A series of constitutive expression vectors to accurately measure the rate of DNA transposition and correct for auto-inhibition

Abstract

AbstractBackgroundTransposable elements (TEs) form a diverse group of DNA sequences encoding functions for their own mobility. This ability has been exploited as a powerful tool for molecular biology and genomics techniques. However, their use is sometimes limited because their activity is auto-regulated to allow them to cohabit within their hosts without causing excessive genomic damage. To overcome these limitations, it is important to develop efficient and simple screening assays for hyperactive transposases.ResultsTo widen the range of transposase expression normally accessible with inducible promoters, we have constructed a set of vectors based on constitutive promoters of different strengths. We characterized and validated our expression vectors with Hsmar1, a member of the mariner transposon family. We observed the highest rate of transposition with the weakest promoters. We went on to investigate the effects of mutations in the Hsmar1 transposase dimer interface and of covalently linking two transposase monomers in a single-chain dimer. We also tested the severity of mutations in the lineage leading to the human SETMAR gene, in which one copy of the Hsmar1 transposase has contributed a domain.ConclusionsWe generated a set of vectors to provide a wide range of transposase expression which will be useful for screening libraries of transposase mutants. We also found that mutations in the Hsmar1 dimer interface provides resistance to overproduction inhibition in bacteria, which could be valuable for improving bacterial transposon mutagenesis techniques.