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Team:Harvard/Split
From 2009.igem.org
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<b>Theoretical Description and Purpose: </b>The impetus behind the split-luciferase system came from our initial goal to create a cellular blackboard, one that could be written on using a red laser and erased with a far-red laser. While the two-hybrid system offered us the flexibility of being able to alter gene expression, that was at a significant cost: time delay. A response from the yeast would not be evident for many minutes or even hours because of the lag time in gene expression: the PhyB/PIF3 would have to bind to DNA, induce transcription, and the transcription product would then have to be translated. We devised the split-luciferase system as a means to avoid that problem and thus create a blackboard with instant on/off capabilities. | <b>Theoretical Description and Purpose: </b>The impetus behind the split-luciferase system came from our initial goal to create a cellular blackboard, one that could be written on using a red laser and erased with a far-red laser. While the two-hybrid system offered us the flexibility of being able to alter gene expression, that was at a significant cost: time delay. A response from the yeast would not be evident for many minutes or even hours because of the lag time in gene expression: the PhyB/PIF3 would have to bind to DNA, induce transcription, and the transcription product would then have to be translated. We devised the split-luciferase system as a means to avoid that problem and thus create a blackboard with instant on/off capabilities. | ||
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<b>What is a split reporter system?</b>In the past several years a number of split-protein reporter systems have been developed, most famously the split-GFP reporter for protein-protein interaction. However, for these experiments we elected to use a split-luciferase reporter system. Luciferase is the enzyme used by Photinus pyralis, the firefly, to create bioluminescence, used for communication and attracting mates. The split-luciferase system is based on the fact that when luciferase is cut into two portions, and each attached to a protein of interest, when the interaction of those proteins of interest brings the two halves of the luciferase into close enough proximity, this results in reconstitution of the functional enzyme. In the presence of the luciferin substrate, bioluminescence will be produced. | <b>What is a split reporter system?</b>In the past several years a number of split-protein reporter systems have been developed, most famously the split-GFP reporter for protein-protein interaction. However, for these experiments we elected to use a split-luciferase reporter system. Luciferase is the enzyme used by Photinus pyralis, the firefly, to create bioluminescence, used for communication and attracting mates. The split-luciferase system is based on the fact that when luciferase is cut into two portions, and each attached to a protein of interest, when the interaction of those proteins of interest brings the two halves of the luciferase into close enough proximity, this results in reconstitution of the functional enzyme. In the presence of the luciferin substrate, bioluminescence will be produced. | ||
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<b>Where we stand. </b>We have constructed all of the plasmids needed to test this system, and are in the process of doing the necessary transfections. Fusion protein constructs were made under the control of a high expression promoter, TEF1, in an integrating vector. | <b>Where we stand. </b>We have constructed all of the plasmids needed to test this system, and are in the process of doing the necessary transfections. Fusion protein constructs were made under the control of a high expression promoter, TEF1, in an integrating vector. | ||
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Revision as of 03:57, 22 October 2009
Split Luciferase/***********************************************************************************************************************/
Theoretical Description and Purpose: The impetus behind the split-luciferase system came from our initial goal to create a cellular blackboard, one that could be written on using a red laser and erased with a far-red laser. While the two-hybrid system offered us the flexibility of being able to alter gene expression, that was at a significant cost: time delay. A response from the yeast would not be evident for many minutes or even hours because of the lag time in gene expression: the PhyB/PIF3 would have to bind to DNA, induce transcription, and the transcription product would then have to be translated. We devised the split-luciferase system as a means to avoid that problem and thus create a blackboard with instant on/off capabilities.
 ,What is a split reporter system?In the past several years a number of split-protein reporter systems have been developed, most famously the split-GFP reporter for protein-protein interaction. However, for these experiments we elected to use a split-luciferase reporter system. Luciferase is the enzyme used by Photinus pyralis, the firefly, to create bioluminescence, used for communication and attracting mates. The split-luciferase system is based on the fact that when luciferase is cut into two portions, and each attached to a protein of interest, when the interaction of those proteins of interest brings the two halves of the luciferase into close enough proximity, this results in reconstitution of the functional enzyme. In the presence of the luciferin substrate, bioluminescence will be produced. Although the split-GFP system is better characterized, the split-luciferase system has a very important advantage: it is reversible and instantaneous. Upon dissociation of the proteins of interest, the split-luciferase proteins also dissociate, resulting in cessation of bioluminescence. Thus, bioluminescence in a split-luciferase system is a real-time indicator of protein association. In the context of a cell-to-cell communication system, this has important implications. In theory, if the N-terminal portion of luciferase is fused to PhyB, and the C-terminal portion of luciferase is fused to PIF3 and the fusion proteins exposed to red light, this should result in association of the PhyB/PIF3 complex. Once the luciferase halves are in close enough proximity, they should be able to reconstitute the functional enzyme, resulting in induction of bioluminescence in response to exposure to red light. This bioluminescence can then be turned off by conversion of PhyB back to the Pr form via exposure to far-red light. What can we do with a split reporter system?The result of this system would be a real-time cell-to-cell communication system. If a red luciferase is used, this would result in the amplification of the signal within and between cells, as the bioluminescence from the red luciferase would cause the interconversion of other molecules of PhyB and thus induce more luciferase to bioluminesce. An interesting side effect of the split-luciferase is that the reconstituted protein tends to have a redshift of 10 nm, which would increase emission in the maximal absorption range of PhyB. This would make the split-luciferase system even more suited to signal amplification than the two-hybrid system. As green luciferase is approximately 10 times brighter than red luciferase, if green luciferase is used, the signal would be much brighter. This would make the green variant of the split-luciferase system well suited to use in an instantaneous writable/erasable cellular blackboard, as outlined in our section “Cellular Blackboard.” Where we stand. We have constructed all of the plasmids needed to test this system, and are in the process of doing the necessary transfections. Fusion protein constructs were made under the control of a high expression promoter, TEF1, in an integrating vector.  , |
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