Team:DTU-Denmark/Project

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<title>Welcome to the DTU iGEM wiki!</title>
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     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark" >Home</a></font> </td>
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     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark" >Home</a></font></td>
     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark/Team" >The Team</a> </font></td>
     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark/Team" >The Team</a> </font></td>
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     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark/Project" >The Project</a> </font></td>
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     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark/Project" >The Project</a>  
     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark/Parts" >Parts submitted</a> </font></td>
     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark/Parts" >Parts submitted</a> </font></td>
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     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark/Modelling">Modelling</a></font> </td>
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     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark/Results">Results</a></font> </td>
     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark/Notebook" title="Day to day lab activity">Notebook</a>  
     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark/Notebook" title="Day to day lab activity">Notebook</a>  
   <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark/Blog">Blog</a></font> </td>
   <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark/Blog">Blog</a></font> </td>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Basics">Basics</a></li><br>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Background">Synthetic Biology</a></li><br>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Regulatory_sytems">Regulatory Systems</a></li><br>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Regulatory_sytems">Regulatory Systems</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Switch">The Switch</a></li><br>
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<ul><font size="2">
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Team1">Anti-Repressor group</a></li><br>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Regulatory_sytems#lambda">Lambda Phage</a></li>
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<li ><a href="https://2010.igem.org/Team:DTU-Denmark/Team2">Anti-Terminator group</a></li><br>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Regulatory_sytems#gifsy">Gifsy Phages</a></li>
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</font></ul>
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<br>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Switch">The Switch</a></li>
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<ul><font size="2">
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Switch#Biological_Switch">Biological Switches</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Switch#Bistable_Switches">Bistable Switches</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Switch#Design">Design of our Bi[o]stable Switch</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Switch#Engineering">Step-wise Engineering of the Switch</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Switch#Applications">Applications</a></li>
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</font></ul>
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<br><li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL">Synthetic Promoter Library</a>
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<ul><font size="2">
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL#standard">The DTU SPL Standard</a>
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<ul>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL#strategy">Strategy</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL#design">Primer Design</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL#protocol">Protocol</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL#advantages">Advantages</a></li>
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<h1>Background</h1>
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</ul></font>
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<h3>What is Synthetic Biology?</h3>
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</li><br>
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<p align="justify">Not many people have heard of Synthetic Biology. Synthetic biology essentially aims to utilize natures tricks to design and build artificial biological systems for engineering purposes as well as a way to get a better understanding of why biological systems are set up as they are. The term “synthetic biology” was first used on genetically engineered bacteria that were created with recombinant DNA technology. Parts from natural biological systems are taken, characterized and simplified and used as a component of a highly unnatural, engineered, biological system. The term was then used when referring to when organic synthesis is used to generate artificial molecules that mimic natural molecules such as enzymes. </p>
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<li ><a href="https://2010.igem.org/Team:DTU-Denmark/Modelling">Modeling</a>
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There are two types of synthetic biologists:
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Modelling#Approach">Modeling Approach</a></li>
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<li>those who deal with re-designing and fabricating existing biological systems.</li>
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<li>those who deal with designing and fabricating biological components that do not already exist in the real world. </li>
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<p align="justify">Synthetic biology provides us with a new perspective from which we can understand and ultimately utilize life for our own benefits. [1,2,3]</p><br><br>
 
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<h1>Introduction</h1>
 
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<h3>Aim</h3>
 
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<p align="justify">The goal of our project is to enable colonies of ''E. coli'' bacteria to transition between production of two different reporter proteins. In our system, switching between states will be induced by exposing the bacteria to light. Each of the states will have a specific frequency associated with it. There are multiple potential applications for biologicals "switches" such as these, this includes the improved control of production of additives in industrial biotechnological processes.</p>
 
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<h3>Project Concept</h3>
 
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<p align="justify">As previously stated, the main goal of our project is to design a bistable switch. The switching between the two states will be controlled by the introduction of two different wavelengths of light, each wavelength responsible for the induction of a different state. As a proof of concept, we’re using fluorescent proteins as reporter genes which makes it easy to observe and characterise the system. In principle, however, any reporter gene can be used.</p>
 
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<p align="justify">Our original project concept revolved around using light-receptors to instigate the switch between the two stable state. It was thought that the production of the first reporter protein would be induced by red light (660 nm). At the same time, production of the other reporter will be suppressed by a coexpressed repressor. Conversely, production of the second reporter would be induced by blue light (470 nm). Bistability of the system is achieved by using two repressors which negatively regulate each other’s expression. This enables the system to sustain state without continuous input, i. e. once production of a reporter protein is initiated, it will persist until the system is forced into the other state.</p>
 
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<img src="https://static.igem.org/mediawiki/2010/f/ff/DTU_Project_illustration_1.png" width="570px"  align="center"> </img>
 
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<p align="justify">Our project concept has since changed to using two different carbohydrate sources as a means of switching between the two stable states. This means that the state in which the bacteria will be found depends on which one of two carbohydrate sources it was last exposed.</p>
 
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<b>Project Concept</b><br><br>
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<p align="justify">As previously stated, the main goal of our project is to design a bistable switch. We want to enable bacteria to transition between two stable states. In our system, switching between states will be induced by two different inputs and each of the states will have a specific output associated with it.</p>
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<p align="justify">Our original project concept evolved around building a switch that we could turn on and off continuously. Not only did we want the switch to be able to switch states, but we also wanted it to be able to stay in a certain state without having to induce it constantly. Several designs were discussed, for example using light at different wavelengths to induce the system.</p>
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</body>
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<p align="justify">During the last couple of years several attempts have been made to construct bistable switches. One switch design is a one input, two outputs stable switch. It has a stable output but it looses the switching ability and 90% of the individuals in the population are killed when the switch is induced by UV-light (Lou, C. et al.,2010). Another mechanism tested has been a flipases system where the DNA is inverted by specific recognition sites. The system was found to function but was limited by the robustness of the flipase systems and knowledge about their function (Ham, T.S. et al.,2008). Another general problem with the construction of synthetic switches is the loss of function over time (Canton, B. et al.,2008). The limited function and stability of existing switches also limit the application to short time spans. Based on these problems we saw the untapped potential in designing a novel biological switch.</p>
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</html>
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<p align="justify">Our switch design is a complex regulatory system, which is induced with the help of input plasmids carrying inducible promoters.  However due to the complexity of the design of the bistable switch it was out of the scope of this project to construct the entire switch. Therefore focus was put on characterizing the key regulatory subparts needed for successful switch function. Characterizing subparts also enable future teams to use them in other contexts.</p>
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== Micro fermentor systems ==
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<table class="https://static.igem.org/mediawiki/2010/9/9a/Intro_switch.png" align="center">
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description of the biolector
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<caption align="bottom"><p align="justify"><b>Figure 1</b>: A simplified illustration of our bistable switch.</p></caption>
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and a few references
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<tr><td><img src="https://static.igem.org/mediawiki/2010/9/9a/Intro_switch.png"  width="400px"></td></tr>
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</table><br>
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<font color="#990000" face="arial" size="5">
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<br> 
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<b>Design of our switch</b><br><br>
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  </font>
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== Synthetic promoter library (SPL) ==
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<p align="justify"> We have set up the complete design for a bistable switch. The main design criteria has been that the switch should be able to toggle  back and forth between states, stay in its induced state until it receives another input and remain stable through subsequent generations. These criteria imply that:
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How does it work, examples, what have it been used to characterize?
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<ul>
-
how do you construct it? Figures and illustrations to explain.
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<li>It should be designed without induction by UV-light.</li>
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Figures to explain our use? And example on our specific design primer sequences illustration on the double stranded DNA, with BB - prefix suffix.
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<li>It should not be based on essential native regulatory mechanisms.</li>
 +
<li>It should be possible to incorporate into the genome for stable replication, and function in subsequent generations.</li>
 +
</ul>
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<p align="justify">A simplified version of our switch design can be illustrated using a basic  SR (Set-Reset) flip flop circuit used when representing electronic circuits. It provides feedback from its outputs to its inputs and is commonly used in memory circuits to store data bits. The term flip-flop relates to the actual operation of the device, as it can be "Flipped" into one logic state or "Flopped" back into another <a href="http://www.electronics-tutorials.ws/sequential/seq_1.html" target="_blank"> (reference)</a>. For further description on the logical behavior and requirements of switches see the <a href="https://2010.igem.org/Team:DTU-Denmark/Modelling" target="_blank">modeling section</a>.</p>
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<table class="https://static.igem.org/mediawiki/2010/5/53/SRflipflop.png" align="center">
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<caption align="bottom"><p align="justify"><b>Figure 2</b>: SR flip-flop switch.</p></caption>
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<tr><td><img src="https://static.igem.org/mediawiki/2010/5/53/SRflipflop.png"  width="200px"></td></tr>
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</table><br>
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<p align="justify">The switch design is based on phage regulatory systems. We used the repressor/anti-represor system from the Gifsy phages and an anti-termination system from the lambda-phage.</p>
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<p align="justify"> The switch has three levels of regulatory mechanisms to ensure a stable expression and tight control and thereby creating a robust bistable switch (see Figure 3):
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<ol>
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<li>The first level is negative feed back control - repression of the uninduced state of the switch.</li>
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<li>The second level is a positive feed back mechanism with a threshold level that when triggered will induce the third level of regulation - antitermination allows third level to be induced.</li>
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<li>The third regulatory level is a positive feed back mechanism stabilizing the expression of the winning state by, anti-repression of the repression from the loosing states.</li>
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</ol></p>
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== Presentation ==
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<table class="https://static.igem.org/mediawiki/2010/1/14/Threestages.png" align="center">
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Figures and illustrations - explanations
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<caption align="bottom"><p align="justify"><b>Figure 3</b>: Simplified representation of the regulatory mechanisms: [<b>1</b>] negative feed back control of opposite side. [<b>2</b>] positive feed back trigger mechanism for side commitment. [<b>3</b>] positive feed back mechanism, by canceling the opposite sides repressor.</p></caption>
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<tr><td><img src="https://static.igem.org/mediawiki/2010/1/14/Threestages.png"  width="400px"></td></tr>
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</table><br>
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<p align="justify">For an in depth description of the function and origin of the regulatory parts have a look into the <a href=" https://2010.igem.org/Team:DTU-Denmark/Switch" target="_blank">switch</a> section.</p>
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<p align="justify">One important feature of the switch is the strength of the promoters. For the switch to work properly we need promoters of equal strength. To solve this problem we utilized a <a href=" https://2010.igem.org/Team:DTU-Denmark/SPL" target="_blank">synthetic promoter library</a>, enabling us to generate a library of promoters with a wide variety of different strengths.</p>
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== Designing the switch - selecting parts==
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<font color="#990000" face="arial" size="5">
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(modeling)
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<br> 
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Requirements before a biological switch functions. On the paper and theoretically.
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<b>Characterizing phage regulatory mechanisms</b><br><br>
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Components of the switch
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   </font>
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We have decided not to use cI, why? The hong kong paper flaws!! (REF)   we did not use UV-activation why?  To have a stable system we did not what to use cI and UV-regulatory systems as they can impose problems with the genetic stability.
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==== Selecting N protein and nut site ====
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<p align="justify">The main regulatory parts of the switch are the repressor/antirepressor system from the Gifsy phages and the anti-terminator system from lambda phage, see Figure 4, Figure 5 and Figure 6.</p>
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In the end, after evaluating what component pair to use we selected λ N-protein and nut-site. Different nut-sites N-protein systems have been identified and investigated (REFFF), the nutsites for λ-phage and p21, p22, are the best described (REFFF) comparison of the antiterminator effect have not been charfully investigated, as emphasis have been on function and interacting parts. we wanted to selected the nutsite with a strong consistent anti-terminator effect. But as this was not well defined continues work was done with the lambda nut side because more articles and knowledge was available, for potential trouble shooting and improvement of the system interaction and dynamic.
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What have been described is that the N-nut-site pair have specific function and thus the λ-N-protein was used for continues, construction of the switch.
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==== Flourescene protein ====
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<table class="https://static.igem.org/mediawiki/2010/c/c8/DTU_anitsystem.png" align="center">
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Why were the shown proteins selected.
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<caption align="bottom"><p align="justify"><b>Figure 4</b>: Graphical presentation of the repressor part of our regulatory system from the Gifsy phages.</p></caption>
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<tr><td><img src="https://static.igem.org/mediawiki/2010/c/c8/DTU_anitsystem.png"  width="225px"></td></tr>
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==== The Biolector ====
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<table class="https://static.igem.org/mediawiki/2010/2/26/Repressor_antirepressor.png" align="center">
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selected filters in releation to flourescence protein<br>
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<caption align="bottom"><p align="justify"><b>Figure 5</b>: Graphical presentation of the anti-repressor part of our regulatory system from the Gifsy phages.</p></caption>
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The filters for the biolector is ordered individually to fit the required needs and proteins. we decided to order the filters we needed for this application.  
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<tr><td><img src="https://static.igem.org/mediawiki/2010/2/26/Repressor_antirepressor.png"  width="350px"></td></tr>
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Filters applied for this experimentWe chose
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<table class="https://static.igem.org/mediawiki/2010/a/a4/DTU_repressorsystem.png" align="center">
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RED:
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<caption align="bottom"><p align="justify"><b>Figure 6</b>: Graphical presentation of the anti-terminator part of our regulatory system from the lambda phage.</p></caption>
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Green:
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<tr><td><img src="https://static.igem.org/mediawiki/2010/a/a4/DTU_repressorsystem.png"  width="180px"></td></tr>
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= Characterizing BBricks as parts of the switch =
 
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'''(Materials and Methods)''' section<br>
 
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Mainly the hard control of the switch is due to a double regulation system build on a both terminator-anti-terminator and repressor anti-repressor regulation. It was out of the scope of this project to construct the entire theoretical developed switch, and characterize the fully constructed switch. Have focused on characterizing the two regulatory systems individually. This was done in order to investigate if the responses were satisfactory to use in a future complete switch construction. (????  By getting the regulatory mechanism of the subparts we further, by modeling, could conclude constraints for successful function of the system and other subparts.
 
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In this section we describe the design of and the experimental setup used to characterize the subparts of the system and our bio-bricks.
 
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== Anti-terminator function ==
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<p align="justify">As a proof of concept for the regulatory mechanisms, we constructed plasmids that were able to test the regulatory mechanism and strength of the two systems. We used low copy number plasmids and fluorescent proteins as reporters. For more information about the experimental setup and characterization results of the Repressor - Anti-Repressor system please click <a href="https://2010.igem.org/Team:DTU-Denmark/Repressor_Section" target="_blank">here</a> and for the Terminator - Anti-Terminator system please click <a href="https://2010.igem.org/Team:DTU-Denmark/AntiTermination_Section" target="_blank">here</a>.</p>
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(experimental work)
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==== selecting supparts ====
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<p align="justify">The key parts of the regulatory systems have been tested and are available as BioBricks through the parts registry. See the <a href=" https://2010.igem.org/Team:DTU-Denmark/Parts" target="_blank">parts</a> page for a list of available parts. </p>
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Why were these supparts chosen ?<br>
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AIM
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==== Design and experimental setup ====
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<font color="#990000" face="arial" size="5">
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'''presentation - Figure of setup and explanation'''
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<br> 
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<b>Conclusion</b><br><br>
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  </font>
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==== Materials and methods ====
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<p align="justify">We have shown that the Gifsy repressor system has a sufficient tight expression and control to be used in the future construction of biological switches.</p>
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HOW ? what plasmids and why, what measurering method and why?
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refer to the notebook page with protocols - and actual info from lab.
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'''mRNA-stability'''
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<p align="justify">We have set up the frame work for testing anti-terminator function, but further characterization is needed before it can be applied in standard regulatory systems.</p>
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when introducing non-coding sequences problems will acour  with rna-degredation of RNAP is not attracted to the area, to fast degredation, unwanted steam loops. (Reference to the terminator screening plasmids for BB)
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'''Synthetic promoter library (SPL)'''<br>
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<p align="justify">Furthermore we have designed and demonstrated the functionality of a Synthetic Promoter Library, compatible with the BioBrick standard. We have also developed a standard for integrating a BioBrick compatible Synthetic Promoter Library in bacteria in order to fine-tune the expression of BioBrick parts and devices.</p>
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How does it work, examples, what have it been used to characterize?
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how do you construct it? Figures and illustrations to explain.
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Figures to explain our use? And example on our specific design primer sequences illustration on the double stranded DNA, with BB - prefix suffix.
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==== Results ====
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<p align="justify">We hope that this work will inspire future teams to take up the challenge of constructing a genetic bistable switch. They can easily benefit from the new DTU Synthetic Promoter Library standard and our submitted BioBricks.</p>
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comments to the results and reference to the BB pages with info and results.
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== Repressor function ==
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(experimental work)
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==== selecting supparts ====
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Why were these supparts chosen ?<br>
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AIM
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==== Design and experimental setup ====
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'''presentation - Figure of setup and explanation'''
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==== Materials and methods ====
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HOW ? what plasmids and why, what measurering method and why?
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refer to the notebook page with protocols - and actual info from lab.
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==== Results ====
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comments to the results and reference to the BB pages with info and results.
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== References ==
== References ==
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* (Gottesman et.al. 2002) Gottesman. Max E, Nudler. Evgeny, 2002  ”Transcription termination and anti-termination in E.coli”  Genes to cells.  (a good introduction review to termination function)
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* Canton, B. et al. Refinement and standardization of synthetic biological parts and devices. Nature Biotechnology 26, 787-793, (2008)
-
 
+
* Ham, T.S. et al. Design and construction of a double inversion recombination Switch for Heritable Sequential Genetic Memory. PloS ONE 3(7),(2008)
-
* (Franklin et.al. 1989) NC Franklin, JH Doelling - Am Soc Microbiol "Overexpression of N antitermination proteins of bacteriophages lambda, 21, and P22: loss of N protein specificity." - Journal of bacteriology, 1989
+
* Lou, C. et al. Synthesizing a novel genetic sequential logic circuit: a push-on push-off switch. Mol Syst Biol 6, (2010)
-
 
+
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* (Jensen 2004) Ole Nørregaard Jensen, “Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry,” Current Opinion in Chemical Biology 8, no. 1 (February 2004): 33-41.
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* [1] http://syntheticbiology.org/FAQ.html
* [1] http://syntheticbiology.org/FAQ.html
* [2]http://www.nature.com.globalproxy.cvt.dk/nrg/journal/v6/n7/execsumm/nrg1637.html
* [2]http://www.nature.com.globalproxy.cvt.dk/nrg/journal/v6/n7/execsumm/nrg1637.html
* [3]http://www.nature.com.globalproxy.cvt.dk/msb/journal/v2/n1/full/msb4100073.html
* [3]http://www.nature.com.globalproxy.cvt.dk/msb/journal/v2/n1/full/msb4100073.html
 +
* [4]www.partsregistry.org

Latest revision as of 03:58, 28 October 2010

Welcome to the DTU iGEM wiki!


Project Concept

As previously stated, the main goal of our project is to design a bistable switch. We want to enable bacteria to transition between two stable states. In our system, switching between states will be induced by two different inputs and each of the states will have a specific output associated with it.

Our original project concept evolved around building a switch that we could turn on and off continuously. Not only did we want the switch to be able to switch states, but we also wanted it to be able to stay in a certain state without having to induce it constantly. Several designs were discussed, for example using light at different wavelengths to induce the system.

During the last couple of years several attempts have been made to construct bistable switches. One switch design is a one input, two outputs stable switch. It has a stable output but it looses the switching ability and 90% of the individuals in the population are killed when the switch is induced by UV-light (Lou, C. et al.,2010). Another mechanism tested has been a flipases system where the DNA is inverted by specific recognition sites. The system was found to function but was limited by the robustness of the flipase systems and knowledge about their function (Ham, T.S. et al.,2008). Another general problem with the construction of synthetic switches is the loss of function over time (Canton, B. et al.,2008). The limited function and stability of existing switches also limit the application to short time spans. Based on these problems we saw the untapped potential in designing a novel biological switch.

Our switch design is a complex regulatory system, which is induced with the help of input plasmids carrying inducible promoters. However due to the complexity of the design of the bistable switch it was out of the scope of this project to construct the entire switch. Therefore focus was put on characterizing the key regulatory subparts needed for successful switch function. Characterizing subparts also enable future teams to use them in other contexts.

Figure 1: A simplified illustration of our bistable switch.



Design of our switch

We have set up the complete design for a bistable switch. The main design criteria has been that the switch should be able to toggle back and forth between states, stay in its induced state until it receives another input and remain stable through subsequent generations. These criteria imply that:

  • It should be designed without induction by UV-light.
  • It should not be based on essential native regulatory mechanisms.
  • It should be possible to incorporate into the genome for stable replication, and function in subsequent generations.

A simplified version of our switch design can be illustrated using a basic SR (Set-Reset) flip flop circuit used when representing electronic circuits. It provides feedback from its outputs to its inputs and is commonly used in memory circuits to store data bits. The term flip-flop relates to the actual operation of the device, as it can be "Flipped" into one logic state or "Flopped" back into another (reference). For further description on the logical behavior and requirements of switches see the modeling section.

Figure 2: SR flip-flop switch.


The switch design is based on phage regulatory systems. We used the repressor/anti-represor system from the Gifsy phages and an anti-termination system from the lambda-phage.

The switch has three levels of regulatory mechanisms to ensure a stable expression and tight control and thereby creating a robust bistable switch (see Figure 3):

  1. The first level is negative feed back control - repression of the uninduced state of the switch.
  2. The second level is a positive feed back mechanism with a threshold level that when triggered will induce the third level of regulation - antitermination allows third level to be induced.
  3. The third regulatory level is a positive feed back mechanism stabilizing the expression of the winning state by, anti-repression of the repression from the loosing states.

Figure 3: Simplified representation of the regulatory mechanisms: [1] negative feed back control of opposite side. [2] positive feed back trigger mechanism for side commitment. [3] positive feed back mechanism, by canceling the opposite sides repressor.


For an in depth description of the function and origin of the regulatory parts have a look into the switch section.

One important feature of the switch is the strength of the promoters. For the switch to work properly we need promoters of equal strength. To solve this problem we utilized a synthetic promoter library, enabling us to generate a library of promoters with a wide variety of different strengths.


Characterizing phage regulatory mechanisms

The main regulatory parts of the switch are the repressor/antirepressor system from the Gifsy phages and the anti-terminator system from lambda phage, see Figure 4, Figure 5 and Figure 6.

Figure 4: Graphical presentation of the repressor part of our regulatory system from the Gifsy phages.


Figure 5: Graphical presentation of the anti-repressor part of our regulatory system from the Gifsy phages.


Figure 6: Graphical presentation of the anti-terminator part of our regulatory system from the lambda phage.


As a proof of concept for the regulatory mechanisms, we constructed plasmids that were able to test the regulatory mechanism and strength of the two systems. We used low copy number plasmids and fluorescent proteins as reporters. For more information about the experimental setup and characterization results of the Repressor - Anti-Repressor system please click here and for the Terminator - Anti-Terminator system please click here.

The key parts of the regulatory systems have been tested and are available as BioBricks through the parts registry. See the parts page for a list of available parts.


Conclusion

We have shown that the Gifsy repressor system has a sufficient tight expression and control to be used in the future construction of biological switches.

We have set up the frame work for testing anti-terminator function, but further characterization is needed before it can be applied in standard regulatory systems.

Furthermore we have designed and demonstrated the functionality of a Synthetic Promoter Library, compatible with the BioBrick standard. We have also developed a standard for integrating a BioBrick compatible Synthetic Promoter Library in bacteria in order to fine-tune the expression of BioBrick parts and devices.

We hope that this work will inspire future teams to take up the challenge of constructing a genetic bistable switch. They can easily benefit from the new DTU Synthetic Promoter Library standard and our submitted BioBricks.

References

  • Canton, B. et al. Refinement and standardization of synthetic biological parts and devices. Nature Biotechnology 26, 787-793, (2008)
  • Ham, T.S. et al. Design and construction of a double inversion recombination Switch for Heritable Sequential Genetic Memory. PloS ONE 3(7),(2008)
  • Lou, C. et al. Synthesizing a novel genetic sequential logic circuit: a push-on push-off switch. Mol Syst Biol 6, (2010)
  • [1] http://syntheticbiology.org/FAQ.html
  • [2]http://www.nature.com.globalproxy.cvt.dk/nrg/journal/v6/n7/execsumm/nrg1637.html
  • [3]http://www.nature.com.globalproxy.cvt.dk/msb/journal/v2/n1/full/msb4100073.html
  • [4]www.partsregistry.org