pShuffle: A Plasmid for in vitro Evolution

Multi-gene shuffling is a powerful method used to combine and optimize attributes of various proteins. Here we report on the design and construction of the plasmid “pShuffle” which is suited for a variety of in vitro DNA-recombination techniques. The multiple cloning site (MCS) of pShuffle was designed to allow for the cloning of genes as well as their expression under control of either a lac- or a T7-promoter. As a specific feature, this MCS allows for the fusion of special linker sequences to both ends of cloned genes. After subsequent DNA-recombination steps, these linkers facilitate reamplification of generated gene variants, and thus may be used to construct clone libraries for activity screenings. The suitability of pShuffle for multi-gene shuffling applications was further shown with a set of styrene monooxygenase genes originating from proteo- and actinobacteria

Crystallization and preliminary characterization of chloromuconolactone dehalogenase from Rhodococcus opacus 1CP

Chloroaromatic compounds are often very persistent environmental pollutants. Nevertheless, numerous bacteria are able to metabolize these compounds and to utilize them as sole energy and carbon sources. ıt Rhodococcus opacus} 1CP is able to degrade several chloroaromatic compounds, some of them {ıt via} a variation of the 3-chlorocatechol branch of the modified {ıt ortho}-cleavage pathway. This branch in {ıt R. opacus} differs from that in {ıt Proteobacteria} in the inability of the chloromuconate cycloisomerase to dehalogenate. Instead, a unique enzyme designated as chloromuconolactone dehalogenase (ClcF) is recruited. ClcF dehalogenates 5-chloromuconolactone to {ıt cis}-dienelactone and shows a high similarity to muconolactone isomerases (EC 5.3.3.4). However, unlike the latter enzymes, it is unable to catalyse the isomerization of muconolactone to 3-⁻oxoadipate enollactone. In order to characterize the catalytic mechanism of this unusual dehalogenase, the enzyme was crystallized and subjected to X-ray structural analysis. Data sets to up to 1.65Å resolution were collected from two different crystal forms using synchrotron radiation. Crystal form I (space group {ıt P}2{\sb 1}) contained 40 subunits in the asymmetric unit, whereas ten subunits were present in crystal form II (space group {ıt P}2{\sb 1}2{\sb 1}2{\sb 1). The self-rotation function revealed the orientations of the molecular symmetry axes of the homodecamer of 52 symmetry