Abstract: Split inteins are powerful tools for seamless ligation of synthetic split proteins. Yet, their use remains limited because the already intricate split site identification problem is often complicated by the requirement of extein junction sequences. To address this, we augment a mini-Mu transposon-based screening approach and devise the intein-assisted bisection mapping (IBM) method. Read more >>

Keywords: protein engineering; MuA transposase; split protein-intein fusion; synthetic biology; split intein.

The phage Mu DNA transposition system provides a versatile species non-specific tool for molecular biology, genetic engineering and genome modification applications. Mu transposition is catalyzed by MuA transposase, with DNA cleavage and integration reactions ultimately attaching the transposon DNA to target DNA. To improve the activity of the Mu DNA transposition machinery, we mutagenized MuA protein and screened for hyperactivity-causing substitutions using an in vivo assay. Read more >>

Keywords: MuA Transposase, hyperactive enzyme, mutant, transposon, genome engineering

Cover: Two views of the Mu transpososome structure superimposed on one of the papilated E. coli colonies used in a screen to select hyperactive variants of the transposase protein. Structure pictures were made with Pymol (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.).
For more information see article by Rasila, T.S. et al., pages 4649–4661 in this issue.

Abstract: Archaea, like bacteria, use type IV pili to facilitate surface adhesion. Moreover, archaeal flagella—structures required for motility—share a common ancestry with type IV pili. While the characterization of archaeal homologs of bacterial type IV pilus biosynthesis components has revealed important aspects of flagellum and pilus biosynthesis and the mechanisms regulating motility and adhesion in archaea, many questions remain.   Read more >>

Keywords: Haloferax volcanii; adhesion; archaellum; archaeon; biofilm; chemotaxis; flagellum; swimming motility; transposon mutagenesis; type IV pilus.

Archaea share fundamental properties with bacteria and eukaryotes. Yet, they also possess unique attributes, which largely remain poorly characterized. Haloferax volcanii is an aerobic, moderately halophilic archaeon that can be grown in defined media. It serves as an excellent archaeal model organism to study the molecular mechanisms of biological processes and cellular responses to changes in the environment. Read more >>

Keywords: Gene discovery; Haloferax volcanii; Halophilic archaea; Insertion mutant library; MuA transposase.

Archaea constitute the third domain of life, but studies on their physiology and other features have lagged behind bacteria and eukarya, largely due to the challenging biology of archaea and concomitant difficulties in methods development. The use of genome-wide en masse insertion mutagenesis is one of the most efficient means to discover the genes behind various biological functions, and such a methodology is described in this chapter for a model archaeon Haloferax volcanii.  Read more >>

Keywords: Gene discovery; Haloferax volcanii; Halophilic archaea; Insertion mutant library; MuA transposase.

Keywords: archaea; hypochlorite; oxidative stress; proteasome; redox-active.