We narrowed to 1,082 results for: TOR

...responsible for storage.  If datasets are too large to transfer to a journal repository, they are likely...publishers. Nonetheless, the initial release caused a storm as the organization left open the question of how...for advice about the scope of the policy. End of story? Not quite. The PLOS policy on data access is a...statistical methods, omission of some data, etc.  The firestorm that followed implementation of the policy comprised... with limited acknowledgement of the initial contributors. This is probable to some extent - but that ...tools and assumptions to reveal findings that the originator was oblivious to, no matter how much time they...they had. It could be argued that data generators may, in light of the policy, hold onto data longer before...

... years ago and developed cloning vectors to harness it. These vectors, called pKIL18 and pKIL19, contained.... If, however, an investigator were to successfully clone an insert into the vector, the ccdB reading ...cells that contained non-recombinant vectors (re-ligated empty vectors, for instance) would still express...ccdB makes cloning easier by selecting against vectors that did not take up your insert. But exactly how...ccdB accomplish this? Let’s start with a brief history of the gene and how molecular biologists have harnessed...potent toxin... The ccdB gene, located on the F sex factor plasmid of E. coli, is part of a toxin-antitoxin...from Bernard, P., et al. showing the pKIL 18/19 vectors and demonstrating the concept of restriction enzyme-mediated...

.... Viral vectors undergo DNA rearrangements. Depending on the composition of the viral vector and the ... researchers prefer to store freshly harvested virus at 4 °C instead of storing it briefly at -80 °C. ...of a recombinant adenoviral vector: optimization of conditions for storage, transport and delivery." Japanese...about Lentiviral Vectors Additional Resources on the Addgene.org Check out Our Viral Vector Guide Pages ...perform complex biological tasks, both of which are factors in the technical challenges they can present. Many... one point or another. After amplifying a viral vector, collecting virus, and transducing target cells...the repeat sequences present in the LTR, the vector may undergo genomic rearrangements during replication...

... space is the Victoria Makerspace in Canada, which I belong to. The team at the Victoria Makerspace is..., we intend to focus on motor neuron knockouts so that we can perturb factors related to worm motion. ...student and then instructor at Singularity University at NASA Ames, and an instructor at Biocurious.org...was contributed by Derek Jacoby from Makerspace Victoria, CA Over the decades science has become increasingly... of a bigger movement in a variety of research sectors to provide open source technologies and spaces ... are constantly moving so we need good ways to monitor and track them. We have a number of microscopes...instance, we use a mutant known as CX51 that exhibits motor defects above 25 degrees. Computer scientists and...

...counterselection vector is the pDS132 sacB-based allelic exchange vector. This vector contains sacB, an...This post was contributed by Jacob Lazarus, a postdoctoral researcher at Harvard. There’s an astounding...selecting for an antibiotic resistance marker from the vector (abR, Figure 1). The resolution step (“double crossover...leads to the desired mutation and excision of the vector backbone. This step requires “counter-selection...colonies isolated are those where the allelic exchange vector has undergone single crossover and integrated into...recombineering (Khetrapal et al., 2015). They created vectors for Lambda Red that contained one of eight bacterial...aeruginosa. These toxins were a mix of Type VI secreted effector toxins, toxin-antitoxin type toxins, and phage...

... to be nice, the mentor fails to effectively mentor the underrepresented - the mentor over praised and...received a doctoral degree (or equivalent) and is engaged in a temporary and defined period of mentored advanced...to your peers and other mentors who can help you. You Can Talk to Your Editors I wish I had been more...well-meaning mentors often fail to judge the work of underrepresented groups under their mentorship with the...research community to develop a plan to make postdoctoral pay/benefits commensurate with work training...image below). Slide of information gathered by Postdoctoral Associations around Boston - From Panel Session... staff scientists. This results in a glut of postdoctoral trainees who, even though they are aware that...

...This also means that between two types, the Cas effector may have the same name but be completely different... understand. Classes are based on the main Cas effector; types are based on main function and composition...Class 2. Class 1 CRISPR systems use multi-Cas effectors, referred to as Cascade (CRISPR-associated complex...complexes, while Class 2 use single protein Cas effectors, like Cas9. The Cascade complex doesn’t usually...specific CRISPR type but often recruits the main effector complex after binding to a target. Class 1 is ...the lab. This can be attributed to the multi-Cas effector, Cascade. Developing tools using a Cascade complex...the CRISPR types and is thought to be the common ancestor of all other CRISPR systems. Type III systems ...

...Interaction with receptors: Understanding how fluorescent dyes interact with specific receptors is key, especially...especially for G-protein-coupled receptors (GPCRs). Mutations in receptor sites can affect the fluorescence...Throughout their history, fluorescent dyes have enabled the visualization of both organic tissues and... intensity during exposure to light, is another factor to consider. If your application requires longer...exposure, you’ll want a more photostable fluorophore. Factors such as exciting the fluorophore at peak wavelength...It's important to consider how dyes can influence receptor conformation and signaling activity​​ (Sridharan...varying degrees of β-arrestin-2 recruitment to receptors, indicating their agonistic nature. The potency...

... to all of our generous, sharing depositors. Over 3,000 laboratories are sharing plasmids (with 16 labs...sponsored the capital construction of our new BL2+ laboratory and the infrastructure capabilities to produce...currently sharing viral preps) through the Addgene repository. Without these scientists and the support of ...blessed to have an incredibly supportive Board of Directors at Addgene. Our Board helps keep our focus on ...helping thousands of sharing scientists from laboratories all over the world. With this reach and exposure...new team to push this project forward. The viral vector project team develops methods, performs extensive...generates  educational materials, and provides viral vector technical service (details about virus production...

...different assays, not just transcription factors. The second vector is a homology directed repair donor plasmid...system is so flexible. Other investigators can easily adapt these vectors for use with their favorite ...in Huntsville explains it, a major goal in his laboratory is to understand the function of the non-coding...focused considerable attention on transcription factors, starting with an established method called ChIP-seq...antibodies to recognize and bind transcription factors directly, but that’s a slow-going process and it...effort to track down every last one of those gene regulatory proteins and the places in the genome where they...is to look at all 1,500 or 1,600 transcription factors and figure out where they bind to the genome. We...

...controlled transcription factor and a ZIF268/progesterone controlled transcription factor as well as reporters...transcription factors in series and independently changes inducer levels. That is, if transcription factor 1 controls...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles... to drug inhibition, with TRIM-away targeting of mTOR producing similar results to rapamycin inhibition... super-resolution microscopy.” Endosomes are historically difficult to study in living cells using conventional...the Hell lab designed their super-resolution pH indicators (SRpHi) biosensors to meet all of them. These...Ford Listen to the Synthetic Yeast Transcription Factors Podcast Segment Researchers from the El-Samad lab...

...in epidermal growth factor receptor (EGFR) with the ClonTracer library and monitored population dynamics...paths to become resistant to epidermal growth factor receptor inhibition." Nature medicine (2016). PubMed...Library, deposited by Carrie Bhang, a research investigator in the In Vivo Pharmacology group at Novartis...mammalian cells through lentiviral infection and to monitor their abundance and clonal dynamics over time using...drug resistance, Bhang says “the [drug] resistance story panned out quickly,” and was summarized in a recent.... The library consists of a pool of lentiviral vectors with roughly 73 million semi-random, 30 bp DNA ... abundance of each barcoded clone can then be monitored over time by sequencing the barcodes in the population...

...recombination step, the effector is expressed. In cells where the stop cassette remains, the effector is not expressed...inactivate any read-through transcripts of the effector gene. Effector genes, any gene that you want to control...organisms by replacing the transcription factor binding site and effector in the construct. In Drosophila, the...T5 neurons by placing the calcium indicator GCaMP6f in the effector position, as well as to optogenetically...CsChrimson) attached to a fluorescent indicator (tdTomato), as the effector. They also expanded the SPARC system...A predictable number of cells will express the effector or retain the stop cassette following recombination...Drosophila (Fig. 1):  GAL4-UAS, a transcription factor binding site. GAL4 expressed in the fly binds to...

...G base editor (ABE) followed based on similar principles (Gaudelli et al., 2017). These editors did not... universities, private companies, and federal regulators. But the speed is impressive on a larger scale...2013; reviewed in Lander, 2016). The first base editor was reported three years later in 2016 (Komor et...access were all challenges for these early base editors. Further innovations were needed! Minimizing off-target...important addition. Improved activity of adenine base editors The first ABE was about 50% efficient in human ...mean better chances of identifying a successful editor. For this application, ABE8e turned out to be most...Targeting the right PAM Expanding PAM preferences Base editors can only access a short sequence, between four ...

...of pExpression vectors) plasmids. The type IIS restriction sites used in these vectors are of low abundance...of the many useful kits you can order from the repository: FX Cloning Kit BIOFAB Plasmid Set pCri System...to produce the constructs you need. Expression vectors within this kit contain a variety of promoters ...insects, and even Xenopus oocytes. The expression vectors additionally contain various combinations of protein...blocker isolated from a designed repeat protein combinatorial library by in vivo functional screen." Scientific...Series Confronted with the problem of creating vectors for predictable protein expression in bacteria,...this so-called bicistronic design (BCD). The 49 vectors in this kit can be used to drive production of ...

...genes with minimal effort. The versatility of Cas effectors allows for a wide variety of screens, including...packaging capacity and stable expression of lentiviral vectors make them an attractive option. However, safety... particularly in vivo ones. In those cases, AAV vector screens may be AAVantageous!       In vivo, in ...delivery method for in vivo CRISPR screens. AAV vectors can be made at high titers equal to those achieved... customization that is unmatched by other viral vector delivery methods. Lentivirus has limited packaging...packaging options, with most lentiviral vectors being packaged using VSV-G. In contrast, AAV can be packaged...psuedotypes to further improve the tropism of AAV vectors, enhancing their affinity for specific cell and...

...controlled by still other regulatory factors: enhancers, boundary elements, insulators, and silencers; however...tetracycline operators. Transcription can be turned on or off depending on what tet transactivator is used.... controlled by lac operator. Can be induced by IPTG. Commonly found in pET vectors. Very tightly regulated...the promoter (RNA polymerase binding site) and operators (response elements). Promoters are about 100 to...binding of the RNA polymerase and transcription factors, therefore promoters play a large role in determining...we will discuss these in a future post on viral vector parts. Promoter specificity Aside from choosing...Strong mammalian expression from human elongation factor 1 alpha Constitutive  Tends to give consistent ...

...I’ve learned the value in finding mentors wherever I go. This story by Dr. Erika Moore in Science Careers....351.6276.994 Moore E (2021) One mentor isn’t enough. Here’s how I built a network of mentors. Science. https://doi.org...undergraduate years. We each have a unique “origin story,” but taking the first step into the world of academic...lot of help from my peers, professors, and other mentors, and four years after I started college, I will...postdoc” is, and that “PI” stood for principal investigator. In basic science (as opposed to translational...in a lab. Position Common tasks Principle investigator (PI) Runs the lab Writes a lot of grants Varying...teaches graduate and/or undergraduate classes Postdoctoral researcher (postdoc) Getting more scientific...

...homology to the vector or neighboring fragment. Then you would amplify the fragments and vector by PCR, verify...linearized vector, the Cas9 enzyme along with a specific gRNA was used to cleave the 22kb vector. When followed...and then assembled into a vector using Gibson assembly. In this case the vector was PCR amplified to contain...be used in basic cloning of an insert into your vector of choice as shown in the figure below. To start...you want to insert your gene of interest into a vector with a large tag at the N-terminus, but you don...don’t have the tag already included in the vector you want to use. If you inserted these two pieces of ...gene of interest and the tag sequences into the vector in one step without scars as depicted below. First...

...plasmids are formed. Viral vectors are prone to recombination In lentiviral vectors, recombination often occurs...problems that I help troubleshoot involves viral vectors and plasmids with repeating sequences. Repeating.... Over time, bacteria containing the recombined vector backbone will also outgrow bacteria that contain...into two smaller plasmids. The ~3.7 kb recombined vector backbone contains the antibiotic resistance gene...samples? Plasmid recombination can affect viral vectors (LTRs and ITRs), plasmids with multiple polyA sites...recommendations from the depositing lab. If your vector is large (>10 kb) or has a low copy number, try...that in Stbl3 bacterial cultures with lentiviral vectors, the flat white colonies contained the full plasmid...

...initial reversal drive cannot restore the exact original sequence, it can restore the original protein-coding...are eliminated by natural selection, gene drives distort inheritance in their favor and consequently can...carrying a synthetic gene drive were to escape the laboratory, the drive could eventually spread through the...mistake would necessarily extend far beyond the laboratory and seriously damage public trust in scientists...gene drive is also the easiest: don't use a DNA vector that encodes both Cas9 and sgRNA. What if I want... can spread through populations of transgenic laboratory organisms but not wild organisms. For example...synthetic sequence will only spread in transgenic laboratory populations with that sequence2. Both methods...

... College of Medicine. Her doctoral research was conducted in the laboratory of Erik Lee Snapp and focused...the cytoplasm. That is, unfortunately, some investigators realize too late that they have missed the impact...environment where it will localize [4]. Most investigators are surprised to learn that FPs are often poorly...environments, including the organelles that compose the secretory pathway, endocytic vesicles, mitochondria, lysosomes...residues form inappropriate disulfide bonds in the secretory pathway (Figure 1) [4,6–8]. Additionally, FPs ...and a sequence intended to localize them to the secretory pathway, a dark pool that is mislocalized to the...misfolded, blocked from proceeding through the secretory pathway, and retained in the ER. FPs have been...

...then easy to package in low-capacity vectors like AAV, a popular vector due to its low immunogenicity. But...RNA-editing Cas13 enzymes have taken the CRISPR world by storm. Like RNA interference, these enzymes can knock ...in size and are easy to package in low-capacity vectors! These small, but mighty type VI-D enzymes are ...looked for a CRISPR repeat array near a putative effector nuclease. The Cas13d proteins they identified ...identified other advantages, including Cas13d-specific regulatory proteins and high targeting efficiency, both ...WYL domain-containing proteins may be broader regulators of Cas13d activity. This property makes WYL proteins...Konermann et al.: CasRx is a highly potent, specific editor in mammalian cells Not all Cas13d proteins are ...

...therapeutics such as  (Chimeric Antigen Receptors) CAR T cells and PD-1 modulators. An exciting development in immunotherapy...methodology with T cell receptor engineering (Ref 5)  as described by Blue Flame Addgene depositor Professor Michel...Professor Karin about alcohol and obesity as driving factors for chronic inflammation which in turn suppresses...self-limiting effects kick in trying to resolve the inflammatory state by inducing immunosuppressive measures...upregulation of the immunosuppressive cell surface receptor PD-1. This immunosuppressive environment in turn...tumor and immune cells in real time. Addgene depositor Professor Max Krummel from UCSF showed some impressive...Throughout the conference aspirin and the use of COX inhibitors was discussed and Professor Caetano Reis e Sousa...

...  Find the scrambled shRNA vector. pLKO.1 - TRC cloning vector - This vector from the David Root lab is...you! We would like to thank all of our depositors and requestors for making their reagents available and...opening our UK office in 2014, to starting our viral vector service in 2016...we’re excited to help scientists...look back at some of the contributions from our depositors. Below are the 15 top requested plasmids over... be used with 2nd or 3rd generation lentiviral vectors and an envelope expressing plasmid, such as our...lentiCRISPR v2 - This 3rd generation lentiviral transfer vector also from the Zhang lab is used to deliver Cas9...your gRNA of interest into mammalian cells. This vector was the top requested plasmid of 2015. What makes...

...adenine base editor Earlier this year, Keith Joung’s lab developed SECURE cytosine base editors, which decrease... Nature Biotechnology Find the base editors   Single base editor that induces C-to-T and A-to-G mutations...developed a base editor that combines the abilities of C-to-T and A-to-G base editors into one protein...pyogenes Cas9. They called this editor Target-ACE (adenine and cytosine editor). Target-ACE exhibits increased...research! This time: CRISPRi in Burkholderia Base editors CRISPR toolkit for epitope tagging Lentiviral capsid-based...APOBEC1. Now, they’ve engineered SECURE adenine base editor variants using a protein-truncation strategy with...a target site compared to a mixture of two base editor enzymes in mammalian cells. Read the paper in ...

...activation of keratinocytes, which release inflammatory mediators that trigger the activation of dendritic...recruitment of neutrophils. Though there are IL-36 receptor inhibitors in development, there’s still a lot of mysteries...Generalized pustular psoriasis is a chronic autoinflammatory skin disease estimated to affect 0.27 to 4.6...actually get out of keratinocytes and induce their effector functions. It’s an important process that we need...IL-36 cytokines are a potent inducer of an autoinflammatory loop: keratinocytes both respond to and release...when he was studying thoracic aortic disease. “My mentors Dr. Dianna Milewicz, Dr. Carolyn Bondy, and Dr....with TS (Trolle et al., 2016). Using lentiviral vectors from Addgene, the lab will express these candidate...

...point.   What makes a story? A story is a narrative connecting cause and effect. Stories can and should be... or notes. Listen to their stories and narratives (see below “Tell a story”) and note how they build suspense...hear material delivered in the form of a story. Using stories is one of the best ways to make you and ...me and wished I had been more careful. 3. Tell a story We are biologically programmed to listen and remember...presentations engaging and memorable. If you read my story above about the presentation faux pas, you are more... striking images, creative ideas, and engaging stories as you encounter or experience them. Drop content... all graduate students and postdocs form peer mentoring groups to practice giving talks and getting feedback...

...editing assemblies are sensor-actuators, either transcriptional regulators or chromatin-modifying enzymes...subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These articles...s chromatin status into account (Fig 1a). The laboratory of Karmella Haynes at Emory recently contributed...contributed a set of plasmids to the repository that will help epigenetic researchers custom design their own...Examples of histone-modifying enzymes and reader-effector proteins. b) Golden Gate assembly (backbones available...with examples of potential synthetic chromatin mediators and probes. Image from Priode et al., 2021. ...potential tools fall into two categories: probes and editors. A probe array may consist of at least two sensor...

...lab has developed AAV vectors to selectively visualize excitatory and inhibitory synapses in the brain...combining vectors expressing different fluorescent proteins targeting inhibitory or excitatory synapses... find in this post: Tools for labeling excitatory inhibitory synaptic proteins Fluorescent protein system...New from the viral service Labeling excitatory and inhibitory synaptic proteins with FingRs By Jason... proteins PSD95 and gephyrin to label excitatory and inhibitory synapses, respectively. Once expressed...subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These articles... cycle in vivo, particularly if the cells are migratory or in deep tissues. To resolve this issue, Shangqin...

...efficiencies of integrating viral vectors. New Sleeping Beauty transposase vectors Even more recently, Eric ...developed two new types of vectors for the SB transposase system - one set of vectors for robust constitutive...transposase as a nonviral vector for CD19-directed chimeric antigen receptor (CAR) therapy. SB is a valuable...of DNA that can move around in a genome. In a laboratory setting, transposons can be used to both introduce...cells’ defense mechanisms. The use of non-viral vectors, like transposons, avoids many, though not all,...use of viruses is also important for social and regulatory reasons. 1.) The transposon consists of a mirrored... expression (pSBbi derivatives) and one set of vectors for inducible expression (pSBtet derivatives). ...

...chair of the postdoctoral engagement committee and active member of the university's Postdoctoral Association...science on April 22, 2017. While experiments (and editors) can move slowly, news about possible broad changes...in the veracity of its work. Alina Chan is a postdoctoral researcher at Harvard Medical School working...dabbles in science policy, public outreach, and restorative justice.  Follow her on twitter @sung_vanessa...health depends on it. Deanna McLafferty is an editorial assistant and activist. Washington, DC, USA 38.9072...to join the public conversation and to tell the stories behind our research.  That's why I marched for ...Michigan, where programs like the Great Lakes Restoration Initiative are at risk of major cuts and where...

...combinations of promoters, terminators, solubility and purification tags, connectors, cDNAs, and more to find...site-specific recombinases, regulatory elements, developmental regulators, and lots of viral components...workhorse yeast — K. phaffii (formerly known as Pichia pastoris). If your goal is protein isolation, then the ...kit has a focus on infrastructure with shuttle vectors, several delivery systems which can be engineered...engineered into the TUs, and even viral gene silencing vectors. The Plant Parts III kit has a focus on transformation... examine the effects of different promoters, terminators, enhancers, and cross-cassette interactions in...effects of protein degradation on chimeric antigen receptor signaling (Kim et al., 2024). The EMMA Toolkit...

...about being an editor - that I learned on the job. The day-to-day work of a science editor In my role as...sorts of different editors. Do you like science journalism? Take a look at our news editors. A fondness for...at our multimedia producer and online editor. And, the copyeditors deal with things like word choice, punctuation...contributed by guest writer Pamela J. Hines PhD, Senior Editor at Science Magazine. Although we only walk one ...be like? Then I spotted an opening for being an editor at Science magazine. That one I just could not ...as an editor at Science, my various interests come together as useful and productive, forming a different...science journalists, or policy makers. The term editor covers a wide variety of careers and jobs. At Science...

...might not be a big factor for you. Know who's boss You sign on to a lab for the mentoring only to find out...choose a mentor that sees you only as a paper making machine. Choose one that is a true mentor, supporting...rotation was with a mid-sized lab with an experienced mentor. The lab was very, very quiet. Every time I asked...some of these questions in the sections below. Mentoring relationships can help identify your priorities...not worked for you in the past. For all of the factors I touch on below, the first question to ask yourself...“which ones matter to me?”  and hone in on the factors that will make the most difference for your success...? Will a postdoc or lab manager be your actual mentor? Is there a chance the supervisor will be moving...

...him a more effective motivator (“be like Tony Robbins!” he encourages faculty mentors.)  Collaboration Maximizes...technical and pedagogical resources, offers mentoring for instructors, and provides opportunities for students...in a year-long biochemistry laboratory : Part II – research-based laboratory – a semester-long research...becoming just the fifth repeat Nobel Prize winner in history. Interestingly, he also adds to the disproportionately...this trend as evidence that the best teachers and mentors pass on more than just technical knowledge, shaping...responsibilities left him with limited time for one-on-one mentoring. His solution? Bringing his research into the ...work identifying new genes required for stem and progenitor cell maintenance in planarians in Developmental...

...has a great team of moderators who facilitated scheduling, filtered out inflammatory questions, and even...and the moderators were on top of any comments that were at all trollish. Kudos to the moderators for keeping...trigger phrase these days). Redditors ask serious questions What the moderators did not delete or suppress...incredibly important to find good mentors and incentivize good mentorship: AMA mechanics: Be prepared! The...Addgene Executive director, Joanne Kamens, recently participated in a Reddit AMA (short for “Ask Me Anything...lists of forums on particular topics. Volunteer moderators set the rules for these forums and try to keep...careers, women in STEM, work-life negotiation, and mentorship, we decided to have her participate in an AMA...

...empty control vector would be Plasmid A sans shRNA, or backbone Y alone. The empty vector control allows... In this context, the empty vector control shows any effect of the vector/backbone itself on gene expression...hypothesis: proper controls! For every experiment, an investigator needs a standard against which the results can...variable you are testing and not some other unintended factor. Control plasmids are used to minimize any effects...planning your experiment includes determining what factors need to be controlled for in order to eliminate...replicate controls.  Transfection controls: empty vector and internal control A transfection control measures...any effects of the transfection itself (i.e., the vector used in transfection, transfection reagent, or ...

...few inventory items offered domestically, but by the end of 2016, we expanded our viral inventory to 25... is a dual-vector system, meaning it is typically used in conjunction with a second vector that delivers...constructs (i.e., using a dual-vector system as opposed to a single-vector system) has several benefits.... lentiCas9-Blast Most of Addgene’s lentiviral inventory is based on CRISPR tools, though we do provide...LentiCas9-blast LentiCas9-blast is a Cas9 expression vector developed by Feng Zhang’s lab at the Broad Institute...such as lentiGuide-Puro. The alternative single-vector systems typically express Cas9 and a gRNA from ...compared to separate transductions with a single-vector system). Tips for using this plasmid Cas9 expression...

... Set up a table of contents in the top directory: A directory is a file system in the computer and is...experiment its own directory: In addition to output files, this is also where you can store any power point...in C++ and Java Command line history: Tracking command line history by recording all commands entered...analysis like a wet lab experiment I use the text editor TextWrangler to record the following, which are...name for a folder. For instance, your Desktop directory contains the folders and files that you see on...on your desktop. The location of your master directory or folder for all of your analyses is up to you...README.txt document is invaluable. In the top directory of every experiment, immediately write a text ...