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Team:Harvard/Blackboard
From 2009.igem.org
Potential Projects with Other Reporters
CELLULAR BLACKBOARD The beauty of a synthetic light regulated gene expression circuit lies in its ability to execute bidirectional expression. Switching on by red light and off by far red light enables a wide range of applications for this elegant yeast two hybrid system. Output monitoring through a bacterial blackboard may in addition to providing an exquisite proof-of-principle system, additionally generate a powerful technique to visualize spatiotemporal control of gene expression. This genetic circuit seeks much inspiration from naturally occurring molecules present in nature. To construct the light sensing properties of the system, we resort to finding the suitable match that nature has provided through evolution. Plants are known to harness the energy from light to carry out various catabolic reactions necessary to mediate survival. One of such proteins, Phytochrome B possesses a robust, photon-induced light sensing domain that is able to convert extracellular light stimuli to mediate transcription-dependent and independent signaling. Once activated, PhyB is known to interact with the Phytochrome Interacting Factor 3 (PIF3) through a well characterized binding event. To engineer the requisite parts of this system, we have fused the PhyB protein with the DNA binding domain of our target element. The complementary half of our system involves a PIF3 fusion with the target element activation domain. Hence, upon light stimulation, PhyB-PIF3 interaction will ensue, resulting in the proximal placement of the DNA binding domain to the activation domain. Ultimately, this results in subsequent expression of the target luciferase gene that lies directly downstream of the promoter. Because PhyB possesses both excitatory and inhibitory behavior in the presence of red and far red light, respectively, such a system can easily be manipulated as a switch. It is our goal to create a lawn of bacteria several millimeters thick. Upon irradiation with a red light source, the area corresponding to light irradiation will glow to the emission intensity of its respective luciferase. Due to the switching capabilities of the phytochrome, incident irradiation with far red light will conversely result in attenuation of this signal, serving as an effective “eraser” to the blackboard. RED LIGHT DISTRICT The basic idea of the yeast red light district is to have haploid yeast communicate with each other via light signals. Haploid yeast come in two mating types Mat A and MAT alpha. The HO endonculease gene allows yeast to switch mating types. Since only yeast of different mating types will mate with each other, this change in mating type is necessary in order to allow neighboring yeast to mate. We are going to use the same pulse of red light (used as an input in our bacterial blackboard) to act as a stimulus that will prompt expression of the red luciferase gene. The emitted red light which will result from the expression of the red luciferase gene will then activate the yeast two hybrid system. In this system the two phytochromes PhyB and Pif 3 will come together in order to trigger a conformational change* which will then allow for the expression of the HO endonuclease gene. Expression of this particular gene will then result in a mating type switch, thereby allowing the yeast to mate with each other. We can use yeast complementation tests to see whether or not the red light has induced a mating type switch and mating. Since we are using red light to induce mating we call this our “yeast red light district.” **** INSERT yeast mating switch picture **** |
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