...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...

...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...

...: 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...

...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...

...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...

...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...

...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...

... 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...

... 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...