Protein switches identified from diverse insertion libraries created using S1 nuclease digestion of supercoiled‐form plasmid DNA

• DOI: 10.1002/bit.23224
• Publication Date: 2011-05-26T14:33:13Z
• Authors: Jennifer Tullman; Gurkan Guntas; Matthew Dumont; Marc Ostermeier
• Keywords: Nuclease; Micrococcal nuclease; Insert (composites); Inverted repeat; Bacterial conjugation

We demonstrate that S1 nuclease converts supercoiled plasmid DNA to unit-length, linear dsDNA through the creation of a single, double-stranded break in molecule. These breaks occur not only origin replication near inverted repeats but also at wide variety locations throughout plasmid. exhibits this activity under conditions typically employed for nuclease's single-stranded activity. Thus, digestion DNA, unlike analogous with DNaseI, effectively halts after first break. This property makes easier construction large domain insertion libraries which goal is insert used create library circularly permuted TEM1 β-lactamase gene was inserted containing encoding Escherichia coli ribose binding protein. Gene fusions encode allosteric switch proteins modulates catalytic were isolated from using combination genetic selection and screen.