Using ColE1-derived RNA I for suppression of a bacterially encoded gene: implication for a novel plasmid addiction system

Abstract

The use of plasmid DNA for gene therapeutical purposes is a novel technology with advantages and drawbacks. One of the required improvements is to avoid antibiotic resistance genes or other additional sequences for selection within the plasmid. Here, we describe an alternative approach to equip a ColE1 plasmid with a regulatory function within the cell, which could be used for selection of plasmid carrying cells. No additional sequences are required, since the mechanism is based on RNA/RNA antisense interaction involving the naturally occurring RNA I derived from the plasmid's origin of replication. The plasmid replicational regulatory network was linked to the transcriptional regulatory network of an engineered target gene, present on the bacterial chromosome. Thus, gene suppression of a reporter could be achieved by mere presence of the ColE1-type plasmid pBR322. Proof of this concept was shown in shaker-flask experiments and fed-batch fermentation processes. The strategy of regulating gene expression by plasmid replication implicates a novel strategy for plasmid selection, as the gene to be suppressed could be toxic or growth hampering, providing advantage to plasmid carrying host cells.