...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...

...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...fluorescent protein (Mahmoudian et al., n.d.). 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...

...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 ...

... 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...

...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...