Understanding Rules for Engineering Large Genomically Integrated Mammalian Gene Circuits
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Understanding Rules for Engineering Large Genomically Integrated Mammalian Gene Circuits , Aldulaimi NB , Piepergerdes TC , O’Connell RW , Bashor CJ, Endowing mammalian cells with therapeutic phenotypes is an important goal for synthetic biology research. The current goal is to develop a framework for the combinatorial assembly, genomic integration, and testing arrays of up to 10 genes. TypeIIs-based cloning allows rapid generation of a modular library of individual expression units containing unique combinations of parts: chromatin insulator, promoter, open reading frame, RNA stabilization domain, and terminator. We are interested in determining rules for performance and compatibility of these parts. Here we focus on testing combinations of chromatin insulators and promoters within a solitary mCherry-expression unit, first by transient transfection into mammalian cells, and then by integration into specific loci. We hypothesize that chromatin insulator-promoter pairs will exhibit differential expression which may or may not be dependent on their position within a tandem gene array. Understanding these context rules will help us predict tunable multi-gene circuitry that can support engineered therapeutic function. This project was completed with contributions from Caleb Bashor from the Bioengineering Department, Rice University.