An inducible and titratable site-specific in vivo recombination system in Escherichia coli

Site-specific recombination systems, such as Cre-lox, have notably enhanced spatial and temporal control of gene expression, and cell labeling techniques like the Brainbow method. Some applications, such as biosensors, might benefit from in vivo recombination events at low rates that are tunable with an external inducer. Developing such a system is challenging due to the high efficiency of DNA recombinases. In Saccharomyces cerevisiae, this has been obtained by fusing the Cre recombinase to the estradiol-binding domain (EBD) which sequesters the Cre in the cytoplasm until estradiol is provided. Once the Cre is in the nucleus, it is able to cleave DNA between two lox sites located on the genome. The development of tunable, low-rate recombination systems for prokaryotes has been very limited, and achieved only through light-inducible systems. We developed and characterized a Cre/lox system that allows us to perform in vivo recombineering at tunable, low rates in Escherichia coli, using chemically inducible promoters with low leakage and a degradation tag on the Cre recombinase. This system can be introduced on either plasmids or the E. coli chromosome. Future applications include controlled and reproducible cell conversion in evolutionary experiments and the development of recombination-based biosensors.