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Slootweg, EJ, Keller, HJ, Hink, MA, Borst, JW, Bakker, J and Schots, A (2006) Fluorescent T7 display phages obtained by translational frameshift. Nucleic Acids Res. 34:e137
Lytic phages form a powerful platform for the display of large cDNA libraries and offer the possibility to screen for interactions with almost any substrate. To visualize these interactions directly by fluorescence microscopy, we constructed fluorescent T7 phages by exploiting the flexibility of phages to incorporate modified versions of its capsid protein. By applying translational frameshift sequences, helper plasmids were constructed that expressed a fixed ratio of both wild-type capsid protein (gp10) and capsid protein fused to enhanced yellow fluorescent protein (EYFP). The frameshift sequences were inserted between the 3' end of the capsid gene and the sequence encoding EYFP. Fluorescent fusion proteins are only formed when the ribosome makes a -1 shift in reading frame during translation. Using standard fluorescence microscopy, we could sensitively monitor the enrichment of specific binders in a cDNA library displayed on fluorescent T7 phages. The perspectives of fluorescent display phages in the fast emerging field of single molecule detection and sorting technologies are discussed.
Bacterial Proteins/analysis; Bacterial Proteins/genetics; Bacteriophage T7/genetics; Capsid Proteins/analysis; Capsid Proteins/genetics; Escherichia coli/genetics; Fluorescent Dyes/analysis; Frameshifting, Ribosomal; Genetic Vectors; Luminescent Proteins/analysis; Luminescent Proteins/genetics; Microscopy, Fluorescence; Peptide Library; Plasmids/genetics; Protein Interaction Mapping/methods; Recombinant Fusion Proteins/analysis; Recombinant Fusion Proteins/biosynthesis; Recombinant Fusion Proteins/genetics; Temperature; Virion/chemistry
|Gene product||Qualifier||GO Term||Evidence Code||with/from||Aspect||Extension||Notes||Status|
|GO:0039620: T=7 icosahedral viral capsid||
This is a T7 capsid protein (gp10) that is used in combination with a enhanced yellow fluorescent protein (EYFP). The protein gp10 is with in a T7 phage. When combining this phage with the fluorescent protein, the phage only allows a proportion of the capsid protein to be used. In the paper they described out they made a frameshift that allowed bacterial ribosome to read the reading frame and hit a stop codon after the capsid protein. The frame then shifts to -1 and allows the bacterial ribosomes to continue reading the fluorescent protein. This creates T7 fluorescent phages that allow us to see the interaction between displayed proteins and their binding partners with in the T7 phage. The evidence is in figure 1 of the paper.
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