Site-Directed Genome Integration via Recombinase-Mediated Cassette Exchange (RMCE) in Escherichia coli

Abstract

The gold standard for successful genome integration in Escherichia coli is the homologous recombination by the bacteriophage-inspired lambda Red system. This method uses the bacteriophage lambda Red recombination proteins to promote homologous recombination between a target DNA sequence and a DNA fragment, which is introduced into the bacterial cell by electroporation. It allows researchers to create specific genetic changes in bacterial genomes, making it a valuable tool for studies in microbiology and biotechnology. However, this system is not without limitations, which are characteristic of its working mechanism and remain to present challenges. The most formidable constraints stem from nucleotide sequences that contain self-homology or homologies to the host genome. These instances lead to uncontrolled homologous recombination events, consequently hindering the desired integration event. Furthermore, handling very large fragments can also be problematic, although, in many instances, this can be overcome by multiple lambda Red integrations in a row. In this study, we illustrate that the limitations associated with the lambda Red system can be overcome through the application of recombinase-mediated cassette exchange (RMCE). This enables the genome integration of larger and more complex DNA fragments and facilitates new research opportunities.

Keywords: BL21(DE3); RMCE; genome integration; homologous recombination; recombinant protein production; synthetic biology.

Figure 1

Schematic illustration of the preparation of the attTN7 site in the BL21(DE3)::FRT_RS I-SceI_FRT3 strain by lambda Red homologous recombination.

Figure 2

(a) Map of the designed plasmid pSG4, containing the heat-sensitive Rep101 protein, the ORI pSC101, the antibiotic resistance gene BleoR, and the arabinose inducible polycistronic gene expression system containing the gene Flp and the endonuclease enzyme I-SceI. Behind the two genes, two rrnB terminators are placed to stop transcription. Between the FRT and FRT3 sites, an example cassette for integration is illustrated. (b) Schematic illustration of recombinase-mediated cassette exchange induced by the pSG4 vector; screening primers are located on the genome outside of the FRT sides.

Figure 3

(a) Schematic illustration of the dsfGFP model protein expression construct located between the two FRT sites. (b) Screening of 16 colonies on day 4 of the RMCE genome integration protocol.

Figure 4

(a) Schematic illustration of the dSpRY-MCP-SoxS expression construct located between the two FRT sites. (b) Screening of 16 colonies on day 4 of the RMCE genome integration protocol resulted in 3 out of 16 positive colonies.

Figure 5

(a) Schematic illustration of the Fab1 light and heavy chain expression construct located between the two FRT sites. (b) Screening of 16 colonies on day 4 of the RMCE genome integration protocol.

Figure 6

(a) Schematic illustration of triple T7 sfGFP expression construct located between the two FRT sites. (b) Screening of 16 colonies on day 4 of the RMCE genome integration protocol.

Figure 7

(a) Schematic illustration of the T7 sfGFP expression construct and the lacI repressor gene located between the two FRT sites. (b) The screening of 16 colonies on day 4 of the RMCE genome integration protocol.