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<h4 style = "text-align: center;text-indent:0px; color:purple"> ORTHOGONAL BACTERIAL COMMUNICATION WITH LUMINESCENCE & LIGHT-ACTIVATED TRANSCRIPTION FACTORS </h4>
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<h1>OUR PROJECT</h1>
 
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<h4 style = "color: blue"> INTRODUCTION: </h4>
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<p> Although they are small, bacteria are capable of surviving in complex networks and harsh environments. In order to be able to do so effectively, bacterial species often depend on quorum sensing as a means of regulating gene expression based on population density. This means of communication has been studied in a species of bacteria known as Vibrio fisheri. The crux of the mechanism lies in a chemical produced by the bacteria known as AHL. This molecule, developed through transcription of the LuxI gene, binds to a transcriptional regulator only at high population levels and activates transcription of the “lux box.” Expression of the lux box causes the bacteria to produce luminescence (Popham & Stevens).</p>
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<p>Although quorum sensing through a chemical like AHL is incredibly effective, there are a few limitations when it comes to applying this type of system to more complex synthetic biology projects. Namely, a chemical output is pervasive and diffuses throughout the whole medium. Therefore, it is not possible to only target communication and response from a certain region of bacteria. Additionally, in order for communication to occur between two bacterium, they have to share the same environment. </p>  
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<img src="https://static.igem.org/mediawiki/2015/9/9d/Photocoupler2015Penn.png" alt />
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      <p> Communication between cells is instrumental in coordinating population-level activity. In a process known as "quorum sensing," bacteria both secrete and sense autoinducer signaling molecules to enable synchronization of group gene expression paradigms. The synthetic biology community has rapidly adopted these quorum signaling pathways for use in programmed circuitry. However, chemical signals must diffuse between sender and receiver cells, limiting such communication to a common environment. In electronics, when electrical signals must be transferred between two circuits operating at incompatible voltages, electrical engineers use optocouplers, components that transfer information between isolated circuits via light. The 2015 Penn iGEM team presents a biological analog of the optocoupler, a cell-to-cell communication system in which a "sender" cell generates light via bioluminesence and a "receiver" cell expresses photoreceptors to enable light-dependent physiological responses. We show that light elicits a response in light-sensitive receivers and illuminated potential applications for this alternative form of cell communication.</p>
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<h4 style = "color: blue">PROJECT GOALS </h4>
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<p> The 2015 iGEM Team proposes that these caveats can be addressed by accomplishing light based communication of bacterial cells. This type of communication would involve one cell producing light and when the population density is high enough, a second receiver cell would respond to it and express a certain gene. This type of a circuit design could lead to various novel synthetic biology advancements as light can be localized as well as shine through boundaries.</p>  
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<h4 style = "color: blue">CIRCUIT DESIGN</h4>
 
  
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<p> The receiver cell will contain plasmid pDawn. In the presence of light from an external light source, the YFI gene will phosphorylate FixJ will in repress the expression of the lambda repressor. This will then allow for the expression of the gene in the multiple cloning site. A reporter gene such as lacZ will be cloned into the multiple cloning site. The first experiment will test this circuit design in order to determine if light produced externally (artificially) is sufficient to trigger the light-activated transcription factor.</p>
 
  
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<h1>BREAKDOWN</h1>
 
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<p>The sender cell will contain a constitutive promoter that is continuously expressed followed by the lux box. The lux box, responsible for luminescence contains the genes LuxC, D, A, B, E and G. Collectively, the genes encode for luciferase, the substrate tetradecanal and increase light output. The receiver cell plasmid will be cloned into this construct in order to ensure that the light produced by the lux box is sufficient to activate the light-activated transcription factor.</p>
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<td width="33.33%"><a href="https://2015.igem.org/Team:Penn/Receiver"><img id = "daicon" src="https://static.igem.org/mediawiki/2015/c/c0/Pennigem_receiver_15.png" height="85%" width="85%"></a></td>
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<td width="33.33%"><a href="https://2015.igem.org/Team:Penn/Communication"><img id = "daicon" src="https://static.igem.org/mediawiki/2015/f/fc/Pennigem_comm_15.png" height="85%" width="85%"></a></td>
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<h1>HUMAN PRACTICES</h1>
 
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<p> If both the previously described fast fail experiments are successful, the following further experiments can be performed to examine to further the development and understanding of light based communication: </p>
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<li> The distance at which the light source can be separated from the transcription factor with production of lacZ</li>
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<li> Impact of a strong constitutive promoter </li>
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<a href="https://2015.igem.org/Team:Penn/Educational_Toolbox"><img src="https://static.igem.org/mediawiki/2015/c/c3/Whatwillstandasatransferfunctionfornow3.png"></a></td>
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<li> A tag added to luciferase at the C-terminus to direct it to the membrane</li>
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      <p> Analagous to our project goals, the Penn iGEM 2015 team has worked to further illuminate the parallels between genetic circuits and electrical circuits with the addition of the PennTunes Toolbox. Just as there is a distinct change in response when replacing an electric circuit component, the various genetic parts included in this toolbox clearly demonstrate the divergence in expression level due to a change in genetic part (promoter, RBS, etc). As an example, we have characterized one of the inverter strains in the toolbox that exhibits the relationship in the figure to the right.
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</div></div> <!--These are the closing tags for div id="mainContainer" and div id="contentContainer". The corresponding opening tags appear in the template that is {{included}} at the top of this page.-->
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<p>Our aim is to increase the reach of synthetic biology by providing the tools and infrastructure that will make biotechnology more accessible in educational settings. Find out more about our vision of the future for synthetic biology and biotechnology tools by clicking on the graph! </p>
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<h1>Our Team</h1>
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Latest revision as of 19:40, 16 September 2015

University of Pennsylvania iGEM

OUR PROJECT

Communication between cells is instrumental in coordinating population-level activity. In a process known as "quorum sensing," bacteria both secrete and sense autoinducer signaling molecules to enable synchronization of group gene expression paradigms. The synthetic biology community has rapidly adopted these quorum signaling pathways for use in programmed circuitry. However, chemical signals must diffuse between sender and receiver cells, limiting such communication to a common environment. In electronics, when electrical signals must be transferred between two circuits operating at incompatible voltages, electrical engineers use optocouplers, components that transfer information between isolated circuits via light. The 2015 Penn iGEM team presents a biological analog of the optocoupler, a cell-to-cell communication system in which a "sender" cell generates light via bioluminesence and a "receiver" cell expresses photoreceptors to enable light-dependent physiological responses. We show that light elicits a response in light-sensitive receivers and illuminated potential applications for this alternative form of cell communication.

BREAKDOWN



HUMAN PRACTICES

Analagous to our project goals, the Penn iGEM 2015 team has worked to further illuminate the parallels between genetic circuits and electrical circuits with the addition of the PennTunes Toolbox. Just as there is a distinct change in response when replacing an electric circuit component, the various genetic parts included in this toolbox clearly demonstrate the divergence in expression level due to a change in genetic part (promoter, RBS, etc). As an example, we have characterized one of the inverter strains in the toolbox that exhibits the relationship in the figure to the right.

Our aim is to increase the reach of synthetic biology by providing the tools and infrastructure that will make biotechnology more accessible in educational settings. Find out more about our vision of the future for synthetic biology and biotechnology tools by clicking on the graph!

Our Team