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...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...