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Team:Harvard/Comm
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<p><b> PROJECT BACKGROUND: Bacteria to yeast communication as a means to bridge a physically separated canonical lac operon using light </b></p> | <p><b> PROJECT BACKGROUND: Bacteria to yeast communication as a means to bridge a physically separated canonical lac operon using light </b></p> | ||
<p><b>Project Overview: </b> Communication requires three components: a sender, a means of communication, and a receiver. In the case of our bacteria-to-yeast optical signaling system, our signal senders are E. coli bacteria, expressing a red variant of luciferase from the firefly Photinus pyralis. Expression of this protein is controlled by IPTG induction. </p> | <p><b>Project Overview: </b> Communication requires three components: a sender, a means of communication, and a receiver. In the case of our bacteria-to-yeast optical signaling system, our signal senders are E. coli bacteria, expressing a red variant of luciferase from the firefly Photinus pyralis. Expression of this protein is controlled by IPTG induction. </p> | ||
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<p> The second component required is a means of communication. The means of communication in our system is the red bioluminescence produced by the E. coli. This optical signal is received by the yeast, which engineered to be light-sensitive via the expression of a two-hybrid system incorporating a phytochrome (PhyB) from the plant Arabidopsis thaliana, and its interacting factor PIF3. Red light causes a change in conformation of the PhyB protein which allows it to interact with its interacting factor PIF3. </p> | <p> The second component required is a means of communication. The means of communication in our system is the red bioluminescence produced by the E. coli. This optical signal is received by the yeast, which engineered to be light-sensitive via the expression of a two-hybrid system incorporating a phytochrome (PhyB) from the plant Arabidopsis thaliana, and its interacting factor PIF3. Red light causes a change in conformation of the PhyB protein which allows it to interact with its interacting factor PIF3. </p> | ||
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<p> In a two hybrid system, one of these proteins of interest is fused to a Gal4 activation domain and one is fused to a Gal4 DNA binding domain. When these two domains are brought into close enough proximity via the interaction of the PhyB and PIF3, they result in activation of transcription of a gene of our choice under control of the Gal1 promoter, in this case, the lacZ gene. </p> | <p> In a two hybrid system, one of these proteins of interest is fused to a Gal4 activation domain and one is fused to a Gal4 DNA binding domain. When these two domains are brought into close enough proximity via the interaction of the PhyB and PIF3, they result in activation of transcription of a gene of our choice under control of the Gal1 promoter, in this case, the lacZ gene. </p> | ||
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<p> The lacZ gene was chosen as the readout for our system because it is simple to assay for its expression, and because it endowed our system with a very interesting property: this system not only allows for interspecies optical communication, but enables us to optically bridge a physically separated canonical lac operon using light as a trans-acting factor. In other words, we have separated the induction portion and the expression portions of lac operon expression both spatially and temporally, using light as a means to bridge the separation over time and space. </p> | <p> The lacZ gene was chosen as the readout for our system because it is simple to assay for its expression, and because it endowed our system with a very interesting property: this system not only allows for interspecies optical communication, but enables us to optically bridge a physically separated canonical lac operon using light as a trans-acting factor. In other words, we have separated the induction portion and the expression portions of lac operon expression both spatially and temporally, using light as a means to bridge the separation over time and space. </p> | ||
Revision as of 23:43, 21 October 2009
Bacteria-to-Yeast Communication/***********************************************************************************************************************/PROJECT BACKGROUND: Bacteria to yeast communication as a means to bridge a physically separated canonical lac operon using light Project Overview: Communication requires three components: a sender, a means of communication, and a receiver. In the case of our bacteria-to-yeast optical signaling system, our signal senders are E. coli bacteria, expressing a red variant of luciferase from the firefly Photinus pyralis. Expression of this protein is controlled by IPTG induction. The second component required is a means of communication. The means of communication in our system is the red bioluminescence produced by the E. coli. This optical signal is received by the yeast, which engineered to be light-sensitive via the expression of a two-hybrid system incorporating a phytochrome (PhyB) from the plant Arabidopsis thaliana, and its interacting factor PIF3. Red light causes a change in conformation of the PhyB protein which allows it to interact with its interacting factor PIF3. In a two hybrid system, one of these proteins of interest is fused to a Gal4 activation domain and one is fused to a Gal4 DNA binding domain. When these two domains are brought into close enough proximity via the interaction of the PhyB and PIF3, they result in activation of transcription of a gene of our choice under control of the Gal1 promoter, in this case, the lacZ gene. The lacZ gene was chosen as the readout for our system because it is simple to assay for its expression, and because it endowed our system with a very interesting property: this system not only allows for interspecies optical communication, but enables us to optically bridge a physically separated canonical lac operon using light as a trans-acting factor. In other words, we have separated the induction portion and the expression portions of lac operon expression both spatially and temporally, using light as a means to bridge the separation over time and space. In summary: bacteria communicate the presence of IPTG in their culture to the yeast cells via red light. The red light is produced by luciferase in the bacteria. The yeast absorb the red light using a plant phytochrome. In response to this light, the bacteria produce beta-galactosidase, which can then be assayed for with an X-gal or ONPG assay. |
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