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Nucleic Acids Research Advance Access originally published online on October 13, 2006
Nucleic Acids Research 2006 34(20):e137; doi:10.1093/nar/gkl600
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Nucleic Acids Research, 2006, Vol. 34, No. 20 e137
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Fluorescent T7 display phages obtained by translational frameshift

Erik J. Slootweg*, Hans J.H.G. Keller, Mark A. Hink2, Jan Willem Borst2, Jaap Bakker1 and Arjen Schots

Laboratory of Molecular Recognition and Antibody Technology, Wageningen University Binnenhaven 5, 6709 PD, Wageningen, The Netherlands 1 Department of Nematology, Wageningen University Binnenhaven 5, 6709 PD, Wageningen, The Netherlands 2 Microspectroscopy Centre, Wageningen University Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands

*To whom correspondence should be addressed. Tel: +31 317482427; Fax: +31 317484254; Email: erik.slootweg{at}wur.nl

Received February 28, 2006. Revised August 2, 2006. Accepted August 3, 2006.

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.


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