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Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
Published on: March 16, 2011
New Escherichia coli WP2 tester strains highly sensitive to reversion by oxidative mutagens
M Blanco1, A Urios, A Martínez
1Instituto de Investigaciones Citológicas, Fundación Valenciana de Investigaciones Biomédicas, Amadeo de Saboya 4, 46010 Valencia, Spain.blanco@ochoa.fib.es
Abstract:
New Escherichia coli strains have been added to the WP2 mutagenicity test for the specific detection of oxidative mutagens. Strain IC203 derives from WP2 uvrA/pKM101 and is highly sensitive to oxidative stress due to a deficiency in the OxyR function. Following exposure to t-butyl hydroperoxide (BuOOH) or menadione (MD), but not to 4-nitroquinoline 1-oxide (4NQO), strain IC203 (oxyR) shows increased mutability with respect to the oxyR+ parent. The advantage that the OxyR deficiency confers on IC203 strain in detecting oxidative mutagens is not obtained with strains deficient in either katG or ahpCF, two OxyR-regulated genes. Strain IC206, a derivative of WP2 uvrA carrying a deletion of the umuDC genes and deficient in the MutY glycosylase, has also been added to the WP2 test for the detection of SOS-independent mutations promoted by 8-oxoguanine lesions. Induction of these mutations was observed after treatment with BuOOH, but not after MD or 4NQO exposure. The two new strains, IC203 and IC206, can be useful for the screening of mutations resulting from oxidative stress as well as in studies on antioxidants preventing mutagenesis.
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