![]() This is difficult to achieve using existing technologies, which typically use conventional molecular biology techniques to fuse gfp to the 5′ or 3′ end of the target gene –. The ideal strategy for imaging studies is to employ fully functional fluorescent fusion proteins produced from a gene in its native chromosomal context. Furthermore, even modest overproduction of some proteins, particularly those involved in signal transduction and cell division, can have deleterious effects on cell viability and on cellular architecture. Overexpression of Bacillus subtilis MinC causes it to accumulate at the cell poles, , although when produced under its native expression controls MinC localizes to midcell. A two-fold overexpression of a partially functional GFP-SpoIIQ fusion protein changes its localization. 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. 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. 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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