Markerless Gene Replacement Plasmids for E. coli
Inventors: Frederick Blattner, Gyorgy Posfai, Vitaliy Kolisnychenko, Zsuzsa Bereczki
The Wisconsin Alumni Research Foundation (WARF) is seeking commercial partners interested in developing a simple and efficient gene replacement method that allows targeted modifications of E. coli DNA sequences.
Microbial genome sequencing projects uncover large numbers of new genes. Functional analyses of these genes require targeted modifications of particular DNA sequences in their chromosomal locations using mutant alleles constructed in vitro. For delivery of a mutant allele into a cell, the allele is typically carried on either a suicide plasmid or a linear DNA fragment.
UW-Madison researchers have discovered a simple and efficient gene replacement method that permits targeted introduction of markerless deletions, insertions and point mutations into the E. coli chromosome. In this method, the mutant allele is carried on a circular plasmid that integrates into the chromosome at a homologous locus, resulting in a direct duplication. Resolution of this cointegrate via intramolecular recombination is controlled by introducing a unique double-stranded break into the chromosome by the meganuclease I-SceI. The enzyme recognizes an 18-base pair sequence and generates a double-stranded break with a four-base 3` hydroxyl overhang. The method can be used in recombination-proficient E. coli and produces markerless replacements at high efficiency.
- Functional analyses of genes discovered via microbial genome sequencing projects
- Potentially useful in numerous strains of bacteria
- Does not leave selectable marker behind
- No special growth conditions needed – method is relatively insensitive to culture medium and temperature
- Efficiency of resolution increased by two to three orders of magnitude because I-SceI cleavage serves as both a selection tool and a stimulator of the resolution process