SSA-like and MMEJ-like recombination events in a transgenic construct with in-cis programmed self-eliminating components in different E. coli strains.

OBJECTIVE: DNA repair pathways have been leveraged for genetic engineering strategies, including self-eliminating transgenes in insects. In effort towards establishing a self-eliminating transgenic system that leaves desired cargoes like antibodies or anti-viral molecules in Aedes aegypti, steps were taken to generate a plasmid with direct repeats and other necessary transgenic components. Here, we present an account of series of recombination events in E. coli that were encountered during the building of a multi-component plasmid for achieving a cargo-leaving self-eliminating transgenic system in Ae. aegypti.

RESULTS: Step-wise construction of the multi-component plasmid in Stellar™ E. coli competent cells achieved successful modifications of a parent plasmid from a previous study by removing some DNA fragments, incorporating direct repeats and incorporating fluorescent marker gene cassettes (3xP3-DsRed and 3xP3-BFP) in desired locations. However, recombination events resulting in precise and repeatable deletions of small and large DNA fragments were obtained following incorporation of a target site for the in-cis-located I-SceI homing endonuclease into the plasmid. These events were also observed using other E. coli strains like SURE and NEB® Stable competent cells, although one clone from the NEB® Stable cells eventually yielded the expected plasmid construct. Analysis of the incorrect plasmids revealed that SSA-like and MMEJ-like DNA repair pathways appeared responsible for the recombination events encountered, suggesting that Aedes aegypti regulatory elements used for control of the homing endonuclease might be leaky in E. coli.