Engineering large functional plasmids for biosafety

• A strategy for recombineering of intact large plasmids with non-antibiotic markers is presented.
• Designed for allowing biosafety requirements to be met for such plasmids.
• Approach is demonstrated using the R995 + SPI-1 and R995 + SPI-2 plasmids.
• The new constructs are functional, defective for conjugation, and transferred via electroporation.
• The new constructs are stably maintained under conditions where original plasmids are not.

Large bacterial plasmid constructs (generally 25–100 kb, but can be greater), such as those engineered with DNA encoding specific functions such as protein secretion or specialized metabolism, can carry antibiotic resistance genes and/or conjugation systems that typically must be removed before use in medical or environmental settings due to biosafety concerns. However, a convenient in vivo recombineering approach for intact large plasmids to sequentially remove multiple different genes using non-antibiotic selection methods is not described in the literature to our knowledge. We developed strategies and reagents for convenient removal of antibiotic resistance markers and conjugation genes while retaining non-antibiotic-based plasmid selection to increase practical utility of large engineered plasmids. This approach utilizes targeted lambda Red recombination of PCR products encoding the trpE and asd genes and as well as FLP/FRT-mediated marker removal. This is particularly important given that use of restriction enzymes with plasmids of this size is extremely problematic and often not feasible. This report provides the first example of the trpE gene/tryptophan prototrophy being used for recombineering selection. We applied this strategy to the plasmids R995 + SPI-1 and R995 + SPI-2 which encode cloned type III secretion systems to allow protein secretion and substrate delivery to eukaryotic cells. The resulting constructs are functional, stably maintained under conditions where the original constructs are unstable, completely defective for conjugative transfer, and transferred via electroporation.