Design strategies for molecular switches in mammalian cells

Synthetic biology is an emerging field that is involved in the design and construction of artificial gene circuits that can be used to encode novel functions or study endogenous gene circuits inside living cells. In recent years, synthetic biology has been applied successfully in a variety of applications. Continued progress in the field of synthetic biology relies on the development of novel molecular switches that are robust and modular.;Translational repression based RNA switches are promising building blocks for the construction of artificial gene circuits that can encode novel functions inside living cells. Translational repression based switches in living cells have been previously demonstrated. However, no specific design strategy for the construction of novel translational repression based switches has been described. In this work, we developed a design strategy for the construction of novel translational repression based switches. Specifically, we designed a translational repression based switch using the well- characterized interaction between Bovine Immunodeficiency Virus (BIV) Tat peptide and TAR RNA and obtained 7-fold repression in the presence of BIV Tat peptide. We also developed a NAND logic gate in living mammalian cells using the translational repression based switches as building blocks.;Optogenetics involves the use of genetically encoded, light-sensitive proteins to control and study various cellular processes. In most animal cells, visible light is an orthogonal signal with minimal off-target effects. Visible light also provides a high degree of spatiotemporal control. Thus, light provides a significant advantage over small molecule drugs that have been previously used to control cellular processes. In this thesis, we describe the development of a novel protein that can be used to induce light activated target protein clustering. In another study, we report the progress on the construction of an optogenetic Tobacco Etch Virus (TEV) protease that exhibits TEV protease activity only in the presence of blue light.;In the future work section of this thesis, we suggest design strategies for the construction of NOR logic gates using translational repression based switches as building blocks. Further, we also suggest strategies to identify a light-dependent TEV protease.