Positive selection screens are challenging, but essential, components of isolating functional mutants in large libraries, and evolving proteins. Such screens must be selective enough to ensure that only functional variants are propagated, but not too stringent that it is always lethal. To screen a DNA endonuclease library, we develop and optimize three selection screens: a ccdB-based selection screen, a chloramphenicol selection screen, and a competing endonuclease selection screen. In the first selection system, we use ccdB which encodes a toxin that induces lethal double strand DNA breaks in E. coli. We program endonuclease cleavage of the ccdB gene on a plasmid with the hypothesis that all surviving mutants display some cleavage activity proportional to the number of surviving mutants. First, we titrated expression of ccdB to determine optimal expression levels and times for effective selection of functional mutants. However, the high toxicity of ccdB selected for cells that spontaneously cured the plasmid independent of endonuclease activity after 5 h. To reduce spontaneous curing, we expressed ccdB from a lower copy plasmid and tuned relative expression levels of endonuclease and ccdB. In the second selection system, we use a repressible chloramphenicol resistance gene, targeting the repressor with an endonuclease. Successful cleavage of the repressor yields chloramphenicol resistance which can be verified by counting colonies grown in the presence of chloramphenicol. Our third selection system employs competing endonucleases, each with an associated resistance marker. Each endonuclease is targeted to the other endonuclease, and successful cleavage linearizes the plasmid thereby removing the selection marker. Growing colonies in the presence of each selection marker clearly shows the more efficient endonuclease. Moreover, endonuclease expression can be tuned if the competing endonuclease is found to be more efficient than the one of interest. We are now employing these systems to evolve programmable DNA endonucleases.

Positive selection screens for DNA endonuclease activity

2019 Spring National Meeting

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