We narrowed to 1,082 results for: TOR

...Despite its relatively recent development, the history of optogenetics begins almost 50 years ago with...Wick and Krook-Magnuson, 2018). Optogenetic FLEx vectors contain a strong promoter upstream of an opsin ...Cre-dependent viruses, such as the optogenetic FLEx vectors in combination with transgenic animals or rAAVs...example, Taylor and colleagues used rAAV FLEx-based vectors to study the neural circuitry behind the mechanisms...paper, de Jong and colleagues used rAAV FLEx-based vectors to answer this question, mapping and characterizing...spatio-temporal precision and reversible modulation of FLEx vectors combined with the use of several Cre transgenic...motivated behaviors (de Jong et al., 2019). FLEx vectors represent, therefore, an ideal partner to optogenetics...

... learning. 1. Create a peer mentoring group We don’t all get the mentoring support we need, and most of...to access great mentorship is by recruiting your own peers and classmates! Your mentors don’t need to be...Own Peer Mentoring Group: A How-To Guide for Scientists.   Once in-person, peer-mentoring groups ... of us could use more mentors to listen, guide and support us during training and throughout our careers...goals.  I’ve been surprised to see that virtual mentoring groups seem to be as successful as in person groups...? You could do a talk virtually for your peer mentoring group (see above). However, now that we are all...initiative. In fact, hear from Addgene’s former blog editor Tyler Ford on how initiative landed him at Addgene...

...transcriptional regulator to modulate expression at that locus. The use of regulators allows a greater...shifted in recent years toward tinkering with regulatory networks and multiplex gene control. However,...approaches are often met with the challenges of regulatory bottlenecks and their scope is limited by the...Far removed from the bio-industry’s vision of ‘biofactories’, most successes in metabolic engineering have...us one step closer to the vision of metabolic biofactories by demonstrating the use of CRISPR-based RNA...scaffolds that can recruit various transcriptional regulators (Zalatan et al. 2015).  These scaffolds are stretches...levels dependent upon the type of transcriptional regulator or the number of designed scaffold binding sites...

...Fluorescent Proteins: Plasmids and Resources Addgene's repository contains thousands of fluorescent protein plasmids...conformational changes within a protein. Biosensors Monitor small biomolecules or other physiological intracellular...small molecules to activate or inhibit engineered receptors that affect cell signalling pathways. Luciferase...

... at some of the stories behind the names. We also have many plasmids in our repository that you can use...to a specific set of DNA-binding transcription factors to repress the expression of genes primarily involved...Cancer Institute! Sonic Hedgehog, an important regulator of embryonic development The largest conference...the hedgehog signalling pathway, which is a key regulator of all bilateral animal development. SHH plays...publication, Ivics et al 1997. Spaghetti Squash, a regulator of cellular contraction You may think that a gene...vessels). Spaghetti squash (Sqh) is the Drosophila Regulatory Light Chain (RMLC) of nonmuscle myosin II (Karess... Jordan and Karess, 1997), which contains the regulatory regions that are necessary for myosin activity...

...exactly how postdoctoral salaries are changing. Transparent salary information for postdoctoral positions...institutions set their postdoctoral salary scales to the NRSA scale (Figure 21, National Postdoctoral Association...was contributed by Future of Research Executive Director, Gary McDowell. On Dec 1st, the threshold at which...when the ruling was issued, a joint blog post by Director of the NIH Francis Collins and Secretary of Labor...paid. Institutional salary policies are one indicatator of how salaries may changes due to the FLSA. ...as having postdocs, representing ~85% of the postdoctoral workforce. Based on our communications with ...according to the NSF data, account for 18% of the postdoctoral workforce in the sciences. The Dana Farber Cancer...

...the activator - the CRY2 photolyase homology region (CRY2PHR) fused to a transcriptional activator domain...directed by the guide RNA (gRNA), while the CRY2PHR-activator fusion floats freely, depicted in the figure below...and CIB1 proteins heterodimerize and move the activator into position to activate gene transcription. ...manner. With their light-activated CRISPR/Cas9 effector (LACE) system, the Gersbach lab utilized a similar...fusion of full-length CRY2 and the transcriptional activator domain VP64. Using this system in HEK293T cells...photoswitchable proteins derived from the fungal photoreceptor, Vivid (VVD, N. crassa) (Kawano et al., 2015...transcription, they fused BldD to the transcriptional activators p65 and VP64 which in turn transcribes N-Cas9...

... and progenitor cells. Now with the DAP array, it is possible to use the state-of-art base editor, prime...prime editor, or any CRISPR-based gene editors, for correcting mutations or installing functional genes...thought to enable MBE by using Cas12a fused base editors (dCas12a-BE). However, the poor editing efficiencies...they found led them back towards Cas9 fused base editors (nCas9-BE), as Cas9 is mechanistically more favored...number of gRNAs that can be packaged into viral vectors for potential in vivo applications or multiplex...array.7 Using DAP arrays with a variety of base editors, the Gao Lab successfully demonstrated up to 31...unprecedented number of genes with a single viral vector.   Fig. 1: Schematic of multiplex gene ...

...fragments of DNA at specific locations. The Cas effector lacks nuclease activity, instead recruiting the... and type V-K. Type I systems use a type I Cas effector, composed of several separate Cas enzymes that...target site. Type V-K systems use a type V-K Cas effector, Cas12k, and three transposase components (TnsB...

...proteins are derived from bacterial phytochrome photoreceptors. Spectrally distinct fluorescent iRFP variants...Localization Luciferase Plasmid Collection Viral Vector Packaging Service Credits Contributing Authors ...

...adjacent fragment or vector. 5′ overhangs are generated on all the fragments and the vector, specifying the... together through transformation, so the cloned vector must be ligated. But in LIC, longer single-stranded...due to the absence of the other dNTPs. Once the vector and insert are (separately) digested to create ...received an important update, courtesy of Addgene depositor Stephen Elledge. His new method, named sequence...homologous recombination. SLIC inserts and linearized vectors must have homologous DNA sequences (20–60 bp) on...complete plasmid. Besides being compatible with any vector, the SLIC protocol itself is flexible. For one ...digestion (no dNTPs needed), allows both insert and vector to be digested in the same tube. Alternatively,...

...calcium indicator (Shemetov et al., 2021). In addition to FRET, intensiometric calcium indicators NIR-GECOs...proteins (NIR FPs) were derived from natural photoreceptors incorporating biliverdin IXα (BV) autocatalytically... derived from bacterial phytochrome (BphPs) photoreceptors have higher apparent cellular brightness than...were derived from cyanobacteriochrome (CBCR) photoreceptors (Oliinyk et al., 2019). They were engineered...performance, and applications of near-infrared calcium indicators in this recent review (Shcherbakova et al., 2021...encoded near-infrared fluorescent calcium ion indicator. Nat Methods 16, 171-174 (2019). Matlashov, M..... A near-infrared genetically encoded calcium indicator for in vivo imaging. Nat Biotechnol 39, 368-377...

...genes. We’ve created enough regulatory elements, CRISPRs, repressors, activators. We’ve been putting together...CRISPR/Cas genetic circuits are linked to the regulatory networks within E. coli host cells. ...in building genetic circuits is getting enough regulatory proteins that don’t interfere with each other...programmable that you can make a very large number of regulators. That basically means larger genetic circuits...that could conceivably be as large as natural regulatory networks. Addgene: What kinds of things could... pattern, shape. All of that is controlled by regulatory networks in the plant. If we want to access that...CRISPR is how much easier it is to work with than regulatory proteins. There are still things we haven’t solved...

...Insert Vector Type Promoter PI Return to top Additional Resources Optogenetics Guide Viral Vector Packaging...optogenetics plasmids. ID Plasmid Description Gene/Insert Vector Type Promoter Eligible for Viral Packaging PI Return...

...massive amount of information regarding which regulatory proteins, signaling molecules, mutations, and...little is known about the order or timing of these factors. Recombinase-based state machines (RSMs), which...In the absence of extra recombinase-specific co-factors (which should be excluded from the system), these...paper (1). Lastly, it is possible to build gene regulatory RSMs (GRSMs, the subset of RSMs that regulate...also demonstrates that genetic parts (genes, terminators, and promoters) may be interweaved onto a GRSM...parts used in this paper (recognition sites, terminators, promoters, and genes) were built with flanking...them. We recommend using strong, directional terminators and promoters with buffer sequences on their ...

...Mice can be engineered by inserting a targeting vector containing the gene of interest into a zygote, ...microinjection of DNA or infection with a viral vector containing the gene construct can be used to insert...generated via homologous recombination. A targeting vector is electroporated into embryonic stem cells and...engineer knockin mice. Co-injection of a targeting vector containing homologous ends to the breakpoint promotes...promotes homologous recombination of the targeting vector. CRISPR/Cas9-based systems have been developed ...the expression of a particular gene. Targeting vectors used to create knockout mice include a reporter...standing technique is the injection of a targeting vector into embryonic stem cells, which are then injected...

...Looking for a mentor or want to start a peer mentoring group? "What Makes a Good Mentor?" Form Your Own...form a peer mentoring group to get real feedback Write whenever you can, find supportive editors so your ... Own Peer Mentoring Group: A How-To Guide for Scientists Download our eBook: Mentoring for Scientists ...your PhD or perhaps you have almost completed a postdoctoral position… or two. You have learned a lot. Whether... scientists, create and stick to timelines Be a mentor in a formal program Start something (a journal,... plan a conference Recruit many supporters and mentors that will speak for you (and you for them) Communication...

...the tool, and develop new applications for magnetoresponsive proteins. Motivated by curiosity Although...Maria dreamed about the possibilities of a magnetoresponsive fluorescent protein. Controlling an optogenetic...experiments to see if any small molecules act as a cofactor (among others, flavins are very effective) and...self-labeling FlavinTag made a reliable one-component magnetoresponsive system that could be expressed and purified...thrilled to see that MagLOV’s “astonishing” magnetoresponse was clearly visible whether expressed in E....or in purified form, and didn’t need any added cofactors.      Figure 2: Fluorescence intensity...respond to magnetic fields?” and “What are magnetoresponsive fluorescent proteins good for?”, and while...

...following: the elevator pitch, talks, the poster session, networking, and social media. The elevator pitch Before...research, gathered up your data into an exciting story, and are ready to present your findings at a conference... Before the conference Practice your “elevator pitch,” the 30 - 60 second blurb you will use to introduce...work you are familiar with. You can tweak your elevator pitch slightly if you know a bit more about the...or talk. After the conference After using your elevator pitch several times at the conference, you might...might notice some trends. Were there parts of your elevator pitch where the other person seemed confused, ...questions. If you or your lab identifies a potential collaborator from the meeting, reach out to them, pointing...

...: A knockout targeting vector designed to insert a resistance gene. The vector contains a neomycin resistance... homologous sequence using a specially designed vector that contains sequence homology to the locus of... of the targeted gene have been cloned into the vector on either side of an antibiotic resistance gene...Figure 2). Figure 2: A knockout targeting vector designed to insert and later remove a resistance... the sequence to be inserted is cloned into the vector between the homologous sequences together with ... Figure 3: A knockout/knock-in targeting vector designed to insert GFP into a given locus. ...earlier. In this case, you design your targeting vector such that a set of three LoxP sites flank the resistance...

...Bacillus subtilis and the yeast Pichia pastoris. The improved secretory capacities of these microbes make ...this information, you can then pick an expression vector and expression system. If you find that your protein...not fold well, you should screen a few different vectors to see which gives you the most functional protein...coli: purifying from Bacillus subtilis and Pichia pastoris pCri also includes plasmids designed for protein...producing difficult-to-fold proteins. In addition, P. pastoris has the capacity to glycosylate proteins and may...peptide (SP) that routes the protein through the secretory pathway to improve folding and disulfide bond ...coli, the use of a thioredoxin A (TRX)-containing vector can improve disulfide bond formation. pCri also...

...include the laboratory personnel, the serum, the cell culture media, water baths, incubators, etc. Contamination...another. Multiple cell lines should not be stored in a single incubator since it increases the risk of cross-contamination... Addgene's viral vectors articles Find tips and tricks for working with viral vectors Resources on Addgene.org...Contamination! Tissue culture media often have indicators included in the media that turns yellow from ...proliferating shortly. Once they are present in the laboratory environment, they are difficult to eradicate....using expired products or reagents that have been stored for a longer time. Use fresh media as much as possible...tighten the flask cap before placing them in the incubators. This will also solve the problem of contamination...

... voltage indicator: JEDI-2P  by: Brian O'Neill  The François St-Pierre lab and collaborators have successfully...found an indicator that is optimized for 2-photon imaging of voltages. This includes monitoring of fast... plasmids, antibodies, and viral preps in the repository through our hot plasmids articles. Think we missed...what you'll find in this post: Improved voltage indicator: JEDI-2P Novel class of light-gated potassium...successfully developed an improved voltage indicator (GEVI) called JEDI-2P. Using a custom, high-throughput... available as Cre-dependent, EF1ɑ-driven viral vectors (AAV1) - and you can also find several other constructs...screen for improved genetically-encoded voltage indicators (GEVIs) based on the ASAP sensors. The resultant...

...petunia SL enzymatic receptor DAD2. SLs induce a conformational change of the receptor which modulates the...subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These articles...inducible CRISPR inhibition (CRISPRi). A) Lentiviral vector design. B) Strategy for leak-free transcriptional...detection of SLs. The authors also showed that the receptor-based fluorescent biosensor has a high sensitivity...biosensor based on the parasitic Striga hermonthica SL receptor HTL7. This novel type of biosensors could be used...  Design and mechanism of action of the receptor-based SL fluorescent biosensor. Image from Chesterfield...CRISPR plasmids are always being added to the repository. To find all of the CRISPR plasmids available...

... to deliver larger cargo such as base editors and prime editors. While using AAV to deliver large transgenes...problems with Cas9 delivery. Adeno-associated viral vectors (AAV) are commonly used for in vivo gene delivery...SpCas9) and a gRNA together (~4.2 kb) into an AAV vector is challenging due to its packaging capacity of...feasible, it leaves little room for additional regulatory elements. Feng Zhang's group previously packaged...packaged Cas9 and multiple gRNAs into separate AAV vectors, increasing overall packaging capacity but necessitating...for Cas9. However, to allow for more room for regulatory sequences while still fitting Cas9 and its gRNAs...Split inteins have also been used to express base editors. References Chew WL, Tabebordbar M, Cheng JKW...

...including the lung, the heart, and the abdomen. Long story short, when asbestos is breathed in (or less frequently... for years, or they are exposed even one time. Doctors and researchers don’t entirely understand why some...do, but we do know that there are some genetic factors involved. For example, multiple studies show that... being done to see what, if any, other genetic factors may be in play. However, there is no question that...asbestos exposure remains the single-most important factor in developing mesothelioma. Asbestos, and certain...Early detection, immunotherapy, and angiogenesis inhibitors New research is also going into two other areas...exposed to asbestos Tests like these will allow doctors not only to detect mesothelioma much earlier, thus...

... Daniel Mejías Sánchez, PhD, traductor  L. Denise Roche, PhD, MD, editora, [email protected] The ...with distributors across the world to help increase global access to materials in our repository. This...  Daniel Mejías Sánchez, PhD, translator  Denise Roche, PhD, MD, editor, [email protected] la traducción, Addgene contrató a una traductora científica profesional, L. Denise Roche, para...el acceso global a los materiales de nuestro repositorio. Este increíble regalo permitirá que más personas...Addgene contracted with a professional scientific translator, L. Denise Roche, to edit the translation. We...

...skeptical reader of historical fiction (I can personally attest to this!). Following history from the Ice Age...relevant science."     - Randall Carter, Contracts Administrator Inferior: How Science Got Women Wrong - and ...and the New Research That's Rewriting the Story by Angela Saini "This book is a smart and carefully presented...science of race."     - Joanne Kamens, Executive Director  I Contain Multitudes by Ed Yong "Microbes are... Tsang, Science Communications and Marketing Coordinator Non-science favorites  The Mistborn series by...another secret."     - Marcy Patrick, Associate Director of Quality Control Jonathan Strange & Mr. Norrell...Norrell by Susanna Clarke "A quasi-historical/fantasy fiction. Two dueling magicians in the 1800s in England...

...you can usually see a director sitting in their chair not watching their actors live on set, but peering...about getting in close to mic them up. Monitor: I then set up a monitor connected to the wireless transmitter...camera, OR, monitor from that distance.  Build: We got a wheeled stand and mounted a monitor to it. It ... their mask. But while the camera is here, the operator is in a different room.   Design: The previous...peering at a 7-inch wireless monitor. Wireless video technology has gotten very impressive, but due to costs...the beating heart of Addgene. It’s why we exist, storing plasmids, and distributing them to labs that need...Cycle 5 - The Future! Figure 5: The mobile monitor and teleprompter being repurposed for a different...

... addition, there was an entire story-telling session organized by Story Collider and which featured a ...new knockout mice, using adenoviral vectors to deliver IGF receptors to the brain, and probing how fatty...to make efficiently. For select viral vectors in the repository, you can instead have Addgene make the...the different parts of the brain stimulated by stories. In retrospect, I should have expected this given...helmets, gait tracking technologies, and brain monitors. Walking around the poster presentation area, ...available from Addgene. While super useful, these vectors require a bit of expertise and can be difficult... into neuroscience, you could look to our viral vector pages on the Addgene website and on the Addgene...

... 1. Testing kinase inhibitors Zhou et al examined the ability of kinase inhibitors to alter Raf1-MEK-ERK...kinase inhibitors, psMEK1 and ERK KTR-mRuby2 expressing cells were incubated with an inhibitor of choice...signaling pathways, scientists frequently use growth factors or serum to stimulate a pathway of interest, but...endogenous ERK, and its localization serves as an indicator of MEK1 activity. When MEK1 is active, it phosphorylates...localization of ERK KTR-mRuby2 determined. If the inhibitor targeted MEK1 or its downstream target ERK, the...the KTR remained in the nucleus. But if the inhibitor targeted upstream members of the pathway (Raf1, ..., which frequently occurs when serum or growth factors are used as a stimulus. This system also allows...

...one 30-minute reaction. The destination vector and entry vector(s) are placed in a single tube containing... appear in the final construct. The destination vector contains sites with complementary overhangs that...TGGA and 3’ overhang TCCG can be ligated into a vector containing those overhangs. Entry DNA overhangs...enzyme and ligase. Although the original destination vector + insert may spontaneously religate, this transient...for multiple DNA components (promoters, genes, terminators, etc) to be assembled in the correct order in...assembly of multiple DNA fragments to create TAL effector nucleases. These plasmids were designed to utilize...efficiently clone up to 7 gRNAs into one destination vector, making multiplexing easy.  Disadvantages of Golden...

...  Expanding our viral vector service Addgene has offered in-stock viral vectors since 2016, with the goal...Packaged on Request, an expansion of our viral vectors service, has officially launched! As of today, ...3,000 eligible AAV cargo plasmids in the Addgene repository to be packaged in your choice of five different...easy access to high quality, commonly used viral vectors. Of course, many scientists also need easy access...access to high quality, not so commonly used viral vectors. That’s where AAV Packaged on Request comes in.... making it easier to go from selection to viral vector. To start the AAV packaged on request process, ...for you to dig into. Check our blog post on viral vector production and quality control, our AAV Packaged...

...plasmids, antibodies, viral preps, and more in the repository through our Hot Plasmids articles. Here's what...cell mitochondrial transfer tracker Red calcium indicators PinkyCaMP and FRCaMPi Micropeptide killswitch...doi.org/10.1073/pnas.0510511103      Red calcium indicators PinkyCaMP and FRCaMPi By Mike Lacy Many popular...popular genetically encoded calcium indicators (GECIs) are available, but red GECIs have been limited compared...sensitive soma-localized red fluorescent calcium indicator for in vivo imaging of neuronal populations at...macromolecules to serve cellular functions. Along with collaborators, the Denes Hnisz Lab previously discovered that...the killswitch (GFP-nb–KS) is delivered using a vector that also expresses mCherry to mark transfected...

...Susanna Bachle, Addgene the nonprofit plasmid repository 49:45 0 1:18:45 - Reagent Development - Steven...Alex Tucker (Ginkgo Bioworks) Pamela Hines (Senior Editor at Science) Edward J. Hall (Professor of Philosophy...sequenced and quality controlled plasmids and viral vectors. When working with a plasmid from Addgene, you ...negative” data We all want to be able to tell a good story when presenting scientific findings. This makes ...of information and, from a SciComm perspective, stories with links of causation from initial hypothesis...validation are easier to understand. The need for great stories has, however, made it difficult to publish so-called...original hypotheses. Looking for novelty in our stories, we are additionally averse to producing papers...

...industry, non-profit, and government sectors.  Within these sectors, you could start your own company, ...based on any or all the contents of this post.  An attorney should be contacted for advice on specific legal... as law, medical/technical writing, clinical, regulatory, product development, business development, consulting...career paths in academia as well as the non-profit sector are transferrable to industry such as technology...greater scientific field, potential customers, investors, or other relevant entities or individuals. A ...peer reviewer for journals, acting as a journal editor) of the work of those in your field. Original contributions...papers that you should start contacting journal editors to request to be a reviewer. Proactivity is very...

...for drafting my stories. In total, I prepare 5-6 stories for an interview, since one story usually has more...Instead, I preferred to prepare stories to answer interview questions. Story telling shares many of the same...of the theme of my interview stories as the characteristic which the story demonstrates I possess.   Bullet...helpful for my interviews in industry. Prepare stories to tell in your interview The STAR interview method...answer: a setting, a conflict, and a resolution. Stories usually have a theme too, which is something not...of questions. Writing a rough outline of these stories helped me remember them for the interview.  Prepare...

...time: Functional genomics research in an introductory laboratory course. CBE-Life Sci. Educ. 17, ar1. (2018...experiences to undergraduate students (1, 2), but educators face significant challenges in designing suitable...could provide the basis for student projects in introductory labs. Genome projects use computational methods...research questions. The idea is straightforward. Instructors first select a multi-gene pathway to study and...yeasts are thought to have diverged from a common ancestor ~350 million years ago. Over time, the two genomes...species into the pYES2.1/V5-His yeast overexpression vector. This plasmid contains origins of replication and...research skills (8). A complete set of student and instructor resources is available at our Investigations ...

...proteins for use as FLIM-FRET acceptors  Listen to the FLIM-FRET acceptor podcast segment Article contributed...FLIM-FRET acceptors here. See Addgene's FRET reference page for many other common FRET vectors. Murakoshi...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles...termied soCoChR, by fusing a fragment of the kainate receptor subunit to the C-terminus of the protein. Unlike...soCoChR-expressing cells. Finally, using synaptic receptor blockers to distinguish real and artifactual postsynaptic...placesTagRFP-T and Dronpa in close proximity. By monitoring changes in TagRFP-T fluorescence intensity at...Murakoshi lab has developed two new FLIM-FRET acceptors, ShadowG, a novel dark green fluorescent protein...

...including a handful of plasmids that have been in the repository for many years, has now grown into a collection... spike protein to bind ACE2, the host cellular receptor. To more easily study the spike variants, scientists...Choe’s lab identified that ACE2 is the functional receptor for SARS-CoV. The lab also found that cathepsins...have an important role in viral entry, which are inhibitors of SARS-CoV-2 entry and become upregulated during...pandemic, the Krogan lab at UCSF generated lentiviral vectors expressing SARS-CoV-2 open reading frames and identified...proteins which all but one are cloned into pLVX vectors (for lentiviral packaging) and contain an IRES-...above, some of these plasmids have been in the repository for many years while many were developed during...