Fig. 3.

Target- and self-curing of plasmids by in vivo meganuclease digestion with pQURE vectors. (A) Synthetic control of plasmid replication for target- and self-curing. In pQURE vectors, the gene encoding TrfA (which binds to the vegetative origin of replication, oriV) is under transcriptional control of the XylS/Pm expression system. pQURE vectors can only replicate in the presence of 3-methylbenzoate (3-mBz), effector of the XylS transcriptional regulator. The Pm promoter also drives the expression of the gene encoding the I-SceI meganuclease. When I-SceI is expressed, the meganuclease introduces double-strand breaks in any DNA molecule containing its recognition site. In the example, a target plasmid carrying an engineered I-SceI restriction site is recognized by the meganuclease and subjected to in vivo digestion—resulting in selective plasmid loss. (B)P. putida KT2440 harboring plasmid pS6313·GFPs was co-transformed with vector pQURE1·H (XylS/Pm→trfA, Amp^R). During the first curing step (indicated as 1), cell were grown in LB medium in the presence of 2 ​mM 3-mBz for 18 ​h and aliquots were plated onto solid LB medium. A single colony displaying an msfGFP^– phenotype (indicative of loss of plasmid pS6313·GFPs) was inoculated into fresh LB medium without any additives, and grown for 18 ​h (shown as 2). After plating aliquots of this suspension, the percentage of plasmid-free cells as well as the fraction of P. putida cells carrying plasmids pS6313·GFPs and/or pQURE1·H was calculated as indicated in Fig. 1. (C) Segregational stability of vector pQURE6·H (XylS/Pm→trfA, Gm^R) in P. putida KT2440 over 7–8 generations in LB medium cultures added with varying concentrations (concn.) of 3-mBz in the μM range. The specific (Sp) red fluorescence of these cultures was calculated as indicated in Fig. 1. Bars represent the mean value of either the percentage of plasmid-containing cells or the Sp red fluorescence in each culture ​± ​standard deviation of quadruplicate measurements from at least three independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)