Abstract
We have developed two bacterial one-hybrid systems for interrogating and selecting zinc finger-DNA interactions. Our systems utilize two plasmids: a zinc finger-plasmid containing the gene for the zinc finger fused to a fragment of the alpha subunit of RNA polymerase and a reporter plasmid where the zinc finger-binding site is located upstream of a reporter gene-either the gene encoding the green fluorescent protein (GFP) or chloramphenicol acetyltransferase (CAT). Binding of the zinc finger domain to the target binding site results in a 10-fold increase in chloramphenicol resistance with the CAT reporter and an 8- to 22-fold increase in total cell fluorescence with the GFP reporter. The CAT reporter allows for sequence specific zinc fingers to be isolated in a single selection step whereas the GFP reporter enables quantitative evaluation of libraries using flow cytometry and theoretically allows for both negative and positive selection. Both systems have been used to select for zinc fingers that have affinity for the motif 5- GGGGCAGAA-3 from a library of approximately 2 x 105 variants. The systems have been engineered to report on zinc finger-DNA binding with dissociation constants less than about 1μM in order to be most applicable for evaluating binding specificity in an in vivo setting.
Keywords: Incremental truncation, flow cytometry, one-hybrid system, zinc fingers
Combinatorial Chemistry & High Throughput Screening
Title: A Bacterial One-Hybrid Selection System for Interrogating Zinc Finger- DNA Interactions
Volume: 9 Issue: 4
Author(s): Sundar Durai, Allen Bosley, Alice B. Abulencia, Srinivasan Chandrasegaran and Marc Ostermeier
Affiliation:
Keywords: Incremental truncation, flow cytometry, one-hybrid system, zinc fingers
Abstract: We have developed two bacterial one-hybrid systems for interrogating and selecting zinc finger-DNA interactions. Our systems utilize two plasmids: a zinc finger-plasmid containing the gene for the zinc finger fused to a fragment of the alpha subunit of RNA polymerase and a reporter plasmid where the zinc finger-binding site is located upstream of a reporter gene-either the gene encoding the green fluorescent protein (GFP) or chloramphenicol acetyltransferase (CAT). Binding of the zinc finger domain to the target binding site results in a 10-fold increase in chloramphenicol resistance with the CAT reporter and an 8- to 22-fold increase in total cell fluorescence with the GFP reporter. The CAT reporter allows for sequence specific zinc fingers to be isolated in a single selection step whereas the GFP reporter enables quantitative evaluation of libraries using flow cytometry and theoretically allows for both negative and positive selection. Both systems have been used to select for zinc fingers that have affinity for the motif 5- GGGGCAGAA-3 from a library of approximately 2 x 105 variants. The systems have been engineered to report on zinc finger-DNA binding with dissociation constants less than about 1μM in order to be most applicable for evaluating binding specificity in an in vivo setting.
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Cite this article as:
Durai Sundar, Bosley Allen, Abulencia B. Alice, Chandrasegaran Srinivasan and Ostermeier Marc, A Bacterial One-Hybrid Selection System for Interrogating Zinc Finger- DNA Interactions, Combinatorial Chemistry & High Throughput Screening 2006; 9 (4) . https://dx.doi.org/10.2174/138620706776843147
DOI https://dx.doi.org/10.2174/138620706776843147 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
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