1. Engineering Synthetic Mammalian Expression Vectors 2. A Bacterial Strategy to Link Calcium Influx to Cell Survival

Abstract

(1) The field of synthetic biology is rapidly growing, empowered by advancements in Molecular Biology. To express genes of interest, scientists exploit plasmids engineered for bacterial or mammalian expression. Existing plasmids carry superfluous DNA that decreases transformation and transfection efficiencies. Here, we present a novel set of mammalian expression vectors with different selection markers and tunable expression levels. Despite being substantially smaller than traditional vectors, these minimized plasmids display similar levels of gene expression.This set of novel mammalian expression vectors should be useful for a broad range of modern applications. (2) DREAM is a mammalian calcium-dependent repressor of gene expression, which binds to a downstream DNA sequence, called DRE. The DREAM/DRE system offers a unique way of linking gene expression to calcium influx (44), but the utility of this system has not been assessed in bacteria. Here we develop a simple bacterial cell growth assay, where cell growth is prevented by the expression of SacB. This cell growth assay is then exploited to assess the utility of the DREAM/DRE system in bacteria. Unexpectedly, the DRE sequence by itself represses the expression of SacB, which can then be de-repressed by the co-expression of DREAM. Should this recovery of SacB expression maintain calcium-dependence, the DREAM/DRE system could be exploited in future directed strategies to evolve calcium-permeable ion channels.