![]() Overexpression can also cause misleading protein localization. Co-expressing tagged and untagged proteins is a frequently-used solution that makes it impossible to use PALM/STORM techniques to quantify the number of molecules at a particular location, since the complex will be a mixture of untagged and tagged protein. subtilis engulfment proteins, which cause synergistic engulfment defects and GFP fusions to FtsZ, which are temperature sensitive in most species, including B. Examples of partially functional fusion proteins include GFP fusions to the B. Studies of protein localization in living cells are often compromised by protein overproduction or by partially functional fusion proteins (reviewed by, ). ![]() ![]() However, achieving the maximum gain from these methods requires that the behavior of the fluorescently-tagged fusion protein accurately represents that of the native protein. The combination of Photo Activated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) provides a ten-fold gain in spatial resolution and allows individual proteins to be counted –. Recent advances in optical microscopy enable fluorescently tagged proteins to be observed with subdiffraction-limited spatial resolution and outstanding temporal resolution. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.įunding: This research was supported by a grant from the National Institutes of Health (GM 57045) and from the University of California at San Diego Academic Senate. Received: SeptemAccepted: DecemPublished: January 15, 2010Ĭopyright: © 2010 Gregory et al. Sandler, University of Massachusetts, United States of America TAGIT thereby faciliates the isolation of fully functional insertions of fluorescent proteins into target proteins expressed from the native chromosomal locus as well as potentially useful partially functional proteins.Ĭitation: Gregory JA, Becker EC, Jung J, Tuwatananurak I, Pogliano K (2010) Transposon Assisted Gene Insertion Technology (TAGIT): A Tool for Generating Fluorescent Fusion Proteins. coli chromosome without producing the elongated cells frequently observed when functional LacI-GFP fusions are used in chromosome tagging experiments. Several of these latter GFP insertions localize to lacO arrays integrated in the E. We identified fully functional GFP insertions and partially functional insertions that bind DNA but fail to repress the lacZ operon. We here use TAGIT to generate a library of GFP insertions in the Escherichia coli lactose repressor (LacI). Libraries can be screened to identify GFP insertions that maintain target protein function at native expression levels, allowing more trustworthy localization studies. The resulting gfp insertions maintain target gene reading frame (to the 5′ and 3′ of gfp) and are integrated at the native chromosomal locus, thereby maintaining native expression signals. ![]() TAGIT is a modified Tn5 transposon that uses Kan R to select for insertions on the chromosome or plasmid, β-galactosidase to identify in-frame gene fusions, and Cre recombinase to excise the kan and lacZ genes in vivo. We constructed a transposon ( transposon assisted gene insertion technology, or TAGIT) that allows the random insertion of gfp (or other genes) into chromosomal loci without disrupting operon structure or regulation.
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