Reaction Routes Enabled By Reversibly Acting Tyr-Recombinases and A Unidirectional Ser-Integrase
Fig. 2 shows, in sections A-B, the mode integration/resolution and inversion (INT/RES and INV) depend on the orientation of recombinase target sites (RTS), among these pairs of attP and attB. Section C indicates, in a streamlined fashion, the way recombinase-mediated cassette exchange (RMCE) can be reached by synchronous double-reciprocal crossovers (rather than integration, followed by resolution). Of note is the way reversible Flp-integration/resolution is modulated by 48 bp (in place of 34 bp minimal) FRT versions: the extra 13 bp arm serves as a Flp "landing path" contributing to the formation of the synaptic complex, both in the context of Flp-INT and Flp-RMCE functions (see the respective equilibrium situations). While it is barely possible to prevent the (entropy-driven) reversion of integration in section A for Cre and hard to achieve for Flp, RMCE can be completed if the donor plasmid is provided at an excess due to the bimolecular character of both the forward- and the reverse reaction. Posing both FRT sites in an inverse manner will lead to an equilibrium of both orientations for the insert (green arrow). In contrast to Flp, the Ser integrase PhiC31 (bottom representations) leads to unidirectional integration, at least in the absence of an recombinase-directionality (RDF-)factor. Relative to Flp-RMCE, which requires two different ("heterospecific") FRT-spacer mutants, the reaction partner (attB) of the first reacting attP site is hit arbitrarily, such that there is no control over the direction the donor cassette enters the target (cf. the alternative products). Also different from Flp-RMCE, several distinct RMCE targets cannot be mounted in parallel, owing to the lack of heterospecific (non-crossinteracting) attP/attB combinations.
Read more about this topic: Site-specific Recombinase Technology
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