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Chalmers Gothenburg iGEM 2016

PROJECT

Design and Idea

Introduction

Lung cancer is one of the most common cancer forms today and due to the lack of good detection methods, it is one of the deadliest. A suggested approach for detection of the disease have been to use specific volatile organic compounds (VOCs) that have been found in elevated levels in the breath of lung cancer patients.

The aim of this project is to detect two of these VOCs with a biosensor made of Saccharomyces cerevisiae. The VOCs will be detected by G-protein coupled receptors (GPCRs) which will activate the pheromone pathway and initiate mating between a- and α-cells. The output, a colony color change, will be achieved by partially deleting the ADE2 gene with a CRISPR/Cas9 system in the fused cell. The colony color change in the biosensor will indicate presence of VOCs in the patient’s exhaled breath.

Background

Lung cancer is the cancer form with the highest death toll, causing 1.69 million deaths globally in 2015 [1]. One reason for the high death toll is the late detection of the cancer, due to symptoms starting to appear first in the later stages.

Three different stages are used to characterize lung cancer; I, II and III. The magnitude of the disease is based on size of the tumor and the extent of spreading to lymph nodes and other organs in the body [2]. A majority of the lung cancer cases today are diagnosed in stage III with a 5-year survival rate of 5-14%. An earlier detection at stage I or II would increase the figure to 45-49% which clearly show that early detection is a key to increase the chances of surviving lung cancer [3].

At the appearance of the first symptoms, a chest X-ray is used today to scan for tumors. The scan can indicate the position and size of the tumors. Despite being a non-invasive procedure, it is accompanied by limitations such as cost, time, expertise and sensitivity required for X-ray examinations. Due to these limitations X-ray is not suitable for mass screening of lung cancer [4]. Therefore, there is a demand for new diagnostic tools with mass screening potential.

One of the most promising approaches for early diagnosis is the use of volatile organic compounds (VOCs) as biomarkers. Some specific VOCs can be detected in higher concentrations in the breath of lung cancer patients compared to healthy individuals. The cancer cells have an increased oxidative rate that results in oxidative stress products found in the breath of patients. The low concentration of VOCs, pmol/l, has provided a challenge in using this approach for detection and no efficient screening method is yet available [5].

Studies have shown that there is not only one compound that can be used as a biomarker in the breath of cancer patients [5]. An optimal diagnostic tool would therefore be able to detect multiple VOCs in order to decrease the number of false positive diagnoses. One example of a diagnostic tool is a biosensor, which is a biological-based system that can detect the presence of a target analyte and convert it into a signal. A biosensor can be developed with many different species, antibodies or enzymes. In this case, a yeast-based biosensor is a suitable approach and some of the advantages are the ease of genetic manipulation, as well as the cheap and easy cultivation [6].

To create this novel, non-invasive diagnostic tool, the budding yeast Saccharomyces cerevisiae will be used as a biosensor. The yeast cells will have two xenogeneic olfactory receptors incorporated. These can bind the biomarkers that are present in the breath of lung cancer patients, opening up for the possibility to detect the cancer before any symptoms appears.

The basic design

The aim of this project is to detect two VOCs that have been found in elevated levels in the breath of lung cancer patients, using a biosensor made of Saccharomyces cerevisiae. Two of the most commonly found VOCs; butanone and n-octanal, are chosen for detection [7, 8]. The VOCs will be detected by G-protein coupled receptors (GPCRs) with the ability to activate the pheromone pathway in yeast and thereby initiate mating between a- and α-cells. The final output will be a color change due to deletion of the ADE2 gene using gRNA and Cas9. The colony color change will indicate the presence of the target VOCs, and thereby also lung cancer.

Signal

Natively, yeast have GPCRs regulating the cell mating through activation of the pheromone pathway. These GPCRs, STE2 and STE3, are present respectively in the two different mating types of yeast, a and α. STE2 and STE3 bind pheromones from the other mating type and activate the pheromone pathway which enables mating. In our system STE2 and STE3 will be replaced with the GPCRs RatI7 and Olfr1258 which detect the two VOCs butanone and n-octanal respectively [7,8]. When the GPCRs sense the VOCs, the pheromone pathway will be activated which will result in expression of both the native mating genes and cloned Cre recombinase in both cell types. Binding of butanone and n-octanal will therefore lead to mating of cells, but solely if both VOCs are present.

Switch

In each mating type, a system will be integrated consisting of a promoter in the wrong direction, surrounded by mutated LoxP sites, followed by genes expressing either gRNAs or Cas9, see Figure 2. Presence of the two VOCs will lead to, besides mating, activation of the respective genes since Cre recombinase will turn the promoters in to the right direction. The loxP sites are mutated, preventing the sites from inverting back to their original direction. This ensures that the promoter is inverted into the correct direction upon GPCR activation and remains in that position [9].

AND-gate

For expression of the gRNAs, the promoter pSNR52 will be used. Together with polymerase III, the pSNR52 promoter will not translate the gRNAs into proteins [10]. Two different gRNAs will be expressed by the same promoter and the two gRNAs will be linked together with two ribozymes; HDV and Hammerhead. After expression, the ribozymes will cut themselves out and leave the two gRNAs as separate sequences [11]. For the other mating type, the strong constitutive promoter pTEF1 will be used to express the Cas9 protein. Since the gRNAs and the Cas9 protein are expressed in cells of different mating types, the lack of combination will will not disrupt the adenine synthesis in haploid cells.

After mating, the gRNAs and Cas9 will both be present in the newly formed diploid cell and assemble into a functional unit. The gRNAs will guide the Cas9 protein to two sites in the ADE2 gene, creating two different double strand breaks in the gene. The double stranded breaks will lead to a partial disruption of the ADE2 gene and an accumulation of a red intermediate during adenine synthesis [12]. The color shift of the cells from white to red will function as a diagnostic response.

References

[1] Cancer [Internet]. World Health Organization; 2017 [Cited 2017-06-13]. Available at: http://www.who.int/mediacentre/factsheets/fs297/en/
[2] Non-Small Cell Lung Cancer Stages [Internet]. American Cancer Society; 2016 [Updated 2017-03-02; Cited 2017-06-26]. Available at: https://www.cancer.org/cancer/non-small-cell-lung-cancer/detection-diagnosis-staging/staging.html
[3] Non-Small Lung Cancer Survival Rates, by Stage [Internet]. American Cancer Society; 2016 [Updated 2016-05-16; Cited 2017-06-13]. Available at: https://www.cancer.org/cancer/non-small-cell-lung-cancer/detection-diagnosis-staging/survival-rates.htm
[4] Lungcancer [internet]. Cancerfonden; 2015 [Cited 2017-06-07] Availiable at: https://www.cancerfonden.se/om-cancer/lungcancer
[5] D’Amico A, Pennazza G, Santonico M, Martinelli E, Roscioni C, Galluccio G, Paolesse R, and Di Natale C. An investigation on electronic nose diagnosis of lung cancer. Lung Cancer, 68(2):170–176, 2010.
[6] French C, de Mora K, Joshi N, Elfick A, Haseloff J, and Ajioka J. Synthetic biology and the art of biosensor design. The Science and Applications of Synthetic and Systems Biology:Workshop Summary, 2011.
[7] Minic J, Persuy M-A, Godel E, Aioun J, Connerton I, Salesse R, and Pajot-Augy E. Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening. FEBS Journal, 272(2):524-537, 2005.
[8] Suzuki Y and Shimono K. Deciphering the receptor repertoire encoding specific odorants by time-lapse single-cell array cytometry. Scientic Reports, 6(19934):1-9, 2015.
[9] Carter Z and Delneri D. New generation of loxp-mutated deletion cassettes for the genetic manipulation of yeast natural isolates. Yeast, 27(9):765-775, 2010.
[10] DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, and Church GM. Genome engineering in saccharomyces cerevisiae using crispr-cas systems. Nucleic Acids Research, 41(7):4336-4343, 2013.
[11] Fujita T and Fujii H. Applications of engineered dna-binding molecules such as tal proteins and the crispr/cas system in biology research. International Journal of Molecular Sciences, 16(10):23143-23164, 2015.
[12] Ugolini S and Bruschi CV. The red/white colony color assay in the yeast saccharomyces cerevisiae: epistatic growth advantage of white ade8-18, ade2 cells over red ade2 cells. Current Genetics, 30(6):485-492, 1996.
[13] Kofahl, B. and Klipp, E., Modelling the dynamics of the yeast pheromone pathway. Yeast, 21: 831–850, 2004

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.icon-prev{margin-left:-10px}.carousel-control .glyphicon-chevron-right,.carousel-control .icon-next{margin-right:-10px}.carousel-caption{right:20%;left:20%;padding-bottom:30px}.carousel-indicators{bottom:20px}}.btn-group-vertical>.btn-group:after,.btn-group-vertical>.btn-group:before,.btn-toolbar:after,.btn-toolbar:before,.clearfix:after,.clearfix:before,.container-fluid:after,.container-fluid:before,.container:after,.container:before,.dl-horizontal dd:after,.dl-horizontal dd:before,.form-horizontal .form-group:after,.form-horizontal .form-group:before,.modal-footer:after,.modal-footer:before,.modal-header:after,.modal-header:before,.nav:after,.nav:before,.navbar-collapse:after,.navbar-collapse:before,.navbar-header:after,.navbar-header:before,.navbar:after,.navbar:before,.pager:after,.pager:before,.panel-body:after,.panel-body:before,.row:after,.row:before{display:table;content:" "}.btn-group-vertical>.btn-group:after,.btn-toolbar:after,.clearfix:after,.container-fluid:after,.container:after,.dl-horizontal dd:after,.form-horizontal .form-group:after,.modal-footer:after,.modal-header:after,.nav:after,.navbar-collapse:after,.navbar-header:after,.navbar:after,.pager:after,.panel-body:after,.row:after{clear:both}.center-block{display:block;margin-right:auto;margin-left:auto}.pull-right{float:right!important}.pull-left{float:left!important}.hide{display:none!important}.show{display:block!important}.invisible{visibility:hidden}.text-hide{font:0/0 a;color:transparent;text-shadow:none;background-color:transparent;border:0}.hidden{display:none!important}.affix{position:fixed}@-ms-viewport{width:device-width}.visible-lg,.visible-md,.visible-sm,.visible-xs{display:none!important}.visible-lg-block,.visible-lg-inline,.visible-lg-inline-block,.visible-md-block,.visible-md-inline,.visible-md-inline-block,.visible-sm-block,.visible-sm-inline,.visible-sm-inline-block,.visible-xs-block,.visible-xs-inline,.visible-xs-inline-block{display:none!important}@media (max-width:767px){.visible-xs{display:block!important}table.visible-xs{display:table!important}tr.visible-xs{display:table-row!important}td.visible-xs,th.visible-xs{display:table-cell!important}}@media (max-width:767px){.visible-xs-block{display:block!important}}@media (max-width:767px){.visible-xs-inline{display:inline!important}}@media (max-width:767px){.visible-xs-inline-block{display:inline-block!important}}@media (min-width:768px) and (max-width:991px){.visible-sm{display:block!important}table.visible-sm{display:table!important}tr.visible-sm{display:table-row!important}td.visible-sm,th.visible-sm{display:table-cell!important}}@media (min-width:768px) and (max-width:991px){.visible-sm-block{display:block!important}}@media (min-width:768px) and (max-width:991px){.visible-sm-inline{display:inline!important}}@media (min-width:768px) and (max-width:991px){.visible-sm-inline-block{display:inline-block!important}}@media (min-width:992px) and (max-width:1199px){.visible-md{display:block!important}table.visible-md{display:table!important}tr.visible-md{display:table-row!important}td.visible-md,th.visible-md{display:table-cell!important}}@media (min-width:992px) and (max-width:1199px){.visible-md-block{display:block!important}}@media (min-width:992px) and (max-width:1199px){.visible-md-inline{display:inline!important}}@media (min-width:992px) and (max-width:1199px){.visible-md-inline-block{display:inline-block!important}}@media (min-width:1200px){.visible-lg{display:block!important}table.visible-lg{display:table!important}tr.visible-lg{display:table-row!important}td.visible-lg,th.visible-lg{display:table-cell!important}}@media (min-width:1200px){.visible-lg-block{display:block!important}}@media (min-width:1200px){.visible-lg-inline{display:inline!important}}@media (min-width:1200px){.visible-lg-inline-block{display:inline-block!important}}@media (max-width:767px){.hidden-xs{display:none!important}}@media (min-width:768px) and (max-width:991px){.hidden-sm{display:none!important}}@media (min-width:992px) and (max-width:1199px){.hidden-md{display:none!important}}@media (min-width:1200px){.hidden-lg{display:none!important}}.visible-print{display:none!important}@media print{.visible-print{display:block!important}table.visible-print{display:table!important}tr.visible-print{display:table-row!important}td.visible-print,th.visible-print{display:table-cell!important}}.visible-print-block{display:none!important}@media print{.visible-print-block{display:block!important}}.visible-print-inline{display:none!important}@media print{.visible-print-inline{display:inline!important}}.visible-print-inline-block{display:none!important}@media print{.visible-print-inline-block{display:inline-block!important}}@media print{.hidden-print{display:none!important}}
+/*# sourceMappingURL=bootstrap.min.css.map */
+</style>   <!------------------ bootstrap.min.css STOP ---------------------------------->
-<div class="column full_size">
+<style type="text/css">  /*------------------ style_170803.css START ----------------------------------*/
-<h1>Description</h1>
-<p>Tell us about your project, describe what moves you and why this is something important for your team.</p>
+@import url(https://fonts.googleapis.com/css?family=Rubik|Lato);
-<h5>What should this page contain?</h5>
+* {
-<ul>
+  margin:0;
-<li> A clear and concise description of your project.</li>
+  padding:0;
-<li>A detailed explanation of why your team chose to work on this particular project.</li>
+}
-<li>References and sources to document your research.</li>
-<li>Use illustrations and other visual resources to explain your project.</li>
-</ul>
+body {
+  background-color: #e6e6e6;
+  background: -webkit-linear-gradient(left, rgba(1,0,0,0.2), rgba(1,0,0,0.05), rgba(1,0,0,0.2)); /* For Safari 5.1 to 6.0 */
+  background: -o-linear-gradient(right, rgba(1,0,0,0.2), rgba(1,0,0,0.05), rgba(1,0,0,0.2)); /* For Opera 11.1 to 12.0 */
+  background: -moz-linear-gradient(right, rgba(1,0,0,0.2), rgba(1,0,0,0.05), rgba(1,0,0,0.2)); /* For Firefox 3.6 to 15 */
+  background: linear-gradient(to right, rgba(1,0,0,0.2), rgba(1,0,0,0.05), rgba(1,0,0,0.2)); /* Standard syntax (must be last) */
+  font-family:'Lato', sans-serif;
+}
-</div>
+/*----------------Overriding dumb wiki stuff----------------------------------*/
-<div class="column full_size" >
+#content {
+    width: 100%;
+    padding: 0;
+    margin-left: 0;
+    margin-top: -12px; /*Couldn't figure out why we got a white bar above all content, so I cheated a bit*/
+}
-<h5>Advice on writing your Project Description</h5>
-<p>
+/*This changes margins that messed with the header navbar*/
-We encourage you to put up a lot of information and content on your wiki, but we also encourage you to include summaries as much as possible. If you think of the sections in your project description as the sections in a publication, you should try to be consist, accurate and unambiguous in your achievements.
+.mw-content-ltr ul, .mw-content-rtl .mw-content-ltr ul {
-</p>
+    margin: 0;
+}
-<p>
+/*This is the forced heading and logo. Why? :( */
-Judges like to read your wiki and know exactly what you have achieved. This is how you should think about these sections; from the point of view of the judge evaluating you at the end of the year.
+#top_title {
-</p>
+    display: none;
+}
-</div>
+/*Something caused the sideMenu to start out displayed instead of hidden. This fixes that.*/
+#sideMenu {
+    display: none;
+}
+/*---------------Index page header (above header nav bar)------------------------------*/
-<div class="column half_size" >
+/*This is for the part above the header navbar with the title and logo*/
+#header_navbar_top {
+  background-color: #000000;
+  /*background: linear-gradient(#1f7d01, #016243);*/
+  /*background-image: url("media/header-bg-test1.jpeg");*/
+  background-image: url("pictures_17/backgrounds/korsvagen_5.png"); /*linear-gradient(to bottom, #7bac78, #016243);*/
+	background-repeat: no-repeat;
+  background-position: center;
+	background-size: 100% 100%; /*60% 100%*/
+	/*border: 3px solid #000000;*/
+	text-align: left;
+	position:top;
+  min-height: 750px;
+	height:auto;
+	background-attachment: fixed;
+  background-position: center;
+  background-repeat: no-repeat;
+  background-size: cover;
-<h5>References</h5>
+  /*padding-right: 10px; /*100--> 250*/
-<p>iGEM teams are encouraged to record references you use during the course of your research. They should be posted somewhere on your wiki so that judges and other visitors can see how you thought about your project and what works inspired you.</p>
+  /*padding-left: 10px; /*250--> 250px*/
+	/*height: 270px;/*220px
+  min-width: 70%; /*minimum width of header. Don't change from 1200px, title fucks up
+  min-height: 70%; /*don't think does anything
+	margin-left: auto; /*The width of the header. Keep on auto to avoid gray edges
+  margin-right: auto; /* --"--
+  padding: 40px;/*Find another way to move the logo down a bit?*/
+  border-bottom: 5px solid transparent; /*A lot of code for the line between the header and the body */
+  -moz-border-image: -moz-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%);*/
+  -webkit-border-image: -webkit-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%)*/
+  border-image: linear-gradient(to right, #000000 0%, #404040, #000000 100%); /*(to right, #016243 0%, #b5deca, #016243 100%);*/
+  border-image-slice: 1;
+  -webkit-touch-callout: none;
+  -webkit-user-select: none;
+  -khtml-user-select: none;
+  -moz-user-select: none;
+  -ms-user-select: none;
+  user-select: none;
+  cursor: default;
-</div>
+}
+/*This is the title of the project*/
+#header_navbar_top_title {
+/*Sizes in px*/
+	display:inline-block;
+	padding-top:650px;
+  padding-right:20%;
+  font-size: 100px;
+  line-height: 45px;
+  color: #c91a03;
+	text-shadow: 3px 3px #000000;
+  font-family:'Lato', cursive; /*Dancing Script, Marck Script (meh), Tangerine (ok), Sacramento (meh), Allura (nej), Parisienne (meh), Montez(meh)*/
+	font-weight: bold;
+	text-align:left;
+	margin-left:10%;
+	/*margin-right:10px;	*/
+	/*For the title on index*/
+	/*background-color: transparent;
+  font-size: 60pt; /*35pt
+	font-family:"Lato", cursive; /*Dancing Script, Marck Script, Tangerine, Sacramento, Allura, Parisienne, Montez
+  color:#c91a03;
+	line-height: 45pt;
+  padding-top: 35%;
+	font-weight: bold;
+	text-align:left;
+	margin-left:200px;*/
+}
-<div class="column half_size" >
+#header_navbar_top_subtitle {
-<h5>Inspiration</h5>
+  color:#c91a03;
-<p>See how other teams have described and presented their projects: </p>
+  font-size: 40pt;
+	line-height: 30pt;
+  padding-top: 1%;
+  padding-bottom: 20px;
+	font-family:"Lato", cursive; /*Dancing Script, Marck Script, Tangerine, Sacramento, Allura, Parisienne, Montez*/
+	text-align:left;
+	margin-left:10%;
+	margin-right:25%;
+	text-shadow: 3px 3px #000000;
+}
-<ul>
+.subheader{
-<li><a href="https://2016.igem.org/Team:Imperial_College/Description">2016 Imperial College</a></li>
+	color:#808080; /*#7e1002;*/
-<li><a href="https://2016.igem.org/Team:Wageningen_UR/Description">2016 Wageningen UR</a></li>
+	text-align:center;
-<li><a href="https://2014.igem.org/Team:UC_Davis/Project_Overview"> 2014 UC Davis</a></li>
+	font-size:50pt;
-<li><a href="https://2014.igem.org/Team:SYSU-Software/Overview">2014 SYSU Software</a></li>
+	padding-top:150px;
-</ul>
+	padding-bottom:200px;
-</div>
+}
+#header_navbar_top_logo {
+  display:inline-block;
+		position: absolute;
+	  width:9vw; /*20% 20%*/
+    height:9vw;
+    right:10%;
+    bottom:12%;
+}
+.overview {
+	padding-bottom:100px;
+}
+/*logo in the right corner*/
+.logo_pos_right{
+	position: fixed;
+	/*position in px*/
+	/*  width:20px;
+    height:20px;
+    right:150px;
+    bottom:150px;*/
+	/*position in percent*/
+	  width:7vw; /*20% 20%*/
+    height:7vw;
+    right:3%;
+    bottom:5%;
+}
+#header_navbar_top_logo {
+  display:inline-block;
+}
+/*--------------------Header navbar--------------------------------------------*/
+/*body {
+    font-family: helvetica;
+	margin:0;
+}*/
+/*The wrapper and the nav bar have to be the same height, or the page will jump when the nav bar sticks.*/
+#header_navbar_wrapper, .header_navbar_ul {
+    height: 70px;
+}
+.header_navbar_ul {
+  list-style-type: none; /*No bullet points*/
+  margin: 0;
+  padding: 0;
+  /*background-image: url("pictures_17/random_pictures/BIRD.jpeg");*/
+  background-repeat: no-repeat;
+  background-color: #c91a03; /* #c91a03*/
+	border-bottom: 2px solid black;
+	top: 0;
+  width: 100%;
+  /*min-width: 1440px; Makes the navbar too big for smaller screens. Keep muted if possible*/
+  z-index:9999;
+  -webkit-touch-callout: none;
+  -webkit-user-select: none;
+  -khtml-user-select: none;
+  -moz-user-select: none;
+  -ms-user-select: none;
+  user-select: none;
+}
+/*Having the header navbar stick to the top of the screen after it reaches that position*/
+.header-nav_stick {
+    position: fixed;
+    width: 100%;
+    top: 0;
+    z-index: 9999;
+}
+/*Current page header highlight*/
+.home #home_link,
+.team #team_link,
+.achievements #achievements_link,
+.HP #HP_link,
+.modelling #modelling_link,
+.notebook #notebook_link,
+.parts #parts_link,
+.project #project_link,
+.safety #safety_link
+{
+    color: white;
+		text-decoration:underline;
+}
+.dropbtn {
+    background-color: transparent;
+    color: white;
+    padding: 8px;
+    /*font-size: 16px;*/
+    border: none;
+    cursor: pointer;
+}
+.dropdown,
+#header_navbar_home {
+    position: relative;
+    display: inline-block;
+    z-index:9999;
+    font-family:'Lato', sans-serif;
+}
+.dropdown a,
+#header_navbar_home a {
+  display: block; /*Displaying the links as block elements makes the whole link
+  area clickable (not just the text), and it allows us to specify padding (and
+  height, width, margins, etc. if you want)*/
+  color: #e6e6e6;/*b5deca*/
+  text-align: left;
+  text-decoration: bold;
+  padding: 8px; /*Since block elements take up the full width available, they
+  cannot float next to each other. Therefore, specify some padding to make them look good*/
+  font-size: 14pt;
+  font-weight:bold;
+  line-height: 54px;
+}
+/*Size settings for the dropdown menu content*/
+.dropdown-content a {
+    line-height: 30px;
+    font-size: 12pt;
+}
+/* Change the link color on hover over either dropdown button, or the dropdown menu items. Also, rounded top borders */
+.header_navbar_ul li:hover > a {
+    background-color: #7e1002; /*#7e1002*/
+    color: white;
+    border-radius: 10px 10px 0 0;
+		text-decoration:none;
+}
+#header_navbar_home a:hover {
+    color: white;
+    border-radius: 10px 10px 0 0;
+		border-bottom: 2px solid black;
+}
+/*Top right rounded highlight for first list element in dropdown menu*/
+.dropdown-content a:first-child:hover {
+    border-radius: 0 10px 0 0;
+}
+/*bottom rounded highlights for last list element in dropdown menu*/
+.dropdown-content a:last-child:hover {
+    border-radius: 0 0 10px 10px;
+}
+.dropdown-content {
+    display: none;
+    position: absolute;
+    background-color: #c91a03;
+    box-shadow: 0px 8px 16px 0px rgba(0,0,0,0.2);
+    width: auto !important; /*to avoid line breaks*/
+    white-space: nowrap; /*to avoid line breaks*/
+    min-width: 200px;
+    border-radius: 0 10px 10px 10px;
+}
+.dropdown-content a {
+    color: white;
+    padding: 12px 16px;
+    text-decoration: none;
+    font-weight: bold;
+    display: block;
+}
+.dropdown-content a:hover {
+    background-color: #7e1002;/*#7e1002*/
+    /*border-radius: 10px 10px 10px 10px; /*Temprorary solution. I'd prefer to only round the corners that touch the corners of the menu*/
+    color: white;
+}
+.dropdown:hover .dropdown-content {
+    display: block;
+}
+/*Styling for <hr> between menu items in the dropdown menus*/
+.header_navbar_ul hr {
+    border: 0;
+    height: 1px;
+    background-image: linear-gradient(to right,  rgba(0, 0, 0, 0), #ccc,  rgba(0, 0, 0, 0));
+    margin-top: 0;
+    margin-bottom: 0;
+}
+/*I don't think this one does anything. Probably because of "".dropdown-content a:hover" covering it.*/
+.dropdown:hover .dropbtn {
+    background-color: #111;
+}
+/*---------------------------Footer with sponsor logos----------------------------*/
+footer hr  {
+    border: 0;
+    height: 0;
+    border-top: 1px solid rgba(0, 0, 0, 0.1);
+    border-bottom: 1px solid rgba(255, 255, 255, 0.3);
+    width: 80%;
+    text-align: center;
+    margin-left: 10%;
+    margin-top: 50px;
+}
+#footer-contact-row {
+  padding-bottom: 50px;
+}
+#footer-contact-row a {
+  color: #999999;
+	font-size: 15pt; /*35pt*/
+  text-decoration: none;
+  padding-left: 15px;
+  padding-right: 15px;
+}
+#footer-contact-row a:hover {
+  color: #c91a03;
+}
+#footer-contact-row hr  {
+    border: 0;
+    height: 0;
+    border-top: 1px solid rgba(0, 0, 0, 0.1);
+    /*border-bottom: 1px solid rgba(255, 255, 255, 0.3);*/
+    width: 30%;
+    text-align: center;
+    margin-left: 35%;
+    padding-bottom: 10px;
+}
+#logorow {
+  padding: 2em;
+}
+.footerimg {
+  max-height: 100px;
+  max-width: 250px;
+}
+.footerlogo {
+  display: inline-block;
+  vertical-align: bottom;
+  padding: 1.5em;
+}
+.footerlogo .on {
+  display: block;
+}
+.footerlogo:hover .on, .footerlogo .off {
+  display:none;
+}
+.footerlogo:hover .off {
+  display:block;
+}
+/* should we use this?
+.img-responsive {
+  display: inline;
+	position: center;
+}
+*/
+/*---------------------------Center page styling---------------------------------*/
+/*The wrap class is used for <div> in order to define a space where the
+side navigation bar and the page content (text, images etc) is kept*/
+.wrap {
+  /*width: 100%; /*auto--> 50%
+  text-align: center;
+  margin: auto; /*auto--> %
+  margin-top: 0px; /*40px --> 0px */
+	width: cover;
+  text-align: center;
+  margin: auto;
+  margin-top: 0px;
+	background:#e6e6e6;
+  background: -webkit-linear-gradient(left, rgba(1,0,0,0.2), rgba(1,0,0,0.05), rgba(1,0,0,0.2)); /* For Safari 5.1 to 6.0 */
+  background: -o-linear-gradient(right, rgba(1,0,0,0.2), rgba(1,0,0,0.05), rgba(1,0,0,0.2)); /* For Opera 11.1 to 12.0 */
+  background: -moz-linear-gradient(right, rgba(1,0,0,0.2), rgba(1,0,0,0.05), rgba(1,0,0,0.2)); /* For Firefox 3.6 to 15 */
+  background: linear-gradient(to right, rgba(1,0,0,0.2), rgba(1,0,0,0.05), rgba(1,0,0,0.2)); /* Standard syntax (must be last) */
+}
+/*Attempt to wrap header*/
+.wrap-header {
+	padding-right: auto; /*100--> 250*/
+  padding-left: auto; /*250--> 250px*/
+	background-color: #e6e6e6;
+}
+/*This is the content of the page, i.e. text images etc. Fits inside <div> with the wrap class*/
+.wrap-content {
+	/*margin-top:75px;
+  padding-right: 25%; /*100--> 450*/ /*25%
+  padding-left: 25%; /*250--> 450px*/ /*25%
+	background-color: transparent;
+	border-style:dashed;*/
+	margin-top:100px;
+	padding-right: 22%;
+  padding-left: 22%;
+	background-color: transparent;
+	/*background-size:50px;*/
+}
+/*Like .wrap-content, except with even margins, for pages where a side navigation bar is superfluous*/
+.wrap-content-no-side-nav {
+  padding-right: 75px;/*50px*/
+  padding-left: 75px;
+}
+.wrap-content, .wrap-content-no-side-nav {
+  padding-top: 25px;
+}
+/*----------------------------Side navigation bar-------------------------------*/
+#side-nav {
+    width: 25%;
+}
+#side-nav nav {
+    /*width: 225px;
+    padding-left: 25px;
+    padding-top:80px;
+    float: left;
+	border-style:dashed;*/
+	  width: 200px;
+    margin-left: 25px;
+    margin-top:140px;
+    float: left;
+		border-style:solid;
+		border-width:2px;
+		border-color:#000000;
+}
+#side-nav a {
+    color: #333;
+    display: block;
+    padding: 10px;
+    text-align: left;
+    text-decoration: none;
+		background: #e6e6e6;
+}
+#side-nav a.active {
+    background: #c91a03;/*before: fff7b5*/
+    color: black;
+	}
+#side-nav .sub-level {
+    padding-left: 30px;
+    /*font-size: 12pt;*/
+}
+#side-nav a:hover {
+  background: #ffffff; /*Before: #dcdbdb;*/
+  color: black;
+	font-weight:bold;
+	text-decoration:underline;
+	text-decoration-color:#c91a03;
+}
+.side-nav_stick {
+    position: fixed;
+    top: 0;
+    z-index: 0;
+    margin-top:120px;
+}
+.hidden-sidenav {
+  opacity: 0;
+}
+#side-nav nav {
+	-webkit-transition: opacity 200ms ease-in-out;
+    -moz-transition: opacity 200ms ease-in-out;
+    -ms-transition: opacity 200ms ease-in-out;
+    -o-transition: opacity 200ms ease-in-out;
+}
+/*---------------------------Profile card styling-------------------------------------*/
+.profile-table {
+  margin: 80px auto 0 auto;
+  border-spacing: 30px;
+}
+.profile-main {
+  height: 350px;
+  width: 300px;
+  position: relative;
+  margin: 20px;
+}
+.profile-container {
+  height: 350px;
+  width: 300px;
+  position: absolute;
+  display: block;
+  top: 0;
+  left: 0;
+	-webkit-border-radius: 5px;
+	-moz-border-radius: 5px;
+	border-radius: 5px;
+	box-shadow: 1px 1px 4px #666;
+	padding: 1%;
+	background: #fffef9;
+	background: rgb(252,255,244); /* Old browsers */
+	background: -moz-linear-gradient(top,  #fffffb 0%, #fffef9 100%); /* FF3.6+ */
+	background: -webkit-gradient(linear, left top, left bottom, color-stop(0%,rgba(252,255,244,1)), color-stop(100%,rgba(219,218,201,1))); /* Chrome,Safari4+ */
+	background: -webkit-linear-gradient(top,  #fffffb 0%,#fffef9 100%); /* Chrome10+,Safari5.1+ */
+	background: -o-linear-gradient(top,  #fffffb 0%,#fffef9 100%); /* Opera 11.10+ */
+	background: -ms-linear-gradient(top,  #fffffb 0%,#fffef9 100%); /* IE10+ */
+	background: linear-gradient(top,  #fffffb 0%,#fffef9 100%); /* W3C */
+	filter: progid:DXImageTransform.Microsoft.gradient( startColorstr='#fcfff4', endColorstr='#dbdac9',GradientType=0 ); /* IE6-9 */
+}
+.profile-image {
+  margin: 15px auto 0 auto;
+  height: 250px;
+  width: 250px;
+  position:relative;
+}
+.profile-content {
+  display: none;
+  width: 300px;
+  margin: 70px auto 0 auto;
+  text-align: justify;
+}
+.profile-container:hover .profile-content {
+  display: block;
+}
+.profile-container:hover .profile-image {
+  position: relative;
+}
+.profile-name {
+  padding-top: 15px;
+  color: #c91a03;
+  font-weight: bold;
+  font-size: 15pt;
+	text-align:center;
+}
+.profiletext {
+  font-size: 12pt;
+  margin-top: 10px;
+  line-height: 150%;
+  text-align: justify;
+}
+/*-------------------------------GENERAL STUFF-------------------------------------*/
+div {
+  text-align: center;
+}
+/*.header is the class used in the header image at the top of pages*/
+.header {
+  font-size: 45pt;
+  color: black;
+  text-align: center;
+  background-color: #000000;
+  background-repeat: repeat;
+  height: auto;
+  line-height: 130px;
+		border-bottom: 3px solid transparent; /*A lot of code for the line between the header and the body */
+  -moz-border-image: -moz-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%);*/
+  -webkit-border-image: -webkit-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%)*/
+  border-image: linear-gradient(to right, #000000 0%, #404040, #000000 100%); /*(to right, #016243 0%, #b5deca, #016243 100%);*/
+  border-image-slice: 1;
+  -webkit-touch-callout: none;
+  -webkit-user-select: none;
+  -khtml-user-select: none;
+  -moz-user-select: none;
+  -ms-user-select: none;
+  user-select: none;
+  cursor: default;
+  }
+.header_Modelling {
+  font-size: 45pt;
+  color: black;
+  text-align: center;
+	position:bottom;
+  background-image: url("pictures_17/backgrounds/chalkboard_2.jpg"); /*, linear-gradient(to bottom, #ffffff, #e6e6e6); /*#0c749c, #145071*/
+  min-height: 750px;
+	height:auto;
+  background-attachment: fixed;
+  background-position: center;
+  background-repeat: no-repeat;
+  background-size: cover;
+	background-color:#000000;
+	border-bottom: 3px solid transparent; /*A lot of code for the line between the header and the body */
+  -moz-border-image: -moz-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%);*/
+  -webkit-border-image: -webkit-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%)*/
+  border-image: linear-gradient(to right, #000000 0%, #404040, #000000 100%); /*(to right, #016243 0%, #b5deca, #016243 100%);*/
+  border-image-slice: 1;
+  -webkit-touch-callout: none;
+  -webkit-user-select: none;
+  -khtml-user-select: none;
+  -moz-user-select: none;
+  -ms-user-select: none;
+  user-select: none;
+  cursor: default;
+}
+.header_Achievements_biobricks {
+  font-size: 45pt;
+  color: black;
+  text-align: center;
+	position:bottom;
+  background-image: url("pictures_17/backgrounds/bricks.jpg"); /*, linear-gradient(to bottom, #ffffff, #e6e6e6); /*#0c749c, #145071*/
+  min-height: 750px;
+	height:auto;
+  background-attachment: fixed;
+  background-position: center;
+  background-repeat: no-repeat;
+  background-size: cover;
+	background-color:#000000;
+	border-bottom: 3px solid transparent; /*A lot of code for the line between the header and the body */
+  -moz-border-image: -moz-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%);*/
+  -webkit-border-image: -webkit-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%)*/
+  border-image: linear-gradient(to right, #000000 0%, #404040, #000000 100%); /*(to right, #016243 0%, #b5deca, #016243 100%);*/
+  border-image-slice: 1;
+  -webkit-touch-callout: none;
+  -webkit-user-select: none;
+  -khtml-user-select: none;
+  -moz-user-select: none;
+  -ms-user-select: none;
+  user-select: none;
+  cursor: default;
+}
+.header_Project_theory {
+  font-size: 45pt;
+  color: black;
+  text-align: center;
+	position:bottom;
+  background-image: url("pictures_17/backgrounds/pen_paper.jpg"); /*, linear-gradient(to bottom, #ffffff, #e6e6e6); /*#0c749c, #145071*/
+  min-height: 750px;
+	height:auto;
+  background-attachment: fixed;
+  background-position: center;
+  background-repeat: no-repeat;
+  background-size: cover;
+	background-color:#000000;
+	border-bottom: 3px solid transparent; /*A lot of code for the line between the header and the body */
+  -moz-border-image: -moz-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%);*/
+  -webkit-border-image: -webkit-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%)*/
+  border-image: linear-gradient(to right, #000000 0%, #404040, #000000 100%); /*(to right, #016243 0%, #b5deca, #016243 100%);*/
+  border-image-slice: 1;
+  -webkit-touch-callout: none;
+  -webkit-user-select: none;
+  -khtml-user-select: none;
+  -moz-user-select: none;
+  -ms-user-select: none;
+  user-select: none;
+  cursor: default;
+}
+.header_Project_constructs {
+  font-size: 45pt;
+  color: black;
+  text-align: center;
+	position:bottom;
+  background-image: url("pictures_17/backgrounds/DNA.jpg"); /*, linear-gradient(to bottom, #ffffff, #e6e6e6); /*#0c749c, #145071*/
+  min-height: 750px;
+	height:auto;
+  background-attachment: fixed;
+  background-position: center;
+  background-repeat: no-repeat;
+  background-size: cover;
+	background-color:#000000;
+	border-bottom: 3px solid transparent; /*A lot of code for the line between the header and the body */
+  -moz-border-image: -moz-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%);*/
+  -webkit-border-image: -webkit-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%)*/
+  border-image: linear-gradient(to right, #000000 0%, #404040, #000000 100%); /*(to right, #016243 0%, #b5deca, #016243 100%);*/
+  border-image-slice: 1;
+  -webkit-touch-callout: none;
+  -webkit-user-select: none;
+  -khtml-user-select: none;
+  -moz-user-select: none;
+  -ms-user-select: none;
+  user-select: none;
+  cursor: default;
+}
+.header_Team_students {
+  font-size: 45pt;
+  color: black;
+  text-align: center;
+	position:bottom;
+  background-image: url("pictures_17/team_photos_2017/group_photo_stairs_bw.jpg"); /*, linear-gradient(to bottom, #ffffff, #e6e6e6); /*#0c749c, #145071*/
+  min-height: 750px;
+	height:auto;
+  background-attachment: fixed;
+  background-position: center;
+  background-repeat: no-repeat;
+  background-size: cover;
+	background-color:#000000;
+	border-bottom: 3px solid transparent; /*A lot of code for the line between the header and the body */
+  -moz-border-image: -moz-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%);*/
+  -webkit-border-image: -webkit-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%)*/
+  border-image: linear-gradient(to right, #000000 0%, #404040, #000000 100%); /*(to right, #016243 0%, #b5deca, #016243 100%);*/
+  border-image-slice: 1;
+  -webkit-touch-callout: none;
+  -webkit-user-select: none;
+  -khtml-user-select: none;
+  -moz-user-select: none;
+  -ms-user-select: none;
+  user-select: none;
+  cursor: default;
+}
+.header_Team_supervisors {
+  font-size: 45pt;
+  color: black;
+  text-align: center;
+	position:bottom;
+  background-image: url("pictures_17/backgrounds/Chemistry_entrance_bw.jpg"); /*, linear-gradient(to bottom, #ffffff, #e6e6e6); /*#0c749c, #145071*/
+  /*min-height: 750px;
+	height:auto;*/
+  background-attachment: fixed;
+  background-position: center;
+  background-repeat: no-repeat;
+  background-size: cover;
+	background-color:#000000;
+	border-bottom: 3px solid transparent; /*A lot of code for the line between the header and the body */
+  -moz-border-image: -moz-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%);*/
+  -webkit-border-image: -webkit-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%)*/
+  border-image: linear-gradient(to right, #000000 0%, #404040, #000000 100%); /*(to right, #016243 0%, #b5deca, #016243 100%);*/
+  border-image-slice: 1;
+  -webkit-touch-callout: none;
+  -webkit-user-select: none;
+  -khtml-user-select: none;
+  -moz-user-select: none;
+  -ms-user-select: none;
+  user-select: none;
+  cursor: default;
+}
+.header_Team_sponsors {
+  font-size: 45pt;
+  color: black;
+  text-align: center;
+	position:bottom;
+  background-image: url("pictures_17/backgrounds/money_bw.jpg"); /*, linear-gradient(to bottom, #ffffff, #e6e6e6); /*#0c749c, #145071*/
+  /*min-height: 750px;
+	height:auto;*/
+  background-attachment: fixed;
+  background-position: center;
+  background-repeat: no-repeat;
+  background-size: cover;
+	background-color:#000000;
+	border-bottom: 3px solid transparent; /*A lot of code for the line between the header and the body */
+  -moz-border-image: -moz-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%);*/
+  -webkit-border-image: -webkit-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%)*/
+  border-image: linear-gradient(to right, #000000 0%, #404040, #000000 100%); /*(to right, #016243 0%, #b5deca, #016243 100%);*/
+  border-image-slice: 1;
+  -webkit-touch-callout: none;
+  -webkit-user-select: none;
+  -khtml-user-select: none;
+  -moz-user-select: none;
+  -ms-user-select: none;
+  user-select: none;
+  cursor: default;
+}
+.header_Team_collaborations {
+  font-size: 45pt;
+  color: black;
+  text-align: center;
+	position:bottom;
+  background-image: url("pictures_17/backgrounds/shaking_hands_darker.jpg"); /*, linear-gradient(to bottom, #ffffff, #e6e6e6); /*#0c749c, #145071*/
+  /*min-height: 750px;
+	height:auto;*/
+  background-attachment: fixed;
+  background-position: center;
+  background-repeat: no-repeat;
+  background-size: cover;
+	background-color:#000000;
+	border-bottom: 3px solid transparent; /*A lot of code for the line between the header and the body */
+  -moz-border-image: -moz-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%);*/
+  -webkit-border-image: -webkit-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%)*/
+  border-image: linear-gradient(to right, #000000 0%, #404040, #000000 100%); /*(to right, #016243 0%, #b5deca, #016243 100%);*/
+  border-image-slice: 1;
+  -webkit-touch-callout: none;
+  -webkit-user-select: none;
+  -khtml-user-select: none;
+  -moz-user-select: none;
+  -ms-user-select: none;
+  user-select: none;
+  cursor: default;
+}
+.header_Team_attributions {
+  font-size: 45pt;
+  color: black;
+  text-align: center;
+	position:bottom;
+  background-image: url("pictures_17/backgrounds/Chalmers_port_4.jpg"); /*, linear-gradient(to bottom, #ffffff, #e6e6e6); /*#0c749c, #145071*/
+  /*min-height: 750px;
+	height:auto;*/
+  background-attachment: fixed;
+  background-position: center;
+  background-repeat: no-repeat;
+  background-size: cover;
+	background-color:#000000;
+	border-bottom: 3px solid transparent; /*A lot of code for the line between the header and the body */
+  -moz-border-image: -moz-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%);*/
+  -webkit-border-image: -webkit-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%)*/
+  border-image: linear-gradient(to right, #000000 0%, #404040, #000000 100%); /*(to right, #016243 0%, #b5deca, #016243 100%);*/
+  border-image-slice: 1;
+  -webkit-touch-callout: none;
+  -webkit-user-select: none;
+  -khtml-user-select: none;
+  -moz-user-select: none;
+  -ms-user-select: none;
+  user-select: none;
+  cursor: default;
+}
+.header_Notebook_journal {
+  font-size: 45pt;
+  color: black;
+  text-align: center;
+	position:bottom;
+  background-image: url("pictures_17/backgrounds/Notebook.jpg"); /*, linear-gradient(to bottom, #ffffff, #e6e6e6); /*#0c749c, #145071*/
+  /*min-height: 750px;
+	height:auto;*/
+  background-attachment: fixed;
+  background-position: center;
+  background-repeat: no-repeat;
+  background-size: cover;
+	background-color:#000000;
+	border-bottom: 3px solid transparent; /*A lot of code for the line between the header and the body */
+  -moz-border-image: -moz-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%);*/
+  -webkit-border-image: -webkit-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%)*/
+  border-image: linear-gradient(to right, #000000 0%, #404040, #000000 100%); /*(to right, #016243 0%, #b5deca, #016243 100%);*/
+  border-image-slice: 1;
+  -webkit-touch-callout: none;
+  -webkit-user-select: none;
+  -khtml-user-select: none;
+  -moz-user-select: none;
+  -ms-user-select: none;
+  user-select: none;
+  cursor: default;
+}
+.header_Notebook_lab_safety {
+  font-size: 45pt;
+  color: black;
+  text-align: center;
+	position:bottom;
+  background-image: url("pictures_17/backgrounds/T--Chalmers-Gothenburg--Safety_form_Fume_hood.jpg"); /*, linear-gradient(to bottom, #ffffff, #e6e6e6); /*#0c749c, #145071*/
+  /*min-height: 750px;
+	height:auto;*/
+  background-attachment: fixed;
+  background-position: center;
+  background-repeat: no-repeat;
+  background-size: cover;
+	background-color:#000000;
+	border-bottom: 3px solid transparent; /*A lot of code for the line between the header and the body */
+  -moz-border-image: -moz-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%);*/
+  -webkit-border-image: -webkit-linear-gradient(left, #000000 0%, #404040, #000000 100%); /*(left, #016243 0%, #b5deca, #016243 100%)*/
+  border-image: linear-gradient(to right, #000000 0%, #404040, #000000 100%); /*(to right, #016243 0%, #b5deca, #016243 100%);*/
+  border-image-slice: 1;
+  -webkit-touch-callout: none;
+  -webkit-user-select: none;
+  -khtml-user-select: none;
+  -moz-user-select: none;
+  -ms-user-select: none;
+  user-select: none;
+  cursor: default;
+}
+.header-text {
+  background-color: transparent;
+  font-size: 80pt; /*35pt*/
+	font-family:"Lato", cursive; /*Dancing Script, Marck Script, Tangerine, Sacramento, Allura, Parisienne, Montez*/
+  color:#c91a03 ;/*Alternatives: #c91a03, #ff0000, #fd4833, #fb2104*/
+	line-height: 45pt;
+  padding-top: 650px;
+	font-weight: bold;
+	text-align:left;
+	margin-left:200px;
+	text-shadow: 5px 5px #000000;
+}
+.header-subtitle {
+  color:#c91a03;
+  font-size: 40pt;
+	line-height: 30pt;
+  padding-top: 1%;
+  padding-bottom: 20px;
+	font-family:"Lato", cursive; /*Dancing Script, Marck Script, Tangerine, Sacramento, Allura, Parisienne, Montez*/
+	text-align:left;
+	margin-left:200px;
+	text-shadow: 2px 2px #000000;
+}
+/*.header-text {
+	display:inline-block;
+	background-color:transparent;
+  font-size: 60pt;
+	font-family:"Lato", cursive; /*Dancing Script, Marck Script, Tangerine, Sacramento, Allura, Parisienne, Montez
+  color:#c91a03;
+	position:absolute;
+	top: 850px;
+  left: 200px;
+  width: 800px;
+  height: 100px;
+	line-height: 45pt;
+	font-weight: bold;
+	text-align:left;
+	margin-right:100%;
+/*
+	padding-top: 10%;
+	margin-left:200px;
+}
+.header-subtitle {
+  color:#c91a03;
+	background-color:transparent;
+  font-size: 30pt;
+  line-height: 20pt;
+  /*padding-top: 8px;
+  padding-bottom: 20px;
+	font-family:"Lato", cursive; /*Dancing Script, Marck Script, Tangerine, Sacramento, Allura, Parisienne, Montez
+	text-align:left;
+	/*margin-left:225px;
+	position:absolute;
+	top: 920px;
+  left: 240px;
+  width: 400px;
+  height: 100px;
+}*/
+/*--------------Basic text and content styling---------------------------------*/
+p {
+  font-size: 12pt;
+  text-align: justify;
+  text-justify: inter-word;
+  /*text-indent: 20px;*/
+  line-height: 200%;
+  margin-bottom: 10px;
+	color:#1a1a1a;
+}
+h2 {
+	text-align:left;
+}
+.h2style {
+	color:#1a1a1a;
+	font-size:30pt;
+	border-bottom: 2px solid transparent; /*A lot of code for the line between the header and the body */
+  -moz-border-image: -moz-linear-gradient(left, #1a1a1a 0%, #808080, #1a1a1a 100%); /*(left, #016243 0%, #b5deca, #016243 100%);*/
+  -webkit-border-image: -webkit-linear-gradient(left, #1a1a1a 0%, #808080, #1a1a1a 100%); /*(left, #016243 0%, #b5deca, #016243 100%)*/
+  border-image: linear-gradient(to right, #1a1a1a 0%, #808080, #1a1a1a 100%); /*(to right, #016243 0%, #b5deca, #016243 100%);*/
+  border-image-slice: 1;
+  -webkit-touch-callout: none;
+  -webkit-user-select: none;
+  -khtml-user-select: none;
+  -moz-user-select: none;
+  -ms-user-select: none;
+  user-select: none;
+  cursor: default;
+}
+h3 {
+  text-align: left;
+  /*margin-top: 10px;*/
+}
+figcaption {
+  color: black;
+  font-size: 11pt;
+  text-align: justify;
+  padding-left: 80px;
+	margin-left:50px;
+  /*background-color: #cccccc;*/
+}
+figcaption b {
+  color: #c91a03;
+}
+.subtitle {
+  text-align:left;
+  padding: 5px;
+  font-size: 20pt !important;
+  background-color: #971402;
+  color: white !important;
+  line-height: 150% !important;
+  font-weight:600 !important;
+}
+.list {
+  margin-left:50px;
+  list-style-position: outside;
+  text-align: justify;
+  text-justify: inter-word;
+  margin-top: 10px;
+  font-size: 12pt;
+  line-height: 125%;
+}
+.sponsor-picture {
+	display: inline-block;
+	border-style:dashed;
+	width: 500px;
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+    </div>
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+    <div class="wrap"><!-- To limit the width of the site-->
+        <div class="header_Project_theory">
+    <div class="header-text">PROJECT</div>
+    <div class="header-subtitle">Design and Idea</div>
+    </div>
+  <!------------------------This is the side navigation bar---------------------------->
+      <aside id="side-nav">
+        <div id="nav-anchor">
+          <nav>
+            <a href="#intro" class="active">Introduction</a>
+            <a href="#background">Background</a>
+            <a href="#basic_design">The basic design</a>
+            <a class="sub-level" href="#signal">Signal</a>
+            <a class="sub-level" href="#switch">The switch</a>
+            <a class="sub-level" href="#AND-gate">The AND-gate</a>
+            <a href="#references">References</a>
+          </nav>
+        </div>
+      </aside>
+      <div class="wrap-content">
+         <div class="target" id="intro">
+         <h4 class="subtitle">Introduction</h4>
+         <p class="text">
+           <b>Lung cancer</b> is one of the most common cancer forms today and due to the lack of good
+           detection methods, it is one of the deadliest. A suggested approach for detection of
+           the disease have been to use specific <b>volatile organic compounds</b> (VOCs) that
+           have been found in elevated levels in the breath of lung cancer patients.
+         </p>
+         <p class=text>
+           <b>The aim</b> of this project is to detect two of these VOCs with a biosensor made of <i>Saccharomyces
+           cerevisiae</i>. The VOCs will be detected by <b>G-protein coupled receptors</b> (GPCRs) which will
+           activate the pheromone pathway and initiate mating between a- and α-cells. The output, a
+           colony color change, will be achieved by partially deleting the ADE2 gene with a CRISPR/Cas9
+           system in the fused cell. The colony color change in the biosensor will indicate presence of
+           VOCs in the patient’s exhaled breath.
+         </p>
+         </div>
+         <div class="target" id="background">
+         <h4 class="sidetitle">Background</h4>
+         <p class="text">
+          <b>Lung cancer</b> is the cancer form with the highest death toll, causing <b>1.69 million
+           deaths</b> globally in 2015 [1]. One reason for the high death toll is the late detection
+           of the cancer, due to symptoms starting to appear first in the later stages.
+         </p>
+         <p class="text">
+           Three different stages are used to characterize lung cancer; I, II and III. The magnitude of
+           the disease is based on size of the tumor and the extent of spreading to lymph nodes
+           and other organs in the body [2]. A majority of the lung cancer cases today are diagnosed
+           in stage III with a <b>5-year survival rate of 5-14%</b>. An earlier detection at stage I or II
+           would increase the figure to <b>45-49%</b> which clearly show that early detection is a key to
+           increase the chances of surviving lung cancer [3].
+           </p>
+          <p class="text">
+           At the appearance of the first symptoms,
+           a <b>chest X-ray</b> is used today to scan for tumors. The scan can indicate the position and size
+           of the tumors. Despite being a non-invasive procedure, it is accompanied by <b>limitations</b> such
+           as cost, time, expertise and sensitivity required for X-ray examinations. Due to these
+           limitations X-ray is not suitable for mass screening of lung cancer [4]. Therefore, there is
+           a demand for new diagnostic tools with mass screening potential.
+         </p>
+         <p class="text">
+           One of the most promising
+           approaches for early diagnosis is the use of <b>volatile organic compounds</b> (VOCs) as biomarkers.
+           Some specific VOCs can be detected in higher concentrations in the breath of lung cancer
+           patients compared to healthy individuals. The cancer cells have an increased oxidative rate
+           that results in oxidative stress products found in the breath of patients. <b>The low concentration</b>
+           of VOCs, pmol/l, has provided a challenge in using this approach for detection and no efficient
+           screening method is yet available [5].
+         </p>
+         <p class="text">
+           Studies have shown that there is <b>not only one</b> compound
+           that can be used as a biomarker in the breath of cancer patients [5]. An optimal diagnostic tool
+           would therefore be able to detect multiple VOCs in order to decrease the number of false positive
+           diagnoses. One example of a diagnostic tool is a <b>biosensor</b>, which is a biological-based system
+           that can detect the presence of a target analyte and convert it into a signal. A biosensor can be
+           developed with many different species, antibodies or enzymes. In this case, a yeast-based biosensor
+           is a suitable approach and some of the advantages are the ease of genetic manipulation, as well as
+           the <b>cheap and easy cultivation</b> [6].
+         </p>
+         <p class="text">
+           To create this novel, non-invasive diagnostic tool, the budding
+           yeast <b><i>Saccharomyces cerevisiae</i></b> will be used as a biosensor. The yeast cells will have two xenogeneic
+           olfactory receptors incorporated. These can bind the biomarkers that are present in the breath of
+           lung cancer patients, opening up for the possibility to <b>detect the cancer</b> before any symptoms appears.
+         </p>
+         </div>
+         <div class="target" id="basic_design">
+         <h4 class="sidetitle">The basic design</h4>
+         <p class="text">
+           The aim of this project is to detect two VOCs that have been found in elevated
+           levels in the breath of lung cancer patients, using a biosensor made of <i>Saccharomyces
+           cerevisiae</i>. Two of the most commonly found VOCs; butanone and n-octanal, are chosen for
+           detection [7, 8]. The VOCs will be detected by G-protein coupled receptors (GPCRs) with the
+           ability to activate the pheromone pathway in yeast and thereby initiate mating between a- and
+           α-cells. The final output will be a color change due to deletion of the ADE2 gene using gRNA
+           and Cas9. The colony color change will indicate the presence of the target VOCs, and thereby
+           also lung cancer.
+         </p>
+           <img src="pictures_17/text_figures/yeast map design.png" alt="Design" style="height:75%; width:75%;">
+         </div>
+        <div class="target" id="signal">
+        <h2 class="h2style">Signal</h2>
+          <p class="text">
+           Natively, yeast have GPCRs regulating the cell mating through activation of the pheromone
+           pathway. These GPCRs, STE2 and STE3, are present respectively in the two different mating
+           types of yeast, a and α. STE2 and STE3 bind pheromones from the other mating type and activate
+           the pheromone pathway which enables mating. In our system STE2 and STE3 will be replaced with
+           the GPCRs RatI7 and Olfr1258 which detect the two VOCs butanone and n-octanal respectively [7,8].
+           When the GPCRs sense the VOCs, the pheromone pathway will be activated which will result in
+           expression of both the native mating genes and cloned Cre recombinase in both cell types. Binding
+           of butanone and n-octanal will therefore lead to mating of cells, but solely if both VOCs are present.
+           </p>
+           <img src="pictures_17/text_figures/Module_1.png" alt="Signal" style="height:75%; width:75%;">
+        </div>
+        <div class="target" id="switch">
+          <h2 class="h2style">Switch</h2>
+          <p class="text">
+           In each mating type, a system will be integrated consisting of a promoter in the wrong direction,
+           surrounded by mutated LoxP sites, followed by genes expressing either gRNAs or Cas9, see Figure 2.
+           Presence of the two VOCs will lead to, besides mating, activation of the respective genes since Cre
+           recombinase will turn the promoters in to the right direction. The loxP sites are mutated, preventing
+           the sites from inverting back to their original direction. This ensures that the promoter is inverted
+           into the correct direction upon GPCR activation and remains in that position [9].
+         </p>
+           <img src="pictures_17/text_figures/Module3.png" alt="Switch" style="height:100%; width:100%;">
+        </div>
+        <div class="target" id="AND-gate">
+         <h2 class="h2style">AND-gate</h2>
+         <p class="text">
+           For expression of the gRNAs, the promoter pSNR52 will be used. Together with polymerase III,
+           the pSNR52 promoter will not translate the  gRNAs into proteins [10]. Two different gRNAs will
+           be expressed by the same promoter and the two gRNAs will be linked together with two ribozymes;
+           HDV and Hammerhead. After expression, the ribozymes will cut themselves out and leave the two gRNAs
+           as separate sequences [11]. For the other mating type, the strong constitutive promoter pTEF1 will
+           be used to express the Cas9 protein. Since the gRNAs and the Cas9 protein are expressed in cells of
+           different mating types, the lack of combination will will not disrupt the adenine synthesis in haploid cells.
+         </p>
+         <img src="pictures_17/text_figures/AND_gate.png" alt="AND-gate" style="height:15%; width:30%;">
+         <p class="text">
+          After mating, the gRNAs and Cas9 will both be present in the newly formed diploid cell and assemble into a
+           functional unit. The gRNAs will guide the Cas9 protein to two sites in the ADE2 gene, creating two different
+           double strand breaks in the gene. The double stranded breaks will lead to a partial disruption of the ADE2 gene
+           and an accumulation of a red intermediate during adenine synthesis [12]. The color shift of the cells from white
+           to red will function as a diagnostic response.
+         </p>
+        </div>
+        <div class="target" id="references">
+         <h4 class="sidetitle">References</h4>
+         <p class="text">
+          [1] Cancer [Internet]. World Health Organization; 2017 [Cited 2017-06-13]. Available at: http://www.who.int/mediacentre/factsheets/fs297/en/ <br>
+          [2] Non-Small Cell Lung Cancer Stages [Internet]. American Cancer Society; 2016 [Updated 2017-03-02; Cited 2017-06-26]. Available at:
+          https://www.cancer.org/cancer/non-small-cell-lung-cancer/detection-diagnosis-staging/staging.html <br>
+          [3] Non-Small Lung Cancer Survival Rates, by Stage [Internet]. American Cancer Society; 2016 [Updated 2016-05-16; Cited 2017-06-13]. Available at:
+          https://www.cancer.org/cancer/non-small-cell-lung-cancer/detection-diagnosis-staging/survival-rates.htm <br>
+          [4] Lungcancer [internet]. Cancerfonden; 2015 [Cited 2017-06-07] Availiable at: https://www.cancerfonden.se/om-cancer/lungcancer <br>
+          [5] D’Amico A, Pennazza G, Santonico M, Martinelli E, Roscioni C, Galluccio G, Paolesse R, and Di Natale C. An investigation on electronic nose
+           diagnosis of lung cancer. Lung Cancer, 68(2):170–176, 2010. <br>
+          [6] French C, de Mora K, Joshi N, Elfick A, Haseloff J, and Ajioka J. Synthetic biology and the art of biosensor design. The Science and Applications
+           of Synthetic and Systems Biology:Workshop Summary, 2011. <br>
+          [7] Minic J, Persuy M-A, Godel E, Aioun J, Connerton I, Salesse R, and Pajot-Augy E.
+          Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening. FEBS Journal, 272(2):524-537, 2005. <br>
+          [8] Suzuki Y and Shimono K. Deciphering the receptor repertoire encoding specific odorants by time-lapse single-cell array cytometry. Scientic Reports, 6(19934):1-9, 2015. <br>
+          [9] Carter Z and Delneri D. New generation of loxp-mutated deletion cassettes for the genetic manipulation of yeast natural isolates. Yeast, 27(9):765-775, 2010. <br>
+          [10] DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, and Church GM. Genome engineering in saccharomyces cerevisiae using crispr-cas systems.
+           Nucleic Acids Research, 41(7):4336-4343, 2013. <br>
+          [11] Fujita T and Fujii H. Applications of engineered dna-binding molecules such as tal proteins and the crispr/cas system in biology research.
+           International Journal of Molecular Sciences, 16(10):23143-23164, 2015.<br>
+          [12] Ugolini S and Bruschi CV. The red/white colony color assay in the yeast saccharomyces cerevisiae: epistatic growth advantage of white ade8-18,
+           ade2 cells over red ade2 cells. Current Genetics, 30(6):485-492, 1996. <br>
+          [13] Kofahl, B. and Klipp, E., Modelling the dynamics of the yeast pheromone pathway. Yeast, 21: 831–850, 2004 <br>
+         </p>
+        </div>
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+            <img src="pictures_17/logotypes/neb_logo2_30percentgray.png" class="footerimg on">
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+          <a href="http://www.eurofins.com/">
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+            <img src="pictures_17/logotypes/eurofins_logo2.png" class="footerimg off">
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+            <img src="pictures_17/logotypes/chalmers_logo2.png" class="footerimg off">
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+        </div>
+        <div class="footerlogo">
+          <a href="http://www.sysbio.se/">
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+        <div class="footerlogo">
+          <a href="https://www.atum.bio/">
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