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Download Engineering Independent Reporters for Multiplexed Light
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Engineering Independent Reporters for Multiplexed Light-Switchable Gene Expression Lucy Zhuang Mentor: Elliot Hui Gene expression in engineered yeast cells can be manipulated using light as a synthetic control input, using the light switchable interaction of photoreceptor protein phytochrome B (PhyB) and phytochrome interacting factor (PIF) to drive a yeast two-hybrid. We hypothesize that the response kinetics of this system can be tuned through control of PhyB/PIF subcellular localization, potentially allowing control of different genes using different temporal patterns of light. Control of PIF localization by synthetic Nuclear Localization Sequences may be confounded by PIF’s small size and possible intrinsic NLS activity. To address this, we generated new vectors to alter PhyB localization instead. We also fused fluorescent protein tags and used live cell microscopy to verify PhyB and PIF localization. Light-activated expression of a LacZ reporter gene was measured. Preliminary tests of the new PhyB/PIF fusions show PhyB-GAD fusions induction of LacZ expression independent of light, suggesting some fusion partners may be incompatible. To troubleshoot this issue, we are exploring alternate protein fusions and the use of Nuclear Export Sequences.
