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...Cas12a for multiplexed gene perturbation The Penn State Protein Ladder Engineered miniature CRISPR-Cas system...anti-HA frankenbodies (Zhao et al. 2019), Tim Stasevich and Hiroshi Kimura’s labs have recently published...onto a well-folded scFv scaffold to improve its stability in cells, and use it in a variety of applications...complementarity determining region of an anti-FLAG scFv and a stable scFv scaffold. (b) Schematic of anti-FLAG-FB-GFP...2021. doi: 10.1038/s41587-020-0600-6. The Penn State Protein Ladder  by: Rachel Leeson This simple, inexpensive...expression and purification. Figure 3: The Penn State Ladder run on SDS-Page gels. A: individually expressed...

...mechanism by which bacteria have gained antibiotic resistance and virulence. Scientists have studied how HGT... Guillemin, 2006) or the presence of specific substances (Meibom et al., 2005). Though not all bacteria... replication-incompetent phage particles with assistance from a ‘helper’ phage prior to transduction. ...expressed but not integrated into the genome and stable cell lines, where foreign DNA is incorporated into...can infer genetic advantages such as antibiotic resistance. Bacterial conjugation as explained by... genetic material can pass through. If you are starting any molecular biology experiment check out Addgene...

... attention on transcription factors, starting with an established method called ChIP-seq. ChIP-seq relies...explains it, a major goal in his laboratory is to understand the function of the non-coding portion of the...transcription factor. It also gives cells antibiotic resistance to neomycin so that you can select the population...We’ve done comparisons of our new system versus standard ChIP-seq for transcription factors known to have...available to anyone for a modest cost without having to start all over or write to us to get them. Even though...

...Konermann explained, was new knowledge about Cas9’s crystal structure. “I thought maybe we could use information...genes that, when turned on, make melanoma cells resistant to treatment with a BRAF inhibitor. (The test ...system and contains 10 sgRNAs for each transcription start site in 15,977 human genes. The transcriptional ...library contains 10 sgRNAs for each transcription start site in those 15,977 human genes. Weissman and his...manipulated at one time. Weissman’s group has made standard protocols available and they are working to further...gene, go to http://sam.genome-engineering.org/. Crystal structure of Cas9 in complex with guide RNA and...

...elsewhere (here and here for instance) [2], [3]. Karl Deisseroth of Stanford University , one of the key...neuroscience research where optogenetics will be a standard tool used in nearly any neuroscience project or...Intramural Research Program (NIH-NIDA IRP), with the stated goal of learning behavioral optogenetics in order...been published at this point.  In brief, some standard opsins (see table above) include Channelrhodopsin2...silence neurons than HR. Delivering your opsin  The standard opsin delivery method is local injection of an...considerations for your optogenetics experiments. Stay tuned!  Many thanks to our guest blogger Derek ...

...incredibly useful transferable skill. Being able to stand up in front of an audience and convey your ideas...you need it, rather than trying to push through a state of panic. Now, if I feel myself getting too nervous...present a paper to our classmates so they could understand it. When my mind went blank, my options were ...sense. As tempting as option two was in my panicked state, only option one would let me accomplish my goal... me.) They happen to everyone! Everybody makes mistakes, encounters technical difficulties, and runs into...you or not going perfectly, you truly are a rock star.  The best thing about public speaking as a scientist...

...and SMASh degron tagging vector! Destabilizing domains Destabilizing domains (DD) are akin to degron ...into the system, it will bind and stabilize the DD, resulting in sustained protein levels. Looking for a ...turning on expression of a new protein is a cinch. Stably introducing the coding region of a gene (cDNA) ...mechanism. When a protein is tagged with a DD, it is unstable and quickly degraded. However, if the relevant...

...blots, for instance, almost always use the indirect approach, while flow cytometry’s standard approach ...targeting the Fc region of an antibody, the so-called ‘constant’ region which is, quite usefully, both conserved...cannot recommend it except in the most dire of circumstances.) Multiplex assays Mo’ proteins, mo’ problems... antibodies used are mouse IgG antibodies, for instance, an anti-mouse secondary will bind to all the ...flexibility in assay development. Researchers who understand how and why to use secondary antibodies will ...

...Schiebinger, PhD, Hinds Professor of History of Science, Stanford University. Sex and gender are critical components...Commission, the US National Science Foundation, and Stanford University—that harnesses the creative power of...Gendered Innovations. This project: 1) develops state-of-the-art methods of sex and gender analysis and...sexes come from, one clue or potential place to start research is by considering sex disparities in disease...important point, however, is that cost at this early stage of research is minimal compared to that of human...trials. And, as Janine Clayton points out, late-stage clinical studies that draw from a body of research...

...each ligation reaction uses a pool of gRNAs as starting material, the ligation products are a diverse ... al., 2014) and microRNAs (Wong et al., 2015). Starting your CombiGEM-CRISPR experiments The first thing...efforts made by various research teams to build and constantly improve gRNA libraries, excellent resources with... Wong et al 2015. Alan Wong is currently an Assistant Professor at the School of Biomedical Sciences... applying new technologies to interrogate and understand complex biological systems. He can be contacted...Timothy K. Lu. "Enhanced killing of antibiotic-resistant bacteria enabled by massively parallel combinatorial...

...to discuss their visions for a successful and sustainable biomedical research sector at our first Future...at this event added to a growing dialogue on the state of a system that, despite all of its successes, ...years of earning their PhDs (3), showing that the ‘standard’ pathway into academia may in fact be just another...Andrew Young School of Policy Studies at Georgia State University, is an expert on the academic labour ...postdocs. Among other involvements, he helped to establish grants through the Sloan foundation for postdoc-related...uses synthetic biology to create tools to better understand human disease.  Outside the lab, he is interested...

... biomedical innovations promise an improved understanding of how our bodies work, new treatments for debilitating...School, has a mission of raising awareness and starting conversations about both the benefits as well ...between these communities and the biomedical establishment. This, along with concerns about the use of ...year, I’ve since returned to pgEd as a full-time staff member for the past three years. pgEd is a small...diverse backgrounds and disciplines, and everyone on staff takes on a variety of projects. With our focus on...submitting op-eds to your local newspapers, or starting your own blog. Even if you do not end up pursuing...

...require protein interactions to remain stable (or to be stabilized) during cell lysis and lysate fractionation...identification techniques rely on protein interactors to stay in close contact with the bait protein throughout...can artificially select against PPIs that aren't stable outside the native cell context. The Split-BioID...expressed in cells via plasmids or by the creation of stable cell lines and these cells are then grown in biotin-containing...characterization of these candidates is critical to understanding how they interact with the bait proteins. Another...

... addition to sequencing plasmids, we have also started using NGS to check the quality of other services...quality data! Addgene's new in-house NGS process To start, the process is only made possible by the use of...quickly create Illumina sequencing libraries. From start to finish the library preparation process only takes... individually assembles the reads into a single FASTA sequence that our QC scientists can easily analyze...performed, optimized as needed, and cleaned up using a standard PCR cleanup kit to remove any template DNA and...as part of our OpenBio Repository sometime soon. Stay posted!     References Guerin K, Rego M, Bourges...

...-fold more photostable and as bright to mVenus, the parent YFP, and is the most photostable YFP to date... were exposed to blue light. Cre-Vvd showed substantially improved activity as compared to other recombinase...addition, Casɸ has a minimal PAM requirement. For instance, for Casɸ-2 the PAM sequence is 5’-TBN-3’ (where....ncbi.nlm.nih.gov/32675376/ mGold, the most photostable yellow fluorescent protein to date By Justin ...contribute to experiments that require high photostability, such as time-lapse imaging or with high light...

...laboratory in a cell-free and biosafe format that was stable for up to a year at room temperature (Pardee et...gene networks for the detection of antibiotic resistance and strain-specific diagnostics for the Ebola...Pardee and Alexander Green! Keith Pardee is an Assistant professor in the Leslie Dan Faculty of Pharmacy...the environment.     Alexander A. Green is an Assistant professor at the Biodesign Center for Molecular...and the School of Molecular Sciences at Arizona State University. His group carries out research in synthetic...self-assembly for applications in health, energy, and sustainability.   References 1. Pardee, Keith, et al. "Rapid...

... Cpf1 nucleases, distant cousins of well-known Cas9. Cpf1 cleaves DNA in a staggered pattern and requires...nt upstream of the PAM site, Cpf1 cleaves in a staggered fashion, creating a 5 nucleotide 5’ overhang 18... 18-23 bases away from the PAM. Cpf1’s staggered cleavage pattern opens up the possibility of directional...efficiency of these two processes will help us better understand where Cpf1 fits into the genome editing landscape...Addgene. 4. Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, Barrangou R, Brouns SJ,...

...101 eBook to get all the background you need to start working with plasmids! What are termination and ...structure of the hairpin-DNA complex is thought to destabilize the transcription complex, initiating cleavage...addition of the poly(A) tail is important for stability of the mRNA, protection from degradation, and ...vectors often employ other non-poly(A) transcript-stabilizing and nuclear export elements such as WPRE (5) ...Maitland NJ. An Internal Polyadenylation Signal Substantially Increases Expression Levels of Lentivirus-Delivered...Addgene.org Browse our Plasmid Reference Pages Just Starting Work in a New Field? Check out Our Plasmid Collections...

... mostly learned through experience and from my mistakes. Here I’ll share what I wish I knew when I first...He left after a semester while the other student stayed until he graduated. Years later I attended a formal...often and clearly As a manager you need to clearly state expectations and goals. Yours and your team’s success... can be hard - but it is the best way to avoid mistakes due to miscommunication and to detect trouble....grateful for the feedback, but at least you can start making changes for the best. Positive feedback is...encourage them to speak their minds. They will make mistakes and you will take responsibility (within reason...

...outside a host cell’s genome. In this state, AAV cargo can be stably expressed in non-dividing cells for...identify new target genes for drug discovery or to understand the intricacies of molecular pathways. These ...available at Addgene. The large packaging capacity and stable expression of lentiviral vectors make them an attractive...first two steps, as no mice are involved. Ex vivo starts with transducing cells with the CRISPR library ...options involve generating strains of mice that stably express Cas effectors in specific tissues, eliminating...