Abstract
Background Transposable elements is an extremely diverse group of genetic elements encoding their own mobility. This ability has been exploited as a powerful tool for molecular biology and genomics techniques. However, transposition activity is regulated by cis and/or trans mechanisms because of the need to co-exist with their host. This represents a limitation to their usage as biotechnological tools. The development of screening assays and the improvement of current ones is therefore needed to find hyperactive transposases.
Results We present in this study an improvement of the well-known papillation assay where in place of an inducible promoter, we designed a set of constitutive promoters cloned into a one or five copies vector in presence or absence of a ribosome binding site. This set of vectors provides a wide range of transposase expression and offers a more uniform expression of the transposase across cells compared to inducible promoters. These constructs can therefore be used to screen for hyperactive transposases or for transposases resistant to overproduction inhibition, a mechanism affecting DNA transposases such as Hsmar1, which decreases the transposition rate when the transposase concentration increases. We characterized and validated our set of vectors with the Hsmar1 transposase and took advantage of our approach to investigate the effects on the transposition rate of inserting mutations in the Hsmar1 dimer interface or of covalently binding two Hsmar1 monomer.
Conclusions This improved papillation assay should be applicable to a wide variety of DNA transposases. It also provides a straightforward approach to screen transposase mutant libraries with a specific expression level to find hypoactive, hyperactive or overproduction inhibition resistant transposases. Our approach could also be useful for synthetic biology as a combination of the wild type or covalently bound Hsmar1 transposase with a library of weak promoters offers the possibility to find promoters expressing on average one or two proteins per cell.