Fluorescent protein-based methods for on-plate screening of gene insertion.

<h4>Background</h4>Unlike the commonly used method of blue-white screening for gene insertion, a fluorescent protein-based screening method offers a gain-of-function screening process without using any co-factors and a gene fusion product with a fluorescent protein reporter that is furth...

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Autores principales:	Stanley S C Wong, Kevin Truong
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2010
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/a06690081efb44bba7d83382faa89679
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spelling	oai:doaj.org-article:a06690081efb44bba7d83382faa896792021-11-18T07:01:51ZFluorescent protein-based methods for on-plate screening of gene insertion.1932-620310.1371/journal.pone.0014274https://doaj.org/article/a06690081efb44bba7d83382faa896792010-12-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21170317/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>Unlike the commonly used method of blue-white screening for gene insertion, a fluorescent protein-based screening method offers a gain-of-function screening process without using any co-factors and a gene fusion product with a fluorescent protein reporter that is further useful in cell imaging studies. However, complications related to protein-folding efficiencies of the gene insert in fusion with fluorescent protein reporters prevent effective on-plate bacterial colony selection leading to its limited use.<h4>Methodology/principal findings</h4>Here, we present three methods to tackle this problem. Our first method promotes the folding of the gene insert by using an N-terminal protein such as calmodulin that is well folded and expressed. Under this method, fluorescence was increased more than 30x over control allowing for enhanced screening. Our second method creates a fluorescent protein that is N-terminal to the gene upon insertion, thereby reducing the dependency of the fluorescent protein reporter on the folding of the gene insert. Our third method eliminates any dependence of the fluorescent protein reporter on the folding of the gene insert by using a stop and start sequence for protein translation.<h4>Conclusions/significance</h4>The three methods together will expand the usefulness of fluorescence on-plate screening and offer a powerful alternative to blue-white screening.Stanley S C WongKevin TruongPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 5, Iss 12, p e14274 (2010)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Stanley S C Wong Kevin Truong Fluorescent protein-based methods for on-plate screening of gene insertion.
description	<h4>Background</h4>Unlike the commonly used method of blue-white screening for gene insertion, a fluorescent protein-based screening method offers a gain-of-function screening process without using any co-factors and a gene fusion product with a fluorescent protein reporter that is further useful in cell imaging studies. However, complications related to protein-folding efficiencies of the gene insert in fusion with fluorescent protein reporters prevent effective on-plate bacterial colony selection leading to its limited use.<h4>Methodology/principal findings</h4>Here, we present three methods to tackle this problem. Our first method promotes the folding of the gene insert by using an N-terminal protein such as calmodulin that is well folded and expressed. Under this method, fluorescence was increased more than 30x over control allowing for enhanced screening. Our second method creates a fluorescent protein that is N-terminal to the gene upon insertion, thereby reducing the dependency of the fluorescent protein reporter on the folding of the gene insert. Our third method eliminates any dependence of the fluorescent protein reporter on the folding of the gene insert by using a stop and start sequence for protein translation.<h4>Conclusions/significance</h4>The three methods together will expand the usefulness of fluorescence on-plate screening and offer a powerful alternative to blue-white screening.
format	article
author	Stanley S C Wong Kevin Truong
author_facet	Stanley S C Wong Kevin Truong
author_sort	Stanley S C Wong
title	Fluorescent protein-based methods for on-plate screening of gene insertion.
title_short	Fluorescent protein-based methods for on-plate screening of gene insertion.
title_full	Fluorescent protein-based methods for on-plate screening of gene insertion.
title_fullStr	Fluorescent protein-based methods for on-plate screening of gene insertion.
title_full_unstemmed	Fluorescent protein-based methods for on-plate screening of gene insertion.
title_sort	fluorescent protein-based methods for on-plate screening of gene insertion.
publisher	Public Library of Science (PLoS)
publishDate	2010
url	https://doaj.org/article/a06690081efb44bba7d83382faa89679
work_keys_str_mv	AT stanleyscwong fluorescentproteinbasedmethodsforonplatescreeningofgeneinsertion AT kevintruong fluorescentproteinbasedmethodsforonplatescreeningofgeneinsertion
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Fluorescent protein-based methods for on-plate screening of gene insertion.

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