Fast, easy and efficient: site-specific insertion of transgenes into Enterobacterial chromosomes using Tn7 without need for selection of the insertion event

BACKGROUND: Inserting transgenes into bacterial chromosomes is generally quite involved, requiring a selection for cells carrying the insertion, usually for drug-resistance, or multiple cumbersome manipulations, or both. Several approaches use phage λ red recombination, which allows for the possibility of mutagenesis of the transgene during a PCR step. RESULTS: We present a simple, rapid and highly efficient method for transgene insertion into the chromosome of Escherichia coli, Salmonella or Shigella at a benign chromosomal site using the site-specific recombination machinery of the transposon Tn7. This method requires very few manipulations. The transgene is cloned into a temperature-sensitive delivery plasmid and transformed into bacterial cells. Growth at the permissive temperature with induction of the recombination machinery leads to transgene insertion, and subsequent growth at the nonpermissive temperature cures the delivery plasmid. Transgene insertion is highly site-specific, generating insertions solely at the Tn7 attachment site and so efficient that it is not necessary to select for the insertion. CONCLUSION: This method is more efficient and straightforward than other techniques for transgene insertion available for E. coli and related bacteria, making moving transgenes from plasmids to a chromosomal location a simple matter. The non-requirement for selection is particularly well suited for use in development of unmarked strains for environmental release, such as live-vector vaccine strains, and also for promoter-fusion studies, and experiments in which every bacterial cell must express a transgene construct

Site-specific Tn7 transposition into the human genome

The bacterial transposon, Tn7, inserts into a single site in the Escherichia coli chromosome termed attTn7 via the sequence-specific DNA binding of the target selector protein, TnsD. The target DNA sequence required for Tn7 transposition is located within the C-terminus of the glucosamine synthetase (glmS) gene, which is an essential, highly conserved gene found ubiquitously from bacteria to humans. Here, we show that Tn7 can transpose in vitro adjacent to two potential targets in the human genome: the gfpt-1 and gfpt-2 sequences, the human analogs of glmS. The frequency of transposition adjacent to the human gfpt-1 target is comparable with the E.coli glmS target; the human gfpt-2 target shows reduced transposition. The binding of TnsD to these sequences mirrors the transposition activity. In contrast to the human gfpt sequences, Tn7 does not transpose adjacent to the gfa-1 sequence, the glmS analog in Saccharomyces cerevisiae. We also report that a nucleosome core particle assembled on the human gfpt-1 sequence reduces Tn7 transposition by likely impairing the accessibility of target DNA to the Tns proteins. We discuss the implications of these findings for the potential use of Tn7 as a site-specific DNA delivery agent for gene therapy

Creating the Capacity to Screen Deaf Women for Perinatal Depression [poster]

There are approximately 1 million Deaf women in the U.S. who depend on American Sign Language (ASL) for communication. Although Deaf women become pregnant and enter motherhood at rates similar to hearing women, Deaf women attend fewer prenatal appointments, receive less information from their physicians, are less satisfied with physician concern and quality of communication, and are less satisfied with their prenatal care. These barriers persist after childbirth, leaving Deaf mothers with little professional support for struggles with postnatal healthcare, breastfeeding, and childcare. Combined with pre-existing mental health disparities observed among members of the Deaf community, such barriers leave Deaf women especially vulnerable to development or exacerbation of depression during the perinatal period (i.e., during pregnancy or within one year postpartum). Expert groups recommend depression screening as a standard of perinatal care - the first critical step to direct women to treatment. Yet, available screening tools are not accessible to Deaf women due to documented low levels of English literacy and health literacy. It is, therefore, critical to develop and validate tools to screen for depression among Deaf perinatal women so they may access the same standard of care as other perinatal women. To address these barriers, our team is conducting a one-year, community-engaged pilot study to develop and perform preliminary psychometric analyses on an ASL translation of the Edinburgh Postnatal Depression Scale (EPDS). During the poster session, we will outline our unique community-engaged research methods, as well as exhibit the first draft of the ASL EPDS

Insertion site preference of Mu, Tn5, and Tn7 transposons

<p>Abstract</p> <p>Background</p> <p>Transposons, segments of DNA that can mobilize to other locations in a genome, are often used for insertion mutagenesis or to generate priming sites for sequencing of large DNA molecules. For both of these uses, a transposon with minimal insertion bias is desired to allow complete coverage with minimal oversampling.</p> <p>Findings</p> <p>Three transposons, Mu, Tn5, and Tn7, were used to generate insertions in the same set of fosmids containing <it>Candida glabrata </it>genomic DNA. Tn7 demonstrates markedly less insertion bias than either Mu or Tn5, with both Mu and Tn5 biased toward sequences containing guanosine (G) and cytidine (C). This preference of Mu and Tn5 yields less uniform spacing of insertions than for Tn7, in the adenosine (A) and thymidine (T) rich genome of <it>C. glabrata </it>(39% GC).</p> <p>Conclusions</p> <p>In light of its more uniform distribution of insertions, Tn7 should be considered for applications in which insertion bias is deleterious.</p

Revealing nascent proteomics in signaling pathways and cell differentiation.

Regulation of gene expression at the level of protein synthesis is a crucial element in driving how the genetic landscape is expressed. However, we are still limited in technologies that can quantitatively capture the immediate proteomic changes that allow cells to respond to specific stimuli. Here, we present a method to capture and identify nascent proteomes in situ across different cell types without disturbing normal growth conditions, using O-propargyl-puromycin (OPP). Cell-permeable OPP rapidly labels nascent elongating polypeptides, which are subsequently conjugated to biotin-azide, using click chemistry, and captured with streptavidin beads, followed by digestion and analysis, using liquid chromatography-tandem mass spectrometry. Our technique of OPP-mediated identification (OPP-ID) allows detection of widespread proteomic changes within a short 2-hour pulse of OPP. We illustrate our technique by recapitulating alterations of proteomic networks induced by a potent mammalian target of rapamycin inhibitor, MLN128. In addition, by employing OPP-ID, we identify more than 2,100 proteins and uncover distinct protein networks underlying early erythroid progenitor and differentiation states not amenable to alternative approaches such as amino acid analog labeling. We present OPP-ID as a method to quantitatively identify nascent proteomes across an array of biological contexts while preserving the subtleties directing signaling in the native cellular environment

Characterization of the TnsD-attTn7 complex that promotes site-specific insertion of Tn7

The bacterial transposon Tn7 is distinguished by its ability to recognize a specific site called attTn7, and insert just downstream of the highly conserved chromosomal glmS gene. TnsD is one of four transposon-encoded polypeptides (TnsABC+D) required for site-specific insertion of Tn7 into attTn7, and is the target site-selector that binds to a highly conserved sequence in the end of the glmS protein coding region. In this study, we identified important nucleotides within this region that are crucial for TnsD-attTn7 interaction. We also probed the regions of TnsD that interact with attTn7 and found that there are important DNA-binding determinants throughout the entire length of the protein, including an amino-terminal CCCH zinc-finger motif. A key role of TnsD is to recruit the non-sequence specific DNA-binding protein TnsC to attTn7; TnsC also interacts with and controls both the TnsA and TnsB subunits of the Tn7 transposase. TnsC stimulates the binding of TnsD to attTn7 in vivo, and TnsCD and TnsD can also interact in the absence of DNA and localize their interaction domains to the N-terminal region of each protein

Montelukast in the treatment of duodenal eosinophilia in children with dyspepsia: Effect on eosinophil density and activation in relation to pharmacokinetics

<p>Abstract</p> <p>Background</p> <p>We have previously demonstrated the clinical efficacy of montelukast in a randomized double-blind controlled cross-over trial in patients with dyspepsia in association with duodenal eosinophilia. The mechanism of this clinical response is unknown but could involve a decrease in eosinophil density or activation.</p> <p>Methods</p> <p>Twenty-four dyspeptic patients 8–17 years of age underwent initial blood sampling and endoscopy with biopsy. Eighteen of these patients had elevated duodenal eosinophil density and underwent repeat blood sampling and endoscopy following 21 days of therapy with montelukast (10 mg/day). The following were determined: global clinical response on a 5-point Lickert-type scale, eosinophil density utilizing H & E staining, eosinophil activation determined by degranulation indices on electron microscopy, and serum cytokine concentrations. On day 21, pharmacokinetics and duodenal mucosal drug concentrations were determined.</p> <p>Results</p> <p>Eighty-three percent of the patients had a positive clinical response to montelukast with regard to relief of pain with 50% having a complete or nearly complete clinical response. The response was unrelated to systemic drug exposure or to mucosal drug concentration. Other than a mild decrease in eosinophil density in the second portion of the duodenum, there were no significant changes in eosinophil density, eosinophil activation, or serum cytokine concentrations following treatment with montelukast. Pre-treatment TNF-α concentration was negatively correlated with clinical response.</p> <p>Conclusion</p> <p>The short-term clinical response to montelukast does not appear to result from changes in eosinophil density or activation. Whether the effect is mediated through specific mediators or non-inflammatory cells such as enteric nerves remains to be determined.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov; NCT00148603</p

A One-Step Strategy for End-Functionalized Donor–Acceptor Conjugated Polymers

A modular and robust method for preparing end-functionalized donor–acceptor (D–A) narrow bandgap conjugated polymers is reported that avoids multistep reactions and postpolymerization modification. The strategy is well-controlled and affords functional materials with predictable molecular weight and high end-group fidelity. To exemplify this synthetic strategy, narrow bandgap conjugated polymers based on PDPP2FT were prepared that contain perylene diimide (PDI) units at the chain-ends. Monte Carlo simulations confirm the high degree of chain-end functionalization while photoluminescence studies reveal the unique photophysical properties of the end-functional polymers with efficient charge transfer occurring between the main polymer chain and PDI end-groups that results exclusively from their covalent linkage

The Lantern Vol. 32, No. 2, January 1966

• Mission 63 • The Kiss • The Dream • Silence is Golden • Self-Pity • The Excuse • Car Eighteen • Clothes Make the Man • The Place of a Just Man • Golden Gods • The Journey • Third Prize Picture • Gone Now • In Vain • Deep Dying • Marnie • Ruthttps://digitalcommons.ursinus.edu/lantern/1088/thumbnail.jp