Difference between revisions of "Team:Penn"
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<h2> Penn iGEM 2015! </h2> | <h2> Penn iGEM 2015! </h2> | ||
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| − | <h4> | + | <h4> ORTHOGONAL BACTERIAL COMMUNICATION WITH LUMINESCENCE & LIGHT-ACTIVATED TRANSCRIPTION FACTORS </h4> |
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| − | <h4> | + | <h4> INTRODUCTION: </h4> |
| − | <p> | + | <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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| + | <h4>PROJECT GOALS </h4> | ||
| + | <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>CIRCUIT DESIGN</h4> | ||
| − | < | + | <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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| + | <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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| + | <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> | + | <li> The distance at which the light source can be separated from the transcription factor with production of lacZ</li> |
| − | <li> | + | <li> Impact of a strong constitutive promoter </li> |
| − | <li> | + | <li> A tag added to luciferase at the C-terminus to direct it to the membrane</li> |
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Revision as of 03:21, 3 June 2015
{
Penn iGEM 2015!
ORTHOGONAL BACTERIAL COMMUNICATION WITH LUMINESCENCE & LIGHT-ACTIVATED TRANSCRIPTION FACTORS
INTRODUCTION:
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).
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.
PROJECT GOALS
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.
CIRCUIT DESIGN
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.
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.
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:
- The distance at which the light source can be separated from the transcription factor with production of lacZ
- Impact of a strong constitutive promoter
- A tag added to luciferase at the C-terminus to direct it to the membrane