MuA-mediated in vitro cloning of circular DNA: transpositional autointegration and the effect of MuB

Abstract

Transposons provide useful tools for genetics and genomics studies, as they can be modified easily for a variety of purposes. In this study, a strategy to clone circular DNA was developed on the basis of an efficient Mu in vitro transposition reaction catalyzed by MuA transposase. The transposon used contains a selectable marker as well as an origin of replication, and in vitro integration of the transposon into circular DNA generates a plasmid that can replicate in E. coli. We show that the substrate stoichiometry plays an important role in the profile of intermolecular versus intramolecular transposition reaction products. Increasing the relative amount of target DNA reduced the frequency of intramolecular products that are non-productive with regard to the developed cloning application. Such autointegration was also reduced in the reactions containing phage Mu-encoded MuB, indicating that this protein can be used for cloning in combination with MuA, and it is particularly useful with a limited amount of target DNA. The developed strategy can now be utilized to clone DNA circles regardless of their origin as long as their size is not prohibitive for transformation.

In vivocloning of β 1-4 endoglucanase gene ofSerratia liquefaciensusing Muduction and itsin silicoanalysis

“…During the replication process it transposes to more than fifty different sites in the genome within an hour (Taylor 1963; Howe and Bade 1975; Bukhari 1976; Groisman and Casadaban 1987b; Groisman and Casadaban 1987a) and frequently cause mutations of the host bacteria (Taylor 1963). High frequency of transposition and DNA packaging property make Mu phage ideal for several genetic procedures including cloning (Akhverdyan et al 2011; Harshey 2014; Pulkkinen et al 2016a; Pulkkinen et al 2016b). The mini-Mu phage has been used: for target sequence specific insertional mutagenesis of the HSV genome (Jenkins et al 1985), to clone nitrofuran reductase gene into E. coli (Kumar and Jayaraman 1991) and, to identify the regions encoding the structure and regulatory protein gene of tetracycline resistant determinants of Proteus mirabilis (Magalhaes and Castilho 1997).…”