Abstract
BackgroundPlasmids are widely used and essential tools in molecular biology. However, plasmids often impose a metabolic burden and are only temporarily useful for genetic engineering, bio-sensing and characterization purposes. While numerous techniques for genetic manipulation exist, a universal tool enabling rapid removal of plasmids from bacterial cells is lacking.ResultsBased on replicon abundance and sequence conservation analysis, we show that the vast majority of bacterial cloning and expression vectors share sequence similarities that allow for broad CRISPR-Cas9 targeting. We have constructed a universal plasmid-curing system (pFREE) and developed a one-step protocol and PCR procedure that allow for identification of plasmid-free clones within 24 h. While the context of the targeted replicons affects efficiency, we obtained curing efficiencies between 40 and 100% for the plasmids most widely used for expression and engineering purposes. By virtue of the CRISPR-Cas9 targeting, our platform is highly expandable and can be applied in a broad host context. We exemplify the wide applicability of our system in Gram-negative bacteria by demonstrating the successful application in both Escherichia coli and the promising cell factory chassis Pseudomonas putida.ConclusionAs a fast and freely available plasmid-curing system, targeting virtually all vectors used for cloning and expression purposes, we believe that pFREE has the potential to eliminate the need for individualized vector suicide solutions in molecular biology. We envision the application of pFREE to be especially useful in methodologies involving multiple plasmids, used sequentially or simultaneously, which are becoming increasingly popular for genome editing or combinatorial pathway engineering.
Highlights
Plasmids are widely used and essential tools in molecular biology
We show that our system enables fast and efficient curing of all major plasmid replicons used in modern molecular biology laboratories and can be applied in a broad phylogenetic context
We first explored the distribution of cloning vector replicons by performing a BLAST search of selected replicons against all bacterial plasmids with full nucleotide sequences available in the Addgene plasmid repository [29] (Fig. 1)
Summary
Plasmids are widely used and essential tools in molecular biology. plasmids often impose a metabolic burden and are only temporarily useful for genetic engineering, bio-sensing and characterization purposes. While numerous techniques for genetic manipulation exist, a universal tool enabling rapid removal of plasmids from bacterial cells is lacking Since their discovery in the early 1950s, plasmids have played a pivotal role in the advancement of molecular biology, and form the basis for DNA cloning and gene expression in modern biotechnology [1]. A large proportion of naturally occurring plasmids replicate through the use of replication (Rep) proteins that act in a self-inhibitory fashion to control plasmid copy-number [10] These include the replicons of pBBR1, RK2 and RSF1010 that are found in cloning vectors and function in a broad host context [11]. The Rep protein based pSC101 vector was the first to be used for recombinant gene
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