, that light proceeds from a bacterial culture that has been excited.<br />
<br />
<br />
Its principle of operation is as follows:<br />
First, an electrical signal is sent from Arduino to a LED to activate it. This LED has a specific wavelength to excite the fluorescent protein present in the microbial culture. <br />
<br />
<br />
When these bacteria are shining, we receive that light intensity with a photodiode, and transform it into electrical current. Now, we have an electrical current that is proportional with the light and therefor to the concentration of fluorescent protein present in the culture. We have to be careful with the orientation between the LED and the photodiode because interferences may occur in the measurement due to light received by photodiode but not emitted by the fluorescence in the culture but scattered light emitted by the LED. Also, a proper band-pass filter has been placed between culture and photodiode to allow only the desired wavelengths to pass through.<br />
<br />
<br />
[[File:Fluorimetro1.gif|center]]<br />
<br />
The electric current coming from the photodiode has a very small amplitude, thereby we have to amplify it and translate it into a electric voltage with an electronic trans-impedance amplifier. We can choose the gain of this amplification changing the value of resistances of the circuit. The resistances must be properly chosen to select the desired amplification gain. Excessive amplification will not only amplify the signal but also amplify the noise, which is no desired in the measurement. <br />
<br />
[[File:Fluorimetro2.jpg|center|400px]]<br />
<br />
<html><br />
<center><br />
<br><br />
<iframe width="420" height="315" src="http://www.youtube.com/embed/kBtt37xpkgk" frameborder="0" allowfullscreen></iframe><br><br />
</center><br />
</html><br />
<br />
Finally, the output voltage is sent to Arduino, and it will translate that in a digital signal.<br />
<br />
[[File:Fluorimetro3.jpg|center|400px]]<br />
<br />
The original idea was taken from [1].<br />
<br />
[1] Benjamin T. Wigton etal. ''Low-Cost, LED Fluorimeter for Middle School, High School, and Undergraduate Chemistry Lab'', J. Chem. Educ. 2011, 88, 1182-1187.<br />
</div></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/talking
Team:Valencia Biocampus/talking
2012-09-26T19:59:12Z
<p>Lujemomo: /* Fluorimeter */</p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
<br />
<ol><br />
<br><br />
<center><br />
<div id="Titulos"><br />
<h2>Talking Interfaces</h2><br />
<!-- ya cambiaré esto de sitio pero no lo borréis please!--><br />
<br><br />
<br />
<div id="PorDefecto"><br />
=== '''THE PROCESS''' ===<br />
<br><br />
<br />
The main objective of our project is to accomplish a verbal communication with our microorganisms.<br />
To do that, we need to establish the following process:<br />
<br><br />
<html><br />
<br />
<!-- FLASH--><br />
<br />
</html><br />
<br />
<!--<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/0/07/ProcesoEn2.png" height="450" width="780"><br />
</html><br />
--><br />
<ol><br />
<li>The input used is a voice signal (question), which will be collected by our voice recognizer.<br />
<li>The voice recognizer identifies the question and, through the program in charge of establishing the communication, its corresponding identifier is written in the <br />
assigned port of the arduino.<br />
<li>The software of the arduino reads the written identifier and, according to it, the corresponding port is selected, indicating the flourimeter which wavelength has <br />
to be emitted on the culture. There are four possible questions (q), and each of them is associated to a different wavelength. <br />
<li>The fluorimeter emits light (Bioinput), exciting the compound through optic filters.<br />
<li>Due to the excitation produced, the compound emits fluorescence (BioOutput), which is measured by the fluorimeter with a sensor.<br />
<li>This fluorescence corresponds to one of the four possible answers (r: response).<br />
<li>The program of the arduino identifies the answer and writes its identifier in the corresponding port.<br />
<li>The communication program reads the identifier of the answer from the port.<br />
<li>"Espeak" emits the answer via a voice signal (Output).<br />
</ol><br />
<br><br><br />
You can see an explicative animation of these process here:<br><br><br />
<html><br />
<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=4,0,2,0" width="550" height="300" align="middle"><br />
<param name=wmode value="transparent"><br />
<param name=movie value="https://static.igem.org/mediawiki/2012/0/06/Animacion_ordenador.swf"><br />
<param name=quality value=high><br />
<embed src="https://static.igem.org/mediawiki/2012/0/06/Animacion_ordenador.swf" wmode=transparent quality=high pluginspage="http://www.macromedia.com/shockwave/download/index.cgi?P1_Prod_Version=ShockwaveFlash" type="application/x-shockwave-flash" width="550" height="300" align="middle"><br />
</embed><br />
</object> <br />
</html><br />
<br><br><br><br />
<br />
<br />
<br><br />
In the following sections we analyse in detail the main elements used in the process: <br />
<ul> <br />
<li><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/talking#Voice_recognizer"><b>Voice recognizer</b></a></html><br />
<li><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/talking#Arduino"><b>Arduino</b></a></html><br />
<li><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/talking#Fluorimeter"><b>Fluorimeter</b></a></html><br />
</ul><br />
<br><br />
<br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
=== '''Voice recognizer''' ===<br />
<br />
<html><br />
<center><br />
<br><br />
<iframe width="420" height="315" src="http://www.youtube.com/embed/nhM99FoQYSg" frameborder="0" allowfullscreen></iframe><br />
<br><br />
</center><br />
</html><br />
<br />
Julius is a continuous speech real-time recongizer engine. It is based on Markov's interpretation of hidden models. It's opensource and distributed with a BSD licence. Its main platfomorm is Linux and other Unix systems, but it also works in Windows. It has been developed as a part of a free software kit for research in large-vocabulary continuous speech recognition (LVCSR) from 1977, and the Kyoto University of Japan has continued the work from 1999 to 2003.<br />
<!--<br />
Julius es un motor de reconocimiento de habla continua en tiempo real basado en la interpretación de Modelos Ocultos de Markov. Es de código abierto y se distribuye con licencia BSD. Su principal plataforma es Linux y otros sistemas Unix, también funciona en Windows. Ha sido desarrollado como parte de un kit de software libre para investigación en Reconocimiento de habla continua de amplio vocabulario (LVCSR) desde 1977 y el trabajo ha sido continuado por la Kyoto University de Japón de 1999 hasta el 2003.--><br />
<br />
In order to use Julius, it is necessary to create a language model and an acoustic model. Julius adopts the acoustic models and the pronunctiation dictionaries from the HTK software, which is not opensource, but can be used and downloaded for its use and posterior generation of acoustic models. <br />
<!-- <br />
Para usar Julius es necesario crear un modelo de lenguaje y un modelo acústico. Julius adopta los modelos acústicos y los diccionarios de pronunciación del software HTK, que a diferencia de Julius no es opensource, pero que puede ser usado y descargado para su uso y posterior generación de los modelos acústicos. <br />
--><br />
<br><br><br />
<b> Acoustic model </b><br />
----------------------------------<br />
An acoustic model is a file which contains an statistical representation of each of the different sounds that form a word (phoneme). Julius allows voice recognition through continuous dictation or by using a previously introduced grammar. However, the use of continuous dictation carries a problem. It requires an acoustic model trained with lots of voice files. As the amount of sound files containing voices and different texts increases, the ratio of good hits of the acoustic model will improve. This implies several variations: the pronunciation of the person training the model, the dialect used, etc. Nowadays, Julius doesn't count with any model good enough for continuous dictation in English.<br />
<br />
Due to the different problems presented by this kind of recognition, we chose the second option. We designed a grammar using the Voxforce acoustic model based in Markov's Hidden Models.<br />
To do this, we need the following file: <br><br><br />
-file .dict:a list of all the words that we want our culture to recognize and its corresponding decomposition into phonemes.<br />
<br />
<!--<br />
Un modelo acústico es un fichero que contiene una representación estadística de cada uno de los diferentes sonidos que conforman una palabra (fonemas). Julius permite reconocer voz mediante dictado continuo o mediante el uso de una gramática previamente introducida. Sin embargo el uso del dictado continuo conlleva un problema. Para ello es necesario un modelo acústico entrenado con una gran cantidad de ficheros de voz. A mayor número de ficheros de sonido conteniendo voces y textos diferentes mayor ratio de acierto tendrá el modelo acústico. Esto conllevo muchas variaciones: la pronunciación de la persona que entrena el modelo, el dialecto utilizado,... Actualmente, Julius no dispone de un modelo en Inglés lo suficientemente bueno para dictado continuo.<br />
<br />
Debido los diferentes problemas que conlleva este tipo de reconocimiento, nosotros optamos por la segunda opción. Diseñamos una gramática empleando el modelo acústico de Voxforce, basado en modelos ocultos de Markov.<br />
Para ello es necesario el siguiente fichero: <br><br><br />
- fichero.dict: lista de todas las palabras que queremos que reconozca nuestro cultivo y su correspondiente descomposición en fonemas.<br />
--><br />
<br />
<ul><br />
<li>Acoustic Analysis<br><br />
Acoustic models take the acoustic properties of the input signal. They acquire a group of vectors of certain characteristics that will later be compared with a group of <br />
patterns that represent symbols of a phonetic alphabet and return the symbols which resembles them the most. This is the basis of the mathematical probabilistic <br />
process called Hidden Markov Model. The acoustic analysis is based on the extraction of a vector similar to the input acoustic signal with the purpose of applying the <br />
theory of pattern recognition.<br />
<br />
This vector is a parametric representation of the acoustic signal, containing the most relevant information and storing as compressed as possible.<br />
In order to obtain a good group of vectors, the signal is pre-processed, reducing background noise and correlation.<br />
<br><br><br />
<br />
<li>HMM, Hidden Markov Model<br><br />
Hidden Markov Models (HMMs) provide a simple and effective framework<br />
for modelling time-varying spectral vector sequences. As a consequence,<br />
almost all present day large vocabulary continuous speech<br />
recognition systems are based on HMMs.<br><br><br />
<br />
The core of all speech recognition systems consists of a set<br />
of statistical models representing the various sounds of the language to<br />
be recognised. Since speech has temporal structure and can be encoded<br />
as a sequence of spectral vectors spanning the audio frequency range,<br />
the hidden Markov model (HMM) provides a natural framework for<br />
constructing such models.<br><br><br />
<br />
Basically, the input audio waveform from<br />
a microphone is converted into a sequence of fixed size acoustic vectors<br />
Y[1:T] = y1, . . . ,yT in a process called feature extraction. The decoder<br />
then attempts to find the sequence of words w'[1:L] = w1,...,wL which<br />
is most likely to have generated Y , i.e. the decoder tries to find.<br><br><br />
<br />
w' = argmax{P(w|Y)} for all the words w considered<br><br><br />
<br />
operating this (Bayes rule) the likelihood p(Y|w) is determined by the <br />
acoustic model and the prior P(w) is determined by the language model.<br />
<br />
<br><br><br />
</ul><br />
<br />
<!--<br />
<ul><br />
<li>Análisis acústico: <br><br />
Los modelos acústicos toman las propiedades acústicas de la señal de entrada, obtienen un conjunto de vectores de características que después compararán con un <br />
conjunto de patrones que representan los símbolos de un alfabeto fonético y devuelve los símbolos que más se parecen. En esto se basa el proceso matemático <br />
probabilístico llamado Modelo Oculto de Markov. El análisis acústico se basa en la extracción de un vector de características de la señal acústica de entrada con el fin <br />
de aplicar la teoría de reconocimiento de patrones.<br />
<br />
Éste vector es una representación paramétrica de la señal acústica, conteniendo la información más importante y almacenándola de la forma más compacta posible. <br />
Con el fin de extraer un buen conjunto de vectores, la señal es preprocesada reduciendo el ruido y la correlación.<br />
<br><br><br />
<br />
<li>Modelos Ocultos de Markov (HMM, Hidden Markov Model): <br><br />
Hidden Markov Models (HMMs) provide a simple and effective framework<br />
for modelling time-varying spectral vector sequences. As a consequence,<br />
almost all present day large vocabulary continuous speech<br />
recognition systems are based on HMMs.<br><br><br />
<br />
The core of all speech recognition systems consists of a set<br />
of statistical models representing the various sounds of the language to<br />
be recognised. Since speech has temporal structure and can be encoded<br />
as a sequence of spectral vectors spanning the audio frequency range,<br />
the hidden Markov model (HMM) provides a natural framework for<br />
constructing such models.<br><br><br />
<br />
Basically, the input audio waveform from<br />
a microphone is converted into a sequence of fixed size acoustic vectors<br />
Y[1:T] = y1, . . . ,yT in a process called feature extraction. The decoder<br />
then attempts to find the sequence of words w'[1:L] = w1,...,wL which<br />
is most likely to have generated Y , i.e. the decoder tries to find.<br><br><br />
<br />
w' = argmax{P(w|Y)} for all the words w considered<br><br><br />
<br />
operating this (Bayes rule) the likelihood p(Y|w) is determined by the <br />
acoustic model and the prior P(w) is determined by the language model.<br />
<br><br><br />
</ul><br />
--><br />
<br />
<b>Language model</b><br />
---------------------------------<br />
Language model refers to the grammar on which the recognizer will work and specify the phrases that it will be able to identify.<br />
<br />
In Julius, recognition grammar is composed by two separate files:<br><br><br />
<br />
- .voca: List of words that the grammar contains. <br><br />
- .grammar: specifies the grammar of the language to be recognised.<br><br><br />
<br />
Both files must be converted to .dfa and to .dict using the grammar compilator "mkdfa.pl"<br />
The .dfa file generated represents a finite automat. The .dict archive contains the dictionary of words in Julius format.<br />
<br />
<br />
<!--<br />
El modelo de lenguaje hace referencia a la gramática sobre la que el reconocedor va a trabajar y especificará las frases que podrá identificar.<br />
En Julius, la gramática de reconocimiento se compone por dos archivos separados: <br><br><br />
- fichero . voca: listado de las palabras que contendrá la gramática. <br><br />
- fichero .grammar: Se especifica la gramática del lenguaje a reconocer. <br><br><br />
Ambos archivos deben ser convertidos a .dfa y a .dict utilizando el compilador de gramáticas "mkdfa.pl"<br />
El archivo generado .dfa representa un autómata finito y el archivo .dict contiene el diccionario de palabras en el formato de Julius.<br />
--><br />
<br />
<br><br />
<html><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/5/59/Grammar.png" height="230" width="280"><br />
</center><br />
</html><br />
<br><br><br />
<br />
Once the acoustic language and the language model have been defined, all we need is the implementation of the main program in Python.<br />
<br />
<!--<br />
Una vez hemos definido el modelo acústico y el modelo de lenguaje, únicamente falta la implementación del programa principal en Phyton.<br />
En él ejecutamos Julius y a través del reconocimiento de voz identificamos la pregunta realizada al cultivo.<br />
--><br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
=== '''Arduino''' ===<br />
<br />
<br><br />
<br />
[[Image:arduino.png|700 px||center]]<br />
<br />
<br />
<br />
The Arduino Uno is a microcontroller board based on the ATmega328 (datasheet). It has 14 digital input/output pins (of which 6 can be used as PWM outputs), 6 analog inputs, a 16 MHz crystal oscillator, a USB connection, a power jack, an ICSP header, and a reset button. It contains everything needed to support the microcontroller; simply connect it to a computer with a USB cable or power it with a AC-to-DC adapter or battery to get started.<br />
<br />
The Uno differs from all preceding boards in that it does not use the FTDI USB-to-serial driver chip. Instead, it features the Atmega16U2 (Atmega8U2 up to version R2) programmed as a USB-to-serial converter.<br />
<br />
Revision 2 of the Uno board has a resistor pulling the 8U2 HWB line to ground, making it easier to put into DFU mode.<br />
<br />
Revision 3 of the board has the following new features:<br />
<ol><br />
<li>1.0 pinout: added SDA and SCL pins that are near to the AREF pin and two other new pins placed near to the RESET pin, the IOREF that allow the shields to adapt to the voltage provided from the board. In future, shields will be compatible both with the board that use the AVR, which operate with 5V and with the Arduino Due that operate with 3.3V. The second one is a not connected pin, that is reserved for future purposes.<br />
<br />
<li> Stronger RESET circuit.<br />
<li> Atmega 16U2 replace the 8U2.<br />
</ol><br />
<br />
"Uno" means one in Italian and is named to mark the upcoming release of Arduino 1.0. The Uno and version 1.0 will be the reference versions of Arduino, moving forward. The Uno is the latest in a series of USB Arduino boards, and the reference model for the Arduino platform; for a comparison with previous versions, see the index of Arduino boards.<br />
<br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
=== '''Fluorimeter''' ===<br />
<br><br />
A fluorimeter is an electronic device that can read the light and transform it in an electrical signal. In our project, that light proceeds from a bacterial culture that has been excited.<br />
<br />
<br />
Its principle of operation is as follows:<br />
First, an electrical signal is sent from Arduino to a LED to activate it. This LED has a specific wavelength to excite the fluorescent protein present in the microbial culture. <br />
<br />
<br />
When these bacteria are shining, we receive that light intensity with a photodiode, and transform it into electrical current. Now, we have an electrical current that is proportional with the light and therefor to the concentration of fluorescent protein present in the culture. We have to be careful with the orientation between the LED and the photodiode because interferences may occur in the measurement due to light received by photodiode but not emitted by the fluorescence in the culture but scattered light emitted by the LED. Also, a proper band-pass filter has been placed between culture and photodiode to allow only the desired wavelengths to pass through.<br />
<br />
<br />
[[File:Fluorimetro1.gif|center]]<br />
<br />
The electric current coming from the photodiode has a very small amplitude, thereby we have to amplify it and translate it into a electric voltage with an electronic trans-impedance amplifier. We can choose the gain of this amplification changing the value of resistances of the circuit. The resistances must be properly chosen to select the desired amplification gain. Excessive amplification will not only amplify the signal but also amplify the noise, which is no desired in the measurement. <br />
<br />
[[File:Fluorimetro2.jpg|center|400px]]<br />
<br />
<center><br />
<br><br />
<iframe width="420" height="315" src="http://youtu.be/kBtt37xpkgk" frameborder="0" allowfullscreen></iframe><br />
<br><br />
</center><br />
<br />
<br />
Finally, the output voltage is sent to Arduino, and it will translate that in a digital signal.<br />
<br />
[[File:Fluorimetro3.jpg|center|400px]]<br />
<br />
The original idea was taken from [1].<br />
<br />
[1] Benjamin T. Wigton etal. ''Low-Cost, LED Fluorimeter for Middle School, High School, and Undergraduate Chemistry Lab'', J. Chem. Educ. 2011, 88, 1182-1187.<br />
</div><br />
<html><font color="#9F9F9F">Manel yo paso del iGEM</font></html></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/talking
Team:Valencia Biocampus/talking
2012-09-26T19:58:04Z
<p>Lujemomo: /* Fluorimeter */</p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
<br />
<ol><br />
<br><br />
<center><br />
<div id="Titulos"><br />
<h2>Talking Interfaces</h2><br />
<!-- ya cambiaré esto de sitio pero no lo borréis please!--><br />
<br><br />
<br />
<div id="PorDefecto"><br />
=== '''THE PROCESS''' ===<br />
<br><br />
<br />
The main objective of our project is to accomplish a verbal communication with our microorganisms.<br />
To do that, we need to establish the following process:<br />
<br><br />
<html><br />
<br />
<!-- FLASH--><br />
<br />
</html><br />
<br />
<!--<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/0/07/ProcesoEn2.png" height="450" width="780"><br />
</html><br />
--><br />
<ol><br />
<li>The input used is a voice signal (question), which will be collected by our voice recognizer.<br />
<li>The voice recognizer identifies the question and, through the program in charge of establishing the communication, its corresponding identifier is written in the <br />
assigned port of the arduino.<br />
<li>The software of the arduino reads the written identifier and, according to it, the corresponding port is selected, indicating the flourimeter which wavelength has <br />
to be emitted on the culture. There are four possible questions (q), and each of them is associated to a different wavelength. <br />
<li>The fluorimeter emits light (Bioinput), exciting the compound through optic filters.<br />
<li>Due to the excitation produced, the compound emits fluorescence (BioOutput), which is measured by the fluorimeter with a sensor.<br />
<li>This fluorescence corresponds to one of the four possible answers (r: response).<br />
<li>The program of the arduino identifies the answer and writes its identifier in the corresponding port.<br />
<li>The communication program reads the identifier of the answer from the port.<br />
<li>"Espeak" emits the answer via a voice signal (Output).<br />
</ol><br />
<br><br><br />
You can see an explicative animation of these process here:<br><br><br />
<html><br />
<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=4,0,2,0" width="550" height="300" align="middle"><br />
<param name=wmode value="transparent"><br />
<param name=movie value="https://static.igem.org/mediawiki/2012/0/06/Animacion_ordenador.swf"><br />
<param name=quality value=high><br />
<embed src="https://static.igem.org/mediawiki/2012/0/06/Animacion_ordenador.swf" wmode=transparent quality=high pluginspage="http://www.macromedia.com/shockwave/download/index.cgi?P1_Prod_Version=ShockwaveFlash" type="application/x-shockwave-flash" width="550" height="300" align="middle"><br />
</embed><br />
</object> <br />
</html><br />
<br><br><br><br />
<br />
<br />
<br><br />
In the following sections we analyse in detail the main elements used in the process: <br />
<ul> <br />
<li><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/talking#Voice_recognizer"><b>Voice recognizer</b></a></html><br />
<li><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/talking#Arduino"><b>Arduino</b></a></html><br />
<li><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/talking#Fluorimeter"><b>Fluorimeter</b></a></html><br />
</ul><br />
<br><br />
<br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
=== '''Voice recognizer''' ===<br />
<br />
<html><br />
<center><br />
<br><br />
<iframe width="420" height="315" src="http://www.youtube.com/embed/nhM99FoQYSg" frameborder="0" allowfullscreen></iframe><br />
<br><br />
</center><br />
</html><br />
<br />
Julius is a continuous speech real-time recongizer engine. It is based on Markov's interpretation of hidden models. It's opensource and distributed with a BSD licence. Its main platfomorm is Linux and other Unix systems, but it also works in Windows. It has been developed as a part of a free software kit for research in large-vocabulary continuous speech recognition (LVCSR) from 1977, and the Kyoto University of Japan has continued the work from 1999 to 2003.<br />
<!--<br />
Julius es un motor de reconocimiento de habla continua en tiempo real basado en la interpretación de Modelos Ocultos de Markov. Es de código abierto y se distribuye con licencia BSD. Su principal plataforma es Linux y otros sistemas Unix, también funciona en Windows. Ha sido desarrollado como parte de un kit de software libre para investigación en Reconocimiento de habla continua de amplio vocabulario (LVCSR) desde 1977 y el trabajo ha sido continuado por la Kyoto University de Japón de 1999 hasta el 2003.--><br />
<br />
In order to use Julius, it is necessary to create a language model and an acoustic model. Julius adopts the acoustic models and the pronunctiation dictionaries from the HTK software, which is not opensource, but can be used and downloaded for its use and posterior generation of acoustic models. <br />
<!-- <br />
Para usar Julius es necesario crear un modelo de lenguaje y un modelo acústico. Julius adopta los modelos acústicos y los diccionarios de pronunciación del software HTK, que a diferencia de Julius no es opensource, pero que puede ser usado y descargado para su uso y posterior generación de los modelos acústicos. <br />
--><br />
<br />
<b> Acoustic model </b><br />
----------------------------------<br />
An acoustic model is a file which contains an statistical representation of each of the different sounds that form a word (phoneme). Julius allows voice recognition through continuous dictation or by using a previously introduced grammar. However, the use of continuous dictation carries a problem. It requires an acoustic model trained with lots of voice files. As the amount of sound files containing voices and different texts increases, the ratio of good hits of the acoustic model will improve. This implies several variations: the pronunciation of the person training the model, the dialect used, etc. Nowadays, Julius doesn't count with any model good enough for continuous dictation in English.<br />
<br />
Due to the different problems presented by this kind of recognition, we chose the second option. We designed a grammar using the Voxforce acoustic model based in Markov's Hidden Models.<br />
To do this, we need the following file: <br><br><br />
-file .dict:a list of all the words that we want our culture to recognize and its corresponding decomposition into phonemes.<br />
<br />
<!--<br />
Un modelo acústico es un fichero que contiene una representación estadística de cada uno de los diferentes sonidos que conforman una palabra (fonemas). Julius permite reconocer voz mediante dictado continuo o mediante el uso de una gramática previamente introducida. Sin embargo el uso del dictado continuo conlleva un problema. Para ello es necesario un modelo acústico entrenado con una gran cantidad de ficheros de voz. A mayor número de ficheros de sonido conteniendo voces y textos diferentes mayor ratio de acierto tendrá el modelo acústico. Esto conllevo muchas variaciones: la pronunciación de la persona que entrena el modelo, el dialecto utilizado,... Actualmente, Julius no dispone de un modelo en Inglés lo suficientemente bueno para dictado continuo.<br />
<br />
Debido los diferentes problemas que conlleva este tipo de reconocimiento, nosotros optamos por la segunda opción. Diseñamos una gramática empleando el modelo acústico de Voxforce, basado en modelos ocultos de Markov.<br />
Para ello es necesario el siguiente fichero: <br><br><br />
- fichero.dict: lista de todas las palabras que queremos que reconozca nuestro cultivo y su correspondiente descomposición en fonemas.<br />
--><br />
<br />
<ul><br />
<li>Acoustic Analysis<br><br />
Acoustic models take the acoustic properties of the input signal. They acquire a group of vectors of certain characteristics that will later be compared with a group of <br />
patterns that represent symbols of a phonetic alphabet and return the symbols which resembles them the most. This is the basis of the mathematical probabilistic <br />
process called Hidden Markov Model. The acoustic analysis is based on the extraction of a vector similar to the input acoustic signal with the purpose of applying the <br />
theory of pattern recognition.<br />
<br />
This vector is a parametric representation of the acoustic signal, containing the most relevant information and storing as compressed as possible.<br />
In order to obtain a good group of vectors, the signal is pre-processed, reducing background noise and correlation.<br />
<br><br><br />
<br />
<li>HMM, Hidden Markov Model<br><br />
Hidden Markov Models (HMMs) provide a simple and effective framework<br />
for modelling time-varying spectral vector sequences. As a consequence,<br />
almost all present day large vocabulary continuous speech<br />
recognition systems are based on HMMs.<br><br><br />
<br />
The core of all speech recognition systems consists of a set<br />
of statistical models representing the various sounds of the language to<br />
be recognised. Since speech has temporal structure and can be encoded<br />
as a sequence of spectral vectors spanning the audio frequency range,<br />
the hidden Markov model (HMM) provides a natural framework for<br />
constructing such models.<br><br><br />
<br />
Basically, the input audio waveform from<br />
a microphone is converted into a sequence of fixed size acoustic vectors<br />
Y[1:T] = y1, . . . ,yT in a process called feature extraction. The decoder<br />
then attempts to find the sequence of words w'[1:L] = w1,...,wL which<br />
is most likely to have generated Y , i.e. the decoder tries to find.<br><br><br />
<br />
w' = argmax{P(w|Y)} for all the words w considered<br><br><br />
<br />
operating this (Bayes rule) the likelihood p(Y|w) is determined by the <br />
acoustic model and the prior P(w) is determined by the language model.<br />
<br />
<br><br><br />
</ul><br />
<br />
<!--<br />
<ul><br />
<li>Análisis acústico: <br><br />
Los modelos acústicos toman las propiedades acústicas de la señal de entrada, obtienen un conjunto de vectores de características que después compararán con un <br />
conjunto de patrones que representan los símbolos de un alfabeto fonético y devuelve los símbolos que más se parecen. En esto se basa el proceso matemático <br />
probabilístico llamado Modelo Oculto de Markov. El análisis acústico se basa en la extracción de un vector de características de la señal acústica de entrada con el fin <br />
de aplicar la teoría de reconocimiento de patrones.<br />
<br />
Éste vector es una representación paramétrica de la señal acústica, conteniendo la información más importante y almacenándola de la forma más compacta posible. <br />
Con el fin de extraer un buen conjunto de vectores, la señal es preprocesada reduciendo el ruido y la correlación.<br />
<br><br><br />
<br />
<li>Modelos Ocultos de Markov (HMM, Hidden Markov Model): <br><br />
Hidden Markov Models (HMMs) provide a simple and effective framework<br />
for modelling time-varying spectral vector sequences. As a consequence,<br />
almost all present day large vocabulary continuous speech<br />
recognition systems are based on HMMs.<br><br><br />
<br />
The core of all speech recognition systems consists of a set<br />
of statistical models representing the various sounds of the language to<br />
be recognised. Since speech has temporal structure and can be encoded<br />
as a sequence of spectral vectors spanning the audio frequency range,<br />
the hidden Markov model (HMM) provides a natural framework for<br />
constructing such models.<br><br><br />
<br />
Basically, the input audio waveform from<br />
a microphone is converted into a sequence of fixed size acoustic vectors<br />
Y[1:T] = y1, . . . ,yT in a process called feature extraction. The decoder<br />
then attempts to find the sequence of words w'[1:L] = w1,...,wL which<br />
is most likely to have generated Y , i.e. the decoder tries to find.<br><br><br />
<br />
w' = argmax{P(w|Y)} for all the words w considered<br><br><br />
<br />
operating this (Bayes rule) the likelihood p(Y|w) is determined by the <br />
acoustic model and the prior P(w) is determined by the language model.<br />
<br><br><br />
</ul><br />
--><br />
<br />
<b>Language model</b><br />
---------------------------------<br />
Language model refers to the grammar on which the recognizer will work and specify the phrases that it will be able to identify.<br />
<br />
In Julius, recognition grammar is composed by two separate files:<br><br><br />
<br />
- .voca: List of words that the grammar contains. <br><br />
- .grammar: specifies the grammar of the language to be recognised.<br><br><br />
<br />
Both files must be converted to .dfa and to .dict using the grammar compilator "mkdfa.pl"<br />
The .dfa file generated represents a finite automat. The .dict archive contains the dictionary of words in Julius format.<br />
<br />
<br />
<!--<br />
El modelo de lenguaje hace referencia a la gramática sobre la que el reconocedor va a trabajar y especificará las frases que podrá identificar.<br />
En Julius, la gramática de reconocimiento se compone por dos archivos separados: <br><br><br />
- fichero . voca: listado de las palabras que contendrá la gramática. <br><br />
- fichero .grammar: Se especifica la gramática del lenguaje a reconocer. <br><br><br />
Ambos archivos deben ser convertidos a .dfa y a .dict utilizando el compilador de gramáticas "mkdfa.pl"<br />
El archivo generado .dfa representa un autómata finito y el archivo .dict contiene el diccionario de palabras en el formato de Julius.<br />
--><br />
<br />
<br><br />
<html><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/5/59/Grammar.png" height="230" width="280"><br />
</center><br />
</html><br />
<br><br><br />
<br />
Once the acoustic language and the language model have been defined, all we need is the implementation of the main program in Python.<br />
<br />
<!--<br />
Una vez hemos definido el modelo acústico y el modelo de lenguaje, únicamente falta la implementación del programa principal en Phyton.<br />
En él ejecutamos Julius y a través del reconocimiento de voz identificamos la pregunta realizada al cultivo.<br />
--><br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
=== '''Arduino''' ===<br />
<br />
<br><br />
<br />
[[Image:arduino.png|700 px||center]]<br />
<br />
<br />
<br />
The Arduino Uno is a microcontroller board based on the ATmega328 (datasheet). It has 14 digital input/output pins (of which 6 can be used as PWM outputs), 6 analog inputs, a 16 MHz crystal oscillator, a USB connection, a power jack, an ICSP header, and a reset button. It contains everything needed to support the microcontroller; simply connect it to a computer with a USB cable or power it with a AC-to-DC adapter or battery to get started.<br />
<br />
The Uno differs from all preceding boards in that it does not use the FTDI USB-to-serial driver chip. Instead, it features the Atmega16U2 (Atmega8U2 up to version R2) programmed as a USB-to-serial converter.<br />
<br />
Revision 2 of the Uno board has a resistor pulling the 8U2 HWB line to ground, making it easier to put into DFU mode.<br />
<br />
Revision 3 of the board has the following new features:<br />
<ol><br />
<li>1.0 pinout: added SDA and SCL pins that are near to the AREF pin and two other new pins placed near to the RESET pin, the IOREF that allow the shields to adapt to the voltage provided from the board. In future, shields will be compatible both with the board that use the AVR, which operate with 5V and with the Arduino Due that operate with 3.3V. The second one is a not connected pin, that is reserved for future purposes.<br />
<br />
<li> Stronger RESET circuit.<br />
<li> Atmega 16U2 replace the 8U2.<br />
</ol><br />
<br />
"Uno" means one in Italian and is named to mark the upcoming release of Arduino 1.0. The Uno and version 1.0 will be the reference versions of Arduino, moving forward. The Uno is the latest in a series of USB Arduino boards, and the reference model for the Arduino platform; for a comparison with previous versions, see the index of Arduino boards.<br />
<br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
=== '''Fluorimeter''' ===<br />
<br><br />
A fluorimeter is an electronic device that can read the light and transform it in an electrical signal. In our project, that light proceeds from a bacterial culture that has been excited.<br />
<br />
<br />
Its principle of operation is as follows:<br />
First, an electrical signal is sent from Arduino to a LED to activate it. This LED has a specific wavelength to excite the fluorescent protein present in the microbial culture. <br />
<br />
<br />
When these bacteria are shining, we receive that light intensity with a photodiode, and transform it into electrical current. Now, we have an electrical current that is proportional with the light and therefor to the concentration of fluorescent protein present in the culture. We have to be careful with the orientation between the LED and the photodiode because interferences may occur in the measurement due to light received by photodiode but not emitted by the fluorescence in the culture but scattered light emitted by the LED. Also, a proper band-pass filter has been placed between culture and photodiode to allow only the desired wavelengths to pass through.<br />
<br />
<br />
[[File:Fluorimetro1.gif|center]]<br />
<br />
The electric current coming from the photodiode has a very small amplitude, thereby we have to amplify it and translate it into a electric voltage with an electronic trans-impedance amplifier. We can choose the gain of this amplification changing the value of resistances of the circuit. The resistances must be properly chosen to select the desired amplification gain. Excessive amplification will not only amplify the signal but also amplify the noise, which is no desired in the measurement. <br />
<br />
[[File:Fluorimetro2.jpg|center|400px]]<br />
<br />
<center><br />
<br><br />
<iframe width="420" height="315" src="http://www.youtube.com/watch?v=kBtt37xpkgk" frameborder="0" allowfullscreen></iframe><br />
<br><br />
</center><br />
<br />
<br />
Finally, the output voltage is sent to Arduino, and it will translate that in a digital signal.<br />
<br />
[[File:Fluorimetro3.jpg|center|400px]]<br />
<br />
The original idea was taken from [1].<br />
<br />
[1] Benjamin T. Wigton etal. ''Low-Cost, LED Fluorimeter for Middle School, High School, and Undergraduate Chemistry Lab'', J. Chem. Educ. 2011, 88, 1182-1187.<br />
</div><br />
<html><font color="#9F9F9F">Manel yo paso del iGEM</font></html></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/talking
Team:Valencia Biocampus/talking
2012-09-26T19:55:38Z
<p>Lujemomo: /* The process */</p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
<br />
<ol><br />
<br><br />
<center><br />
<div id="Titulos"><br />
<h2>Talking Interfaces</h2><br />
<!-- ya cambiaré esto de sitio pero no lo borréis please!--><br />
<br><br />
<br />
<div id="PorDefecto"><br />
=== '''The process''' ===<br />
<br><br />
<br />
The main objective of our project is to accomplish a verbal communication with our microorganisms.<br />
To do that, we need to establish the following process:<br />
<br><br />
<html><br />
<br />
<!-- FLASH--><br />
<br />
</html><br />
<br />
<!--<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/0/07/ProcesoEn2.png" height="450" width="780"><br />
</html><br />
--><br />
<ol><br />
<li>The input used is a voice signal (question), which will be collected by our voice recognizer.<br />
<li>The voice recognizer identifies the question and, through the program in charge of establishing the communication, its corresponding identifier is written in the <br />
assigned port of the arduino.<br />
<li>The software of the arduino reads the written identifier and, according to it, the corresponding port is selected, indicating the flourimeter which wavelength has <br />
to be emitted on the culture. There are four possible questions (q), and each of them is associated to a different wavelength. <br />
<li>The fluorimeter emits light (Bioinput), exciting the compound through optic filters.<br />
<li>Due to the excitation produced, the compound emits fluorescence (BioOutput), which is measured by the fluorimeter with a sensor.<br />
<li>This fluorescence corresponds to one of the four possible answers (r: response).<br />
<li>The program of the arduino identifies the answer and writes its identifier in the corresponding port.<br />
<li>The communication program reads the identifier of the answer from the port.<br />
<li>"Espeak" emits the answer via a voice signal (Output).<br />
</ol><br />
<br><br><br />
You can see an explicative animation of these process here:<br><br><br />
<html><br />
<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=4,0,2,0" width="550" height="300" align="middle"><br />
<param name=wmode value="transparent"><br />
<param name=movie value="https://static.igem.org/mediawiki/2012/0/06/Animacion_ordenador.swf"><br />
<param name=quality value=high><br />
<embed src="https://static.igem.org/mediawiki/2012/0/06/Animacion_ordenador.swf" wmode=transparent quality=high pluginspage="http://www.macromedia.com/shockwave/download/index.cgi?P1_Prod_Version=ShockwaveFlash" type="application/x-shockwave-flash" width="550" height="300" align="middle"><br />
</embed><br />
</object> <br />
</html><br />
<br><br><br><br />
<br />
<br />
<br><br />
In the following sections we analyse in detail the main elements used in the process: <br />
<ul> <br />
<li><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/talking#Voice_recognizer"><b>Voice recognizer</b></a></html><br />
<li><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/talking#Arduino"><b>Arduino</b></a></html><br />
<li><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/talking#Fluorimeter"><b>Fluorimeter</b></a></html><br />
</ul><br />
<br><br />
<br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
=== '''Voice recognizer''' ===<br />
<br />
<html><br />
<center><br />
<br><br />
<iframe width="420" height="315" src="http://www.youtube.com/embed/nhM99FoQYSg" frameborder="0" allowfullscreen></iframe><br />
<br><br />
</center><br />
</html><br />
<br />
Julius is a continuous speech real-time recongizer engine. It is based on Markov's interpretation of hidden models. It's opensource and distributed with a BSD licence. Its main platfomorm is Linux and other Unix systems, but it also works in Windows. It has been developed as a part of a free software kit for research in large-vocabulary continuous speech recognition (LVCSR) from 1977, and the Kyoto University of Japan has continued the work from 1999 to 2003.<br />
<!--<br />
Julius es un motor de reconocimiento de habla continua en tiempo real basado en la interpretación de Modelos Ocultos de Markov. Es de código abierto y se distribuye con licencia BSD. Su principal plataforma es Linux y otros sistemas Unix, también funciona en Windows. Ha sido desarrollado como parte de un kit de software libre para investigación en Reconocimiento de habla continua de amplio vocabulario (LVCSR) desde 1977 y el trabajo ha sido continuado por la Kyoto University de Japón de 1999 hasta el 2003.--><br />
<br />
In order to use Julius, it is necessary to create a language model and an acoustic model. Julius adopts the acoustic models and the pronunctiation dictionaries from the HTK software, which is not opensource, but can be used and downloaded for its use and posterior generation of acoustic models. <br />
<!-- <br />
Para usar Julius es necesario crear un modelo de lenguaje y un modelo acústico. Julius adopta los modelos acústicos y los diccionarios de pronunciación del software HTK, que a diferencia de Julius no es opensource, pero que puede ser usado y descargado para su uso y posterior generación de los modelos acústicos. <br />
--><br />
<br />
<b> Acoustic model </b><br />
----------------------------------<br />
An acoustic model is a file which contains an statistical representation of each of the different sounds that form a word (phoneme). Julius allows voice recognition through continuous dictation or by using a previously introduced grammar. However, the use of continuous dictation carries a problem. It requires an acoustic model trained with lots of voice files. As the amount of sound files containing voices and different texts increases, the ratio of good hits of the acoustic model will improve. This implies several variations: the pronunciation of the person training the model, the dialect used, etc. Nowadays, Julius doesn't count with any model good enough for continuous dictation in English.<br />
<br />
Due to the different problems presented by this kind of recognition, we chose the second option. We designed a grammar using the Voxforce acoustic model based in Markov's Hidden Models.<br />
To do this, we need the following file: <br><br><br />
-file .dict:a list of all the words that we want our culture to recognize and its corresponding decomposition into phonemes.<br />
<br />
<!--<br />
Un modelo acústico es un fichero que contiene una representación estadística de cada uno de los diferentes sonidos que conforman una palabra (fonemas). Julius permite reconocer voz mediante dictado continuo o mediante el uso de una gramática previamente introducida. Sin embargo el uso del dictado continuo conlleva un problema. Para ello es necesario un modelo acústico entrenado con una gran cantidad de ficheros de voz. A mayor número de ficheros de sonido conteniendo voces y textos diferentes mayor ratio de acierto tendrá el modelo acústico. Esto conllevo muchas variaciones: la pronunciación de la persona que entrena el modelo, el dialecto utilizado,... Actualmente, Julius no dispone de un modelo en Inglés lo suficientemente bueno para dictado continuo.<br />
<br />
Debido los diferentes problemas que conlleva este tipo de reconocimiento, nosotros optamos por la segunda opción. Diseñamos una gramática empleando el modelo acústico de Voxforce, basado en modelos ocultos de Markov.<br />
Para ello es necesario el siguiente fichero: <br><br><br />
- fichero.dict: lista de todas las palabras que queremos que reconozca nuestro cultivo y su correspondiente descomposición en fonemas.<br />
--><br />
<br />
<ul><br />
<li>Acoustic Analysis<br><br />
Acoustic models take the acoustic properties of the input signal. They acquire a group of vectors of certain characteristics that will later be compared with a group of <br />
patterns that represent symbols of a phonetic alphabet and return the symbols which resembles them the most. This is the basis of the mathematical probabilistic <br />
process called Hidden Markov Model. The acoustic analysis is based on the extraction of a vector similar to the input acoustic signal with the purpose of applying the <br />
theory of pattern recognition.<br />
<br />
This vector is a parametric representation of the acoustic signal, containing the most relevant information and storing as compressed as possible.<br />
In order to obtain a good group of vectors, the signal is pre-processed, reducing background noise and correlation.<br />
<br><br><br />
<br />
<li>HMM, Hidden Markov Model<br><br />
Hidden Markov Models (HMMs) provide a simple and effective framework<br />
for modelling time-varying spectral vector sequences. As a consequence,<br />
almost all present day large vocabulary continuous speech<br />
recognition systems are based on HMMs.<br><br><br />
<br />
The core of all speech recognition systems consists of a set<br />
of statistical models representing the various sounds of the language to<br />
be recognised. Since speech has temporal structure and can be encoded<br />
as a sequence of spectral vectors spanning the audio frequency range,<br />
the hidden Markov model (HMM) provides a natural framework for<br />
constructing such models.<br><br><br />
<br />
Basically, the input audio waveform from<br />
a microphone is converted into a sequence of fixed size acoustic vectors<br />
Y[1:T] = y1, . . . ,yT in a process called feature extraction. The decoder<br />
then attempts to find the sequence of words w'[1:L] = w1,...,wL which<br />
is most likely to have generated Y , i.e. the decoder tries to find.<br><br><br />
<br />
w' = argmax{P(w|Y)} for all the words w considered<br><br><br />
<br />
operating this (Bayes rule) the likelihood p(Y|w) is determined by the <br />
acoustic model and the prior P(w) is determined by the language model.<br />
<br />
<br><br><br />
</ul><br />
<br />
<!--<br />
<ul><br />
<li>Análisis acústico: <br><br />
Los modelos acústicos toman las propiedades acústicas de la señal de entrada, obtienen un conjunto de vectores de características que después compararán con un <br />
conjunto de patrones que representan los símbolos de un alfabeto fonético y devuelve los símbolos que más se parecen. En esto se basa el proceso matemático <br />
probabilístico llamado Modelo Oculto de Markov. El análisis acústico se basa en la extracción de un vector de características de la señal acústica de entrada con el fin <br />
de aplicar la teoría de reconocimiento de patrones.<br />
<br />
Éste vector es una representación paramétrica de la señal acústica, conteniendo la información más importante y almacenándola de la forma más compacta posible. <br />
Con el fin de extraer un buen conjunto de vectores, la señal es preprocesada reduciendo el ruido y la correlación.<br />
<br><br><br />
<br />
<li>Modelos Ocultos de Markov (HMM, Hidden Markov Model): <br><br />
Hidden Markov Models (HMMs) provide a simple and effective framework<br />
for modelling time-varying spectral vector sequences. As a consequence,<br />
almost all present day large vocabulary continuous speech<br />
recognition systems are based on HMMs.<br><br><br />
<br />
The core of all speech recognition systems consists of a set<br />
of statistical models representing the various sounds of the language to<br />
be recognised. Since speech has temporal structure and can be encoded<br />
as a sequence of spectral vectors spanning the audio frequency range,<br />
the hidden Markov model (HMM) provides a natural framework for<br />
constructing such models.<br><br><br />
<br />
Basically, the input audio waveform from<br />
a microphone is converted into a sequence of fixed size acoustic vectors<br />
Y[1:T] = y1, . . . ,yT in a process called feature extraction. The decoder<br />
then attempts to find the sequence of words w'[1:L] = w1,...,wL which<br />
is most likely to have generated Y , i.e. the decoder tries to find.<br><br><br />
<br />
w' = argmax{P(w|Y)} for all the words w considered<br><br><br />
<br />
operating this (Bayes rule) the likelihood p(Y|w) is determined by the <br />
acoustic model and the prior P(w) is determined by the language model.<br />
<br><br><br />
</ul><br />
--><br />
<br />
<b>Language model</b><br />
---------------------------------<br />
Language model refers to the grammar on which the recognizer will work and specify the phrases that it will be able to identify.<br />
<br />
In Julius, recognition grammar is composed by two separate files:<br><br><br />
<br />
- .voca: List of words that the grammar contains. <br><br />
- .grammar: specifies the grammar of the language to be recognised.<br><br><br />
<br />
Both files must be converted to .dfa and to .dict using the grammar compilator "mkdfa.pl"<br />
The .dfa file generated represents a finite automat. The .dict archive contains the dictionary of words in Julius format.<br />
<br />
<br />
<!--<br />
El modelo de lenguaje hace referencia a la gramática sobre la que el reconocedor va a trabajar y especificará las frases que podrá identificar.<br />
En Julius, la gramática de reconocimiento se compone por dos archivos separados: <br><br><br />
- fichero . voca: listado de las palabras que contendrá la gramática. <br><br />
- fichero .grammar: Se especifica la gramática del lenguaje a reconocer. <br><br><br />
Ambos archivos deben ser convertidos a .dfa y a .dict utilizando el compilador de gramáticas "mkdfa.pl"<br />
El archivo generado .dfa representa un autómata finito y el archivo .dict contiene el diccionario de palabras en el formato de Julius.<br />
--><br />
<br />
<br><br />
<html><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/5/59/Grammar.png" height="230" width="280"><br />
</center><br />
</html><br />
<br><br><br />
<br />
Once the acoustic language and the language model have been defined, all we need is the implementation of the main program in Python.<br />
<br />
<!--<br />
Una vez hemos definido el modelo acústico y el modelo de lenguaje, únicamente falta la implementación del programa principal en Phyton.<br />
En él ejecutamos Julius y a través del reconocimiento de voz identificamos la pregunta realizada al cultivo.<br />
--><br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
=== '''Arduino''' ===<br />
<br />
<br><br />
<br />
[[Image:arduino.png|700 px||center]]<br />
<br />
<br />
<br />
The Arduino Uno is a microcontroller board based on the ATmega328 (datasheet). It has 14 digital input/output pins (of which 6 can be used as PWM outputs), 6 analog inputs, a 16 MHz crystal oscillator, a USB connection, a power jack, an ICSP header, and a reset button. It contains everything needed to support the microcontroller; simply connect it to a computer with a USB cable or power it with a AC-to-DC adapter or battery to get started.<br />
<br />
The Uno differs from all preceding boards in that it does not use the FTDI USB-to-serial driver chip. Instead, it features the Atmega16U2 (Atmega8U2 up to version R2) programmed as a USB-to-serial converter.<br />
<br />
Revision 2 of the Uno board has a resistor pulling the 8U2 HWB line to ground, making it easier to put into DFU mode.<br />
<br />
Revision 3 of the board has the following new features:<br />
<ol><br />
<li>1.0 pinout: added SDA and SCL pins that are near to the AREF pin and two other new pins placed near to the RESET pin, the IOREF that allow the shields to adapt to the voltage provided from the board. In future, shields will be compatible both with the board that use the AVR, which operate with 5V and with the Arduino Due that operate with 3.3V. The second one is a not connected pin, that is reserved for future purposes.<br />
<br />
<li> Stronger RESET circuit.<br />
<li> Atmega 16U2 replace the 8U2.<br />
</ol><br />
<br />
"Uno" means one in Italian and is named to mark the upcoming release of Arduino 1.0. The Uno and version 1.0 will be the reference versions of Arduino, moving forward. The Uno is the latest in a series of USB Arduino boards, and the reference model for the Arduino platform; for a comparison with previous versions, see the index of Arduino boards.<br />
<br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
=== '''Fluorimeter''' ===<br />
<br />
A fluorimeter is an electronic device that can read the light and transform it in an electrical signal. In our project, that light proceeds from a bacterial culture that has been excited.<br />
<br />
<br />
Its principle of operation is as follows:<br />
First, an electrical signal is sent from Arduino to a LED to activate it. This LED has a specific wavelength to excite the fluorescent protein present in the microbial culture. <br />
<br />
<br />
When these bacteria are shining, we receive that light intensity with a photodiode, and transform it into electrical current. Now, we have an electrical current that is proportional with the light and therefor to the concentration of fluorescent protein present in the culture. We have to be careful with the orientation between the LED and the photodiode because interferences may occur in the measurement due to light received by photodiode but not emitted by the fluorescence in the culture but scattered light emitted by the LED. Also, a proper band-pass filter has been placed between culture and photodiode to allow only the desired wavelengths to pass through.<br />
<br />
<br />
[[File:Fluorimetro1.gif|center]]<br />
<br />
The electric current coming from the photodiode has a very small amplitude, thereby we have to amplify it and translate it into a electric voltage with an electronic trans-impedance amplifier. We can choose the gain of this amplification changing the value of resistances of the circuit. The resistances must be properly chosen to select the desired amplification gain. Excessive amplification will not only amplify the signal but also amplify the noise, which is no desired in the measurement. <br />
<br />
[[File:Fluorimetro2.jpg|center|400px]]<br />
<br />
<html><br />
<center><br />
<br><br />
<iframe width="420" height="315" src="https://www.youtube.com/watch?v=kBtt37xpkgk" frameborder="0" allowfullscreen></iframe><br />
<br><br />
</center><br />
</html><br />
<br />
Finally, the output voltage is sent to Arduino, and it will translate that in a digital signal.<br />
<br />
[[File:Fluorimetro3.jpg|center|400px]]<br />
<br />
The original idea was taken from [1].<br />
<br />
[1] Benjamin T. Wigton etal. ''Low-Cost, LED Fluorimeter for Middle School, High School, and Undergraduate Chemistry Lab'', J. Chem. Educ. 2011, 88, 1182-1187.<br />
</div><br />
<html><font color="#9F9F9F">Manel yo paso del iGEM</font></html></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/talking
Team:Valencia Biocampus/talking
2012-09-26T19:55:07Z
<p>Lujemomo: </p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
<br />
<ol><br />
<br><br />
<center><br />
<div id="Titulos"><br />
<h2>Talking Interfaces</h2><br />
<!-- ya cambiaré esto de sitio pero no lo borréis please!--><br />
<br><br />
<br />
<div id="PorDefecto"><br />
=== '''The process''' ===<br />
<br><br />
<br />
The main objective of our project is to accomplish a verbal communication with our microorganisms.<br />
To do that, we need to establish the following process:<br />
<br><br />
<html><br />
<br />
<!-- FLASH--><br />
<br />
</html><br />
<br />
<!--<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/0/07/ProcesoEn2.png" height="450" width="780"><br />
</html><br />
--><br />
<ol><br />
<li>The input used is a voice signal (question), which will be collected by our voice recognizer.<br />
<li>The voice recognizer identifies the question and, through the program in charge of establishing the communication, its corresponding identifier is written in the <br />
assigned port of the arduino.<br />
<li>The software of the arduino reads the written identifier and, according to it, the corresponding port is selected, indicating the flourimeter which wavelength has <br />
to be emitted on the culture. There are four possible questions (q), and each of them is associated to a different wavelength. <br />
<li>The fluorimeter emits light (Bioinput), exciting the compound through optic filters.<br />
<li>Due to the excitation produced, the compound emits fluorescence (BioOutput), which is measured by the fluorimeter with a sensor.<br />
<li>This fluorescence corresponds to one of the four possible answers (r: response).<br />
<li>The program of the arduino identifies the answer and writes its identifier in the corresponding port.<br />
<li>The communication program reads the identifier of the answer from the port.<br />
<li>"Espeak" emits the answer via a voice signal (Output).<br />
</ol><br />
<br><br><br />
You can see an explicative animation of these process here:<br><br><br />
<html><br />
<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=4,0,2,0" width="550" height="300" align="middle"><br />
<param name=wmode value="transparent"><br />
<param name=movie value="https://static.igem.org/mediawiki/2012/0/06/Animacion_ordenador.swf"><br />
<param name=quality value=high><br />
<embed src="https://static.igem.org/mediawiki/2012/0/06/Animacion_ordenador.swf" wmode=transparent quality=high pluginspage="http://www.macromedia.com/shockwave/download/index.cgi?P1_Prod_Version=ShockwaveFlash" type="application/x-shockwave-flash" width="550" height="300" align="middle"><br />
</embed><br />
</object> <br />
</html><br />
<br><br><br><br />
<br />
<br />
<br><br />
In the following sections we analyse in detail the main elements used in the process: <br />
<ul> <br />
<li><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/talking#Voice_recognizer"><b>Voice recognizer</b></a></html><br />
<li><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/talking#Arduino"><b>Arduino</b></a></html><br />
<li><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/talking#Fluorometer"><b>Fluorometer</b></a></html><br />
</ul><br />
<br><br />
<br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
=== '''Voice recognizer''' ===<br />
<br />
<html><br />
<center><br />
<br><br />
<iframe width="420" height="315" src="http://www.youtube.com/embed/nhM99FoQYSg" frameborder="0" allowfullscreen></iframe><br />
<br><br />
</center><br />
</html><br />
<br />
Julius is a continuous speech real-time recongizer engine. It is based on Markov's interpretation of hidden models. It's opensource and distributed with a BSD licence. Its main platfomorm is Linux and other Unix systems, but it also works in Windows. It has been developed as a part of a free software kit for research in large-vocabulary continuous speech recognition (LVCSR) from 1977, and the Kyoto University of Japan has continued the work from 1999 to 2003.<br />
<!--<br />
Julius es un motor de reconocimiento de habla continua en tiempo real basado en la interpretación de Modelos Ocultos de Markov. Es de código abierto y se distribuye con licencia BSD. Su principal plataforma es Linux y otros sistemas Unix, también funciona en Windows. Ha sido desarrollado como parte de un kit de software libre para investigación en Reconocimiento de habla continua de amplio vocabulario (LVCSR) desde 1977 y el trabajo ha sido continuado por la Kyoto University de Japón de 1999 hasta el 2003.--><br />
<br />
In order to use Julius, it is necessary to create a language model and an acoustic model. Julius adopts the acoustic models and the pronunctiation dictionaries from the HTK software, which is not opensource, but can be used and downloaded for its use and posterior generation of acoustic models. <br />
<!-- <br />
Para usar Julius es necesario crear un modelo de lenguaje y un modelo acústico. Julius adopta los modelos acústicos y los diccionarios de pronunciación del software HTK, que a diferencia de Julius no es opensource, pero que puede ser usado y descargado para su uso y posterior generación de los modelos acústicos. <br />
--><br />
<br />
<b> Acoustic model </b><br />
----------------------------------<br />
An acoustic model is a file which contains an statistical representation of each of the different sounds that form a word (phoneme). Julius allows voice recognition through continuous dictation or by using a previously introduced grammar. However, the use of continuous dictation carries a problem. It requires an acoustic model trained with lots of voice files. As the amount of sound files containing voices and different texts increases, the ratio of good hits of the acoustic model will improve. This implies several variations: the pronunciation of the person training the model, the dialect used, etc. Nowadays, Julius doesn't count with any model good enough for continuous dictation in English.<br />
<br />
Due to the different problems presented by this kind of recognition, we chose the second option. We designed a grammar using the Voxforce acoustic model based in Markov's Hidden Models.<br />
To do this, we need the following file: <br><br><br />
-file .dict:a list of all the words that we want our culture to recognize and its corresponding decomposition into phonemes.<br />
<br />
<!--<br />
Un modelo acústico es un fichero que contiene una representación estadística de cada uno de los diferentes sonidos que conforman una palabra (fonemas). Julius permite reconocer voz mediante dictado continuo o mediante el uso de una gramática previamente introducida. Sin embargo el uso del dictado continuo conlleva un problema. Para ello es necesario un modelo acústico entrenado con una gran cantidad de ficheros de voz. A mayor número de ficheros de sonido conteniendo voces y textos diferentes mayor ratio de acierto tendrá el modelo acústico. Esto conllevo muchas variaciones: la pronunciación de la persona que entrena el modelo, el dialecto utilizado,... Actualmente, Julius no dispone de un modelo en Inglés lo suficientemente bueno para dictado continuo.<br />
<br />
Debido los diferentes problemas que conlleva este tipo de reconocimiento, nosotros optamos por la segunda opción. Diseñamos una gramática empleando el modelo acústico de Voxforce, basado en modelos ocultos de Markov.<br />
Para ello es necesario el siguiente fichero: <br><br><br />
- fichero.dict: lista de todas las palabras que queremos que reconozca nuestro cultivo y su correspondiente descomposición en fonemas.<br />
--><br />
<br />
<ul><br />
<li>Acoustic Analysis<br><br />
Acoustic models take the acoustic properties of the input signal. They acquire a group of vectors of certain characteristics that will later be compared with a group of <br />
patterns that represent symbols of a phonetic alphabet and return the symbols which resembles them the most. This is the basis of the mathematical probabilistic <br />
process called Hidden Markov Model. The acoustic analysis is based on the extraction of a vector similar to the input acoustic signal with the purpose of applying the <br />
theory of pattern recognition.<br />
<br />
This vector is a parametric representation of the acoustic signal, containing the most relevant information and storing as compressed as possible.<br />
In order to obtain a good group of vectors, the signal is pre-processed, reducing background noise and correlation.<br />
<br><br><br />
<br />
<li>HMM, Hidden Markov Model<br><br />
Hidden Markov Models (HMMs) provide a simple and effective framework<br />
for modelling time-varying spectral vector sequences. As a consequence,<br />
almost all present day large vocabulary continuous speech<br />
recognition systems are based on HMMs.<br><br><br />
<br />
The core of all speech recognition systems consists of a set<br />
of statistical models representing the various sounds of the language to<br />
be recognised. Since speech has temporal structure and can be encoded<br />
as a sequence of spectral vectors spanning the audio frequency range,<br />
the hidden Markov model (HMM) provides a natural framework for<br />
constructing such models.<br><br><br />
<br />
Basically, the input audio waveform from<br />
a microphone is converted into a sequence of fixed size acoustic vectors<br />
Y[1:T] = y1, . . . ,yT in a process called feature extraction. The decoder<br />
then attempts to find the sequence of words w'[1:L] = w1,...,wL which<br />
is most likely to have generated Y , i.e. the decoder tries to find.<br><br><br />
<br />
w' = argmax{P(w|Y)} for all the words w considered<br><br><br />
<br />
operating this (Bayes rule) the likelihood p(Y|w) is determined by the <br />
acoustic model and the prior P(w) is determined by the language model.<br />
<br />
<br><br><br />
</ul><br />
<br />
<!--<br />
<ul><br />
<li>Análisis acústico: <br><br />
Los modelos acústicos toman las propiedades acústicas de la señal de entrada, obtienen un conjunto de vectores de características que después compararán con un <br />
conjunto de patrones que representan los símbolos de un alfabeto fonético y devuelve los símbolos que más se parecen. En esto se basa el proceso matemático <br />
probabilístico llamado Modelo Oculto de Markov. El análisis acústico se basa en la extracción de un vector de características de la señal acústica de entrada con el fin <br />
de aplicar la teoría de reconocimiento de patrones.<br />
<br />
Éste vector es una representación paramétrica de la señal acústica, conteniendo la información más importante y almacenándola de la forma más compacta posible. <br />
Con el fin de extraer un buen conjunto de vectores, la señal es preprocesada reduciendo el ruido y la correlación.<br />
<br><br><br />
<br />
<li>Modelos Ocultos de Markov (HMM, Hidden Markov Model): <br><br />
Hidden Markov Models (HMMs) provide a simple and effective framework<br />
for modelling time-varying spectral vector sequences. As a consequence,<br />
almost all present day large vocabulary continuous speech<br />
recognition systems are based on HMMs.<br><br><br />
<br />
The core of all speech recognition systems consists of a set<br />
of statistical models representing the various sounds of the language to<br />
be recognised. Since speech has temporal structure and can be encoded<br />
as a sequence of spectral vectors spanning the audio frequency range,<br />
the hidden Markov model (HMM) provides a natural framework for<br />
constructing such models.<br><br><br />
<br />
Basically, the input audio waveform from<br />
a microphone is converted into a sequence of fixed size acoustic vectors<br />
Y[1:T] = y1, . . . ,yT in a process called feature extraction. The decoder<br />
then attempts to find the sequence of words w'[1:L] = w1,...,wL which<br />
is most likely to have generated Y , i.e. the decoder tries to find.<br><br><br />
<br />
w' = argmax{P(w|Y)} for all the words w considered<br><br><br />
<br />
operating this (Bayes rule) the likelihood p(Y|w) is determined by the <br />
acoustic model and the prior P(w) is determined by the language model.<br />
<br><br><br />
</ul><br />
--><br />
<br />
<b>Language model</b><br />
---------------------------------<br />
Language model refers to the grammar on which the recognizer will work and specify the phrases that it will be able to identify.<br />
<br />
In Julius, recognition grammar is composed by two separate files:<br><br><br />
<br />
- .voca: List of words that the grammar contains. <br><br />
- .grammar: specifies the grammar of the language to be recognised.<br><br><br />
<br />
Both files must be converted to .dfa and to .dict using the grammar compilator "mkdfa.pl"<br />
The .dfa file generated represents a finite automat. The .dict archive contains the dictionary of words in Julius format.<br />
<br />
<br />
<!--<br />
El modelo de lenguaje hace referencia a la gramática sobre la que el reconocedor va a trabajar y especificará las frases que podrá identificar.<br />
En Julius, la gramática de reconocimiento se compone por dos archivos separados: <br><br><br />
- fichero . voca: listado de las palabras que contendrá la gramática. <br><br />
- fichero .grammar: Se especifica la gramática del lenguaje a reconocer. <br><br><br />
Ambos archivos deben ser convertidos a .dfa y a .dict utilizando el compilador de gramáticas "mkdfa.pl"<br />
El archivo generado .dfa representa un autómata finito y el archivo .dict contiene el diccionario de palabras en el formato de Julius.<br />
--><br />
<br />
<br><br />
<html><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/5/59/Grammar.png" height="230" width="280"><br />
</center><br />
</html><br />
<br><br><br />
<br />
Once the acoustic language and the language model have been defined, all we need is the implementation of the main program in Python.<br />
<br />
<!--<br />
Una vez hemos definido el modelo acústico y el modelo de lenguaje, únicamente falta la implementación del programa principal en Phyton.<br />
En él ejecutamos Julius y a través del reconocimiento de voz identificamos la pregunta realizada al cultivo.<br />
--><br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
=== '''Arduino''' ===<br />
<br />
<br><br />
<br />
[[Image:arduino.png|700 px||center]]<br />
<br />
<br />
<br />
The Arduino Uno is a microcontroller board based on the ATmega328 (datasheet). It has 14 digital input/output pins (of which 6 can be used as PWM outputs), 6 analog inputs, a 16 MHz crystal oscillator, a USB connection, a power jack, an ICSP header, and a reset button. It contains everything needed to support the microcontroller; simply connect it to a computer with a USB cable or power it with a AC-to-DC adapter or battery to get started.<br />
<br />
The Uno differs from all preceding boards in that it does not use the FTDI USB-to-serial driver chip. Instead, it features the Atmega16U2 (Atmega8U2 up to version R2) programmed as a USB-to-serial converter.<br />
<br />
Revision 2 of the Uno board has a resistor pulling the 8U2 HWB line to ground, making it easier to put into DFU mode.<br />
<br />
Revision 3 of the board has the following new features:<br />
<ol><br />
<li>1.0 pinout: added SDA and SCL pins that are near to the AREF pin and two other new pins placed near to the RESET pin, the IOREF that allow the shields to adapt to the voltage provided from the board. In future, shields will be compatible both with the board that use the AVR, which operate with 5V and with the Arduino Due that operate with 3.3V. The second one is a not connected pin, that is reserved for future purposes.<br />
<br />
<li> Stronger RESET circuit.<br />
<li> Atmega 16U2 replace the 8U2.<br />
</ol><br />
<br />
"Uno" means one in Italian and is named to mark the upcoming release of Arduino 1.0. The Uno and version 1.0 will be the reference versions of Arduino, moving forward. The Uno is the latest in a series of USB Arduino boards, and the reference model for the Arduino platform; for a comparison with previous versions, see the index of Arduino boards.<br />
<br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
=== '''Fluorimeter''' ===<br />
<br />
A fluorimeter is an electronic device that can read the light and transform it in an electrical signal. In our project, that light proceeds from a bacterial culture that has been excited.<br />
<br />
<br />
Its principle of operation is as follows:<br />
First, an electrical signal is sent from Arduino to a LED to activate it. This LED has a specific wavelength to excite the fluorescent protein present in the microbial culture. <br />
<br />
<br />
When these bacteria are shining, we receive that light intensity with a photodiode, and transform it into electrical current. Now, we have an electrical current that is proportional with the light and therefor to the concentration of fluorescent protein present in the culture. We have to be careful with the orientation between the LED and the photodiode because interferences may occur in the measurement due to light received by photodiode but not emitted by the fluorescence in the culture but scattered light emitted by the LED. Also, a proper band-pass filter has been placed between culture and photodiode to allow only the desired wavelengths to pass through.<br />
<br />
<br />
[[File:Fluorimetro1.gif|center]]<br />
<br />
The electric current coming from the photodiode has a very small amplitude, thereby we have to amplify it and translate it into a electric voltage with an electronic trans-impedance amplifier. We can choose the gain of this amplification changing the value of resistances of the circuit. The resistances must be properly chosen to select the desired amplification gain. Excessive amplification will not only amplify the signal but also amplify the noise, which is no desired in the measurement. <br />
<br />
[[File:Fluorimetro2.jpg|center|400px]]<br />
<br />
<html><br />
<center><br />
<br><br />
<iframe width="420" height="315" src="https://www.youtube.com/watch?v=kBtt37xpkgk" frameborder="0" allowfullscreen></iframe><br />
<br><br />
</center><br />
</html><br />
<br />
Finally, the output voltage is sent to Arduino, and it will translate that in a digital signal.<br />
<br />
[[File:Fluorimetro3.jpg|center|400px]]<br />
<br />
The original idea was taken from [1].<br />
<br />
[1] Benjamin T. Wigton etal. ''Low-Cost, LED Fluorimeter for Middle School, High School, and Undergraduate Chemistry Lab'', J. Chem. Educ. 2011, 88, 1182-1187.<br />
</div><br />
<html><font color="#9F9F9F">Manel yo paso del iGEM</font></html></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/talking
Team:Valencia Biocampus/talking
2012-09-26T19:54:34Z
<p>Lujemomo: </p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
<br />
<ol><br />
<br><br />
<center><br />
<div id="Titulos"><br />
<h2>Talking Interfaces</h2><br />
<!-- ya cambiaré esto de sitio pero no lo borréis please!--><br />
<br><br />
<br />
<div id="PorDefecto"><br />
=== '''The process''' ===<br />
<br><br />
<br />
The main objective of our project is to accomplish a verbal communication with our microorganisms.<br />
To do that, we need to establish the following process:<br />
<br><br />
<html><br />
<br />
<!-- FLASH--><br />
<br />
</html><br />
<br />
<!--<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/0/07/ProcesoEn2.png" height="450" width="780"><br />
</html><br />
--><br />
<ol><br />
<li>The input used is a voice signal (question), which will be collected by our voice recognizer.<br />
<li>The voice recognizer identifies the question and, through the program in charge of establishing the communication, its corresponding identifier is written in the <br />
assigned port of the arduino.<br />
<li>The software of the arduino reads the written identifier and, according to it, the corresponding port is selected, indicating the flourimeter which wavelength has <br />
to be emitted on the culture. There are four possible questions (q), and each of them is associated to a different wavelength. <br />
<li>The fluorimeter emits light (Bioinput), exciting the compound through optic filters.<br />
<li>Due to the excitation produced, the compound emits fluorescence (BioOutput), which is measured by the fluorimeter with a sensor.<br />
<li>This fluorescence corresponds to one of the four possible answers (r: response).<br />
<li>The program of the arduino identifies the answer and writes its identifier in the corresponding port.<br />
<li>The communication program reads the identifier of the answer from the port.<br />
<li>"Espeak" emits the answer via a voice signal (Output).<br />
</ol><br />
<br><br><br />
You can see an explicative animation of these process here:<br><br><br />
<html><br />
<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=4,0,2,0" width="550" height="300" align="middle"><br />
<param name=wmode value="transparent"><br />
<param name=movie value="https://static.igem.org/mediawiki/2012/0/06/Animacion_ordenador.swf"><br />
<param name=quality value=high><br />
<embed src="https://static.igem.org/mediawiki/2012/0/06/Animacion_ordenador.swf" wmode=transparent quality=high pluginspage="http://www.macromedia.com/shockwave/download/index.cgi?P1_Prod_Version=ShockwaveFlash" type="application/x-shockwave-flash" width="550" height="300" align="middle"><br />
</embed><br />
</object> <br />
</html><br />
<br><br><br><br />
<br />
<br />
<br><br />
In the following sections we analyse in detail the main elements used in the process: <br />
<ul> <br />
<li><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/talking#Voice_recognizer.3F"><b>Voice recognizer</b></a></html><br />
<li><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/talking#Arduino.3F"><b>Arduino</b></a></html><br />
<li><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/talking#Fluorometer.3F"><b>Fluorometer</b></a></html><br />
</ul><br />
<br><br />
<br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
=== '''Voice recognizer''' ===<br />
<br />
<html><br />
<center><br />
<br><br />
<iframe width="420" height="315" src="http://www.youtube.com/embed/nhM99FoQYSg" frameborder="0" allowfullscreen></iframe><br />
<br><br />
</center><br />
</html><br />
<br />
Julius is a continuous speech real-time recongizer engine. It is based on Markov's interpretation of hidden models. It's opensource and distributed with a BSD licence. Its main platfomorm is Linux and other Unix systems, but it also works in Windows. It has been developed as a part of a free software kit for research in large-vocabulary continuous speech recognition (LVCSR) from 1977, and the Kyoto University of Japan has continued the work from 1999 to 2003.<br />
<!--<br />
Julius es un motor de reconocimiento de habla continua en tiempo real basado en la interpretación de Modelos Ocultos de Markov. Es de código abierto y se distribuye con licencia BSD. Su principal plataforma es Linux y otros sistemas Unix, también funciona en Windows. Ha sido desarrollado como parte de un kit de software libre para investigación en Reconocimiento de habla continua de amplio vocabulario (LVCSR) desde 1977 y el trabajo ha sido continuado por la Kyoto University de Japón de 1999 hasta el 2003.--><br />
<br />
In order to use Julius, it is necessary to create a language model and an acoustic model. Julius adopts the acoustic models and the pronunctiation dictionaries from the HTK software, which is not opensource, but can be used and downloaded for its use and posterior generation of acoustic models. <br />
<!-- <br />
Para usar Julius es necesario crear un modelo de lenguaje y un modelo acústico. Julius adopta los modelos acústicos y los diccionarios de pronunciación del software HTK, que a diferencia de Julius no es opensource, pero que puede ser usado y descargado para su uso y posterior generación de los modelos acústicos. <br />
--><br />
<br />
<b> Acoustic model </b><br />
----------------------------------<br />
An acoustic model is a file which contains an statistical representation of each of the different sounds that form a word (phoneme). Julius allows voice recognition through continuous dictation or by using a previously introduced grammar. However, the use of continuous dictation carries a problem. It requires an acoustic model trained with lots of voice files. As the amount of sound files containing voices and different texts increases, the ratio of good hits of the acoustic model will improve. This implies several variations: the pronunciation of the person training the model, the dialect used, etc. Nowadays, Julius doesn't count with any model good enough for continuous dictation in English.<br />
<br />
Due to the different problems presented by this kind of recognition, we chose the second option. We designed a grammar using the Voxforce acoustic model based in Markov's Hidden Models.<br />
To do this, we need the following file: <br><br><br />
-file .dict:a list of all the words that we want our culture to recognize and its corresponding decomposition into phonemes.<br />
<br />
<!--<br />
Un modelo acústico es un fichero que contiene una representación estadística de cada uno de los diferentes sonidos que conforman una palabra (fonemas). Julius permite reconocer voz mediante dictado continuo o mediante el uso de una gramática previamente introducida. Sin embargo el uso del dictado continuo conlleva un problema. Para ello es necesario un modelo acústico entrenado con una gran cantidad de ficheros de voz. A mayor número de ficheros de sonido conteniendo voces y textos diferentes mayor ratio de acierto tendrá el modelo acústico. Esto conllevo muchas variaciones: la pronunciación de la persona que entrena el modelo, el dialecto utilizado,... Actualmente, Julius no dispone de un modelo en Inglés lo suficientemente bueno para dictado continuo.<br />
<br />
Debido los diferentes problemas que conlleva este tipo de reconocimiento, nosotros optamos por la segunda opción. Diseñamos una gramática empleando el modelo acústico de Voxforce, basado en modelos ocultos de Markov.<br />
Para ello es necesario el siguiente fichero: <br><br><br />
- fichero.dict: lista de todas las palabras que queremos que reconozca nuestro cultivo y su correspondiente descomposición en fonemas.<br />
--><br />
<br />
<ul><br />
<li>Acoustic Analysis<br><br />
Acoustic models take the acoustic properties of the input signal. They acquire a group of vectors of certain characteristics that will later be compared with a group of <br />
patterns that represent symbols of a phonetic alphabet and return the symbols which resembles them the most. This is the basis of the mathematical probabilistic <br />
process called Hidden Markov Model. The acoustic analysis is based on the extraction of a vector similar to the input acoustic signal with the purpose of applying the <br />
theory of pattern recognition.<br />
<br />
This vector is a parametric representation of the acoustic signal, containing the most relevant information and storing as compressed as possible.<br />
In order to obtain a good group of vectors, the signal is pre-processed, reducing background noise and correlation.<br />
<br><br><br />
<br />
<li>HMM, Hidden Markov Model<br><br />
Hidden Markov Models (HMMs) provide a simple and effective framework<br />
for modelling time-varying spectral vector sequences. As a consequence,<br />
almost all present day large vocabulary continuous speech<br />
recognition systems are based on HMMs.<br><br><br />
<br />
The core of all speech recognition systems consists of a set<br />
of statistical models representing the various sounds of the language to<br />
be recognised. Since speech has temporal structure and can be encoded<br />
as a sequence of spectral vectors spanning the audio frequency range,<br />
the hidden Markov model (HMM) provides a natural framework for<br />
constructing such models.<br><br><br />
<br />
Basically, the input audio waveform from<br />
a microphone is converted into a sequence of fixed size acoustic vectors<br />
Y[1:T] = y1, . . . ,yT in a process called feature extraction. The decoder<br />
then attempts to find the sequence of words w'[1:L] = w1,...,wL which<br />
is most likely to have generated Y , i.e. the decoder tries to find.<br><br><br />
<br />
w' = argmax{P(w|Y)} for all the words w considered<br><br><br />
<br />
operating this (Bayes rule) the likelihood p(Y|w) is determined by the <br />
acoustic model and the prior P(w) is determined by the language model.<br />
<br />
<br><br><br />
</ul><br />
<br />
<!--<br />
<ul><br />
<li>Análisis acústico: <br><br />
Los modelos acústicos toman las propiedades acústicas de la señal de entrada, obtienen un conjunto de vectores de características que después compararán con un <br />
conjunto de patrones que representan los símbolos de un alfabeto fonético y devuelve los símbolos que más se parecen. En esto se basa el proceso matemático <br />
probabilístico llamado Modelo Oculto de Markov. El análisis acústico se basa en la extracción de un vector de características de la señal acústica de entrada con el fin <br />
de aplicar la teoría de reconocimiento de patrones.<br />
<br />
Éste vector es una representación paramétrica de la señal acústica, conteniendo la información más importante y almacenándola de la forma más compacta posible. <br />
Con el fin de extraer un buen conjunto de vectores, la señal es preprocesada reduciendo el ruido y la correlación.<br />
<br><br><br />
<br />
<li>Modelos Ocultos de Markov (HMM, Hidden Markov Model): <br><br />
Hidden Markov Models (HMMs) provide a simple and effective framework<br />
for modelling time-varying spectral vector sequences. As a consequence,<br />
almost all present day large vocabulary continuous speech<br />
recognition systems are based on HMMs.<br><br><br />
<br />
The core of all speech recognition systems consists of a set<br />
of statistical models representing the various sounds of the language to<br />
be recognised. Since speech has temporal structure and can be encoded<br />
as a sequence of spectral vectors spanning the audio frequency range,<br />
the hidden Markov model (HMM) provides a natural framework for<br />
constructing such models.<br><br><br />
<br />
Basically, the input audio waveform from<br />
a microphone is converted into a sequence of fixed size acoustic vectors<br />
Y[1:T] = y1, . . . ,yT in a process called feature extraction. The decoder<br />
then attempts to find the sequence of words w'[1:L] = w1,...,wL which<br />
is most likely to have generated Y , i.e. the decoder tries to find.<br><br><br />
<br />
w' = argmax{P(w|Y)} for all the words w considered<br><br><br />
<br />
operating this (Bayes rule) the likelihood p(Y|w) is determined by the <br />
acoustic model and the prior P(w) is determined by the language model.<br />
<br><br><br />
</ul><br />
--><br />
<br />
<b>Language model</b><br />
---------------------------------<br />
Language model refers to the grammar on which the recognizer will work and specify the phrases that it will be able to identify.<br />
<br />
In Julius, recognition grammar is composed by two separate files:<br><br><br />
<br />
- .voca: List of words that the grammar contains. <br><br />
- .grammar: specifies the grammar of the language to be recognised.<br><br><br />
<br />
Both files must be converted to .dfa and to .dict using the grammar compilator "mkdfa.pl"<br />
The .dfa file generated represents a finite automat. The .dict archive contains the dictionary of words in Julius format.<br />
<br />
<br />
<!--<br />
El modelo de lenguaje hace referencia a la gramática sobre la que el reconocedor va a trabajar y especificará las frases que podrá identificar.<br />
En Julius, la gramática de reconocimiento se compone por dos archivos separados: <br><br><br />
- fichero . voca: listado de las palabras que contendrá la gramática. <br><br />
- fichero .grammar: Se especifica la gramática del lenguaje a reconocer. <br><br><br />
Ambos archivos deben ser convertidos a .dfa y a .dict utilizando el compilador de gramáticas "mkdfa.pl"<br />
El archivo generado .dfa representa un autómata finito y el archivo .dict contiene el diccionario de palabras en el formato de Julius.<br />
--><br />
<br />
<br><br />
<html><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/5/59/Grammar.png" height="230" width="280"><br />
</center><br />
</html><br />
<br><br><br />
<br />
Once the acoustic language and the language model have been defined, all we need is the implementation of the main program in Python.<br />
<br />
<!--<br />
Una vez hemos definido el modelo acústico y el modelo de lenguaje, únicamente falta la implementación del programa principal en Phyton.<br />
En él ejecutamos Julius y a través del reconocimiento de voz identificamos la pregunta realizada al cultivo.<br />
--><br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
=== '''Arduino''' ===<br />
<br />
<br><br />
<br />
[[Image:arduino.png|700 px||center]]<br />
<br />
<br />
<br />
The Arduino Uno is a microcontroller board based on the ATmega328 (datasheet). It has 14 digital input/output pins (of which 6 can be used as PWM outputs), 6 analog inputs, a 16 MHz crystal oscillator, a USB connection, a power jack, an ICSP header, and a reset button. It contains everything needed to support the microcontroller; simply connect it to a computer with a USB cable or power it with a AC-to-DC adapter or battery to get started.<br />
<br />
The Uno differs from all preceding boards in that it does not use the FTDI USB-to-serial driver chip. Instead, it features the Atmega16U2 (Atmega8U2 up to version R2) programmed as a USB-to-serial converter.<br />
<br />
Revision 2 of the Uno board has a resistor pulling the 8U2 HWB line to ground, making it easier to put into DFU mode.<br />
<br />
Revision 3 of the board has the following new features:<br />
<ol><br />
<li>1.0 pinout: added SDA and SCL pins that are near to the AREF pin and two other new pins placed near to the RESET pin, the IOREF that allow the shields to adapt to the voltage provided from the board. In future, shields will be compatible both with the board that use the AVR, which operate with 5V and with the Arduino Due that operate with 3.3V. The second one is a not connected pin, that is reserved for future purposes.<br />
<br />
<li> Stronger RESET circuit.<br />
<li> Atmega 16U2 replace the 8U2.<br />
</ol><br />
<br />
"Uno" means one in Italian and is named to mark the upcoming release of Arduino 1.0. The Uno and version 1.0 will be the reference versions of Arduino, moving forward. The Uno is the latest in a series of USB Arduino boards, and the reference model for the Arduino platform; for a comparison with previous versions, see the index of Arduino boards.<br />
<br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
=== '''Fluorimeter''' ===<br />
<br />
A fluorimeter is an electronic device that can read the light and transform it in an electrical signal. In our project, that light proceeds from a bacterial culture that has been excited.<br />
<br />
<br />
Its principle of operation is as follows:<br />
First, an electrical signal is sent from Arduino to a LED to activate it. This LED has a specific wavelength to excite the fluorescent protein present in the microbial culture. <br />
<br />
<br />
When these bacteria are shining, we receive that light intensity with a photodiode, and transform it into electrical current. Now, we have an electrical current that is proportional with the light and therefor to the concentration of fluorescent protein present in the culture. We have to be careful with the orientation between the LED and the photodiode because interferences may occur in the measurement due to light received by photodiode but not emitted by the fluorescence in the culture but scattered light emitted by the LED. Also, a proper band-pass filter has been placed between culture and photodiode to allow only the desired wavelengths to pass through.<br />
<br />
<br />
[[File:Fluorimetro1.gif|center]]<br />
<br />
The electric current coming from the photodiode has a very small amplitude, thereby we have to amplify it and translate it into a electric voltage with an electronic trans-impedance amplifier. We can choose the gain of this amplification changing the value of resistances of the circuit. The resistances must be properly chosen to select the desired amplification gain. Excessive amplification will not only amplify the signal but also amplify the noise, which is no desired in the measurement. <br />
<br />
[[File:Fluorimetro2.jpg|center|400px]]<br />
<br />
<html><br />
<center><br />
<br><br />
<iframe width="420" height="315" src="https://www.youtube.com/watch?v=kBtt37xpkgk" frameborder="0" allowfullscreen></iframe><br />
<br><br />
</center><br />
</html><br />
<br />
Finally, the output voltage is sent to Arduino, and it will translate that in a digital signal.<br />
<br />
[[File:Fluorimetro3.jpg|center|400px]]<br />
<br />
The original idea was taken from [1].<br />
<br />
[1] Benjamin T. Wigton etal. ''Low-Cost, LED Fluorimeter for Middle School, High School, and Undergraduate Chemistry Lab'', J. Chem. Educ. 2011, 88, 1182-1187.<br />
</div><br />
<html><font color="#9F9F9F">Manel yo paso del iGEM</font></html></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/talking
Team:Valencia Biocampus/talking
2012-09-26T19:53:29Z
<p>Lujemomo: /* THE PROCESS */</p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
<br />
<ol><br />
<br><br />
<center><br />
<div id="Titulos"><br />
<h2>Talking Interfaces</h2><br />
<!-- ya cambiaré esto de sitio pero no lo borréis please!--><br />
<br><br />
<br />
<div id="PorDefecto"><br />
=== '''The process''' ===<br />
<br><br />
<br />
The main objective of our project is to accomplish a verbal communication with our microorganisms.<br />
To do that, we need to establish the following process:<br />
<br><br />
<html><br />
<br />
<!-- FLASH--><br />
<br />
</html><br />
<br />
<!--<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/0/07/ProcesoEn2.png" height="450" width="780"><br />
</html><br />
--><br />
<ol><br />
<li>The input used is a voice signal (question), which will be collected by our voice recognizer.<br />
<li>The voice recognizer identifies the question and, through the program in charge of establishing the communication, its corresponding identifier is written in the <br />
assigned port of the arduino.<br />
<li>The software of the arduino reads the written identifier and, according to it, the corresponding port is selected, indicating the flourimeter which wavelength has <br />
to be emitted on the culture. There are four possible questions (q), and each of them is associated to a different wavelength. <br />
<li>The fluorimeter emits light (Bioinput), exciting the compound through optic filters.<br />
<li>Due to the excitation produced, the compound emits fluorescence (BioOutput), which is measured by the fluorimeter with a sensor.<br />
<li>This fluorescence corresponds to one of the four possible answers (r: response).<br />
<li>The program of the arduino identifies the answer and writes its identifier in the corresponding port.<br />
<li>The communication program reads the identifier of the answer from the port.<br />
<li>"Espeak" emits the answer via a voice signal (Output).<br />
</ol><br />
<br><br><br />
You can see an explicative animation of these process here:<br><br><br />
<html><br />
<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=4,0,2,0" width="550" height="300" align="middle"><br />
<param name=wmode value="transparent"><br />
<param name=movie value="https://static.igem.org/mediawiki/2012/0/06/Animacion_ordenador.swf"><br />
<param name=quality value=high><br />
<embed src="https://static.igem.org/mediawiki/2012/0/06/Animacion_ordenador.swf" wmode=transparent quality=high pluginspage="http://www.macromedia.com/shockwave/download/index.cgi?P1_Prod_Version=ShockwaveFlash" type="application/x-shockwave-flash" width="550" height="300" align="middle"><br />
</embed><br />
</object> <br />
</html><br />
<br><br><br><br />
<br />
<br />
<br><br />
In the following sections we analyse in detail the main elements used in the process: <br />
<ul> <br />
<li><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/talking#Voice_recognizer.3F"><b>Voice recognizer</b></a></html><br />
<li><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/talking#Arduino.3F"><b>Arduino</b></a></html><br />
<li><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/talking#Fluorometer.3F"><b>Fluorometer</b></a></html><br />
</ul><br />
<br><br />
<br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
=== '''VOICE RECOGNIZER''' ===<br />
<br />
<html><br />
<center><br />
<br><br />
<iframe width="420" height="315" src="http://www.youtube.com/embed/nhM99FoQYSg" frameborder="0" allowfullscreen></iframe><br />
<br><br />
</center><br />
</html><br />
<br />
Julius is a continuous speech real-time recongizer engine. It is based on Markov's interpretation of hidden models. It's opensource and distributed with a BSD licence. Its main platfomorm is Linux and other Unix systems, but it also works in Windows. It has been developed as a part of a free software kit for research in large-vocabulary continuous speech recognition (LVCSR) from 1977, and the Kyoto University of Japan has continued the work from 1999 to 2003.<br />
<!--<br />
Julius es un motor de reconocimiento de habla continua en tiempo real basado en la interpretación de Modelos Ocultos de Markov. Es de código abierto y se distribuye con licencia BSD. Su principal plataforma es Linux y otros sistemas Unix, también funciona en Windows. Ha sido desarrollado como parte de un kit de software libre para investigación en Reconocimiento de habla continua de amplio vocabulario (LVCSR) desde 1977 y el trabajo ha sido continuado por la Kyoto University de Japón de 1999 hasta el 2003.--><br />
<br />
In order to use Julius, it is necessary to create a language model and an acoustic model. Julius adopts the acoustic models and the pronunctiation dictionaries from the HTK software, which is not opensource, but can be used and downloaded for its use and posterior generation of acoustic models. <br />
<!-- <br />
Para usar Julius es necesario crear un modelo de lenguaje y un modelo acústico. Julius adopta los modelos acústicos y los diccionarios de pronunciación del software HTK, que a diferencia de Julius no es opensource, pero que puede ser usado y descargado para su uso y posterior generación de los modelos acústicos. <br />
--><br />
<br />
<b> ACOUSTIC MODEL </b><br />
----------------------------------<br />
An acoustic model is a file which contains an statistical representation of each of the different sounds that form a word (phoneme). Julius allows voice recognition through continuous dictation or by using a previously introduced grammar. However, the use of continuous dictation carries a problem. It requires an acoustic model trained with lots of voice files. As the amount of sound files containing voices and different texts increases, the ratio of good hits of the acoustic model will improve. This implies several variations: the pronunciation of the person training the model, the dialect used, etc. Nowadays, Julius doesn't count with any model good enough for continuous dictation in English.<br />
<br />
Due to the different problems presented by this kind of recognition, we chose the second option. We designed a grammar using the Voxforce acoustic model based in Markov's Hidden Models.<br />
To do this, we need the following file: <br><br><br />
-file .dict:a list of all the words that we want our culture to recognize and its corresponding decomposition into phonemes.<br />
<br />
<!--<br />
Un modelo acústico es un fichero que contiene una representación estadística de cada uno de los diferentes sonidos que conforman una palabra (fonemas). Julius permite reconocer voz mediante dictado continuo o mediante el uso de una gramática previamente introducida. Sin embargo el uso del dictado continuo conlleva un problema. Para ello es necesario un modelo acústico entrenado con una gran cantidad de ficheros de voz. A mayor número de ficheros de sonido conteniendo voces y textos diferentes mayor ratio de acierto tendrá el modelo acústico. Esto conllevo muchas variaciones: la pronunciación de la persona que entrena el modelo, el dialecto utilizado,... Actualmente, Julius no dispone de un modelo en Inglés lo suficientemente bueno para dictado continuo.<br />
<br />
Debido los diferentes problemas que conlleva este tipo de reconocimiento, nosotros optamos por la segunda opción. Diseñamos una gramática empleando el modelo acústico de Voxforce, basado en modelos ocultos de Markov.<br />
Para ello es necesario el siguiente fichero: <br><br><br />
- fichero.dict: lista de todas las palabras que queremos que reconozca nuestro cultivo y su correspondiente descomposición en fonemas.<br />
--><br />
<br />
<ul><br />
<li>Acoustic Analysis<br><br />
Acoustic models take the acoustic properties of the input signal. They acquire a group of vectors of certain characteristics that will later be compared with a group of <br />
patterns that represent symbols of a phonetic alphabet and return the symbols which resembles them the most. This is the basis of the mathematical probabilistic <br />
process called Hidden Markov Model. The acoustic analysis is based on the extraction of a vector similar to the input acoustic signal with the purpose of applying the <br />
theory of pattern recognition.<br />
<br />
This vector is a parametric representation of the acoustic signal, containing the most relevant information and storing as compressed as possible.<br />
In order to obtain a good group of vectors, the signal is pre-processed, reducing background noise and correlation.<br />
<br><br><br />
<br />
<li>HMM, Hidden Markov Model<br><br />
Hidden Markov Models (HMMs) provide a simple and effective framework<br />
for modelling time-varying spectral vector sequences. As a consequence,<br />
almost all present day large vocabulary continuous speech<br />
recognition systems are based on HMMs.<br><br><br />
<br />
The core of all speech recognition systems consists of a set<br />
of statistical models representing the various sounds of the language to<br />
be recognised. Since speech has temporal structure and can be encoded<br />
as a sequence of spectral vectors spanning the audio frequency range,<br />
the hidden Markov model (HMM) provides a natural framework for<br />
constructing such models.<br><br><br />
<br />
Basically, the input audio waveform from<br />
a microphone is converted into a sequence of fixed size acoustic vectors<br />
Y[1:T] = y1, . . . ,yT in a process called feature extraction. The decoder<br />
then attempts to find the sequence of words w'[1:L] = w1,...,wL which<br />
is most likely to have generated Y , i.e. the decoder tries to find.<br><br><br />
<br />
w' = argmax{P(w|Y)} for all the words w considered<br><br><br />
<br />
operating this (Bayes rule) the likelihood p(Y|w) is determined by the <br />
acoustic model and the prior P(w) is determined by the language model.<br />
<br />
<br><br><br />
</ul><br />
<br />
<!--<br />
<ul><br />
<li>Análisis acústico: <br><br />
Los modelos acústicos toman las propiedades acústicas de la señal de entrada, obtienen un conjunto de vectores de características que después compararán con un <br />
conjunto de patrones que representan los símbolos de un alfabeto fonético y devuelve los símbolos que más se parecen. En esto se basa el proceso matemático <br />
probabilístico llamado Modelo Oculto de Markov. El análisis acústico se basa en la extracción de un vector de características de la señal acústica de entrada con el fin <br />
de aplicar la teoría de reconocimiento de patrones.<br />
<br />
Éste vector es una representación paramétrica de la señal acústica, conteniendo la información más importante y almacenándola de la forma más compacta posible. <br />
Con el fin de extraer un buen conjunto de vectores, la señal es preprocesada reduciendo el ruido y la correlación.<br />
<br><br><br />
<br />
<li>Modelos Ocultos de Markov (HMM, Hidden Markov Model): <br><br />
Hidden Markov Models (HMMs) provide a simple and effective framework<br />
for modelling time-varying spectral vector sequences. As a consequence,<br />
almost all present day large vocabulary continuous speech<br />
recognition systems are based on HMMs.<br><br><br />
<br />
The core of all speech recognition systems consists of a set<br />
of statistical models representing the various sounds of the language to<br />
be recognised. Since speech has temporal structure and can be encoded<br />
as a sequence of spectral vectors spanning the audio frequency range,<br />
the hidden Markov model (HMM) provides a natural framework for<br />
constructing such models.<br><br><br />
<br />
Basically, the input audio waveform from<br />
a microphone is converted into a sequence of fixed size acoustic vectors<br />
Y[1:T] = y1, . . . ,yT in a process called feature extraction. The decoder<br />
then attempts to find the sequence of words w'[1:L] = w1,...,wL which<br />
is most likely to have generated Y , i.e. the decoder tries to find.<br><br><br />
<br />
w' = argmax{P(w|Y)} for all the words w considered<br><br><br />
<br />
operating this (Bayes rule) the likelihood p(Y|w) is determined by the <br />
acoustic model and the prior P(w) is determined by the language model.<br />
<br><br><br />
</ul><br />
--><br />
<br />
<b>LANGUAGE MODEL</b><br />
---------------------------------<br />
Language model refers to the grammar on which the recognizer will work and specify the phrases that it will be able to identify.<br />
<br />
In Julius, recognition grammar is composed by two separate files:<br><br><br />
<br />
- .voca: List of words that the grammar contains. <br><br />
- .grammar: specifies the grammar of the language to be recognised.<br><br><br />
<br />
Both files must be converted to .dfa and to .dict using the grammar compilator "mkdfa.pl"<br />
The .dfa file generated represents a finite automat. The .dict archive contains the dictionary of words in Julius format.<br />
<br />
<br />
<!--<br />
El modelo de lenguaje hace referencia a la gramática sobre la que el reconocedor va a trabajar y especificará las frases que podrá identificar.<br />
En Julius, la gramática de reconocimiento se compone por dos archivos separados: <br><br><br />
- fichero . voca: listado de las palabras que contendrá la gramática. <br><br />
- fichero .grammar: Se especifica la gramática del lenguaje a reconocer. <br><br><br />
Ambos archivos deben ser convertidos a .dfa y a .dict utilizando el compilador de gramáticas "mkdfa.pl"<br />
El archivo generado .dfa representa un autómata finito y el archivo .dict contiene el diccionario de palabras en el formato de Julius.<br />
--><br />
<br />
<br><br />
<html><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/5/59/Grammar.png" height="230" width="280"><br />
</center><br />
</html><br />
<br><br><br />
<br />
Once the acoustic language and the language model have been defined, all we need is the implementation of the main program in Python.<br />
<br />
<!--<br />
Una vez hemos definido el modelo acústico y el modelo de lenguaje, únicamente falta la implementación del programa principal en Phyton.<br />
En él ejecutamos Julius y a través del reconocimiento de voz identificamos la pregunta realizada al cultivo.<br />
--><br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
=== '''Arduino''' ===<br />
<br />
<br><br />
<br />
[[Image:arduino.png|700 px||center]]<br />
<br />
<br />
<br />
The Arduino Uno is a microcontroller board based on the ATmega328 (datasheet). It has 14 digital input/output pins (of which 6 can be used as PWM outputs), 6 analog inputs, a 16 MHz crystal oscillator, a USB connection, a power jack, an ICSP header, and a reset button. It contains everything needed to support the microcontroller; simply connect it to a computer with a USB cable or power it with a AC-to-DC adapter or battery to get started.<br />
<br />
The Uno differs from all preceding boards in that it does not use the FTDI USB-to-serial driver chip. Instead, it features the Atmega16U2 (Atmega8U2 up to version R2) programmed as a USB-to-serial converter.<br />
<br />
Revision 2 of the Uno board has a resistor pulling the 8U2 HWB line to ground, making it easier to put into DFU mode.<br />
<br />
Revision 3 of the board has the following new features:<br />
<ol><br />
<li>1.0 pinout: added SDA and SCL pins that are near to the AREF pin and two other new pins placed near to the RESET pin, the IOREF that allow the shields to adapt to the voltage provided from the board. In future, shields will be compatible both with the board that use the AVR, which operate with 5V and with the Arduino Due that operate with 3.3V. The second one is a not connected pin, that is reserved for future purposes.<br />
<br />
<li> Stronger RESET circuit.<br />
<li> Atmega 16U2 replace the 8U2.<br />
</ol><br />
<br />
"Uno" means one in Italian and is named to mark the upcoming release of Arduino 1.0. The Uno and version 1.0 will be the reference versions of Arduino, moving forward. The Uno is the latest in a series of USB Arduino boards, and the reference model for the Arduino platform; for a comparison with previous versions, see the index of Arduino boards.<br />
<br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
=== '''Fluorimeter''' ===<br />
<br />
A fluorimeter is an electronic device that can read the light and transform it in an electrical signal. In our project, that light proceeds from a bacterial culture that has been excited.<br />
<br />
<br />
Its principle of operation is as follows:<br />
First, an electrical signal is sent from Arduino to a LED to activate it. This LED has a specific wavelength to excite the fluorescent protein present in the microbial culture. <br />
<br />
<br />
When these bacteria are shining, we receive that light intensity with a photodiode, and transform it into electrical current. Now, we have an electrical current that is proportional with the light and therefor to the concentration of fluorescent protein present in the culture. We have to be careful with the orientation between the LED and the photodiode because interferences may occur in the measurement due to light received by photodiode but not emitted by the fluorescence in the culture but scattered light emitted by the LED. Also, a proper band-pass filter has been placed between culture and photodiode to allow only the desired wavelengths to pass through.<br />
<br />
<br />
[[File:Fluorimetro1.gif|center]]<br />
<br />
The electric current coming from the photodiode has a very small amplitude, thereby we have to amplify it and translate it into a electric voltage with an electronic trans-impedance amplifier. We can choose the gain of this amplification changing the value of resistances of the circuit. The resistances must be properly chosen to select the desired amplification gain. Excessive amplification will not only amplify the signal but also amplify the noise, which is no desired in the measurement. <br />
<br />
[[File:Fluorimetro2.jpg|center|400px]]<br />
<br />
Finally, the output voltage is sent to Arduino, and it will translate that in a digital signal.<br />
<br />
[[File:Fluorimetro3.jpg|center|400px]]<br />
<br />
</div><br />
<html><font color="#9F9F9F">Manel yo paso del iGEM</font></html></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/Results1
Team:Valencia Biocampus/Results1
2012-09-26T19:52:33Z
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<h2>Talking to bacteria</h2><br />
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=== '''Are you hungry?''' ===<br />
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The experiments with our glucose-sensitive construction were carried out by three different ways: (1) containing only glucose as a carbon source, (2) containing galactose as an extra carbon source and (3) containing sodium acetate as an extra carbon source, since we checked in previous tests that low concentrations of glucose compromised cell growth. All tubes had, in addition to the glucose gradation, also IPTG (see information on the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#LACTOSE-INDUCED_PROMOTER"><b>molecular mechanism</b></a></html>). For fluorescence intensity (FI) measures cell growth, i.e. OD600, was taken into account. We worked with a 0D600 close to 0.1 in order to be able to use high sensitivity in the fluorimeter.<br />
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As you can see in the graphs below, <b>the less glucose you have, the more fluorescence you get</b>. And this results replicate well when galactose (Figure 2) or sodium acetate (Figure 3) were added to the medium as supplementary carbon sources. The threshold for a boost in the fluorescence intensity seems to be 0.1 g/L of glucose, since from this concentration the values get really high. Normal values are 10 g/L, and we got the best results when we added 10<sup>-4</sup> glucose concentration of that of the canonical values.<br />
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<table align="center" border="0.01" bordercolor="#transparent" style="background-color:#9F9F9F"><br />
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<td><html><img src="https://static.igem.org/mediawiki/2012/7/73/Glucose_alone.png" width="500" height="250" BORDER=0</a></html></td></tr><br />
<tr><td><b>Figure 1.</b> Fluorescence intensity (FI) normalized by the optical density of the<br> culture (OD) for differents amounts of glucose present in the medium.<br><br><br />
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<td><html><img src="https://static.igem.org/mediawiki/2012/e/e1/Glucose_galactose.png" width="500" height="250" BORDER=0</a></html></td></tr><br />
<tr><td><b>Figure 2.</b> Fluorescence intensity (FI) normalized by the optical density of the<br> culture (OD) for differents amounts of glucose in a medium supplemented with <br>3 g/L galactose.<br><br><br />
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<tr><br />
<td><html><img src="https://static.igem.org/mediawiki/2012/b/b1/Glucose_acetate.png" width="500" height="250" BORDER=0</a></html></td></tr><br />
<tr><td><b>Figure 3.</b> Fluorescence intensity (FI) normalized by the optical density of the<br> culture (OD) for differents amounts of glucose in a medium supplemented with <br>3 g/L sodium acetate.<br><br><br />
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=== '''Do you have enough nitrogen?''' ===<br />
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In order to characterize our nitrogen-sensitive construction, we carried out an experiment as follows: our <i>E.coli</i> strain carrying the ZsYellow1 gene under the control of the glnA promoter was grown on LBA medium (until an OD of 1.5) in order the bacteria to have sufficient amounts of proteins and nitrogen compounds. Then, cells were pelleted and resuspended in a medium lacking nitrogen. After that, values of OD and fluorescence intensity were measured at different times. As a control, we resuspended one of the aliquots in medium containing 10 g/L of ammonium sulphate.<br />
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As expected according to the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#NITROGEN-REGULATED_PROMOTER"><b>underlying molecular mechanism</b></a></html>, <b>nitrogen starvation induces the expression of the fluorescent protein</b>:<br><br />
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<table align="center" border="0.01" bordercolor="#9F9F9F" style="background-color:#9F9F9F"><br />
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<td><html><img src="https://static.igem.org/mediawiki/2012/4/43/NITROGEN_60.png" width="300" height="250" BORDER=0</a></html></td><br />
<td><html><img src="https://static.igem.org/mediawiki/2012/0/0f/Fondo_gris.png" width="30" height="250" BORDER=0</a></html></td><br />
<td><html><img src="https://static.igem.org/mediawiki/2012/8/8d/NITROGEN_150.png" width="300" height="250" BORDER=0</a></html></td><br />
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<tr><td><b>Figure 4.</b> Fluorescence intensity (FI) normalized by the optical density (OD) of the culture. Measures were taken 60 min after the resuspension in a medium containing no nitrogen (pink) or 10 g/L ammonium sulphate (grey).</td><br />
<td></td><br />
<td><b>Figure 5.</b> Fluorescence intensity (FI) normalized by the optical density (OD) of the culture. Measures<br> were taken 150 min after the resuspension in a medium containing no nitrogen (pink) or 10 g/L ammonium sulphate (grey).</td><br />
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</table><br />
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=== '''Can you breathe?''' ===<br />
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In this experiment, our <i>E.coli</i> strain carrying the ZsGreen1 protein under the control of the nirB promoter was grown on LBA under two different conditions: aerobic and anaerobic. Anaerobic conditions were achieved by adding a small volume of sterile oil over the liquid culture (Figure 6), so no oxygen can be taken from the environment. After 2 days of growth, samples were taken from each culture and diluted to an OD of 0.1. After that, fluorescence intensity was measured. <br />
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Our results show that <b>ZsGreen1 expression increases</b> (a 20% approx.) <b>under anaerobic conditions</b>, according to the effect of oxygen on the FNR proteins (see the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#OXYGEN-REGULATED_PROMOTER"><b>molecular mechanism description</b></a></html>).<br />
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<table align="center" border="0.01" bordercolor="#9F9F9F" style="background-color:#9F9F9F"><br />
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<td><html><img src="https://static.igem.org/mediawiki/2012/4/45/AEROBIOSIS_ANAER.png" width="320" height="270" BORDER=0</a></html></td></tr><br />
<tr><br />
<td><b>Figure 6.</b> Fluorescence intensity (FI) normalized<br> by the optical density (OD) of cultures grown under<br> aerobic (grey) or anaerobic (pink) conditions.</td></tr><br />
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</table><br />
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=== '''Are you hot?''' ===<br />
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To test the heat-sensitive construction, an overnight culture of our <i>E.coli</i> strain carrying the AsRed2 gene under the control of groE promoter was set up at 25 ºC. After that, OD was adjusted to 0.15 and several aliquots were transferred to fluorimetry cuvettes. Heat shock was carried out at different temperatures in a water bath. Then, the cuvettes were maintained at room temperature and OD and fluorescence intensity were measured at different time points.<br />
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During the first experiments, we did not get any successful result... As we can see in the following graph, fluorescence emission showed no significant increasement after different heat-shock treatments:<br />
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<table align="center" border="0.01" bordercolor="#9F9F9F" style="background-color:#9F9F9F"><br />
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<td><html><img src="https://static.igem.org/mediawiki/2012/5/59/Hs_cheaters1.png" width="700" height="500" BORDER=0</a></html></td></tr><br />
<tr><br />
<td><b>Figure 7.</b> Fluorescence intensity (FI) normalized by the optical density (OD) of cultures subjected to a 44 ºC heat-shock treatment of different durations. Heat-shock was not applied in the control experiment.</td></tr><br />
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</table><br />
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That was suspicious! Since we were sure that our experimental set-up was fine, we decided to check if our bacteria were actually carrying the right construction (Bac2, that consisted of groE promoter + AsRed2). Then, we found out that we had lying bacteria in the lab! <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Results3"><b>Cheaters</b></a></html>!<br />
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After that, we started to perform new experiments with "honest" bacteria, and good results arrived soon: as shown in the graphs below, we found a correlation between the temperature at which heat shock was carried out and the level of expression of the fluorescent protein. <b>Higher fluorescence intensity was obtained when higher temperatures</b> (ranging from 40 to 44 ºC) <b>were applied</b> (check the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#TEMPERATURE-INDUCED_PROMOTER"><b>molecular mechanism details</b></a></html>). According to our results, it seems that protein expression cannot keep increasing at temperatures higher than 44 ºC, probably due to lethality effects.<br />
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<table align="center" border="0.01" bordercolor="#9F9F9F" style="background-color:#9F9F9F"><br />
<tr><br />
<td><html><img src="https://static.igem.org/mediawiki/2012/c/ca/HS_120.png" width="550" height="300" BORDER=0</a></html></td></tr><br />
<tr><td><b>Figure 8.</b> Fluorescence intensity (FI) normalized by the optical density (OD) of cultures that<br> were subjected to a 10-min heat-shock at different temperatures. Measures were taken 60 min<br> after the shock.<br><br><br />
</td></tr><br />
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<td><html><img src="https://static.igem.org/mediawiki/2012/9/96/HS_60.png" width="550" height="300" BORDER=0</a></html></td></tr><br />
<tr><td><b>Figure 9.</b> Fluorescence intensity (FI) normalized by the optical density (OD) of cultures that<br> were subjected to a 10-min heat-shock at different temperatures. Measures were taken 120 min<br> after the shock.<br />
</td></tr><br />
</table><br />
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=== '''Express your gene!''' ===<br />
<br><br />
In order to test our UV-induced construction, the <i>E. coli</i> strain carrying the ZsGreen1 gene under the control of the lexA promoter was grown on LBA (LB broth supplemented with ampicillin) until an OD value of 0.8. Then, cells were pelleted, resuspended in fresh medium, and 100 uL were spread in agar plates. Once the medium was completely absorbed by the agar (to avoid UV absorption by medium), cells were irradiated inside the <html><a href="http://www.youtube.com/watch?v=fycIw4f1LX8&feature=youtu.be" target="blank"><b>TORDEITOR</b></a></html> (the device we specially designed for this experiment) with different amounts of UV light (lambda=254 nm). To fine tune UV dose, we used a shutter (similar to those used in cameras, click <html><a href="http://www.youtube.com/watch?v=fycIw4f1LX8&feature=youtu.be" target="blank">here</a></html> to see an explicative video) able to produce UV pulses of less than 1 second of duration. After that, cells were harvested from the agar plate with SOC medium supplemented with ampicillin. Finally, OD and fluorescence intensity were measured at different times. An aliquot of the same culture that was not irradiated was used as control.<br />
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As we can see in the graph below, <b>UV exposure does induce the expression of ZsGreen1</b>. Furthermore, longer exposures result in higher levels of protein expression (higher fluorescence intensity was observed, as expected according to the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#UV-INDUCED_PROMOTER"><b>molecular mechanism</b></a></html> of lexA promoter), although lethal effects were observed when doses about 5 seconds were applied.<br />
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<table align="center" border="0.01" bordercolor="#9F9F9F" style="background-color:#9F9F9F"><br />
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<td><html><img src="https://static.igem.org/mediawiki/2012/4/45/UV_3BARRES.png" width="300" height="250" BORDER=0</a></html></td><br />
<td><html><img src="https://static.igem.org/mediawiki/2012/0/0f/Fondo_gris.png" width="30" height="250" BORDER=0</a></html></td><br />
<td><html><img src="https://static.igem.org/mediawiki/2012/e/eb/UV_2BARRES.png" width="300" height="250" BORDER=0</a></html></td><br />
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<tr><td><b>Figure 10.</b> Fluorescence intensity (FI) normalized by the optical density (OD) of cultures that were subjected to UV exposures of different duration in comparison to a control culture (not exposed). Measures were taken 70 min after the exposure.</td><br />
<td></td><br />
<td><b>Figure 11.</b> Fluorescence intensity (FI) normalized by the optical density (OD) of cultures that were subjected to UV exposures of different duration in comparison to a control culture (not exposed). Measures were taken 90 min after the exposure.</td><br />
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</table><br />
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Lujemomo
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2012-09-26T19:30:38Z
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Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/Ethics
Team:Valencia Biocampus/Ethics
2012-09-26T18:36:28Z
<p>Lujemomo: </p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
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<div id="Titulos"><br />
<h2>Human Practices</h2><br />
<br><br />
<br />
<div id="PorDefecto"><br />
<br />
<br />
In the "Talking life" project we did not only want to speak with bacteria, but we also wanted to speak with humans! The HP practices team identified some ethical and social issues that were relevant to our project. We wanted to <br />
communicate such issues and discuss them with a broader peer community, with scientists and other citizens. We made a movie that we used as a vehicle to communicate those issues and to trigger public debate. Slide along the timeline to know the steps we followed in the course of our HP exercise: <br />
<br><br><br />
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<br><br />
If you are fond of our work on Human Practices, download our Ethical issues diary <html><a href="https://static.igem.org/mediawiki/2012/5/50/Hp_diary.pdf"target="_blank"><b>here</b></a></html>!<br><br><br />
<br />
<h2>Talking Life</h2><br />
<br />
Click here to watch the 10 minute film that we made in collaboration with Artefactando. We also acted in the movie and used it in our debates. Based on real ethical concerns but in the form of a fictional story. We envisaged a possible future applications for our talking bacterial cultures and we used the movie to generate debate. The movie initiates debate around three ethical issues: Could we use talking bacteria to care for others? Who should own living technologies? If bacteria can speak, can they also lie?<br><br />
<br><br />
<html><br />
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<br><br><br />
<div id="PorDefecto"><br />
<br>The film was projected and debated five times: <br />
<b><html><a href="https://static.igem.org/mediawiki/2012/3/35/DEBATE_BANYULS_DD.pdf" target="blank" style="color:#0000FF">(1)</a><html/></b> France (Banyuls) to an audience of scientific European students, <b><html><a href="https://static.igem.org/mediawiki/2012/d/d8/DEBATE_BERGEN_DD.pdf" target="blank" style="color:#0000FF">(2)</a><html/></b> Norway (Bergen), in front of a group of sociologists and ethics, <b><html><a href="https://static.igem.org/mediawiki/2012/3/39/Debate_barcelona_DD.pdf" target="blank" style="color:#0000FF">(3)</a><html/></b> Barcelona, to a heterogeneous group of students and workers, <b><html><a href="https://static.igem.org/mediawiki/2012/8/85/Debate_octubre_DD.pdf" target="blank" style="color:#0000FF">(4)</a><html/></b> Valencia, in the cultural center named “Octubre” to a scientific public from professors and doctors to undergraduate students, and <b><html><a href="https://static.igem.org/mediawiki/2012/4/42/DEBATE_Valencia_DD.pdf" target="blank" style="color:#0000FF">(5)</a><html/></b> in the scientific Campus of University of Valencia to a wide biology-related student audience. <br><br>If you are interested in how we did it or you want to know about the main ideas debated click in the different links that you can find above. To know more about other non-planned ethical aspects that came out to debate click <b><html><a href="https://static.igem.org/mediawiki/2012/f/ff/Non-intendedHP_DD.pdf" target="blank" style="color:#0000FF">here</a><html/></b>.<br><br><br />
Below you can find the <html><a href="https://static.igem.org/mediawiki/2012/d/d1/General_Analysis.pdf"> general analysis </a></html> and conclusions we came up with from the five debates: <br><br><br />
<br />
<br />
<br />
<br />
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<br />
<br />
<br />
<h2>Integrated HP: Lying bacteria? </h2><br />
<br />
Since our project consists of talking with microorganisms, we asked ourselves the question “what would happen if they lied to us?” Not consciously, of course, but mutants who give different answers to the same question could be evolutionarily favored in a bacterial culture. Our HP team addressed this issue in an integrated fashion: both in public debates and in the lab. <br />
<br />
Our Human Practices team and topics play a central and integrated role in our iGEM project. Not only did we explore ethical concerns of our activity, but also we coordinated our work in the lab to test our “lying bacteria” vision. We addressed the questions above in two ways:<br><br><br />
<br />
<div id="PorDefecto"><br />
<br><br><br />
1. Debating: We decided to shoot a short film to give insight and a jumping off point to our integrated vision. We discussed it with different audiences in public debates in three different European countries. We then analyzed their comments and response to the short film. We wanted to discuss ethical issues such as: What if this talking technology was used in everyday devices and they lied? How would people react to this? Should the blame be given to companies developing those devices? How frequently would mutants appear? Imagine that this technology was hackable. What ethical issues would arise?<br><br><br />
<br />
2. <html><b><a href="https://2012.igem.org/Team:Valencia_Biocampus/modhp" style="color:#0000FF">Modeling and wetlab</a></b><html/>: Together with the modeling team, we carried out experiments in the wetlab to predict how often those liars or cheaters would appear in cultures and compared the fitness of cheaters against the original culture. <br />
<br />
</div></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/Yeast
Team:Valencia Biocampus/Yeast
2012-09-26T18:35:34Z
<p>Lujemomo: /* THE IDEA */</p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
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<center><br />
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<ol><br />
<div id="Titulos"><br />
<h2>Yeast Subteam</h2><br />
<br />
<br />
<div id="PorDefecto"><br />
=== '''THE IDEA''' ===<br />
<br><br />
Our aim in this part of the project is to detect when the yeast starts fermenting. At the end of the project we will be able to “ask” the yeast if there is still any glucose in the medium or not through the addition of H2O2. Furthermore, we will be able to know for how long the media has been running out of glucose.<br />
In conclusion, this project allows us to know how much time has elapsed since the fermentation began.<br />
<br />
To do this, we are going to use two gene constructions:<br><br><br />
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<br><br><br />
<br />
• '''The ADH2 promoter is fused to the YAP1 protein coding sequence.''' The YAP1 protein is a yeast transcription factor regulator of H2O2 adaptative response. It is stored in the citoplasm in normal conditions and, in the presence of H2O2, it is transported to the nucleus acting as a transcription factor. The ADH2 promoter is activated in the absence of glucose.<br />
<br />
Thus, complete disappearence of glucose triggers the production of YAP1 in the citoplasm, and YAP1 concentration increases if the lack of glucose continues. Note that we are working with a delta-yap1 mutant.<br />
<br />
• '''The TRR promoter is fused to the GFP (Green Fluorescence Protein) coding sequence.''' The green fluorescent protein can be detected by fluorescent emission. The tiorredoxin reductase promoter is activated by two transcriptional factors (YAP1 and SKN7 in the oxidative form). Both only bind to the promoter if H2O2 has been previously added to the culture medium.<br />
<br><br />
<br><br><br />
<br />
=== '''INDUSTRIAL APPLICATIONS''' ===<br />
<html><br />
<br><br />
The main industrial application of our talking yeast is to know the moment at which they started fermentation. This is possible because we can calculate the time passed since the medium ran out of glucose according to the fluorescence intensity. Taking this into account, we could ask the yeast, through the addition of hydrogen peroxide: “How long have you been fermenting?” And the cells will answer telling us how long it has been since all the glucose was consumed. One possible answer could be “I have been fermenting for 1 hour”. <br />
<br><br><br />
There is also a second possible application, but it needs a more detailed explanation: <br />
<br><br><br />
There are several factors affecting yeast ethanol production during<br />
alcoholic fermentation. <i>Saccharomyces cerevisiae</i> seems to have adapted<br />
all along its evolution to optimize its growing rate in environments<br />
that are rich in easily assimilable nutrients, such as sugars and amino<br />
acids.<br><br><br />
There are two important characteristics responsible of <i>S. cerevisiae</i><br />
adaptation to this particular niche. One of them is its capability of<br />
metabolizing glucose and fructose through both the respiration and the<br />
fermentation pathways. The second one is its ability to grow in aerobic<br />
and anaerobic conditions. All of this makes this species exhibit some<br />
metabolic peculiarities, such as the Pasteur and the Crabtree effects.<br />
The Crabtree effect, described in <i>S. cerevisiae</i> and in a few other yeast species, must be taken into account when ethanol production<br />
is being studied or modified during fermentation.<br />
This effect causes that, even under high concentration of<br />
oxygen and with a relatively low amount of glucose, a considerable<br />
fraction of the consumed sugar is used to produce ethanol through the<br />
fermentative pathway. That is why in every industrial process which<br />
requires fermentation it is necessary to provide the optimum amount of<br />
glucose that permits its direct consumption through the fermentative<br />
pathway. We present here one way of monitoring the fate of glucose in the<br />
medium. By adding a small amount of<br />
hydrogen peroxide as a chemical input, we plan to ask our culture: “Is there any glucose left?”, and the culture would answer -according to the amount of<br />
glucose already present in the medium- what concentration would be<br />
necessary to obtain a higher amount of ethanol through the Crabtree<br />
effect.<br />
<br><br />
<br><br><br />
</html><br />
<br />
=== '''MOLECULAR MECHANISMS''' ===<br />
<br />
<br><br />
Click on each plasmid to learn how our constructions work!<br />
<br><br />
<div style="text-align: center;"><br />
<table align="center" border="0.01" bordercolor="#9F9F9F" style="background-color:#9F9F9F"><br />
<tr><br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#Yeast"><br />
<img src="https://static.igem.org/mediawiki/2012/f/fe/Yeast_fermentative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#OXIDATIVE_STRESS_RESPONSE"><br />
<img src="https://static.igem.org/mediawiki/2012/9/9e/Yeast_oxidative.png" width="300" height="280" BORDER=0</a></html></td><br />
<br />
</tr><br />
</table><br />
</div><br />
<br />
<br><br />
Each construction was carried in diferent plasmids for their expression in yeast:<br />
<br><br />
'''YEplac181 carried Fermentative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4893/">click here</a></html>.<br />
<br><br />
'''YEp352 carried Oxidative response construction'''. To see further information about the vector, <html><a href="http://www.addgene.org/vector-database/4867/">click here</a></html>.<br />
<br><br><br><br />
<br />
=== '''EXPERIMENTAL OUTLINE''' ===<br />
<br />
- We ordered the DNA constructions: pADH2-YAP1 protein and pTRR-GFP protein that were cloned in plasmid pUC57 with a bacterial origin of replication. <br />
<br><br />
- We were supplied the Yeplac181 and Yep352 yeast vectors by our laboratory.<br />
<br><br />
- We carried out four transformations of E. coli strain DH5, one for each plasmid DNA (the two constructions and the two vectors), in order to amplify them. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Heat_Shock_Protocol_for_bacteria_transformation"> Transformation Protocol Using Heat Shock </a></html>.<br />
<br><br />
- We obtained several transformants of E. coli in four plates and took some colonies of each DNA (from both constructions and both vectors) and cultured them in liquid medium over night at 37ºC in shaking flasks. <br />
<br><br />
- A day after, we extracted the plasmid DNA. See the Mini-preps protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- We obtained the purified constructions (both in pUC57 plasmid) and the vectors for yeast (YEplac181 and YEp352) also purified. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Protocol_for_Gel_Extraction"> Protocol for Gel Extraction </a></html><br />
<br><br />
- We digested the four DNAs with restriction enzymes EcoRI and PstI in order to obtain compatible ends. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Digestion_Protocol_For_Plasmid_pUC57_.2B_Construction_Using_EcoRI_and_PstI"> Digestion Protocol </a></html>.<br />
<br><br />
- We ligated the pTRR-GFP construction with the Yep352 vector and the ADH2-YAP1 construction with the Yeplac181 vector. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Ligation">ligation Protocol</a></html>.<br />
<br><br />
- The day after that we transformed E. coli with the results of the ligation in order to amplify the final constructions (pTRR-GFP/Yep352 and pADH2-YAP1/YEplac181). We located the recombinant constructs using X-Gal and white/blue selection. <br />
<br><br />
- We took some of these white colonies and cultured them in liquid medium overnight at 37ºC in shaking flasks. <br />
<br><br />
- The day after we extracted the plasmid DNA. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep">Mini-prep Protocol</a></html>.<br />
<br><br />
- After this, we checked the final purified recombinant constructs by electrophoresis, restriction digest and DNA capilar sequencing.<br />
<br><br />
- We introduced the first of the DNA recombinant plasmids in the yeast. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- We selected the transformants by growth in solid and liquid mineral medium attending to the auxotrophic markers and checked the presence of the construction by PCR. See the protocol here.<br />
<br><br />
- We used the transformed yeast obtained at that moment and transformed it with the second recombinant construct. See the yeast transformation protocol. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_transformation">Yeast transformation protocol</a></html>.<br />
<br><br />
- Once again we identified the transformants by growth in selective medium and after that we used a PCR protocol to check the presence of both constructions.<br />
<br><br />
- In order to detect fluorescence, we carried out a protocol to induce the expression of GFP. See the <html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Yeast_Induction_protocol"> Yeast induction protocol </a></html>.<br />
<br><br />
<br />
</div></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/Bacterium
Team:Valencia Biocampus/Bacterium
2012-09-26T18:34:08Z
<p>Lujemomo: /* THE IDEA */</p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
<center><br />
<br />
<ol><br />
<br><br />
<div id="Titulos"><br />
<h2>Bacteria Subteam</h2><br />
<br><br />
<div id="PorDefecto"><br />
<br />
=== '''THE IDEA''' ===<br />
<br><br />
We have developed a simple and fast method of determining "the comfort" of the bacterial culture by asking them four questions related with their metabolic state which are replied in the form of voice answers. Moreover, we have engineered another genetic construction to order them to produce a protein. <br />
Here is an overview of how our bacteria work. For more information look the '''molecular mechanisms''' below.<br />
<br />
<html><br />
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<param name=quality value=high><br />
<embed src="https://static.igem.org/mediawiki/2012/e/e2/Bacteria_Outline_VLC.swf" quality=high pluginspage="http://www.macromedia.com/shockwave/download/index.cgi?P1_Prod_Version=ShockwaveFlash" type="application/x-shockwave-flash" width="800" height="600" align="middle"><br />
</embed><br />
</object> <br />
</html><br />
<br><br><br />
<br />
==='''BACTERIAL SYNTHAXIS'''===<br />
<br><br />
This is the Rosseta stone we used to define both human and bacteria language:<br />
<br />
[[Image:Tabla_synthaxis.png|700 px||center]]<br />
<br><br><br />
<br />
==='''MOLECULAR MECHANISMS'''===<br />
<br><br />
Click on each plasmid to learn more about how our constructions work!<br />
<br><br />
<table align="center" border="0.01" bordercolor="#9F9F9F" style="background-color:#9F9F9F"><br />
<tr><br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#LACTOSE-INDUCED_PROMOTER"><br />
<img src="https://static.igem.org/mediawiki/2012/6/66/Bacteria_hungry.png" width="230" height="200"</a></html></td><br />
<br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#NITROGEN-REGULATED_PROMOTER"><br />
<img src="https://static.igem.org/mediawiki/2012/7/76/Bacteria_nitrogen.png" width="230" height="200"</a></html></td><br />
<br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#OXYGEN-REGULATED_PROMOTER"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f4/Bacteria_oxygen.png" width="230" height="200"</a></html></td><br />
<br />
</tr><br />
<tr> <br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#TEMPERATURE-INDUCED_PROMOTER"><br />
<img src="https://static.igem.org/mediawiki/2012/3/38/Bacteria_hot.png" width="230" height="200"</a></html></td><br />
<br />
<td><html><a href="https://2012.igem.org/Team:Valencia_Biocampus/Molecular#UV-INDUCED_PROMOTER"><br />
<img src="https://static.igem.org/mediawiki/2012/3/3d/Bacteria_expressgene.png" width="230" height="200"</a></html></td><br />
<td><html><br />
<img src="https://static.igem.org/mediawiki/2012/0/0f/Fondo_gris.png" width="230" height="200"</a></html></td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
<br />
=== '''EXPERIMENTAL OUTLINE''' ===<br />
<br><br />
Here you have a graphical overview of the experimental approach we followed:<br />
<br />
[[Image:Outline_ppt.png|700 px||center]]</div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/talking
Team:Valencia Biocampus/talking
2012-09-26T18:33:25Z
<p>Lujemomo: </p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
<br />
<ol><br />
<br><br />
<center><br />
<div id="Titulos"><br />
<h2>Talking Interfaces</h2><br />
<!-- ya cambiaré esto de sitio pero no lo borréis please!--><br />
<br><br />
<html><br />
<object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=4,0,2,0" width="550" height="300" align="middle"><br />
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</embed><br />
</object> <br />
</html><br />
<br><br><br><br />
<br />
<div id="PorDefecto"><br />
=== '''THE PROCESS''' ===<br />
<br><br />
<br />
The main objective of our project is to accomplish a verbal communication with our microorganisms.<br />
To do that, we need to establish the following process:<br />
<br />
<br />
<br />
<br><br />
<html><br />
<br />
<!-- FLASH--><br />
<br />
</html><br />
<br />
<br />
<html><br />
<img src="https://static.igem.org/mediawiki/2012/0/07/ProcesoEn2.png" height="450" width="780"><br />
</html><br />
<br />
<br><br />
<ol><br />
<li>The basic life cycle of our biological agent is based on an input/output process through the use of interfaces.<br />
<li>The input used is a voice signal (question), which will be collected by our voice recognizer.<br />
<li>The voice recognizer identifies the question and, through the program in charge of establishing the communication, its corresponding identifier is written in the <br />
assigned port of the arduino.<br />
<li>The software of the arduino reads the written identifier and, according to it, the corresponding port is selected, indicating the flourimeter which wavelength has <br />
to be emitted on the culture. There are four possible questions (q), and each of them is associated to a different wavelength. <br />
<li>The fluorimeter emits light (Bioinput), exciting the compound through optic filters.<br />
<li>Due to the excitation produced, the compound emits fluorescence (BioOutput), which is measured by the fluorimeter with a sensor.<br />
<li>This fluorescence corresponds to one of the four possible answers (r: response).<br />
<li>The program of the arduino identifies the answer and writes its identifier in the corresponding port.<br />
<li>The communication program reads the identifier of the answer from the port.<br />
<li>"Espeak" emits the answer via a voice signal (Output).<br />
</ol><br />
<br />
<br><br />
In this section we analyse in detail the main element used in the process: <br />
<ul> <br />
<li>Voice recognizer<br />
<li>Arduino<br />
<li>Fluorimeter<br />
</ul><br />
<br><br />
<br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
=== '''VOICE RECOGNIZER''' ===<br />
<br />
<html><br />
<center><br />
<br><br />
<iframe width="420" height="315" src="http://www.youtube.com/embed/nhM99FoQYSg" frameborder="0" allowfullscreen></iframe><br />
<br><br />
</center><br />
</html><br />
<br />
Julius is a continuous speech real-time recongizer engine. It is based on Markov's interpretation of hidden models. It's opensource and distributed with a BSD licence. Its main platfomorm is Linux and other Unix systems, but it also works in Windows. It has been developed as a part of a free software kit for research in large-vocabulary continuous speech recognition (LVCSR) from 1977, and the Kyoto University of Japan has continued the work from 1999 to 2003.<br />
<!--<br />
Julius es un motor de reconocimiento de habla continua en tiempo real basado en la interpretación de Modelos Ocultos de Markov. Es de código abierto y se distribuye con licencia BSD. Su principal plataforma es Linux y otros sistemas Unix, también funciona en Windows. Ha sido desarrollado como parte de un kit de software libre para investigación en Reconocimiento de habla continua de amplio vocabulario (LVCSR) desde 1977 y el trabajo ha sido continuado por la Kyoto University de Japón de 1999 hasta el 2003.--><br />
<br />
In order to use Julius, it is necessary to create a language model and an acoustic model. Julius adopts the acoustic models and the pronunctiation dictionaries from the HTK software, which is not opensource, but can be used and downloaded for its use and posterior generation of acoustic models. <br />
<!-- <br />
Para usar Julius es necesario crear un modelo de lenguaje y un modelo acústico. Julius adopta los modelos acústicos y los diccionarios de pronunciación del software HTK, que a diferencia de Julius no es opensource, pero que puede ser usado y descargado para su uso y posterior generación de los modelos acústicos. <br />
--><br />
<br />
<b> ACOUSTIC MODEL </b><br />
----------------------------------<br />
An acoustic model is a file which contains an statistical representation of each of the different sounds that form a word (phoneme). Julius allows voice recognition through continuous dictation or by using a previously introduced grammar. However, the use of continuous dictation carries a problem. It requires an acoustic model trained with lots of voice files. As the amount of sound files containing voices and different texts increases, the ratio of good hits of the acoustic model will improve. This implies several variations: the pronunciation of the person training the model, the dialect used, etc. Nowadays, Julius doesn't count with any model good enough for continuous dictation in English.<br />
<br />
Due to the different problems presented by this kind of recognition, we chose the second option. We designed a grammar using the Voxforce acoustic model based in Markov's Hidden Models.<br />
To do this, we need the following file: <br><br><br />
-file .dict:a list of all the words that we want our culture to recognize and its corresponding decomposition into phonemes.<br />
<br />
<!--<br />
Un modelo acústico es un fichero que contiene una representación estadística de cada uno de los diferentes sonidos que conforman una palabra (fonemas). Julius permite reconocer voz mediante dictado continuo o mediante el uso de una gramática previamente introducida. Sin embargo el uso del dictado continuo conlleva un problema. Para ello es necesario un modelo acústico entrenado con una gran cantidad de ficheros de voz. A mayor número de ficheros de sonido conteniendo voces y textos diferentes mayor ratio de acierto tendrá el modelo acústico. Esto conllevo muchas variaciones: la pronunciación de la persona que entrena el modelo, el dialecto utilizado,... Actualmente, Julius no dispone de un modelo en Inglés lo suficientemente bueno para dictado continuo.<br />
<br />
Debido los diferentes problemas que conlleva este tipo de reconocimiento, nosotros optamos por la segunda opción. Diseñamos una gramática empleando el modelo acústico de Voxforce, basado en modelos ocultos de Markov.<br />
Para ello es necesario el siguiente fichero: <br><br><br />
- fichero.dict: lista de todas las palabras que queremos que reconozca nuestro cultivo y su correspondiente descomposición en fonemas.<br />
--><br />
<br />
<ul><br />
<li>Acoustic Analysis<br><br />
Acoustic models take the acoustic properties of the input signal. They acquire a group of vectors of certain characteristics that will later be compared with a group of <br />
patterns that represent symbols of a phonetic alphabet and return the symbols which resembles them the most. This is the basis of the mathematical probabilistic <br />
process called Hidden Markov Model. The acoustic analysis is based on the extraction of a vector similar to the input acoustic signal with the purpose of applying the <br />
theory of pattern recognition.<br />
<br />
This vector is a parametric representation of the acoustic signal, containing the most relevant information and storing as compressed as possible.<br />
In order to obtain a good group of vectors, the signal is pre-processed, reducing background noise and correlation.<br />
<br><br><br />
<br />
<li>HMM, Hidden Markov Model<br><br />
Hidden Markov Models (HMMs) provide a simple and effective framework<br />
for modelling time-varying spectral vector sequences. As a consequence,<br />
almost all present day large vocabulary continuous speech<br />
recognition systems are based on HMMs.<br><br><br />
<br />
The core of all speech recognition systems consists of a set<br />
of statistical models representing the various sounds of the language to<br />
be recognised. Since speech has temporal structure and can be encoded<br />
as a sequence of spectral vectors spanning the audio frequency range,<br />
the hidden Markov model (HMM) provides a natural framework for<br />
constructing such models.<br><br><br />
<br />
Basically, the input audio waveform from<br />
a microphone is converted into a sequence of fixed size acoustic vectors<br />
Y[1:T] = y1, . . . ,yT in a process called feature extraction. The decoder<br />
then attempts to find the sequence of words w'[1:L] = w1,...,wL which<br />
is most likely to have generated Y , i.e. the decoder tries to find.<br><br><br />
<br />
w' = argmax{P(w|Y)} for all the words w considered<br><br><br />
<br />
operating this (Bayes rule) the likelihood p(Y|w) is determined by the <br />
acoustic model and the prior P(w) is determined by the language model.<br />
<br />
<br><br><br />
</ul><br />
<br />
<!--<br />
<ul><br />
<li>Análisis acústico: <br><br />
Los modelos acústicos toman las propiedades acústicas de la señal de entrada, obtienen un conjunto de vectores de características que después compararán con un <br />
conjunto de patrones que representan los símbolos de un alfabeto fonético y devuelve los símbolos que más se parecen. En esto se basa el proceso matemático <br />
probabilístico llamado Modelo Oculto de Markov. El análisis acústico se basa en la extracción de un vector de características de la señal acústica de entrada con el fin <br />
de aplicar la teoría de reconocimiento de patrones.<br />
<br />
Éste vector es una representación paramétrica de la señal acústica, conteniendo la información más importante y almacenándola de la forma más compacta posible. <br />
Con el fin de extraer un buen conjunto de vectores, la señal es preprocesada reduciendo el ruido y la correlación.<br />
<br><br><br />
<br />
<li>Modelos Ocultos de Markov (HMM, Hidden Markov Model): <br><br />
Hidden Markov Models (HMMs) provide a simple and effective framework<br />
for modelling time-varying spectral vector sequences. As a consequence,<br />
almost all present day large vocabulary continuous speech<br />
recognition systems are based on HMMs.<br><br><br />
<br />
The core of all speech recognition systems consists of a set<br />
of statistical models representing the various sounds of the language to<br />
be recognised. Since speech has temporal structure and can be encoded<br />
as a sequence of spectral vectors spanning the audio frequency range,<br />
the hidden Markov model (HMM) provides a natural framework for<br />
constructing such models.<br><br><br />
<br />
Basically, the input audio waveform from<br />
a microphone is converted into a sequence of fixed size acoustic vectors<br />
Y[1:T] = y1, . . . ,yT in a process called feature extraction. The decoder<br />
then attempts to find the sequence of words w'[1:L] = w1,...,wL which<br />
is most likely to have generated Y , i.e. the decoder tries to find.<br><br><br />
<br />
w' = argmax{P(w|Y)} for all the words w considered<br><br><br />
<br />
operating this (Bayes rule) the likelihood p(Y|w) is determined by the <br />
acoustic model and the prior P(w) is determined by the language model.<br />
<br><br><br />
</ul><br />
--><br />
<br />
<b>LANGUAGE MODEL</b><br />
---------------------------------<br />
Language model refers to the grammar on which the recognizer will work and specify the phrases that it will be able to identify.<br />
<br />
In Julius, recognition grammar is composed by two separate files:<br><br><br />
<br />
- .voca: List of words that the grammar contains. <br><br />
- .grammar: specifies the grammar of the language to be recognised.<br><br><br />
<br />
Both files must be converted to .dfa and to .dict using the grammar compilator "mkdfa.pl"<br />
The .dfa file generated represents a finite automat. The .dict archive contains the dictionary of words in Julius format.<br />
<br />
<br />
<!--<br />
El modelo de lenguaje hace referencia a la gramática sobre la que el reconocedor va a trabajar y especificará las frases que podrá identificar.<br />
En Julius, la gramática de reconocimiento se compone por dos archivos separados: <br><br><br />
- fichero . voca: listado de las palabras que contendrá la gramática. <br><br />
- fichero .grammar: Se especifica la gramática del lenguaje a reconocer. <br><br><br />
Ambos archivos deben ser convertidos a .dfa y a .dict utilizando el compilador de gramáticas "mkdfa.pl"<br />
El archivo generado .dfa representa un autómata finito y el archivo .dict contiene el diccionario de palabras en el formato de Julius.<br />
--><br />
<br />
<br><br />
<html><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/5/59/Grammar.png" height="230" width="280"><br />
</center><br />
</html><br />
<br><br><br />
<br />
Once the acoustic language and the language model have been defined, all we need is the implementation of the main program in Python.<br />
<br />
<!--<br />
Una vez hemos definido el modelo acústico y el modelo de lenguaje, únicamente falta la implementación del programa principal en Phyton.<br />
En él ejecutamos Julius y a través del reconocimiento de voz identificamos la pregunta realizada al cultivo.<br />
--><br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
=== '''Arduino''' ===<br />
<br />
<br><br />
<br />
[[Image:arduino.png|700 px||center]]<br />
<br />
<br />
<br />
The Arduino Uno is a microcontroller board based on the ATmega328 (datasheet). It has 14 digital input/output pins (of which 6 can be used as PWM outputs), 6 analog inputs, a 16 MHz crystal oscillator, a USB connection, a power jack, an ICSP header, and a reset button. It contains everything needed to support the microcontroller; simply connect it to a computer with a USB cable or power it with a AC-to-DC adapter or battery to get started.<br />
<br />
The Uno differs from all preceding boards in that it does not use the FTDI USB-to-serial driver chip. Instead, it features the Atmega16U2 (Atmega8U2 up to version R2) programmed as a USB-to-serial converter.<br />
<br />
Revision 2 of the Uno board has a resistor pulling the 8U2 HWB line to ground, making it easier to put into DFU mode.<br />
<br />
Revision 3 of the board has the following new features:<br />
<ol><br />
<li>1.0 pinout: added SDA and SCL pins that are near to the AREF pin and two other new pins placed near to the RESET pin, the IOREF that allow the shields to adapt to the voltage provided from the board. In future, shields will be compatible both with the board that use the AVR, which operate with 5V and with the Arduino Due that operate with 3.3V. The second one is a not connected pin, that is reserved for future purposes.<br />
<br />
<li> Stronger RESET circuit.<br />
<li> Atmega 16U2 replace the 8U2.<br />
</ol><br />
<br />
"Uno" means one in Italian and is named to mark the upcoming release of Arduino 1.0. The Uno and version 1.0 will be the reference versions of Arduino, moving forward. The Uno is the latest in a series of USB Arduino boards, and the reference model for the Arduino platform; for a comparison with previous versions, see the index of Arduino boards.<br />
<br />
<!-- <br />
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%<br />
--><br />
<br />
=== '''Fluorimeter''' ===<br />
<br />
A fluorometer is an electronic device that can read the light and transform it in an electrical signal. In our project, that light proceeds from a bacterial culture that has been excited.<br />
<br />
<br />
Its principle of operation is as follows:<br />
First, an electrical signal is sent from Arduino to a LED to activate it. This LED has a specific wavelength to excite the fluorescent protein present in the microbial culture. <br />
<br />
<br />
When these bacteria are shining, we receive that light intensity with a photodiode, and transform it into electrical current. Now, we have an electrical current that is proportional with the light and therefor to the concentration of fluorescent protein present in the culture. We have to be careful with the orientation between the LED and the photodiode because interferences may occur in the measurement due to light received by photodiode but not emitted by the fluorescence in the culture but scattered light emitted by the LED. Also, a proper band-pass filter has been placed between culture and photodiode to allow only the desired wavelengths to pass through.<br />
<br />
<br />
[[File:Fluorimetro1.gif|center]]<br />
<br />
The electric current coming from the photodiode has a very small amplitude, thereby we have to amplify it and translate it into a electric voltage with an electronic trans-impedance amplifier. We can choose the gain of this amplification changing the value of resistances of the circuit. The resistances must be properly chosen to select the desired amplification gain. Excessive amplification will not only amplify the signal but also amplify the noise, which is no desired in the measurement. <br />
<br />
[[File:Fluorimetro2.jpg|center|400px]]<br />
<br />
Finally, the output voltage is sent to Arduino, and it will translate that in a digital signal.<br />
<br />
[[File:Fluorimetro3.jpg|center|400px]]<br />
<br />
</div></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus
Team:Valencia Biocampus
2012-09-26T18:29:02Z
<p>Lujemomo: /* Talking Life */</p>
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<br />
'''Talking life in 150 words'''<br />
<br />
''Do you speak to your bacteria? We do. We have designed, constructed and characterized an inter-specific translator based on light pulses that allows to literally dialogue with microorganisms. We have built seven biobricks with fluorescent proteins under the control of environmentally-sensitive promoters. The process is as follows: human voice messages are electronically- and then light-encoded in excitation wavelengths, and microbial proteins’ emission wavelengths are electronically- and voice-encoded back. We have used this system to find out the fermentative status of budding yeast and to dialogue with ''E. coli'' allowing it to answer questions such as “are you hungry?” The three pillars of our project (human practices, modeling and wetlab) yielded continuous feedback with each other, illustrating an integrated interdisciplinary approach. For example, in human practices, we qualitatively discussed the possibility of cheater mutant (“liers”), which was quantitatively supported by our results in both our modeling simulations and in the wetlab.''<br />
<br />
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Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus
Team:Valencia Biocampus
2012-09-26T18:24:33Z
<p>Lujemomo: /* Talking Life */</p>
<hr />
<div>__NOTOC__ <br />
<br />
<!-- PLANTILLAS HEREDADAS --><br />
<br />
{{:Team:Valencia_Biocampus/banner}}<br />
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<br />
== Talking Life ==<br />
<br />
<html><br />
<object param NAME="wmode" VALUE="transparent" classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=4,0,2,0" width="650" height="414" align="middle"><br />
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</embed><br />
</object> <br />
</html><br />
<br />
<br />
'''Talking life in 150 words'''<br />
<br />
''Do you speak to your bacteria? We do. We have designed, constructed and characterized an inter-specific translator based on light pulses that allows to literally dialogue with microorganisms. We have built seven biobricks with fluorescent proteins under the control of environmentally-sensitive promoters. The process is as follows: human voice messages are electronically- and then light-encoded in excitation wavelengths, and microbial proteins’ emission wavelengths are electronically- and voice-encoded back. We have used this system to find out the fermentative status of budding yeast and to dialogue with ''E. coli'' allowing it to answer questions such as “are you hungry?” The three pillars of our project (human practices, modeling and wetlab) yielded continuous feedback with each other, illustrating an integrated interdisciplinary approach. For example, in human practices, we qualitatively discussed the possibility of cheater mutant (“liers”), which was quantitatively supported by our results in both our modeling simulations and in the wetlab.''<br />
<br />
== Crowdfunding ==<br />
<html><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/DMM-RHnVg7E" frameborder="0" allowfullscreen></iframe><br />
</html><br />
<br />
More information: http://www.indiegogo.com/vlcbiocampusIGEM<br />
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Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/menu2
Team:Valencia Biocampus/menu2
2012-09-26T18:20:57Z
<p>Lujemomo: </p>
<hr />
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<a href="https://2012.igem.org/Team:Valencia_Biocampus/Yeast">Yeast</a><br />
<br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Modeling">Modeling</a><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/talking">Talking Interfaces</a><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Notebook">Notebook</a><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols">Protocols</a><br />
</div><br />
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<br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Results1">Talking to bacteria</a><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Results2">Talking to yeast</a><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Results3">Dissecting cheaters</a><br />
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<a href="https://2012.igem.org/Team:Valencia_Biocampus/Modeling#Modeling_Bacteria">Bacteria</a><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Modeling#Modeling_Yeast">Yeast</a><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/modhp">for Human Practices</a> <br />
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<!-- <a href="https://2012.igem.org/Team:Valencia_Biocampus/Ethics">Ethical issues</a> --><br />
<br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Safety">Safety</a><br />
<a><br></a><br />
<a><br></a><br />
<br />
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<br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Achievements"><br />
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<span class="sdt_active"></span><br />
<span class="sdt_wrap"><br />
<span class="sdt_link">Achievements</span><br />
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Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/menu2
Team:Valencia Biocampus/menu2
2012-09-26T18:20:24Z
<p>Lujemomo: </p>
<hr />
<div><html><br />
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<!-- <a href="https://2012.igem.org/Team:Valencia_Biocampus/Team">The Team</a>--><br />
<!-- <a href="https://2012.igem.org/Team:Valencia_Biocampus/Team2"> Student Members </a> --><br />
<br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/miembros">Student Members </a><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/miembros2"> Advisors </a><br />
<!-- <a href="https://2012.igem.org/Team:Valencia_Biocampus/miembros3"> Instructors </a>--><br />
<!--<a href="https://2012.igem.org/Team:Valencia_Biocampus/c3po">Student Members </a>--><br />
<!-- <a href="https://2012.igem.org/Team:Valencia_Biocampus/c3poAdvisors"> Advisors </a>--><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/c3poInstructors"> Instructors </a><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Universities">The Universities</a><br />
<br />
<!--<a href="https://2012.igem.org/Team:Valencia_Biocampus/Akw">Aknowledgements</a>--><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Akw">Aknowledgements</a><br />
<br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Attributions">Attributions</a><br />
<br />
<!-- <a href="https://2012.igem.org/Team:Valencia_Biocampus/Gallery">Gallery</a> --><br />
<!-- <a href="https://2012.igem.org/Team:Valencia_Biocampus/galeria">Gallery</a> --><br />
<!-- <a href="https://2012.igem.org/Team:Valencia_Biocampus/galeria">Gallery</a> --><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/galeria3">Gallery</a> <br />
<br />
<!-- <a href="https://2012.igem.org/Team:Valencia_Biocampus/contactUs">Contact Us</a> --><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/contactUs">Contact Us</a><br />
</div><br />
</li><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1a/C3po_valencia.png" alt=""/><br />
<span class="sdt_active"></span><br />
<span class="sdt_wrap"><br />
<span class="sdt_link">The Project</span><br />
<span class="sdt_descr">Where ideas get born</span><br />
</span><br />
</a><br />
<div class="sdt_box"><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Project">Overview</a><br />
<br />
<!-- <a href="https://2012.igem.org/Team:Valencia_Biocampus/Bacterium">Bacteria</a> --><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Bacterium">Bacteria</a><br />
<br />
<br />
<!-- <a href="https://2012.igem.org/Team:Valencia_Biocampus/Yeast">Yeast</a> --><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Yeast">Yeast</a><br />
<br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Modeling">Modeling</a><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/talking">Talking Interfaces</a><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Notebook">Notebook</a><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols">Protocols</a><br />
</div><br />
</li><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/b/b4/Biobrick_valencia.png" alt=""/><br />
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<span class="sdt_wrap"><br />
<span class="sdt_link">Results</span><br />
<span class="sdt_descr">Our dialogues</span><br />
</span><br />
</a><br />
<div class="sdt_box"><br />
<br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Results1">Talking to bacteria</a><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Results2">Talking to yeast</a><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Results3">Dissecting cheaters</a><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Biobricks">Submitted Biobricks</a><br />
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<br />
</div><br />
</li><br />
<li><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Modeling"><br />
<img src="https://static.igem.org/mediawiki/2012/7/70/Modeling_menu_vlc.png" alt=""/><br />
<span class="sdt_active"></span><br />
<span class="sdt_wrap"><br />
<span class="sdt_link">Modeling</span><br />
<span class="sdt_descr">Inside knowlegde</span><br />
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<div class="sdt_box"><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Modeling">Introduction</a> <br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Modeling#Modeling_Bacteria">Bacteria</a><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Modeling#Modeling_Yeast">Yeast</a><br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/modhp">for Human Practices</a> <br />
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</li><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/9/9d/Human_valencia.png" alt=""/><br />
<span class="sdt_active"></span><br />
<span class="sdt_wrap"><br />
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<span class="sdt_descr">Talking life</span><br />
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<a href="https://2012.igem.org/Team:Valencia_Biocampus/Ethics">Ethical issues</a><br />
<!-- <a href="https://2012.igem.org/Team:Valencia_Biocampus/Ethics">Ethical issues</a> --><br />
<br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Safety">Safety</a><br />
<a><br></a><br />
<a><br></a><br />
<br />
</div><br />
</li><br />
<li><br />
<br />
<a href="https://2012.igem.org/Team:Valencia_Biocampus/Achievements"><br />
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<span class="sdt_wrap"><br />
<span class="sdt_link">Achievements</span><br />
<span class="sdt_descr">What we did</span><br />
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Lujemomo
http://2012.igem.org/File:Animacion_wiki_introduccion.swf
File:Animacion wiki introduccion.swf
2012-09-26T17:58:32Z
<p>Lujemomo: uploaded a new version of &quot;File:Animacion wiki introduccion.swf&quot;</p>
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Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/Protocols
Team:Valencia Biocampus/Protocols
2012-09-26T17:32:01Z
<p>Lujemomo: /* Mini-prep */</p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estilo}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
<br />
<br><br />
<br />
<ol><p style="font-size:x-large;">Protocols</p><br />
------------<br />
</ol><br />
<ol><br />
__TOC__<br />
<br />
= '''General Protocols''' =<br />
<ol><br />
<br />
== '''Transformation protocols''' ==<br />
<br />
=== '''Heat Shock Protocol for bacteria transformation''' ===<br />
<html><br />
<ol><br />
<br />
<li>Take competent E.coli cells from –80°C freezer.<br />
<li>Turn on water bath to 42°C.<br />
<li>Put 100 ul of competent cells in an Eppendorf tube.<br />
<li>Keep tubes on ice.<br />
<li>Add 50 ng of circular DNA into E.coli cells. Incubate on ice for 10 minutes to thaw competent cells.<br />
<li>Put tube(s) with DNA and E.coli into water bath at 42°C for 45 seconds.<br />
<li>Put tubes back on ice for 2 minutes to reduce damage to the E.coli cells.<br />
<li>Add 1 ml of <a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#SOC_broth_for_bacteria">SOC Broth</a> (with no antibiotic added). Incubate tubes for 1 hour at 37°C.<br />
<li>Spread about 100 ul of the resulting culture on LB plates (with Ampicillin added). Grow overnight.<br />
<li>Pick colonies about 12-16 hours later.<br />
<br />
</ol><br />
</html><br />
<br />
=== '''Yeast transformation''' ===<br />
<br />
<html><br />
<ol><br />
<br />
<li>Prepare a 2 ml preculture in the selection medium of the strain to be transformed.<br />
<li>Inoculate 20 ml of YPD2% for each transformation, in order to get an OD600 = 1 next day.<br />
<ol></html>[[File:Asffsfasasf.png]]<html><br />
<br>Where n is the number of divisions (generation time: 1´5 h for S. cerevisiae).</ol><br />
From now, it is not necessary to work below sterility conditions.<br />
Once reached the OD600 = 1:<br />
<li>Centrifuge at 3000 rpm 5 min.<br />
<li>Wash with sterile water.<br />
<li>Resuspend in 30 ml of LISORB.<br />
<li>Shake at ambient temperature for 30 min.<br />
<li>Centrifuge at 3000 rpm 5 min and resuspend in 1 ml of LISORB. Transfer to an eppendorf tube.<br />
<li>Centrifuge at 3000 rpm 5 min.<br />
<li>Resuspend in 100 μl of LISORB for each transformation. Transfer 100 μL aliquots in different tubes for each transformation.<br />
<li>Add 7 μl of salmon sperm DNA + 1 μl of transforming DNA.<br />
<li>Incubate 10 min at ambient temperature.<br />
<li>Add 260 μl of 40%PEG/LiAc/TE. Mix well.<br />
<li>Incubate 1 h at 30°C.<br />
<li>Add 43 μl of DMSO and give a thermal shock of 5 minutes at 42°C.<br />
<li>Centrifuge at 3000 rpm 5 min.<br />
<li>Wash with 1 ml of sterile water.<br />
<li>Centrifuge at 3000 rpm 5 min.<br />
<li>Resuspend in 0´5 ml of water and plaque:<br />
<ol>-50 μl<br />
<br>-Rest (centrifuge and decant leaving 50-100 μl).</ol><br />
<br />
<br />
</ol><br />
</html><br />
<br />
== '''DNA extraction and purification protocols''' ==<br />
<br />
=== '''Mini-prep''' ===<br />
<br />
<html><br />
<ol><br />
<br />
<li>Different cultures (each one with a different construction), which are growing in a selective media (LB + Ampicillin), get centrifuged at 4500g 5 min.<br />
<li>Supernatant is removed.<br />
<li>The cells can be washed (x2) with a saline solution (PBS) in order to remove impurities.<br />
<li>The pellet is resuspended in 250 μL of Resuspension Solution (RNase A added to it previously. This solution is kept at 4ºC). Important: resuspend it completely.<br />
<li>Transfer the suspension to an eppendorf tube.<br />
<li>Add 250 μL of Lysis Solution.<br />
<li>Mix it inverting the tube 4-6 times <b>(DO NOT VORTEX!)</b> until solution gets viscous and slightly clear. <br />
<ol><b>Important: Do not incubate more than 5 min.</b></ol><br />
<li>Add 350 μL of Neutralization Solution.<br />
<li>Mix it inverting the tube 4-6 times. Incubate in ice for 15-30 min.<br />
<ol>Now if it was necessary, the process could stop here keeping the eppendorf tube in ice.</ol><br />
<li>Centrifuge 10’ (max. rpm) in order to pellet cell debris and chromosomal DNA.<br />
<li>Transfer the supernatant (≈ 800 μL) to the spin column (pipetting to avoid carrying impurities).<br />
<ol><b>Important: DO NOT TRANSFER THE PRECIPITATE!</ol></b><br />
<li>Centrifuge 1’.<br />
<li>Flow-though liquid is removed.<br />
<li>Add 500 μL of Wash Solution (Solution stock has to be perfectly closed, it contains ethanol!).<br />
<li>Centrifuge ≈ 1’.<br />
<li>Flow-though liquid is removed.<br />
<li>14, 15, 16 steps are repeated.<br />
<li>Centrifuge 1’ in order to eliminate residual Wash Solution.<br />
<li>The spin column is transferred into an eppendorf tube (the collection tube is eliminated).<br />
<li>Add 50 μL of Elution Buffer to the center of spin column membrane and let it 5’ getting soaked (it increases the efficiency of process).<br />
<ol><b>Important: DO NOT CONTACT THE COLUMN MEMBRANE WITH THE PIPETTE TIP!</ol></b><br />
<li>Centrifuge ≈ 2’.<br />
<li>To increase the efficiency (≈ 20%) we can get the flow-though liquid and repeat the steps previously described (20 and 21).<br />
<li>The column is discarded and the solution which contains the purified plasmid can be stored in cold.<br />
<br />
</ol><br />
</html><br />
<br />
==='''Protocol for Gel Extraction'''===<br />
<html><br />
<ol><br />
<br />
<li>Cut bands of interest from the agarose gel.<br />
<li>Add 300 uL of Solution L1 for each 100 mg of gel.<br />
<li>Incubate at 50ºC for 15 minutes.<br />
<li>Centrifugate in a 2 ml column at 12000xg for 1 minute.<br />
<li>Re-insert the spin column into the resaver tube and add 500 uL of Buffer L2.<br />
<li>Centrifugate 12000xg for 1 minute.<br />
<li>Discard the flow-through.<br />
<li>Centrifuge 12000xg for 1 minute. <br />
<li>Place the spin column into a new 1.5 mL microfuge tube.<br />
<li>Add 50 uL of mQ water. <br />
<li>Centrifuge 12000xg for 2 minutes.<br />
<br />
</ol><br />
</html><br />
<br />
== '''DNA digestion and ligation protocols''' ==<br />
<br />
<br />
=== '''Digestion Protocol For Plasmid Backbone Using EcoRI and PstI''' ===<br />
<br />
<html><br />
<ol><br />
<table border="1" style="background:transparent"><br />
<tr><br />
<td>DNA linearized plasmid Backbone (25 ng/uL)</td><br />
<td> </td><br />
<td>8 uL </td><br />
</tr><br />
<tr><br />
<td>PstI</td><br />
<td> </td><br />
<td>1 uL </td><br />
</tr><br />
<tr><br />
<td>EcoRI</td><br />
<td> </td><br />
<td>1 uL </td><br />
</tr><br />
<tr><br />
<td>Buffer 10x must be a common buffer for <br />
<br>EcoRI and PstI (e.g. buffer H in Roche system)</td><br />
<td> </td><br />
<td>2.5 uL</td><br />
</tr><br />
<tr><br />
<td>mQ Water</td><br />
<td> </td><br />
<td>x uL</td><br />
</tr><br />
<tr><br />
<td><b>TOTAL</b></td><br />
<td> </td><br />
<td>25 uL</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<br />
Mix by pipetting when both enzymes have been added. Avoid vortexing. Enzymes are kept in cooler or ice throughout all experiments. <br />
<ol><br />
<li>The digestion mixture is kept for 3 hours at 37 ° C<br />
<br />
<li>The mixture is kept for 20 minutes at 80ºC<br />
<br />
</ol><br />
</ol><br />
</html><br />
<br />
=== '''Digestion Protocol For Plasmid pUC57 + Construction Using EcoRI and PstI''' ===<br />
<br />
<html><br />
<ol><br />
<table border="1" style="background:transparent"><br />
<tr><br />
<td>Plasmid DNA</td><br />
<td> </td><br />
<td>96 uL </td><br />
</tr><br />
<tr><br />
<td>PstI</td><br />
<td> </td><br />
<td>2 uL + 2 uL</td><br />
</tr><br />
<tr><br />
<td>EcoRI</td><br />
<td> </td><br />
<td>2 uL + 2 uL</td><br />
</tr><br />
<tr><br />
<td>Buffer 10x must be a common buffer for <br />
<br>EcoRI and PstI (e.g. buffer H in Roche system)</td><br />
<td> </td><br />
<td>12 uL</td><br />
</tr><br />
<tr><br />
<td>mQ Water</td><br />
<td> </td><br />
<td>4 uL</td><br />
</tr><br />
<tr><br />
<td><b>TOTAL</b></td><br />
<td> </td><br />
<td>120 uL</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<br />
The volume used is so high because after digestion we were going to purify the different inserts from an agarose gel. <br>In order to optimize the digestion reaction we follow these steps <br />
<ol><br />
<li>2 uL of EcoRI is added and incubated for 1 hour.<br />
<li>2 uL of EcoRI is added and incubated for 1 hour.<br />
<li>2 uL of PstI is added and incubated for 1 hour.<br />
<li>2 uL of PstI is added and incubated for 1 hour.<br />
<br />
</ol><br />
</ol><br />
</html><br />
<br />
=== '''Ligation''' ===<br />
<br />
<html><br />
<ol><br />
<table border="1" style="background:transparent"><br />
<tr><br />
<td>Plasmid DNA*</td><br />
<td> </td><br />
<td>X uL </td><br />
</tr><br />
<tr><br />
<td>Insert DNA*</td><br />
<td> </td><br />
<td>Y uL </td><br />
</tr><br />
<tr><br />
<td>10X ligase buffer</td><br />
<td> </td><br />
<td>1 uL </td><br />
</tr><br />
<tr><br />
<td>T4 ligase</td><br />
<td> </td><br />
<td>1 uL </td><br />
</tr><br />
<tr><br />
<td>mQ Water</td><br />
<td> </td><br />
<td>8-(X+Y) uL</td><br />
</tr><br />
<tr><br />
<td><b>TOTAL</b></td><br />
<td> </td><br />
<td>10 uL</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<br />
<b>OBSERVATION:</b> The ratio that has to exist between the number of molecules of plasmid DNA and insert is 1:3 (the volumes depends on the concentration of DNAp and the insert). <br />
</ol><br />
</html><br />
<br />
=== '''Colony PCR''' ===<br />
<br />
<html><br />
<ol><br />
<br />
<li>Each colony is taken from the petri dish and <br />
<ol><ol type="a"><br />
<li>resuspended in 15 μl of mQ water in a eppendorf if we are working with bacteria.<br />
<li>resuspended in 15 μl of NaOH 20 mM in a eppendorf if we are working with yeast.<br />
</ol></ol><br />
<li>Incubate for 15 minutes at room temperature.<br />
<li>The PCR mix is prepared as shown:<br />
<ol> 2 ul of yeast DNA solution<br />
<br> 5 ul of 10X PCR buffer<br />
<br> 4 ul of dNTPs 2.5 mM<br />
<br> 2 ul of A oligo<br />
<br> 2 ul of B oligo<br />
<br> 31 ul of water<br />
</ol><br />
<li>Once mixed, 4 ul of 10X TAQ polymerase solution is added.<br />
<li>The PCR reaction program is the next one:<br />
<ol> 94ºC 3 minutes<br />
<br> 30 cycles of:<br />
<ol> 94ºC 1 minutes<br />
<br> 45ºC 1 minutes 30 seconds<br />
<br> 72ºC 2 minutes<br />
</ol><br />
72ºC 10 minutes<br />
<br> 4ºC Hold<br />
</ol><br />
<br />
</ol><br />
</html><br />
<br />
== '''Biobricks protocols''' ==<br />
<br />
<html><br />
<ol><br />
<br />
<li><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Digestion_Protocol_For_Plasmid_Backbone_Using_EcoRI_and_PstI"> Digestion Protocol For Plasmid Backbone Using EcoRI and PstI</a><br />
<li><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Digestion_Protocol_For_Plasmid_pUC57_.2B_Construction_Using_EcoRI_and_PstI"> Digestion Protocol For Plasmid pUC57 + Construction Using EcoRI and PstI </a><br />
<li><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Heat_Shock_Protocol_for_bacteria_transformation"> Transformation Protocol Using Heat Shock </a><br />
<li><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Ligation"> Ligation Protocol </a><br />
<li><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Mini-prep"> Mini-preps. Purification protocol. </a><br />
<li><a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#Protocol_for_Gel_Extraction"> Protocol for Gel Extraction </a><br />
<br />
<br />
</ol><br />
</html><br />
<br />
</ol><br />
= '''Media and solutions protocols''' =<br />
<ol><br />
<br />
=== '''LB broth for bacteria''' ===<br />
<br />
<html><br />
<ol><br />
<br />
<table style="background:transparent"><br />
<tr><br />
<td><b>Bacterial peptone</b></td><br />
<td> </td><br />
<td>1% (p/v)</td><br />
</tr><br />
<tr><br />
<td><b>Yeast extract</b></td><br />
<td> </td><br />
<td>0.5% (p/v)</td><br />
</tr><br />
<tr><br />
<td><b>NaCl</b></td><br />
<td> </td><br />
<td>1% (p/v)</td><br />
</tr><br />
</table><br />
In order to obtain solid LB, add 15 g/L of bacteriologic agar.<br />
<br />
<br />
</html><br />
<br />
==== '''LBA broth for bacteria''' ====<br />
<br />
<html><br />
<ol><br />
<br />
Add 1 mL of ampicilin 100mg/mL (of mQ water) in 1L of <a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#LB_broth_for_bacteria">LB Broth</a><br />
<br />
</ol><br />
</html><br />
<br />
==== '''LB + Chloramphenicol broth for bacteria''' ====<br />
<br />
<html><br />
<ol><br />
<br />
Add 1 mL of Chloramphenicol 34mg/mL (of pure ethanol) in 1L of <a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#LB_broth_for_bacteria">LB Broth</a><br />
<br />
</ol><br />
</ol><br />
</html><br />
<br />
=== '''SOC broth for bacteria''' ===<br />
<br />
<html><br />
<ol><br />
<table style="background:transparent"><br />
<tr><br />
<td><b>Bacterial triptone</b></td><br />
<td> </td><br />
<td>4g</td><br />
</tr><br />
<tr><br />
<td><b>Yeast extract</b></td><br />
<td> </td><br />
<td>1g</td><br />
</tr><br />
<tr><br />
<td><b>NaCl 5M</b></td><br />
<td> </td><br />
<td>0.4 mL</td><br />
</tr><br />
<tr><br />
<td><b>KCl 3M</b></td><br />
<td> </td><br />
<td>0.167 mL</td><br />
</tr><br />
<tr><br />
<td><b>MgSO4 </b></td><br />
<td> </td><br />
<td>2.465 g</td><br />
</tr><br />
<tr><br />
<td><b>MgCl2 </b></td><br />
<td> </td><br />
<td>2.033 mL</td><br />
</tr><br />
<tr><br />
<td><b>Glucose</b></td><br />
<td> </td><br />
<td>3.603 g</td><br />
</tr><br />
<tr><br />
<td><b>Distiled water</b></td><br />
<td> </td><br />
<td>220 mL</td><br />
</tr><br />
</table><br />
Sterilization by filtration<br />
<br />
</ol><br />
</html><br />
<br />
=== '''YP broth for yeast''' ===<br />
<br />
<html><br />
<ol><br />
<br />
<table style="background:transparent"><br />
<tr><br />
<td><b>Bacterial peptone</b></td><br />
<td> </td><br />
<td>2% (p/v)</td><br />
</tr><br />
<tr><br />
<td><b>Yeast extract</b></td><br />
<td> </td><br />
<td>1% (p/v)</td><br />
</tr><br />
</table><br />
In order to obtain solid YP, add 2% of agar before sterilization.<br />
<br />
<br />
</html><br />
<br />
==== '''YPD broth for yeast''' ====<br />
<br />
<html><br />
<ol><br />
<br />
Add x% (p/v) of dextrose or glucose (sterilized by filtration) in <a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#YP_broth_for_yeast">YP Broth</a> after sterilization.<br />
<br />
</ol><br />
</html><br />
<br />
==== '''YPRE broth for yeast''' ====<br />
<br />
<html><br />
<ol><br />
<br />
Add 2% (p/v) of raffinose and 2% (v/v) of ethanol 100% in <a href="https://2012.igem.org/Team:Valencia_Biocampus/Protocols#YP_broth_for_yeast">YP Broth</a> after sterilization.<br />
<br />
</ol><br />
</ol><br />
</html><br />
<br />
=== '''SD broth for yeast''' ===<br />
<br />
<html><br />
<ol><br />
<br />
<table style="background:transparent"><br />
<tr><br />
<td><b>Yeast nitrogen base</b> <br />
<br> w/o aminoacids and w/ amonium persulfate</td><br />
<td> &nbsp; &nbsp; &nbsp;</td><br />
<td>0.67% (p/v)</td><br />
</tr><br />
<tr><br />
<td><b>Glucose</b></td><br />
<td> </td><br />
<td>2% (p/v)</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<b>Aminoacids</b> (leucine, metionine, histidine) and <b>nucleotides</b> (uracil) are added after sterilization in a final concentration of 20 mg/mL.<br />
<br> In order to obtain solid SD, add 2% (p/v) of agar before sterilization.<br><br />
<br />
</ol><br />
</html><br />
<br />
=== '''LISORB solution for yeast transformation''' ===<br />
<html><br />
<b>For 100 mL</b><br />
<br><br />
<ol><br />
<table style="background:transparent"><br />
<tr><br />
<td><b>LiAc 1M</b></td><br />
<td> </td><br />
<td>10 mL</td><br />
</tr><br />
<tr><br />
<td><b>Sorbitol 2.4M</b></td><br />
<td> </td><br />
<td>41.6 mL</td><br />
</tr><br />
<tr><br />
<td><b>TE 100X</b></td><br />
<td> </td><br />
<td>1 mL</td><br />
</tr><br />
</table><br />
Bring to 100 ml of distilled H2O<br />
<br />
</ol><br />
</html><br />
<br />
=== '''40%PEG/LiAc/TE solution for yeast transformation''' ===<br />
<html><br />
<b>For 20 mL</b><br />
<br><br />
<ol><br />
<table style="background:transparent"><br />
<tr><br />
<td><b>LiAc 1M</b></td><br />
<td> </td><br />
<td>2 mL</td><br />
</tr><br />
<tr><br />
<td><b>PEG 3500 50%</b></td><br />
<td> </td><br />
<td>16 mL</td><br />
</tr><br />
<tr><br />
<td><b>TE 100X</b></td><br />
<td> </td><br />
<td>0.2 mL</td><br />
</tr><br />
</table><br />
Bring to 20 ml of distilled H2O<br />
</ol><br />
</html><br />
</ol><br />
<br />
= '''Yeast Induction protocol''' =<br />
<br />
<html><br />
<ol><br />
<br />
<li>Colonies are picked from petri dish and suspended in suplemented SD media.<br />
<li>Incubate overnight<br />
<li>Resuspend in YPD8%<br />
<li>Incubate overnight to 5 OD.<br />
<li>Resuspend in YPRE<br />
<li>Incubate for 8 to 24 hours<br />
<li>Add a final concentration of 0.3 mM of H2O2<br />
<li>Measure OD and fluorescence intensity.<br />
<br />
</ol></div>
Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/Akw
Team:Valencia Biocampus/Akw
2012-09-26T15:36:05Z
<p>Lujemomo: </p>
<hr />
<div>__NOTOC__ <br />
{{:Team:Valencia_Biocampus/banner}}<br />
{{:Team:Valencia_Biocampus/estiloc3po}}<br />
{{:Team:Valencia_Biocampus/menu2}}<br />
<br />
<!-- SECCIÓN CENTRADA --><br />
<br />
<br />
<br />
<center><br />
<div id="Titulos"><br />
<h2>Aknowledgements</h2><br />
<br><br />
<br />
<div id="PorDefecto"><br />
<br />
<br />
We want to thank all those who, in one way or another, made possible the realization of this project, contributing their resources and expertise.<br />
<br />
First, we would like to thank to <b>''Universitat de València, Universitat Politècnica de València''</b> and<b> ''CSIC''</b>, which have given us the opportunity to participate in this wonderful project. They have guided and advised us at all times.The <b>''Càtedra de Divulgació de la Ciència (Universitat de València)''</b> gave us an immeasurable economic support.<br />
<br />
We also would like to thank<b> ''Universitas Bergensis''</b>, which has guided us in everything related to human practices, both in the realization of the short film as in the analysis of the ethical aspects of the project. They have also provided the necessary support for the production and improvement of the presentation, as well as to <b>''Taller d' Audiovisuals de la Universitat de València''</b> which supported with the proper materials to shoot the film, and a special aknowledge to the Valencian actor <b>Alfred Picó</b> to play the role of the watch seller.<br />
<br />
As we promised, we thank our crowdfunding contributors, which until today have helped us with some money tips: <b>Antonio Amate, Sanz-Gil family, Telesforo Gil's Pharmacy, Romero Lina</b> and<b> Albert Brulles</b> among others.<br />
<br />
Last but not least, we would like to gratefully acknowledge the dedication provided by<b> ''Biopolis''</b>, which has helped us when it has been necessary, lending us its guidance, advice, facilities and time, as well as economic support.<br />
<br />
Thank you all for making this wonderful experience possible!<br><br><br />
<br />
[[Image:Valencia biocampus.pic..jpg|700 px||center]]<br />
<br />
<p align="right">Photographer: Miguel Lorenzo.<br></p><br />
<br />
<br />
<br />
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<br />
<br><br />
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<br />
<br><br />
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Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/estiloc3po
Team:Valencia Biocampus/estiloc3po
2012-09-26T15:34:21Z
<p>Lujemomo: </p>
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Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/Akw
Team:Valencia Biocampus/Akw
2012-09-26T15:33:45Z
<p>Lujemomo: </p>
<hr />
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{{:Team:Valencia_Biocampus/estiloc3po}}<br />
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<h2>Aknowledgements</h2><br />
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<div id="PorDefecto"><br />
<br />
<br />
We want to thank all those who, in one way or another, made possible the realization of this project, contributing their resources and expertise.<br />
<br />
First, we would like to thank to <b>''Universitat de València, Universitat Politècnica de València''</b> and<b> ''CSIC''</b>, which have given us the opportunity to participate in this wonderful project. They have guided and advised us at all times.The <b>''Càtedra de Divulgació de la Ciència (Universitat de València)''</b> gave us an immeasurable economic support.<br />
<br />
We also would like to thank<b> ''Universitas Bergensis''</b>, which has guided us in everything related to human practices, both in the realization of the short film as in the analysis of the ethical aspects of the project. They have also provided the necessary support for the production and improvement of the presentation, as well as to <b>''Taller d' Audiovisuals de la Universitat de València''</b> which supported with the proper materials to shoot the film, and a special aknowledge to the Valencian actor <b>Alfred Picó</b> to play the role of the watch seller.<br />
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As we promised, we thank our crowdfunding contributors, which until today have helped us with some money tips: <b>Antonio Amate, Sanz-Gil family, Telesforo Gil's Pharmacy, Romero Lina</b> and<b> Albert Brulles</b> among others.<br />
<br />
Last but not least, we would like to gratefully acknowledge the dedication provided by<b> ''Biopolis''</b>, which has helped us when it has been necessary, lending us its guidance, advice, facilities and time, as well as economic support.<br />
<br />
Thank you all for making this wonderful experience possible!<br><br><br />
<br />
[[Image:Valencia biocampus.pic..jpg|700 px||center]]<br />
<br />
<p align="right">Photographer: Miguel Lorenzo.<br></p><br />
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Lujemomo
http://2012.igem.org/Team:Valencia_Biocampus/estiloc3po
Team:Valencia Biocampus/estiloc3po
2012-09-26T15:33:37Z
<p>Lujemomo: </p>
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position:absolute;<br />
top:15px;<br />
left:15px;<br />
} <br />
<br />
</style><br />
</html></div>
Lujemomo