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Bacterial selection is an indispensable tool for E. coli genetic engineering. Marker genes allow for mutant isolation even at low editing efficiencies. TolC is an especially useful E. coli marker: its presence can be selected for with sodium dodecyl sulfate, while its absence can be selected for with the bactericidal protein ColE1. However, utilization of this selection system is greatly limited by the lack of commercially available ColE1 protein. Here, we provide a simple, plate-based, ColE1 negative-selection protocol that does not require purification of ColE1. Using agar plates containing a nonpurified lysate from a ColE1-production strain, we achieved a stringent negative selection with an escape rate of 10 -7 . Using this powerful negative-selection assay, we then performed the scarless deletion of multiple, large genomic loci (&gt;10 kb), screening only 12 colonies each. We hope this accessible protocol for ColE1 production will lower the barrier of entry for any lab that wishes to harness olC 's dual selection for genetic engineering.

Characterization of ColE1 Production for Robust tolC Plate Dual-Selection in E. coli