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Transposon assisted gene insertion technology (TAGIT): a tool for generating fluorescent fusion proteins.

Gregory JA, Becker EC, Jung J, Tuwatananurak I, Pogliano K - PLoS ONE (2010)

Bottom Line: TAGIT is a modified Tn5 transposon that uses Kan(R) to select for insertions on the chromosome or plasmid, beta-galactosidase to identify in-frame gene fusions, and Cre recombinase to excise the kan and lacZ genes in vivo.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 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Sciences, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT
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. TAGIT is a modified Tn5 transposon that uses Kan(R) to select for insertions on the chromosome or plasmid, beta-galactosidase to identify in-frame gene fusions, and Cre recombinase to excise the kan and lacZ genes in vivo. 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. Libraries can be screened to identify GFP insertions that maintain target protein function at native expression levels, allowing more trustworthy localization studies. We here use TAGIT to generate a library of GFP insertions in the Escherichia coli lactose repressor (LacI). We identified fully functional GFP insertions and partially functional insertions that bind DNA but fail to repress the lacZ operon. Several of these latter GFP insertions localize to lacO arrays integrated in the E. coli chromosome without producing the elongated cells frequently observed when functional LacI-GFP fusions are used in chromosome tagging experiments. 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.

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Analysis of TAGIT-2-constructed GFP insertions in the E. coli lactose repressor LacI.(A) Amino acid sequence and structural features of LacI, with purple helices indicating α−helices and red arrows indicating β-sheets. Black arrowheads indicate the position of non-functional GFP insertions (Repression−, Focus−). Green arrowheads indicate GFP insertions that fail to repress lacZ, but form foci (Repression−, Focus+) when introduced into a strain with the lacO array integrated near the terminus of replication. Blue arrowheads indicate insertions that repress lacZ (Repression+, Focus+). The orange arrowhead indicates the position of the GFP insertion that is out of frame on the 3′ side of GFP. (B) The LacI-GFP insertions accumulate to variable levels. Numbers correspond to the last undisrupted lacI codon before TAGIT. Cells were harvested at an OD600 of ∼0.5, samples prepared and subject to SDS-PAGE. Protein accumulation was determined using in-gel GFP fluorescence (top panel) and the gel was subsequently stained with Coomassie blue to reveal total protein (bottom panel).
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pone-0008731-g002: Analysis of TAGIT-2-constructed GFP insertions in the E. coli lactose repressor LacI.(A) Amino acid sequence and structural features of LacI, with purple helices indicating α−helices and red arrows indicating β-sheets. Black arrowheads indicate the position of non-functional GFP insertions (Repression−, Focus−). Green arrowheads indicate GFP insertions that fail to repress lacZ, but form foci (Repression−, Focus+) when introduced into a strain with the lacO array integrated near the terminus of replication. Blue arrowheads indicate insertions that repress lacZ (Repression+, Focus+). The orange arrowhead indicates the position of the GFP insertion that is out of frame on the 3′ side of GFP. (B) The LacI-GFP insertions accumulate to variable levels. Numbers correspond to the last undisrupted lacI codon before TAGIT. Cells were harvested at an OD600 of ∼0.5, samples prepared and subject to SDS-PAGE. Protein accumulation was determined using in-gel GFP fluorescence (top panel) and the gel was subsequently stained with Coomassie blue to reveal total protein (bottom panel).

Mentions: We performed in vitro transposition with pTAGIT-2 into the lacIq containing plasmids pEB363 and pEB364 (which have lacI inserted in opposite orientations relative to the plasmid backbone) using purified Tn5 transposase [32]. The resulting insertion library was transformed into the pcnB strain KJ622, selecting for KanR on plates containing the β-galactosidase indicator X-gal. DNA sequencing revealed that 100% of the 57 blue colonies purified for sequencing contained insertions in the same reading frame as the target gene, lacI. The 30 unique insertion sites were distributed throughout lacI and provided sufficient coverage of LacI (Figure 2) to compare with previously constructed epitope insertion mutants of LacI [30]. One of these gfp insertions (LacI-144-GFP) was found to be out of frame on the 3′ side of gfp, thereby producing a protein containing the first 144 amino acids of LacI followed by GFP, because the frame shift resulted in a stop codon following the 3′ end of gfp. Thus, the incorporation of 'lacZ into TAGIT allows the rapid and accurate identification of in-frame gfp insertions prior to sequencing, thereby reducing the cost and effort required to identify in-frame insertions.


Transposon assisted gene insertion technology (TAGIT): a tool for generating fluorescent fusion proteins.

Gregory JA, Becker EC, Jung J, Tuwatananurak I, Pogliano K - PLoS ONE (2010)

Analysis of TAGIT-2-constructed GFP insertions in the E. coli lactose repressor LacI.(A) Amino acid sequence and structural features of LacI, with purple helices indicating α−helices and red arrows indicating β-sheets. Black arrowheads indicate the position of non-functional GFP insertions (Repression−, Focus−). Green arrowheads indicate GFP insertions that fail to repress lacZ, but form foci (Repression−, Focus+) when introduced into a strain with the lacO array integrated near the terminus of replication. Blue arrowheads indicate insertions that repress lacZ (Repression+, Focus+). The orange arrowhead indicates the position of the GFP insertion that is out of frame on the 3′ side of GFP. (B) The LacI-GFP insertions accumulate to variable levels. Numbers correspond to the last undisrupted lacI codon before TAGIT. Cells were harvested at an OD600 of ∼0.5, samples prepared and subject to SDS-PAGE. Protein accumulation was determined using in-gel GFP fluorescence (top panel) and the gel was subsequently stained with Coomassie blue to reveal total protein (bottom panel).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2806921&req=5

pone-0008731-g002: Analysis of TAGIT-2-constructed GFP insertions in the E. coli lactose repressor LacI.(A) Amino acid sequence and structural features of LacI, with purple helices indicating α−helices and red arrows indicating β-sheets. Black arrowheads indicate the position of non-functional GFP insertions (Repression−, Focus−). Green arrowheads indicate GFP insertions that fail to repress lacZ, but form foci (Repression−, Focus+) when introduced into a strain with the lacO array integrated near the terminus of replication. Blue arrowheads indicate insertions that repress lacZ (Repression+, Focus+). The orange arrowhead indicates the position of the GFP insertion that is out of frame on the 3′ side of GFP. (B) The LacI-GFP insertions accumulate to variable levels. Numbers correspond to the last undisrupted lacI codon before TAGIT. Cells were harvested at an OD600 of ∼0.5, samples prepared and subject to SDS-PAGE. Protein accumulation was determined using in-gel GFP fluorescence (top panel) and the gel was subsequently stained with Coomassie blue to reveal total protein (bottom panel).
Mentions: We performed in vitro transposition with pTAGIT-2 into the lacIq containing plasmids pEB363 and pEB364 (which have lacI inserted in opposite orientations relative to the plasmid backbone) using purified Tn5 transposase [32]. The resulting insertion library was transformed into the pcnB strain KJ622, selecting for KanR on plates containing the β-galactosidase indicator X-gal. DNA sequencing revealed that 100% of the 57 blue colonies purified for sequencing contained insertions in the same reading frame as the target gene, lacI. The 30 unique insertion sites were distributed throughout lacI and provided sufficient coverage of LacI (Figure 2) to compare with previously constructed epitope insertion mutants of LacI [30]. One of these gfp insertions (LacI-144-GFP) was found to be out of frame on the 3′ side of gfp, thereby producing a protein containing the first 144 amino acids of LacI followed by GFP, because the frame shift resulted in a stop codon following the 3′ end of gfp. Thus, the incorporation of 'lacZ into TAGIT allows the rapid and accurate identification of in-frame gfp insertions prior to sequencing, thereby reducing the cost and effort required to identify in-frame insertions.

Bottom Line: TAGIT is a modified Tn5 transposon that uses Kan(R) to select for insertions on the chromosome or plasmid, beta-galactosidase to identify in-frame gene fusions, and Cre recombinase to excise the kan and lacZ genes in vivo.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 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Sciences, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT
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. TAGIT is a modified Tn5 transposon that uses Kan(R) to select for insertions on the chromosome or plasmid, beta-galactosidase to identify in-frame gene fusions, and Cre recombinase to excise the kan and lacZ genes in vivo. 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. Libraries can be screened to identify GFP insertions that maintain target protein function at native expression levels, allowing more trustworthy localization studies. We here use TAGIT to generate a library of GFP insertions in the Escherichia coli lactose repressor (LacI). We identified fully functional GFP insertions and partially functional insertions that bind DNA but fail to repress the lacZ operon. Several of these latter GFP insertions localize to lacO arrays integrated in the E. coli chromosome without producing the elongated cells frequently observed when functional LacI-GFP fusions are used in chromosome tagging experiments. 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.

Show MeSH
Related in: MedlinePlus