Author
Cao, Jicong
Other Contributors
Kane, Ravi S.; Collins, Cynthia H.; Koffas, Mattheos A. G.; Makhatadze, George I.;
Date Issued
2014-08
Subject
Chemical engineering
Degree
PhD;
Terms of Use
This electronic version is a licensed copy owned by Rensselaer Polytechnic Institute, Troy, NY. Copyright of original work retained by author.;
Abstract
Synthetic biology provides the platform and tools to design artificial regulators to control mRNA translation. In this work, we report a genetically encoded system to regulate mRNA translation using the Pumilio and FBF (PUF) domains in mammalian cells. PUF domain serves as a designable scaffold to recognize specific RNA elements, and the specificity can be altered easily to target any 8-nt RNA. In this system, the gene expression could be varied by over 17-fold when using PUF-based activators and repressors. The specificity of the method was established by using wild-type and mutant PUF domains.; Optogenetics is a technology that allows control of cellular events using visible light as the signal/inducer. We designed an optogenetic system that employs the light-sensitive dimerizing partners from Arabidopsis thaliana, Cryptochrome 2 (CRY2) and Cryptochrome-interacting basic-helix-loop-helix 1 (CIB1), to reconstitute an RNA binding peptide and a translation initiation protein, thereby activating target mRNA translation downstream of the binding sites. Moreover, the combination of the two technologies allows us to construct to a light-inducible gene expression system using PUF domains, which can be used to regulate cellular RNA functions in a light-sensitive manner.; Additionally, we found that PUF domains could also be used to repress mRNA translation in E. coli. Such a system adds an important tool of RNA/protein interaction into the repertoire of tools for genetic circuit construction in E. coli.; Considerable work has focused on the control of gene expression, motivated by both a fundamental interest in biological research as well as by applications ranging from gene therapy to metabolic engineering.;
Description
August 2014; School of Engineering
Department
Dept. of Chemical and Biological Engineering;
Publisher
Rensselaer Polytechnic Institute, Troy, NY
Relationships
Rensselaer Theses and Dissertations Online Collection;
Access
Restricted to current Rensselaer faculty, staff and students. Access inquiries may be directed to the Rensselaer Libraries.;