Latest revision as of 19:44, 27 October 2013

Uppsala iGEM 2013

Chassi

Lactobacillus as the new probiotic chassi for iGEM

There are many challenges for anyone who wants to do synthetic biology in a new less common organism, like getting familiar with new protocols and techniques. But another major problem is that there is a lack of fundamental parts available like plasmids, reporter genes and promoters. A big part of our project has been to develop these basic parts and make them biobrick compatible. This is done in order to open up the Lactobacillus genus as the new probiotic chassi of choice for the iGEM community. We also intend to use it ourselves for doing metabolic engineering in Lactobacillus. For this we have worked to create two shuttle vectors , a library of constitutive promoters and a couple of reporter genes that should be functional in both E. coli and Lactobacillus. E. coli is substantially easier and faster to work with. Therefore the intention is to allow construction and some characterisation to first be done in E. coli, the construct can then later be transformed to Lactobacillus.

Promoters

Different constructs need different levels of expression. We have constructed and characterised a library of constitutive promoters of different strengths. These are based on a consensus sequence from promoters in Lactococcus lactis but have also been shown to work very well in E. coli and Lactobacillus.

Reporter genes

Many techniques and methods require fluorescent reporter genes to function. While some fluorescent proteins work poorly in Lactobacillus we have biobricked a codon-optimized version of mCherry that has been shown to work. This has been done according to assembly standard 25 to allow it to be used as a fusion protein.

Toxin-antitoxin

A major challenge in synthetic biology is that there is a selective pressure against metabolically taxing systems. In nature, many natural plasmids contain genes coding for a toxin and the associated antitoxin. These systems can significantly increase the segregational stability of plasmids and is an easy to use alternative to chromosomal integration for some applications.

Vectors

Perhaps the most important chassi parts we have constructed are shuttle vectors that work both in Lactobacillus and E. coli. Because Lactobacillus is significantly harder to work with and ligations are hard to transform, constructs should first be assembled and prepped in E. coli and then transferred to Lactobacillus with the shuttle vector.

Signal peptides

Lactobacillus is a great genus for the secretion of proteins and contains several secretory pathways. In order for a protein to be exported it needs to be tagged with a short amino acid sequence at the N-terminus called a signal peptide. We have synthesised a signal peptide that can be assembled with a protein of choice.

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+<title>Uppsala iGEM 2013</title>
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+			<div id="igem_logo"><a href="https://2013.igem.org/Team:Uppsala"><img class="igem_logo" src="https://static.igem.org/mediawiki/2013/e/e0/Uppsala2013_IGEMlogo.png"></a></div>
+			<div id="igem"><a href="https://2013.igem.org/Main_Page"><img class="igem" src="https://static.igem.org/mediawiki/2013/b/b8/Uppsala2013_Uppsala_IGEM_log_blue.png"></a></div>
+<img class="slogan" src="https://static.igem.org/mediawiki/2013/a/ad/Uppsala2013_Header.png">
+	</div>
+		<!-- Contains the "nav div" and the stripe background for the navigation menu -->
+		<div id="navbar">
+		<!-- Contains the navigation menu with list elements, buged on local server, works on mediawiki!!-->
+			<div id="nav">
+				<div id="cow-box"><a href="https://2013.igem.org/Team:Uppsala"><img class="home-knapp" src="https://static.igem.org/mediawiki/2013/e/ef/Uppsala2013_Home.png"><a/></div>
 				<ul>
-				<li><a href="https://2013.igem.org/Team:Uppsala/project" id="list_type1"><img class="nav-text" src="https://static.igem.org/mediawiki/2013/9/92/Project.png"></a>
+				<li><a href="https://2013.igem.org/Team:Uppsala/project" id="list_type1"><img class="nav-text" src="https://static.igem.org/mediawiki/2013/a/aa/Uppsala2013_Project.png"></a>
 					<ul>
-						<li><a href="https://2013.igem.org/Team:Uppsala/overview">Overview</a></li>
 						<li><a href="https://2013.igem.org/Team:Uppsala/chassi">Chassi</a>
 							<ul>
@@ Line 9: / Line 70: @@
 								<li><a href="https://2013.igem.org/Team:Uppsala/promoters">Promoters</a></li>
 								<li><a href="https://2013.igem.org/Team:Uppsala/reporter-genes">Reporter genes</a></li>
-								<li><a href="https://2013.igem.org/Team:Uppsala/affinity-tags">Toxin-antitoxin system</a></li>
+								<li><a href="https://2013.igem.org/Team:Uppsala/toxin-antitoxin-system">Toxin-antitoxin system</a></li>
 								<li><a href="https://2013.igem.org/Team:Uppsala/vectors">Vectors</a></li>
                                                                  <li><a href="https://2013.igem.org/Team:Uppsala/signal-peptide">Signal peptide</a></li>
@@ Line 16: / Line 77: @@
                                                  <li><a href="https://2013.igem.org/Team:Uppsala/metabolic-engineering">Metabolic engineering</a>
                                                      <ul>
-                                                                 <li><a href="https://2013.igem.org/Team:Uppsala/p-coumaric-acid">P-coumeric acid</a></li>
+                                                                 <li><a href="https://2013.igem.org/Team:Uppsala/p-coumaric-acid">p-Coumaric acid</a></li>
                                                                  <li><a href="https://2013.igem.org/Team:Uppsala/resveratrol">Resveratrol</a></li>
 								<li><a href="https://2013.igem.org/Team:Uppsala/lycopene">Lycopene</a></li>
 								<li><a href="https://2013.igem.org/Team:Uppsala/betacarotene">β-carotene</a></li>
-								<li><a href="https://2013.igem.org/Team:Uppsala/astraxantin">Astraxantin</a></li>
+                                                                <li><a
-								<li><a href="https://2013.igem.org/Team:Uppsala/zeaxantin">Zeaxantin</a></li>
+href="https://2013.igem.org/Team:Uppsala/saffron">Saffron</a></li>
+								<li><a href="https://2013.igem.org/Team:Uppsala/astaxanthin">Astaxanthin</a></li>
+								<li><a href="https://2013.igem.org/Team:Uppsala/zeaxanthin">Zeaxanthin</a></li>
 							</ul></li>
 </li>
-						<li><a href="https://2013.igem.org/Team:Uppsala/chromoproteins">Chromoprotein</a></li>
+                                                <li><a
-						<li><a href="https://2013.igem.org/Team:Uppsala/safety">Safety experiments</a></li>
+href="https://2013.igem.org/Team:Uppsala/miraculin">Miraculin</a></li>
-						<li><a href="https://2013.igem.org/Team:Uppsala/result">Result</a></li>
+						<li><a href="https://2013.igem.org/Team:Uppsala/chromoproteins">Chromoproteins</a></li>
+						<li><a href="https://2013.igem.org/Team:Uppsala/safety-experiment">Safety experiment</a></li>
+						<li><a href="https://2013.igem.org/Team:Uppsala/results">Results</a></li>
 						</ul></li>
-				<li><a href="https://2013.igem.org/Team:Uppsala/modeling" id="list_type1"><img class="nav-text" src="https://static.igem.org/mediawiki/2013/3/3c/Modeling.png"></a>
+				<li><a href="https://2013.igem.org/Team:Uppsala/modeling" id="list_type1"><img class="nav-text" src="https://static.igem.org/mediawiki/2013/6/63/Uppsala2013_Modeling.png"></a>
 					<ul>
-						<li><a href="https://2013.igem.org/Team:Uppsala/resveratrol-pathway">Resveratrol pathway </a></li>
+						<li><a href="https://2013.igem.org/Team:Uppsala/P-Coumaric-acid-pathway">Kinetic model</a></li>
-						<li><a href="https://2013.igem.org/Team:Uppsala/carotenoids-pathway">Carotenoids pathway </a></li>
+						<li><a href="https://2013.igem.org/Team:Uppsala/modeling-tutorial">Modeling tutorial </a></li>
+<li><a href="https://2013.igem.org/Team:Uppsala/toxicity-model">Toxicity model</a></li>
 					</ul></li>
-				<li><a href="https://2013.igem.org/Team:Uppsala/parts" id="list_type2"><img class="nav-text" src="https://static.igem.org/mediawiki/2013/7/79/Parts.png"></a></li>
+				<li><a href="https://2013.igem.org/Team:Uppsala/parts" id="list_type2"><img class="nav-text" src="https://static.igem.org/mediawiki/2013/e/eb/Uppsala2013_parts.png"></a></li>
-				<li><a href="https://2013.igem.org/Team:Uppsala/team" id="list_type2"><img class="nav-text" src="https://static.igem.org/mediawiki/2013/1/14/213124236426467234638642.png"></a>
+				<li><a href="https://2013.igem.org/Team:Uppsala/team" id="list_type2"><img class="nav-text" src="https://static.igem.org/mediawiki/2013/a/a7/Uppsala2013_Team.png"></a>
 					<ul>
 						<li><a href="https://2013.igem.org/Team:Uppsala/resveratrol-group">Resveratrol group</a></li>
 						<li><a href="https://2013.igem.org/Team:Uppsala/carotenoid-group">Carotenoid group</a></li>
 						<li><a href="https://2013.igem.org/Team:Uppsala/chassi-group">Chassi group</a></li>
-						<li><a href="https://2013.igem.org/Team:Uppsala/advisors">Advisors</a></li>
+                                                <li><a href="https://2013.igem.org/Team:Uppsala/advisors">Advisors</a></li>
 					</ul></li>
-				<li><a href="https://2013.igem.org/Team:Uppsala/hp" id="list_type3"><img class="nav-text" src="https://static.igem.org/mediawiki/2013/f/f5/HP.png"></a>
+				<li><a href="https://2013.igem.org/Team:Uppsala/human-practice" id="list_type3"><img class="nav-text"       src="https://static.igem.org/mediawiki/2013/b/b8/Uppsala2013_HP.png"></a>
 					<ul>
-						<li><a href="https://2013.igem.org/Team:Uppsala/Yoghurtproducts">Yoghurt products</a></li>
+						<li><a href="https://2013.igem.org/Team:Uppsala/yoghurt">Yoghurt +</a></li>
-					<li><a href="https://2013.igem.org/Team:Uppsala/synbioday">SynBio Day</a></li>
+					<li><a href="https://2013.igem.org/Team:Uppsala/synbioday">SynBioDay</a></li>
-						<li><a href="https://2013.igem.org/Team:Uppsala/bioethics">Bioethics</a></li>
+						<li><a href="https://2013.igem.org/Team:Uppsala/biosafety-and-ethics">Biosafety and ethics</a></li>
-						<li><a href="https://2013.igem.org/Team:Uppsala/safety">Bioethics and Safety</a></li>
+						<li><a href="https://2013.igem.org/Team:Uppsala/public-opinion">Public opinion </a></li>
-						<li><a href="https://2013.igem.org/Team:Uppsala/society">Society and Outreach</a></li>
+                                                <li><a href="https://2013.igem.org/Team:Uppsala/Outreach">High school & media </a></li>
-						<li><a href="https://2013.igem.org/Team:Uppsala/highschool-outreach">High School Outreach</a></li>
+<li><a href="https://2013.igem.org/Team:Uppsala/bioart">BioArt</a></li>
-                                                <li><a href="https://2013.igem.org/Team:Uppsala/outreach">Media Outreach</a></li>
+<li><a href="https://2013.igem.org/Team:Uppsala/LactonutritiousWorld">A LactoWorld</a></li>
+<li><a href="https://2013.igem.org/Team:Uppsala/killswitches">Killswitches</a></li>
+<li><a href="https://2013.igem.org/Team:Uppsala/realization">Patent</a></li>
 					</ul></li>
-				<li><a href="https://2013.igem.org/Team:Uppsala/attribution" id="list_type4"><img class="nav-text" src="https://static.igem.org/mediawiki/2013/8/8d/Attributions.png"></a></li>
+				<li><a href="https://2013.igem.org/Team:Uppsala/attribution" id="list_type4"><img class="nav-text" src="https://static.igem.org/mediawiki/2013/5/5d/Uppsala2013_Attributions.png"></a></li>
-				<li><a href="https://2013.igem.org/Team:Uppsala/notebook" id="list_type3"><img class="nav-text" src="https://static.igem.org/mediawiki/2013/9/9f/Notebook_Uppsala_button.png"></a>
+				<li><a href="https://2013.igem.org/Team:Uppsala/notebook" id="list_type3"><img class="nav-text" src="https://static.igem.org/mediawiki/2013/3/36/Uppsala2013_Notebook.png"></a>
                                       <ul>
-                                         <li><a href="https://2013.igem.org/Team:Uppsala/protocol">Protocol</a></li>
+                                         <li><a href="https://2013.igem.org/Team:Uppsala/safety-form">Safety form</a></li>
-                                         <li><a href="https://2013.igem.org/Team:Uppsala/lab-journal">Lab journal</a></li>
+                                         <li><a href="https://2013.igem.org/Team:Uppsala/protocols">Protocols</a></li>
                                       </ul></li>
 				</ul>
+			</div>
+		</div>
+	<div id="main_content"> <!-- Put content here -->
+        <h1 class="main-title"> Chassi </h1>
+<h1 class="main-title_no_border"> Lactobacillus as the new probiotic chassi for iGEM </h1>
+There are many challenges for anyone who wants to do synthetic biology in a new less common organism, like getting familiar with new protocols and techniques. But another major problem is that there is a lack of fundamental parts available like plasmids, reporter genes and promoters. A big part of our project has been to develop these basic parts and make them biobrick compatible. This is done in order to open up the Lactobacillus genus as the new probiotic chassi of choice for the iGEM community. We also intend to use it ourselves for doing metabolic engineering in Lactobacillus. For this we have worked to create two <a href="https://2013.igem.org/Team:Uppsala/vectors"> shuttle vectors </a>, a library of <a href="https://2013.igem.org/Team:Uppsala/promoters"> constitutive promoters </a> and a couple of <a href="https://2013.igem.org/Team:Uppsala/reporter-genes"> reporter genes </a> that should be functional in both E. coli and Lactobacillus.
+  E. coli is substantially easier and faster to work with. Therefore the intention is to allow construction and some characterisation to first be done in E. coli, the construct can then later be transformed to Lactobacillus.
+	<div id="shortcut_pic">
+		<div id="chassi_pic_container_a">
+			<div id="chassi_pic_promoter">
+<a href="https://2013.igem.org/Team:Uppsala/promoters"> <img class="chassi_pic" src="https://static.igem.org/mediawiki/2013/9/98/Uppsala2013_Promoter_new.png" onmouseover="this.src='https://static.igem.org/mediawiki/2013/e/e2/Uppsala2013_PromoterPushed.png'" onmouseout="this.src='https://static.igem.org/mediawiki/2013/9/98/Uppsala2013_Promoter_new.png'" /> </a>
+</div>
+			<div id="chassi_pic_reporter">
+<a href="https://2013.igem.org/Team:Uppsala/reporter-genes"> <img class="chassi_pic" src="https://static.igem.org/mediawiki/2013/c/cb/Uppsala2013_Reporter_new.png" onmouseover="this.src='https://static.igem.org/mediawiki/2013/0/0e/Uppsala2013_ReporterPushed.png'" onmouseout="this.src='https://static.igem.org/mediawiki/2013/c/cb/Uppsala2013_Reporter_new.png'" /> </a>
+ </div>
+			<div id="chassi_pic_toxin">
+<a href="https://2013.igem.org/Team:Uppsala/toxin-antitoxin-system"> <img class="chassi_pic" src="https://static.igem.org/mediawiki/2013/8/8a/Uppsala2013_Toxin_new.png" onmouseover="this.src='https://static.igem.org/mediawiki/2013/2/2c/Uppsala2013_ToxinPushed.png '" onmouseout="this.src='https://static.igem.org/mediawiki/2013/8/8a/Uppsala2013_Toxin_new.png'" /> </a>
+</div>
+		</div>
+		<div id="chassi_pic_container_b">
+			<div id="chassi_pic_signal">
+<a href="https://2013.igem.org/Team:Uppsala/signal-peptide"> <img class="chassi_pic" src="https://static.igem.org/mediawiki/2013/f/f3/Uppsala2013_Signal_new.png" onmouseover="this.src='https://static.igem.org/mediawiki/2013/0/0c/Uppsala2013_SignalPushed.png'" onmouseout="this.src='https://static.igem.org/mediawiki/2013/f/f3/Uppsala2013_Signal_new.png'" /> </a>
+</div>
+			<div id="chassi_pic_vector">
+<a href="https://2013.igem.org/Team:Uppsala/vectors"> <img class="chassi_pic" src="https://static.igem.org/mediawiki/2013/6/6a/Uppsala2013_Vector_new.png" onmouseover="this.src='https://static.igem.org/mediawiki/2013/9/96/Uppsala2013_VectorPushed.png'" onmouseout="this.src='https://static.igem.org/mediawiki/2013/6/6a/Uppsala2013_Vector_new.png'" /> </a>
+</div>
+		</div>
+	</div> <!-- Closing div for shortcut_pic -->
+	<div id="container_mini_pic">
+<a href="https://2013.igem.org/Team:Uppsala/promoters">
+		<div class="text_box_in_the_box" id="promoter_div">
+			<div id="promoter_title"> <h3> Promoters </h3> </div>
+			<div class="promoter_pic"> <img class="chassi_mini_pic" src="https://static.igem.org/mediawiki/2013/f/f7/Uppsala2013_Promoter_mini.png"> </div>
+</a>
+			<div id="promoter_text"> Different constructs need different levels of expression. We have constructed and characterised a library of constitutive promoters of different strengths. These are based on a consensus sequence from promoters in Lactococcus lactis but have also been shown to work very well in E. coli and Lactobacillus.
+<div id="chassi_mini_images"> <a href="https://2013.igem.org/Team:Uppsala/promoters#b1"> <img class="chassi_mini_images" src="https://static.igem.org/mediawiki/2013/7/72/Uppsala2013_Facspic.png"></a> </div>
+</div>
+		</div>
+		<div class="text_box_in_the_box" id="reporter_div">
+<a href="https://2013.igem.org/Team:Uppsala/reporter-genes">
+			<div id="reporter_title"> <h3> Reporter genes </h3> </div>
+			<div class="reporter_pic"> <img class="chassi_mini_pic" src="https://static.igem.org/mediawiki/2013/c/ca/Uppsala2013_Reporter_mini.png"> </div>
+</a>
+			<div id="reporter_text"> Many techniques and methods require fluorescent reporter genes to function. While some fluorescent proteins work poorly in Lactobacillus we have biobricked a codon-optimized version of mCherry that has been shown to work. This has been done according to assembly standard 25 to allow it to be used as a fusion protein.
+<div id="chassi_mini_images"> <a href="https://2013.igem.org/Team:Uppsala/reporter-genes#b1"> <img class="chassi_mini_images" src="https://static.igem.org/mediawiki/2013/3/39/MCherry.png"> </a> </div>
+</div>
+		</div>
+		<div class="text_box_in_the_box" id="toxin_div">
+<a href="https://2013.igem.org/Team:Uppsala/toxin-antitoxin-system">
+			<div id="toxin_title"> <h3> Toxin-antitoxin </h3> </div>
+			<div class="toxin_pic"> <img class="chassi_mini_pic" src="https://static.igem.org/mediawiki/2013/1/12/Uppsala2013_toxin-antitoxin_symbol_mini.png"> </div>
+</a>
+			<div id="toxin_text"> A major challenge in synthetic biology is that there is a selective pressure against metabolically taxing systems. In nature, many natural plasmids contain genes coding for a toxin and the associated antitoxin. These systems can significantly increase the segregational stability of plasmids and is an easy to use alternative to chromosomal integration for some applications.
+<div id="chassi_mini_images"> <a href="https://2013.igem.org/Team:Uppsala/toxin-antitoxin-system#b1"> <img class="chassi_mini_images" src="https://static.igem.org/mediawiki/2013/0/0f/Uppsala2013_Toxin_antitoxin_system_with_Gene_X.png"></a> </div>
+</div>
+		</div>
+		<div class="text_box_in_the_box" id="vector_div">
+<a href="https://2013.igem.org/Team:Uppsala/vectors">
+			<div id="vector_title"> <h3> Vectors </h3> </div>
+			<div class="vector_pic"> <img class="chassi_mini_pic" src="https://static.igem.org/mediawiki/2013/5/5c/Uppsala2013_Vector_mini.png"></a> </div>
+</a>
+			<div id="vector_text"> Perhaps the most important chassi parts we have constructed are shuttle vectors that work both in Lactobacillus and E. coli. Because Lactobacillus is significantly harder to work with and ligations are hard to transform, constructs should first be assembled and prepped in E. coli and then transferred to Lactobacillus with the shuttle vector.<br><br>
+<div id="chassi_mini_images"> <a href="https://2013.igem.org/Team:Uppsala/vectors#b1"> <img class="chassi_mini_images" src="https://static.igem.org/mediawiki/2013/9/92/Shuttle-vector_pSBLbC.png"></a> </div>
+</div>
+		</div>
+		<div class="text_box_in_the_box" id="signal_div">
+<a href="https://2013.igem.org/Team:Uppsala/signal-peptide">
+			<div id="signal_title"> <h3> Signal peptides </h3> </div>
+			<div class="signal_pic"> <img class="chassi_mini_pic" src="https://static.igem.org/mediawiki/2013/4/40/Uppsala2013_Signal_peptide_symbol.png"> </div>
+</a>
+			<div id="signal_text"> Lactobacillus is a great genus for the secretion of proteins and contains several secretory pathways. In order for a protein to be exported it needs to be tagged with a short amino acid sequence at the N-terminus called a signal peptide. We have synthesised a signal peptide that can be assembled with a protein of choice.
+<div id="chassi_mini_images"> <a href="https://2013.igem.org/Team:Uppsala/signal-peptide#b1"> <img class="chassi_mini_images" src="https://static.igem.org/mediawiki/2013/2/22/Uppsala2013_Miraculin_%28with_signal_sequence%29.png"></a> </div>
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