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We narrowed to 1,033 results for: ELL

Showing: 481 - 500 of 1033 results

  1. CRISPR Antimicrobials

    Type
    Blog Post
    ...reach essentially every cell or else small pools of antibiotic resistant cells could repopulate the environment...strategies are nonspecific - they harm any bacterial cell without a resistance gene, allowing resistant bacteria...antibiotics target one of three essential processes: cell wall synthesis, DNA replication, or protein synthesis...eliminated from the bacterium, which may also induce cell death. CRISPR also enables increased specificity...Treating a mixed population, represented here by cells of different colors, specifically targets bacteria...bacteria. Targeting a genomic sequence results in cell death. Plasmid targeting may result in plasmid loss...and antibiotic resensitization, or may also cause cell death. Image from Beisel et al., licensed under ...

  2. Zebrafish as a Model for Behavior: Swimming into the Optogenetic Spotlight

    Type
    Blog Post
    ... light sensitive proteins (opsins) to manipulate cell activity, enables researchers to silence or incite...organism because their larval stage lends itself well to studies of neuroscience. The larvae of zebrafish...channels that allow charged particles to cross the cell membrane when exposed to light. Neuronal firing ...channel LiGluR both cause ion movement that results in cell depolarization. They therefore function as a neuron...in negative chloride ions which hyperpolarize the cell. It therefore functions as a neuron “off switch....stimulus. While the escape response neural pathway is well characterized, there is much left to be discovered...They found that photostimulating the Kolmer-Agduhr cells was sufficient to trigger the symmetrical tail movement...

  3. SunTag and Fluorescent Imaging

    Type
    Blog Post
    ...to amplify fluorescent signals. Named for the "stellar explosion SUperNova," SunTag can help you turn ...time to get enough signal. This workaround risks cellular phototoxicity and eliminates the possibility of...potential to form aggregates and may be toxic to the cell. Here comes the SunTag How does SunTag fix these...enables single molecule tracking  within living cells without affecting protein function, thereby creating...creating a single-molecule reporter of intracellular processes. Initially, Tanenbaum et al. observed some...attempted single-molecule imaging deep inside the cell, in the nucleus and cytoplasm. Again, they found...SunTag constructs would avoid negative effects on cell physiology. Having seen that mitochondrial tracker...

  4. Plasmids 101: Antibiotic Resistance Genes

    Type
    Blog Post
    ... out of 10,000 cells on average! Without some means of quickly determining which cells successfully received...detail selection in non-bacterial cells such as yeast or mammalian cells.   Name Class Mode of Action...Inhibits cell wall synthesis Bactericidal 100-200 ug/mL Carbenicillin beta-lactam Inhibits cell wall ...the bacterium's perspective – a plasmid-containing cell must replicate the plasmid in addition to its own...easily detect plasmid-containing bacteria when the cells are grown on selective media, and provides those...kill bacteria (bactericidal) or slow growth/prevent cell division (bacteriostatic); however, the distinction...witin 1 month for optimal efficiency. Beware of satellite colonies! Carbenicillin is more stable than Ampicillin...

  5. To Codon Optimize or Not: That is the Question

    Type
    Blog Post
    ...mammalian cells. This was the first codon optimized membrane protein expressed in mammalian cells. In this...sequence. That means there are 61 possible tRNAs. Cells however, may not express all 61 of these tRNAs and...may be found at very different levels (Mauro and Chappell, 2014). Due to this variation in tRNA expression... are also optimized for expression in mammalian cells. For example, Henry Lester’s lab optimized the C...sites and even affect protein function (Mauro and Chappell, 2014). Different rates of translation by different...and in biotechnology and therapeutics (Mauro and Chappell 2014).  Ultimately, it is important to take a ...//doi.org/10.1186/s12859-017-1793-7 Dresios J, Chappell SA, Zhou W, Mauro VP (2005) An mRNA-rRNA base-...

  6. Fluorescent Proteins 101: Aptamer Fluorophores

    Type
    Blog Post
    ... SELEX could be used to classify DNA aptamers as well, and over the course of the next two decades, these..., peptides, amino acids, drugs, metal ions, and cells (even pathogenic bacteria). That range is possible...with such techniques can be problematic, however. Well designed aptamers can help reduce such background... over half a century; the Addgene blog has an excellent primer and tips from experts on FRET techniques... combination of SELEX and fluorescence-activated cell sorting (FACS) of E. coli transformed with aptamer...and FACS, Broccoli is inherently more stable in cells. The new aptamer also has a higher melting temperature...cassettes, thereby extending their use into mammalian cells. They’ve also dimerized Broccoli to further enhance...

  7. Tips for CRISPR Gene Editing in Mice

    Type
    Blog Post
    ...expanded since its adaptation for use in mammalian cells in 2012-2013. Researchers are now using this system...successfully adapted for use in screening experiments as well). Therefore, if you’ve started down the CRISPR path...500bp is best) and select the organism. It will then tell you the available gRNA targets in that region. The...plasmids containing sgRNA sequences into HEK293T cells. The fluorescence intensity was classified into ...is then a simple matter of transfecting HEK293T cells with your modified pCAG-EGXXFP plasmid along with...incubation, you assess the level of fluorescence in your cells under a fluorescence microscope. The higher the ...you’ve validated your gRNAs for use in mammalian cells, it’s time to use them in mouse embryos. When researchers...

  8. Plasmids 101: Origin of Replication

    Type
    Blog Post
    ...to reproduce itself as it must to survive within cells. The replicons of plasmids are generally different...sequences are generally high in As and Ts. Why, you ask? Well, A-T base pairs are held together with two hydrogen...’s sake, we've ignored those used in eukaryotic cells and viruses and focused only on those found in bacteria...pMB1 (which comes in a few slightly different but well known derivatives), pSC101, R6K, and 15A. Not all...example, the pMB1 ori maintains about 20 copies per cell, while pUC – which differs by only two mutations...mutations – will produce as many as 700 copies per cell. Figure 1: A plasmid map showing the standard ...positives? Will you have only one plasmid type in your cells at a time? Do you want to make a lot of your plasmid...

  9. Antibody Neutralization Response Against Pseudoviruses Expressing SARS-CoV-2 Spike Protein Variants

    Type
    Blog Post
    ...expressing target cells. Any neutralized pseudoviruses would be unable to get into the target cells and this can...Khan K, Bernstein M, Balazs AB, Gosnell BI, Hanekom W, Moosa M-YS, Lessells RJ, de Oliveira T, Sigal A (2021...disease severity and survival. Cell 184:476-488.e11. https://doi.org/10.1016/j.cell.2020.12.015 Garcia-Beltran...vaccine-induced humoral immunity. Cell 184:2372-2383.e9. https://doi.org/10.1016/j.cell.2021.03.013 Additional resources...variant), B1.1.298, and B.1.429 (Epsilon), nearly as well as they could for the wild-type. However, variants...vaccines and allows the virus to gain entry into human cells via human ACE2 receptors. In their assay, the pseudoviruses...

  10. Rewiring Metabolic Circuitry with CRISPR RNA Scaffolds [Video]

    Type
    Blog Post
    ...coordinated by the eukaryotic cell for the execution of many complex cellular behaviors, such as differentiation...avoiding toxic intermediate buildup as well as controlling cell fate decisions. By using an orthogonal...or metabolism. Our attempts to manipulate these cellular behaviors often fall short with the generation...bottlenecks and their scope is limited by the lack of well characterized inducible promoters. Far removed from...for sequence-specific control of gene expression. Cell, 152(5), 1173-1183. (2013). PubMed. Zalatan, J...Transcriptional Programs with CRISPR RNA Scaffolds. Cell, 160(1), 339-350 (2015). PubMed. Find the Plasmids...crispr/qi/ Plasmids described in  Zalatan et al. Cell  (2015) can be found here: https://www.addgene.org...

  11. Typing CRISPR Systems

    Type
    Blog Post
    ...CRISPR-Cas Immunity. Cell, 161(5), 1164–1174. https://doi.org/10.1016/j.cell.2015.04.027 Stella, G., & Marraffini...and is able to target and edit RNA in mammalian cells. Type IV Type IV is the middle child (like me!) ...and more. Type II Type II is the most popular and well-known type, as this is where Cas9 falls. The mechanism...mechanism of type II is, unsurprisingly, well understood, and the first taught when introducing CRISPR ...., Belanto, J. J., Verdine, V., Cox, D. B. T., Kellner, M. J., Regev, A., Lander, E. S., Voytas, D. F....Suberski, A. J., Mears, K. S., Demircioglu, F. E., Moeller, L., Kocalar, S., Oshiro, R., Makarova, K. S., ...Gootenberg, J. S., Abudayyeh, O. O., Franklin, B., Kellner, M. J., Joung, J., & Zhang, F. (2017). RNA editing...

  12. Hot Plasmids - September 2019 - CRISPR, Auxin Inducible Degradation, MerMAID Channelrhodopsins, and Genome Looping

    Type
    Blog Post
    ...plant protein TIR1 in non-plant cells, which interacts with the cells’ endogenous ubiquitin ligase, the...able to translate AID to yeast, as well as mammalian and human cell lines (Nishimura et al., 2009, Natsume...degradation using AID Article contributed by Alyssa Cecchetelli Auxin Inducible Degradation (AID) uses plant ...based method to tag endogenous proteins in HCT116 cells with mAID. Using this method, the lab was able to... protein of interest to create conditional human cell lines. Lastly, the Kanemaki lab demonstrated that...the target sites adjacent to an NG PAM in human cells. The increased versatility of the engineered SpCas9... just a 2-D linear molecule snaking through our cells’ nuclei--our chromatin is comprised of thousands...

  13. K. phaffii: Rising to the Occasion

    Type
    Blog Post
    ...path.   Yeast as a model organism Yeast are single cell, eukaryotic organisms formally classified as fungi...and S. cerevisiae (budding). Yeast make for an excellent model organism for a few reasons: Affordable...yeast? It’s actually not new at all, but it’s not as well-known in the general research community. Phillips...methanol as its sole source of carbon and energy (as well as glucose or glycerol). K. phaffii is only distantly...similarity to the ancient yeast strains and metazoan cells.   Applications for K. phaffii There’s a number ...protein bang for your media buck - it grows at higher cell densities than most of its counterparts, yielding...studies Peroxisomes are small, membrane-bound organelles essential for sequestering certain enzymes and...

  14. Visualizing Translation at the Single Molecule Level

    Type
    Blog Post
    ... of all of the mRNA bound-ribosomes in a cell. When cellular mRNAs are digested with ribonucleases, ribosomes...Regulating translation is key to cellular function, especially during development or stress. With ribosome...explore the complexity of translational control in cellular physiology. Ribosome profiling Ribosome profiling...the fluorescent signal: untranslated RNAs appear yellow, and translated RNAs appear red. To enable this...are bound to the reporter, causing it to appear yellow. The act of translation pushes NLS-PCP-GFP off ...the effects of 5’ UTR variants in translation of cell cycle protein Emi1, showing that ~80% of transcripts...powerful tools for studying translation in various cell types and physiological states. References 1. Ingolia...

  15. R Bodies: Membrane-Rupturing Microscopic Tools

    Type
    Blog Post
    ... can rupture E. coli spheroplasts (cells which have had their cell walls removed with lysozyme). This ... blogger Jessica Polka, a Postdoctoral Research Fellow with Pamela Silver.  Most types of biological motion...distance? And can we understand such a structure well enough to engineer it to suit our purposes? In ...distance? And can we understand such a structure well enough to engineer it to suit our purposes? These...protein fusions allowed us to image R bodies inside cells, and these constructs may be useful to future scientists...applications. For example, you could use them to lyse cells (and surrounding membranes) in keeping with their...penetrate other barriers (like vasculature or extracellular matrices) in response to pH changes. Beyond...

  16. Antibodies 101: Introduction to Immunofluorescence

    Type
    Blog Post
    ...interest live within a cell,” “Does this disease change the architecture of my cells,” or “How does this ...IF), is an immunoassay that brings to light the cellular world. The technique allows you to ask questions...this mutation impact the types of cells found in my tissue.” It is based on the same principles as other ... visualize a target of interest within an intact cell, tissue, or, in some cases, a whole organism. But...joined to fluorescent molecules are applied to a cell or tissue sample where they bind to their targets...to visualize a target within an intact sample, a cell in the case of ICC or a tissue in the case of IHC...concentration and incubation time for this step as well. Preservation and Imaging Similar to Sample ...

  17. Tips for Getting a Faculty Position

    Type
    Blog Post
    ...patterning of cells and tissues is fundamental to development. I'm interested in how cells find their way...competitive CV You don't necessarily need a Nature/Cell/Science paper. You do need multiple first/corresponding...quality journals. It helps to come from the lab of a well-known investigator. If you only have middle author...minimize jargon. Equally importantly, you really are selling yourself. A sales pitch that requires reading two...that I had a flair for communication was rapidly dispelled. She would ask me, "Is this what you mean? Are... foot in the door. Your ability to interview and sell a vision for your contribution to a Department's...remember the days of modest postdoc salaries all too well. If there's a group to make money from, postdocs...

  18. Antibodies 101: Flow Cytometry Controls

    Type
    Blog Post
    ...and beads that are unable to bind to them: like cells but with far more predictability. The beads create...The benefit of beads is that they do not require cells from your experiment, and they are non-specific....flow cytometer, you generally want your positive cells to read between 104 and 105 in order to have enough...the plot to see all of your positive and negative cells. This can be achieved by adjusting your voltage ...of your experimental samples, combine it into one well or tube, and stain it with every marker you are ...gating schematic to easily determine where your cells of interest are (B).   Using FMO controls In...markers or commonly used markers that define broader cell populations (e.g., CD4, CD8b, CD11b, CD19), are ...

  19. Plasmids 101: Screening Strategies Used in Plasmid Cloning

    Type
    Blog Post
    ...activity. These cells, presumably the cell you want, cannot break down X-gal and are white. Cells that don’t...screening strategy as well. In a screen, you aren’t killing off a portion of the cells like in a selection... the plasmid you’re looking for out of the many cells that don’t. One way to begin the search is by using...using selection strategies, where only cells that have gained or lost a specific gene survive (ex: antibiotic...selection. Instead, all cells survive and you will need to sort through them to find the desired clone. Do your...bacteria that have taken up this vector, but it won’t tell you whether the vector contains the gRNA insert ... plates containing X-gal allows you to identify cells that took up the vector and distinguish between ...

  20. Hot Plasmids December 2018 - C. elegans RNAi, TurboID, Detecting Viral Outbreaks, and Protein Binding Specificity Tools

    Type
    Blog Post
    ...fundamental approach to studying membranes, organelles, and other subcellular compartments is via proteomics--the...gene knockdown Article contributed by Alyssa Cecchetelli  Listen to the C. elegans RNAi podcast segment... the gene being targeted via RNAi. Recently the Vellai lab has created an improved RNAi vector that leads...incorporated into the dsRNA necessary for RNAi. The Vellai lab’s new plasmid, T444T, contains T7 terminators...interaction networks of specific proteins in living cells. Until recently, the enzymes most frequently used...but requires H2O2, which can be toxic in living cells. BioID was derived from biotin ligase and uses far...detect viral outbreaks Article contributed by Izzy Mueller  Listen to the viral detection podcast segment ...
Showing: 481 - 500 of 1033 results