Construction of pSM201v: A broad host range replicative vector based on shortening of RSF1010

Abstract

Despite possessing attractive features such as autotrophic growth on minimal media, industrial applications of cyanobacteria are hindered by a lack of genetic manipulative tools. There are two important features that are important for an effective manipulation: a vector which can carry the gene, and an induction system activated through external stimuli, giving us control over the expression. In this study, we describe the construction of an improved RSF1010-based vector as well as a temperature-inducible RNA thermometer. RSF1010 is a well-studied incompatibility group Q (IncQ) vector, capable of replication in most Gram negative, and some Gram positive bacteria. Our designed vector, named pSM201v, can be used as an expression vector in some Gram positive and a wide range of Gram negative bacteria including cyanobacteria. An induction system activated via physical external stimuli such as temperature, allows precise control of overexpression. pSM201v addresses several drawbacks of the RSF1010 plasmid; it has a reduced backbone size of 5189 bp compared to 8684 bp of the original plasmid, which provides more space for cloning and transfer of cargo DNA into the host organism. The mobilization function, required for plasmid transfer into several cyanobacterial strains, is reduced to a 99 bp region, as a result that mobilization of this plasmid is no longer linked to the plasmid replication. The RNA thermometer, named DTT1, is based on a RNA hairpin strategy that prevents expression of downstream genes at temperatures below 30 °C. Such RNA elements are expected to find applications in biotechnology to economically control gene expression in a scalable manner.