Team:Bielefeld-Germany/Biosafety/Biosafety System

From 2013.igem.org

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For this approach we combined two common Biosafety-ideas, an auxotrophy and a toxic gene product, in one device. So the constructed Biosafety-System takes the best of this two approaches and is characterized by a double kill-swtich system. This double kill-switch mechanism provides additional a higher plasmid stability and a higher resistance towards undesirable mutations. In one sentence: Our Biosafety-System is safe!
For this approach we combined two common Biosafety-ideas, an auxotrophy and a toxic gene product, in one device. So the constructed Biosafety-System takes the best of this two approaches and is characterized by a double kill-swtich system. This double kill-switch mechanism provides additional a higher plasmid stability and a higher resistance towards undesirable mutations. In one sentence: Our Biosafety-System is safe!
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==Theory==
==Theory==
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===TetOR Alive===
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Our three biosafety systems are all based on an induced repressor system. As shown in the illustration below an inducer activates a promoter which expresses the repressor gene which delays a second promoter in front of a gene. This inducer can be a substrate like L-rhamnose or a product which the organism produces and delays the expression of a gene. This is an advantage because the organism only produces products when it is necessary so the metabolic stress is lower. This delay can be executed in different ways. For example the repressor araC in our [https://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_S araCtive] system regulates negatively the pBAD promoter by building up a dimer, which builds a DNA loop and inhibits the binding of the RNA-polymerase. So this example stands for a structural change of the DNA. This function can be found in our [https://2013.igem.org/Team:Bielefeld-Germany/Biosafety/Biosafety_System_L Lac of Growth] system.
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[[File:IGEM Bielefeld 2013 Biosafety System M 2.png|340px|thumb|left|System M in the MFC: In this case the mikroorganism is in the MFC with sufficient L-rhamnose. It comes to an expression of TetR which blocks TetO by binding and alr which switches L-alanine to D-alanine. Because of the fact that TetR blocks TetO the RNase Ba can't expressed.]]
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Another example for a second way the repressor acts is a direct interaction between the repressor and the operator. The repressor and the operator build a complex which the RNA polymerase can’t overcome. So the repressor blocks the RNA polymerase.
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[[File:IGEM Bielefeld 2013 Biosafety System M ohne Rhamnose+ 2.png|340px|thumb|right|System M outside of the MFC: In this case the mikroorganism could get out of the MFC by damage or incorrect handling. Outside of the MFC there isn't enough L-rhamnose. So TetR doesn't block TetO anymore so the degradation process is induced by activating the TetO and the ensuing expression of RNase Ba. E.coli dies.]]
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===Lac of growth===
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[[File:IGEM Bielefeld 2013 Biosafety System L 2.png|340px|left|thumb|System L in the MFC: In this case the mikroorganism is in the MFC with sufficient L-rhamnose. It comes to an expression of lacI which blocks the lac-promoter by binding and alr which switches L-alanine to D-alanine. Because of the fact that lacI blocks the lac-promoter the RNase Ba can't expressed.]]
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[[File:IGEM Bielefeld 2013 Biosafety System L ohne Rhamnose 2.png|340px|thumb|right|System L outside of the MFC: In this case the mikroorganism could get out of the MFC by damage or incorrect handling. Outside of the MFC there isn't enough L-rhamnose. So... E.coli dies.]]
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===araCtive===
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[[File:IGEM Bielefeld 2013 Biosafety System S+ 2.png|340px|thumb|left|System S in the MFC: In this case the mikroorganism is in the MFC with sufficient L-rhamnose. It comes to an expression of araC which blocks the arabinose-promoter by binding and alr which switches L-alanine to D-alanine. Because of the fact that araC blocks the arabinose-promoter the RNase Ba can't expressed.]]
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[[File:IGEM Bielefeld 2013 Biosafety System S ohne Rhamnose 2.png|340px|thumb|right|System S outside of the MFC: In this case the mikroorganism could get out of the MFC by damage or incorrect handling. Outside of the MFC there isn't enough L-rhamnose. So araC doesn't block the arabinose-promotor any more and RNase Ba can be expressed.E.coli dies.]]
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[[File:IGEM Bielefeld 2013 Biosafety allg. Repressor 2.png|600px|thumb|center|FigureX: General illustration of the repressor activity.]]
==Results==
==Results==

Revision as of 10:47, 4 October 2013



Biosafety System


Overview

IGEM Bielefeld 2013 Biosafety E.coli bewaffnet safe 2..png

Biosafety is an essential aspect when taking part in iGEM especially when you work with living organisms which could possibly get out of your application by damage or incorrect handling. In order to counter this problem there exist useful systems to prevent the bacteria from escaping or killing the bacteria when they are outside of the application. To complement this archive we constructed not only one system but also three systems which differ in leakiness and strength. For this approach we combined two common Biosafety-ideas, an auxotrophy and a toxic gene product, in one device. So the constructed Biosafety-System takes the best of this two approaches and is characterized by a double kill-swtich system. This double kill-switch mechanism provides additional a higher plasmid stability and a higher resistance towards undesirable mutations. In one sentence: Our Biosafety-System is safe!







Theory

Our three biosafety systems are all based on an induced repressor system. As shown in the illustration below an inducer activates a promoter which expresses the repressor gene which delays a second promoter in front of a gene. This inducer can be a substrate like L-rhamnose or a product which the organism produces and delays the expression of a gene. This is an advantage because the organism only produces products when it is necessary so the metabolic stress is lower. This delay can be executed in different ways. For example the repressor araC in our araCtive system regulates negatively the pBAD promoter by building up a dimer, which builds a DNA loop and inhibits the binding of the RNA-polymerase. So this example stands for a structural change of the DNA. This function can be found in our Lac of Growth system.
Another example for a second way the repressor acts is a direct interaction between the repressor and the operator. The repressor and the operator build a complex which the RNA polymerase can’t overcome. So the repressor blocks the RNA polymerase.

FigureX: General illustration of the repressor activity.

Results

References

  • Autoren (Jahr) Titel [Link|Paper Ausgabe: Seiten].








Contents

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