GFP MUTAGENESIS AMPLIFICATION: USE OF A FLUORESCENCE-ANTIBIOTIC RESISTANCE FUSION DUAL REPORTER CONSTRUCT TO PROVIDE QUANTITATIVE AND HIGHLY SENSITIVE DETECTION OF MUTATIONS

摘要

A reversion mutation assay that is unique in providing a quantitative readout for mutagenesis. This assay is based on the creation of a functional GFP-β-lactamase fusion protein as a reporter providing both antibiotic resistance and fluorescence. This dual reporter is placed in a multicopy plasmid to increase the number of targets, with a reversion site at the N-terminus. Rare mutations at the reversion site allow read-through of the fusion protein, producing both beta-lactamase (providing antibiotic resistance) and GFP (emitting fluorescence). In the presence of carbenicillin, beta-lactamase production confers a selective advantage that allows amplification of mutant plasmids, raising the level of fluorescence emitted by GFP to levels that are detectable by fluorimetry. A window of time can be found where fluorescence is proportional to the number of mutation events at the reversion site, making fluorescence a quantitative measure of mutagenesis. Quantitative (as opposed to binary) detection of mutations allows substantial savings in test sample. This has applications in drug discovery, allowing high-throughput screening for DNA-targeting compounds and early pre-screening of leads for potential carcinogenic activity. The increased sensitivity of this assay also facilitates monitoring complex environmental samples.