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.

Keywords: Cyanobacteria; Plasmid vector; RNA thermometer.

Graphical abstract

Fig. 1

Vector map of pSM201v, rep genes were derived from the RSF1010 backbone and placed under the control of the native P4 promoter, the mob genes were replaced by the RP4 oriT reducing the size of the vector, while maintaining the ability to transfer via mobilization with an E. coli mating helper strain.

Fig. 2

Electrophoresis on 1.5% agarose digestion of pSM201v isolated from Synechococcus elongatus PCC 7942 with EcoRI, HindIII, BamHI, and PsuI. (The uncropped image is available as supplementary figure “Gel image 2018-11-23.tif”).

Fig. 3

Minimum free energy structure of DTT1.

Fig. 4

Expression of RFP in E. coli DH5α under the control of DTT1 at various temperatures, 25 °C and 30 °C were unable to induce the expression of RFP, while temperature of 37 °C and above allowed the expression of RFP.

Fig. 5

Fluorescence microscopy images of GFP induced and GFP uninduced PCC 7942 trans- formed with pSM201v-DTT1-GFP, red cells indicate the autofluorescence caused by chlorophyll of cyanobacteria. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)