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BioSpotlight
 
Patrick Lo, Ph.D. and Kristie Nybo, Ph.D.
BioTechniques, Vol. 53, No. 5, November 2012, p. 269
Full Text (PDF)

Titer counts

Phage display enables researchers to identify interactions of protein sequences fused to a phage coat protein expressed on the viral surface. Displayed sequences can be selected based on their desired binding properties, following which the associated virus is propagated in a bacterial host for isolation and purification. It is common to repeat the selection steps three or more times for enrichment, with the phage concentration determined after each propagation step. Phage concentration is commonly calculated using the plating method, where Escherichia coli are infected with phage, mixed in soft agar, plated, and the resulting plaques or colonies counted. Although this is an effective means of quantification, the procedure is lengthy, requiring an overnight incubation period. UV photometry can be used as a quicker approach to assess phage concentrations, but this method requires complete removal of impurities that absorb UV light and adversely affect readings. In this issue of BioTechniques, Lehmusvuori et al. from the University of Turku, Finland describe a new approach for quantifying bacteriophage using lanthanide fluorescence probes, a technology previously developed by the authors for other applications, and demonstrate its effectiveness using the M13 phage display system. The method requires two non-fluorescent oligonucleotide probes, one carrying a lanthanide ion and the other an antenna probe. When the probes hybridize to adjacent positions on the chloramphenicol acetyl-transferase gene, a selectable marker on the M13 phagemid single stranded DNA, the lanthanide ion and antenna probe form a highly fluorescent lanthanide chelate complex that can be measured as an indication of the concentration of ssDNA in solution. After only four hours in culture, phage could be detected in supernatant, demonstrating a significant time savings for this important step in phage display technology compared to the plating method. Measurements were highly reliable, and even more accurate than phage counts obtained using absorbance measurements. Although demonstrated with the M13 phage display system, this methodology should be applicable to quantification of other ssDNA phage species.



See “Homogenous M13 bacteriophage quantification assay using switchable lanthanide fluorescence probes.

Having a BLAST (on your own)

The first step in identifying any novel DNA or protein sequence is to perform a sequence similarity search against databases of known sequences to establish identity or find sequence homologs. The principal algorithm used for such similarity searches is the Basic Local Alignment Search Tool (BLAST), which is conveniently done online using the National Center for Biotechnology Information (NCBI) server, in conjunction with various publicly available databases such as GenBank and UniProt. Under certain circumstances, however, researchers may prefer to carry out offline BLAST searches on a local computer against their own databases of proprietary or unpublished sequences. While NCBI provides stand-alone, command line versions of BLAST that can be freely downloaded and installed on a personal computer, these are not particularly user-friendly, especially for biological researchers lacking computer expertise. There are two graphical front ends with extensive features and options for the BLAST command line software currently available, but these are restricted to Unix-based systems. Windows users unfortunately are restricted to a pair of graphical user interfaces (GUIs) that suffer from several major limitations. In this issue, P. Santiago-Sotelo and J.H. Ramirez-Prado at the Centro de Investigación Científica de Yucatán (Mexico) describe prfectBLAST, an operating system-independent GUI for the NCBI stand-alone suite of the new BLAST+ command line executables. prfectBLAST is free, open-source, completely self-contained, and coded in Java version 6, allowing it to be executed on multiple platforms (including Windows, Mac OS X, and Linux). In addition, the software does not require installation or any dependencies, so it can be run from an external flash or hard drive if desired. Its graphical layout and choice of options was designed to mirror the NCBI web interface for BLAST+ searches, and all BLAST+ parameters can be easily changed through the GUI. Searches using any of the BLAST+ programs are carried out against locally stored, user-customized databases and can be run in the computer background; several searches can be carried out concurrently. prfectBLAST is ideally suited for novice and casual users, yet flexible enough to allow experts to fully utilize all the advanced options available for BLAST+.

See “prfectBLAST: a platform independent portable front end for the command terminal BLAST+ stand-alone suite.