Expanding phage display technology with luminescence

EMBO Conference on Viruses of Microbes III: Structure and Function - from Molecules to Communities (Programme and Abstract Book)[Excerpt] The -omics Era has contributed to the disclosure of genes coding for known and novel biologically active peptides. The interaction of those peptides with desired targets can be studied using phage display, mostly using phagemids as vectors. However, complex and time-consuming procedures are usually needed for purification and validation of desired phages. So, a vector that would present the capacity to easily confirm and detect a certain funct ion would be a step forward in display technology. [...]info:eu-repo/semantics/publishedVersio

Rational design of bacteriophages as a platform for cancer therapy

The aim of this work is to engineer a bacteriophage-based platform to specifically target, invade and control cancer

A new look at bacteriophage phylogenomics

Bacteriophages or phages are viruses that only infect bacteria. The International Committee on Taxonomy of Viruses classified these viruses in accordance with the morphology of their free virion particles and type and size of their genome. This system fails on the classification of several phages, which have their genome already sequenced. It also requires a morphological analysis by transmission electron microscopy, which is very expensive and time consuming [1]. In 2002 Rohwer and Edward proposed the only sequence-based system existing up to this moment. Thus, it is of utmost importance to develop new systems for bacteriophage classification that take into consideration the genomic and proteomic information already available [2]. The purpose of this study is to establish a new method for the classification of phage based on the genetic information available. The principal objective is to cluster the bacteriophages in different family and types. Phylogenetic analysis was carried out using all 670 available genomes sequences deposited in GenBank database. Sequences were aligned using the T-coffee program [3].A genetic marker for the construction of the phylogenetic tree was designed by creating a concatenate of different gene products that presented the highest similarity. In other words, the most conserved gene products were used to form a broader genetic marker. The method allows the use of a single, created genetic marker to classify unknown phages with existing phage types and families.A comparison to existing methods is discussed

Development of a phage-based biosensor to detect salmonella in food stuff

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Bacteriophage endolysins as a response to emerging foodborne pathogens

Continuous reports on foodborne outbreaks and increasing prevalence of antibiotic-resistant bacteria call for the development of novel preservation techniques that assure the safety of food products. Bacteriophage endolysins are highly active antibacterial peptidoglycan hydrolases that have evolved over millions of years to become the ultimate weapon against bacteria, with potential to be used as a food preservative. We here give an overview of all distinct endolysins encountered so far, we discuss their inherent qualities and review their role in preventing and controlling foodborne pathogens, divulging their potential for future applications.This work was supported by a grant from the Portuguese Foundation for Science and Technology in the scope of the Projects PTDC/AGR-ALI/100492/2008 and PTDC/AGR-ALI/121057/2010. Hugo Oliveira is paid by the FCT grant SFRH/BD/63734/2009

Revisiting phage therapy: new applications for old resources

The success of phage therapy is dependent on the development of strategies able to overcome the limitations of bacteriophages as therapeutic agents, the creation of an adequate regulatory framework, the implementation of safety protocols, and the acceptance by the general public. Many approaches have been proposed to circumvent phages intrinsic limitations but none have proved to be completely satisfactory. In this review we present the major hurdles of phage therapy and the solutions proposed to circumvent them. A thorough discussion on the advantages and drawbacks of these solutions is provided and special attention is given to genetic modification of phages as an achievable strategy to shape bacteriophages to exhibit desirable biological properties.The authors thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013 and the Project 'BioHealth - Biotechnology and Bioengineering approaches to improve health quality, Ref. NORTE-07-0124-FEDER-000027' cofunded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, and FEDER. F.L.N. also acknowledges the FCT for grant SFRH/BD/86462/2012

Magnesium aminoclays as plasmid delivery agents for non-competent Escherichia coli JM109 transformation

Magnesium aminoclays were synthesized and used to transform non-competent Escherichia coli JM109 using the exogenous plasmid pUC19. The structure determined for the Mg aminoclays is analogous to 2:1 trioctahedral smectites such as talc, with an approximate composition R8Si8Mg6O16(OH)4, where R = CH2CH2NH2, morphologically arranged in layered sheets. Mg aminoclays were employed as a cationic vehicle that enabled the passage of plasmids across the cell envelope and led to genetic modification of the host. A stock solution of 10 mg/mL of Mg aminoclays was prepared, mixed with E. coli JM109 and pUC19 plasmid, and spread over Petri dishes containing lysogeny broth (LB), isopropyl ?-D-1-thiogalactopyranoside (IPTG), 5-bromo-4-chloro-3-indolyl-?-D-galactopyranoside (X-gal), ampicillin and various concentrations of agar (14%). The transformation efficiency obtained was higher for 1% and 2% agar even though transformation also occurred at agar concentrations of 3% and 4%. The optical density of E. coli JM109 and spreading time were also adjusted, favoring transformation when cells were used in their exponential growth phase (OD600 = 1.0) and spread for 90 s. Transformation was confirmed by the growth of blue colonies in LB/IPTG/X-gal/agar Petri dishes containing ampicillin, by regrowth of biomass in liquid media containing ampicillin and by agarose gel electrophoresis of the linearized pUC19 plasmid that followed plasmidic DNA extraction from 4 blue colonies. The maximum transformation efficiency achieved was 7.0 × 103 CFU/?g pUC19. This transformation approach proved to be suitable for a convenient, cost-effective, room-temperature, risk-free and rapid transformation of non-competent E. coli JM109.This study was supported by the Portuguese Foundation for Science and Technology (FCT) and the European Community fund FEDER, through Program COMPETE, under the scope of the Projects FCOMP-01- 0124-FEDER-007025 (PTDC/AMB/68393/2006), PEst-OE/EQB/LA0023/2013, UID/FIS/04650/2020, RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the Project “BioEnv - Biotech nology and Bioengineering for a sustainable world”. The authors acknowledge the fellowship SFRH/BD/71661/2010 awarded to Gabriel Mendes under the scope of the MIT-Portugal Program. The authors also thank Paul Brown and Takuya Harada for the help in obtaining TEM images.info:eu-repo/semantics/publishedVersio

Engineering of specific bacteriophages for early diagnosis of Alzheimer′s disease

Alzheimer’s disease (AD) is the most common neurodegenerative disease affecting a large proportion of the human population worldwide with great impact on social and economic level. At molecular level, AD is characterized by an increased deposition of plaques, which consist of amyloid-beta however, it is not the amyloid-beta in plaques, but amyloid-beta in soluble oligomeric form that impairs synaptic function and memory encoding. The limitations imposed by the blood-brain barrier (BBB) have hindered the development of new diagnostic/therapeutic techniques. Also, AD-treatments that target plaques have proven to be ineffective, therefore it is important to find diagnostic and therapeutic tools that selectively target amyloid-beta in oligomeric form. Peptie ligands that selectively recognize AB-oligomers are available, however they are not able to cross the BBB. To overcome this limitation, the development and application of viruses has become a very interesting tool. Bacteriophages (or phages – virus that only infect bacterial cells) can bypass the BBB and can be genetically and chemically manipulated in order to recognize and target specific biomarkers commonly used for AD diagnostic. The present work describes the development of a bacteriophage-based system that can be capable of diagnose AD at an early stage by shuttling amyloid-beta specific ligands across the BBB. Phages were genetically engineered with two peptide sequences described to selectively recognize amyloid-beta oligomers in order to target and visualize amyloid-beta aggregates in the brain. Future work will be devoted to test this system in AD-mouse models for diagnosis purposes at an early stage of the disease. If successful, this approach will provide the neuroscience community with a promising tool for AD early diagnose