Statistical noise from recombinant plasmids can be abated via complementation of a ribosomal protein gene deletion.

Abstract

The phenotypes conferred by recombinant plasmids upon host cells often exhibit variability between replicate populations. This statistical noise is mostly a consequence of adaptive evolution in response to fitness burdens imposed by the plasmids themselves. We developed a novel strategy, 'ribosome pegging', to exclude common unwanted mutations that benefit host cells at the expense of heterologous gene expression. Plasmids that constitutively co-expressed the fluorescent reporter tagRFP and ribosomal protein L23 (rplW) were used to transform Escherichia coli cells that lacked the essential chromosomal rplW gene. Cells within the population that expressed too little L23, or too much, were evidently inviable. Ribosome pegging obviates the need for antibiotics, thus facilitating the deployment of recombinant bacteria in uncontrolled environments. We show that ribosome-pegged E. coli carrying a plasmid that constitutively expresses L23 and an artificially evolved enzyme protects fruit flies from otherwise toxic doses of the insecticide malathion.