Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication

Directed evolution is a powerful tool to improve the characteristics of biomolecules. Here we present a protocol for the intracellular evolution of proteins with distinct differences and advantages in comparison with established techniques. These include the ability to select for a particular function from a library of protein variants inside cells, minimizing undesired coevolution and propagation of nonfunctional library members, as well as allowing positive and negative selection logics using basally active promoters. A typical evolution experiment comprises the following stages: (i) preparation of a combinatorial M13 phagemid (PM) library expressing variants of the gene of interest (GOI) and preparation of the Escherichia coli host cells; (ii) multiple rounds of an intracellular selection process toward a desired activity; and (iii) the characterization of the evolved target proteins. The system has been developed for the selection of new orthogonal transcription factors (TFs) but is capable of evolving any gene—or gene circuit function—that can be linked to conditional M13 phage replication. Here we demonstrate our approach using as an example the directed evolution of the bacteriophage λ cI TF against two synthetic bidirectional promoters. The evolved TF variants enable simultaneous activation and repression against their engineered promoters and do not cross-react with the wild-type promoter, thus ensuring orthogonality. This protocol requires no special equipment, allowing synthetic biologists and general users to evolve improved biomolecules within ~7 weeks

Identification of a collagen type I adhesin of Bacteroides fragilis

Bacteroides fragilis is an opportunistic pathogen which can cause life threatening infections in humans and animals. The ability to adhere to components of the extracellular matrix, including collagen, is related to bacterial host colonisation. Collagen Far Western analysis of the B. fragilis outer membrane protein (OMP) fraction revealed the presence two collagen adhesin bands of ∼ 31 and ∼ 34 kDa. The collagen adhesins in the OMP fraction were separated and isolated by two-dimensional SDS-PAGE and also purified by collagen affinity chromatography. The collagen binding proteins isolated by both these independent methods were subjected to tandem mass spectroscopy for peptide identification and matched to a single hypothetical protein encoded by B. fragilis NCTC 9343 (BF0586), conserved in YCH46 (BF0662) and 638R (BF0633) and which is designated in this study as cbp1 (collagen binding protein). Functionality of the protein was confirmed by targeted insertional mutagenesis of the cbp1 gene in B. fragilis GSH18 which resulted in the specific loss of both the ∼ 31 kDa and the ∼ 34 kDa adhesin bands. Purified his-tagged Cbp1, expressed in a B. fragilis wild-type and a glycosylation deficient mutant, confirmed that the cbp1 gene encoded the observed collagen adhesin, and showed that the 34 kDa band represents a glycosylated version of the ∼ 31 kDa protein. Glycosylation did not appear to be required for binding collagen. This study is the first to report the presence of collagen type I adhesin proteins in B. fragilis and to functionally identify a gene encoding a collagen binding protein

A visão administrativa do enfermeiro no macrossistema hospitalar: um estudo reflexivo El punto de vista administrativo del enfermero en el macro sistema hospitalario: un estudio reflexivo The nurse's administrative point of view in the hospitalar macro system: a reflexive study

Objetivou-se neste estudo investigar a visão administrativa do enfermeiro na gerência do macrossistema hospitalar, com base na Teoria Contingencial de Administração. Participaram do estudo 09 enfermeiras administradoras de serviços de enfermagem, de 06 hospitais de grande porte localizados na cidade de Fortaleza, Ceará. Os dados foram coletados mediante entrevista livre no período de agosto a dezembro de 2002. Os resultados apontam para uma visão administrativa do enfermeiro centrada em dois itens da administração geral: Atividades de Liderança e de Controle. As entrevistas revelaram que a administração de enfermagem no Macro Sistema Hospitalar não foi percebida como uma tarefa fácil pelo grupo, porém factível. A dificuldade maior pareceu centrar-se na falta de uma reflexão voltada para as práticas administrativas próprias do enfermeiro.<br>El objetivo de este estudio fue el de investigar la visión administrativa del enfermero en actividades de gerencia dentro del macrossistema administrativo hospitalar, sustentado en la Teoría Contingencial de Administración. Participaron del estudio nueve enfermeras administradoras de servicios de enfermería provenientes de seis hospitales de gran importancia localizados en la ciudad de Fortaleza/Ceará. Entre los meses de agosto a diciembre de 2001 fueron colectados los datos, a través de una entrevista libre. Los resultados indicaron que la visión administrativa del enfermero se centra en el desarrollo de actividades de Liderazgo y de Control. También pudo notarse que la administración de enfermería, no es percibida como una tarea fácil en el Macro Sistema Hospitalar. La mayor dificultad parece concentrarse en la ausencia de reflexiones sobre las practicas administrativas por parte del enfermero.<br>The study aimed at investigating the nurses' administration points of view concerning managing activities in the hospital macro-system, based on the Administration Contingent Theory. A group of 9 nurses responsible for the administration of nursing services at 6 reference hospitals in Fortaleza/Ceara, participated of the study. Data were assessed by means of free interview techniques, from August to December 2001. The results suggest that the administrative approach focused the development of Leadership and Control activities. The interview outcomes reveal that nursing administration in a Hospital Macro-system was not perceived as an easy task, but feasible nevertheless. The greatest difficulty seemed to be due to a poor reflexive practice about the nurse's administrative attributions