Browse
Submit Data
Databases
API
Help

Dataset Information

20 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Stable, site-specific fluorescent tagging constructs optimized for burkholderia species.

ABSTRACT: Several vectors that facilitate stable fluorescent labeling of Burkholderia pseudomallei and Burkholderia thailandensis were constructed. These vectors combined the effectiveness of the mini-Tn7 site-specific transposition system with fluorescent proteins optimized for Burkholderia spp., enabling bacterial tracking during cellular infection.

SUBMITTER: Norris MH

PROVIDER: S-EPMC2976199 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Stable, site-specific fluorescent tagging constructs optimized for burkholderia species.

Norris Michael H MH Kang Yun Y Wilcox Bruce B Hoang Tung T TT

Applied and environmental microbiology 20100917 22

Several vectors that facilitate stable fluorescent labeling of Burkholderia pseudomallei and Burkholderia thailandensis were constructed. These vectors combined the effectiveness of the mini-Tn7 site-specific transposition system with fluorescent proteins optimized for Burkholderia spp., enabling bacterial tracking during cellular infection. ...[more]

PMID: 20851961

Similar Datasets

Site-specific chromosomal integration of large synthetic constructs.

Project description:We have developed an effective, easy-to-use two-step system for the site-directed insertion of large genetic constructs into arbitrary positions in the Escherichia coli chromosome. The system uses lambda-Red mediated recombineering accompanied by the introduction of double-strand DNA breaks in the chromosome and a donor plasmid bearing the desired insertion fragment. Our method, in contrast to existing recombineering or phage-derived insertion methods, allows for the insertion of very large fragments into any desired location and in any orientation. We demonstrate this method by inserting a 7-kb fragment consisting of a venus-tagged lac repressor gene along with a target lacZ reporter into six unique sites distributed symmetrically about the chromosome. We also demonstrate the universality and repeatability of the method by separately inserting the lac repressor gene and the lacZ target into the chromosome at separate locations around the chromosome via repeated application of the protocol.

| S-EPMC2847246 | biostudies-literature

Toward site-specific, homogeneous and highly stable fluorescent silver nanoclusters fabrication on triplex DNA scaffolds.

Project description:A new strategy to create site-specific, homogeneous, and bright silver nanoclusters (AgNCs) with high-stability was demonstrated by triplex DNA as template. By reasonable design of DNA sequence, homogeneous Ag(2) cluster was obtained in the predefined position of CG.C(+) site of triplex DNA. This strategy was also explored for controlled alignment of AgNCs on the DNA nanoscaffold. To the best of our knowledge, this was the first example to simultaneously answer the challenges of excellent site-specific nucleation and growth, homogeneity and stability against salt of DNA-templated AgNCs.

| S-EPMC3439878 | biostudies-other

Tagging the proteasome active site ?5 causes tag specific phenotypes in yeast.

Project description:The efficient and timely degradation of proteins is crucial for many cellular processes and to maintain general proteostasis. The proteasome, a complex multisubunit protease, plays a critical role in protein degradation. Therefore, it is important to understand the assembly, regulation, and localization of proteasome complexes in the cell under different conditions. Fluorescent tags are often utilized to study proteasomes. A GFP-tag on the ?5 subunit, one of the core particle (CP) subunits with catalytic activity, has been shown to be incorporated into proteasomes and commonly used by the field. We report here that a tag on this subunit results in aberrant phenotypes that are not observed when several other CP subunits are tagged. These phenotypes appear in combination with other proteasome mutations and include poor growth, and, more significantly, altered 26S proteasome localization. In strains defective for autophagy, ?5-GFP tagged proteasomes, unlike other CP tags, localize to granules upon nitrogen starvation. These granules are reflective of previously described proteasome storage granules but display unique properties. This suggests proteasomes with a ?5-GFP tag are specifically recognized and sequestered depending on physiological conditions. In all, our data indicate the intricacy of tagging proteasomes, and possibly, large complexes in general.

| S-EPMC7582879 | biostudies-literature

Hetero-oligomeric tagging diminishes non-specific aggregation of target proteins fused with Anthozoa fluorescent proteins.

Project description:The tendency for tetramerization is the main disadvantage in the green fluorescent protein homologues from Anthozoa species. We report a universal method called hetero-oligomeric tagging, which diminishes troublesome consequences of tetramerization of Anthozoa-derived fluorescent proteins (FP) in intracellular protein labelling. This approach is based on the co-expression of the FP-tagged protein of interest together with an excess of free non-fluorescent FP mutant. The resulting FP heterotetramers contain only a single target polypeptide and, therefore, can be considered pseudo-monomeric. Feasibility of the method has been demonstrated with a red FP fused with cytoplasmic beta-actin or tubulin-binding protein Tau34. In addition, heterotetramers appeared to be a unique model for biophysical characterization of Anthozoa FPs in pseudo-monomeric state.

| S-EPMC1223255 | biostudies-other

Site-Specific Incorporation of a Photoactivatable Fluorescent Amino Acid.

Project description:Photoactivatable fluorophores are emerging optical probes for biological applications. Most photoactivatable fluorophores are relatively large in size and need to be activated by ultraviolet light; this dramatically limits their applications. To introduce photoactivatable fluorophores into proteins, recent investigations have explored several protein-labeling technologies, including fluorescein arsenical hairpin (FlAsH) Tag, HaloTag labeling, SNAPTag labeling, and other bioorthogonal chemistry-based methods. However, these technologies require a multistep labeling process. Here, by using genetic code expansion and a single sulfur-for-oxygen atom replacement within an existing fluorescent amino acid, we have site-specifically incorporated the photoactivatable fluorescent amino acid thioacridonylalanine (SAcd) into proteins in a single step. Moreover, upon exposure to visible light, SAcd can be efficiently desulfurized to its oxo derivatives, thus restoring the strong fluorescence of labeled proteins.

| S-EPMC8011588 | biostudies-literature

Improved plant transformation vectors for fluorescent protein tagging.

Project description:Fluorescent protein labelling technologies enable dynamic protein actions to be imaged in living cells and can also be used in conjunction with other methods such as Forster resonance energy transfer and biomolecular fluorescence complementation. In this report, we describe the generation of a series of 23 novel GATEWAY-compatible vectors based on pGreenII and pDH51 backbones with the latest fluorescent protein tags (Cerulean, EGFP and Venus) and the choice of three in planta selection markers. These vectors can be obtained from the Nottingham Arabidopsis Stock Centre (N9819-N9846) and should be a powerful tool box for transgenic research in plants.

| S-EPMC2522295 | biostudies-literature

Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei.

Project description:Here, we constructed stable, chromosomal, constitutively expressed, green and red fluorescent protein (GFP and RFP) as reporters in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei. Using bioinformatic approaches and other experimental analyses, we identified P0253 and P1 as potent promoters that drive the optimal expression of fluorescent reporters in single copy in B. anthracis and Burkholderia spp. as well as their surrogate strains, respectively. In comparison, Y. pestis and its surrogate strain need two chromosomal copies of cysZK promoter (P2cysZK) for optimal fluorescence. The P0253-, P2cysZK-, and P1-driven GFP and RFP fusions were first cloned into the vectors pRP1028, pUC18R6KT-mini-Tn7T-Km, pmini-Tn7-gat, or their derivatives. The resultant constructs were delivered into the respective surrogates and subsequently into the select agent strains. The chromosomal GFP- and RFP-tagged strains exhibited bright fluorescence at an exposure time of less than 200 msec and displayed the same virulence traits as their wild-type parental strains. The utility of the tagged strains was proven by the macrophage infection assays and lactate dehydrogenase release analysis. Such strains will be extremely useful in high-throughput screens for novel compounds that could either kill these organisms, or interfere with critical virulence processes in these important bioweapon agents and during infection of alveolar macrophages.

| S-EPMC4234255 | biostudies-literature

Versatile protein tagging in cells with split fluorescent protein.

Project description:In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh ?-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications.

| S-EPMC4802074 | biostudies-literature

Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs.

Project description:We describe a multistep method for endogenous tagging of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs). A monomeric EGFP (mEGFP) fusion tag and a constitutively expressed mCherry fluorescence selection cassette were delivered in tandem via homology-directed repair to five genes not expressed in hiPSCs but important for cardiomyocyte sarcomere function: TTN, MYL7, MYL2, TNNI1, and ACTN2. CRISPR/Cas9 was used to deliver the selection cassette and subsequently mediate its excision via microhomology-mediated end-joining and non-homologous end-joining. Most excised clones were effectively tagged, and all properly tagged clones expressed the mEGFP fusion protein upon differentiation into cardiomyocytes, allowing live visualization of these cardiac proteins at the sarcomere. This methodology provides a broadly applicable strategy for endogenously tagging transcriptionally silent genes in hiPSCs, potentially enabling their systematic and dynamic study during differentiation and morphogenesis.

| S-EPMC6522946 | biostudies-literature

Site-specific fluorescent labeling of nascent proteins on the translating ribosome.

Project description:As newly synthesized proteins emerge from the ribosome, they interact with a variety of cotranslational cellular machineries that facilitate their proper folding, maturation, and localization. These interactions are essential for proper function of the cell, and the ability to study these events is crucial to understanding cellular protein biogenesis. To this end, we have developed a highly efficient method to generate ribosome-nascent chain complexes (RNCs) site-specifically labeled with a fluorescent dye on the nascent polypeptide. The fluorescent RNC provides real-time, quantitative information on its cotranslational interaction with the signal recognition particle and will be a valuable tool in elucidating the role of the translating ribosome in numerous biochemical pathways.

| S-EPMC3189723 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data