...very useful in the event that you will not be physically present at your poster at all times, so other...readers can navigate your poster and follow the logical progression of your work, and you can accomplish...offers free poster templates, although most are vertical.  Large central box. Another commonly used and...presentation. Portrait. This style displays a poster vertically rather than horizontally. Instead of displaying.... Shapes. In this type of poster, written and graphical information are inserted into larger circles or... or ovals, that are arranged in some logical pattern across the poster. This style has the advantage of...

...Mother Nature’s toolbox. Natural plasmids, also called environmental plasmids, are all around us! A plasmid...carrying genes for bacterial toxins like colicin typically also carry the necessary resistance genes to help...cells survive their own secretions. Pro tip! Historically, colicin-producing plasmids have been referred...which strains of E. coli contain this F plasmid is critical for successful Gateway cloning. In addition, scientists... Vogel, T. M., & Simonet, P. (2001). Laboratory-scale evidence for lightning-mediated gene transfer in...Characteristics and Potential Applications in Biotechnology. Critical Reviews in Biotechnology, 24(4), 155–208. https...

... be used for measuring protein expression or localization in cells, or even single-particle tracking of...named according to the number of strands in the canonical beta-barrel structure. Most frequently, the proteins...fluorescence complementation analysis under physiological conditions. Shyu YJ, Liu H, Deng X, Hu CD. Biotechniques...fluorescent proteins as tags and biosensors for multiscale imaging. Shcherbakova et al. Nat Commun. 2016...split-FP options. While the BiFC interaction is typically irreversible and requires some time for chromophore...

...histone-derived peptide) which can localise the construct specifically to the histone protein of interest...becomes. This prevents the cellular machinery from physically interacting with the genes in the compacted region...grandchildren; in this study,epigenetic changes were statistically significantly associated with reduced life-span... Chi-Wang, Cindy Y. Jao, and Alice Y. Ting. "Genetically encoded fluorescent reporters of histone methylation...methylation in living cells." Journal of the American Chemical Society 126.19 (2004): 5982-5983. PubMed PMID:...

...several ways. Below is a table listing some of critical differences (adapted from Zhou and Melton, 2008...this process. Moreover, it is much easier to genetically modify cells during the reprogramming process...This opens up a wide range of possibilities in clinical situations. In cases where the objective is to...Kushi) Mukherjee is the Editor of Trends in Pharmacological Sciences, a Cell Press reviews journal. She...from Massachusetts General Hospital and Harvard Medical School. Connect with her on LinkedIn @ https://...PMCID: PMC2854424. 23. Burridge, P.W., et al., Chemically defined generation of human cardiomyocytes. Nat...pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proc Natl Acad Sci U S A, 2012....

...Chavez, researchers at the Wyss Institute for Biologically Inspired Engineering. CRISPR/Cas9 technology...has facilitated the development of a simple and scalable means of making targeted genetic edits. Cas9 is... a gene of interest, they have been shown to dramatically alter levels of expression. Cas9’s ability to...Lance-Byrne is a graduate student at the University of California, Santa Cruz, where her research is focused on...Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease." Nature biotechnology ...Options for CRISPR Genotyping Cas9 Activators: A Practical Guide Additional Resources on Addgene.org Check...

...systems. FPs have been used to track protein localization, cell structure, intracellular trafficking, ...tagged proteins can lead to improper protein localization, protein aggregation, or disruption of normal...Also, it remains challenging to screen for the genetically modified strains that contain your newly tagged...recently developed a modular plasmid assembly toolkit called SapTrap to improve CRISPR genomic tagging in C....Protein Tagging. Beyond simply monitoring protein localization and activity, CRISPR tagging systems can be ...endogenous proteins for studying protein expression, localization, and protein-protein interactions. Additionally...

...antibodies for every potential new target and basically impossible if you wanted to distinguish between...C-terminus post-translationally, which turned out to be critical for antibody recognition. As such, the peptide...interest and proteins tagged with it needed to be chemically treated before the antibody could detect them...investigating mechanisms of endoplasmic reticulum localization, Munro and Pelham casually introduced the Myc-tag... recognized by its antibody without additional chemical modification steps and when the epitope was within... of new versions of Myc1-9E10 mentioned above basically involve taking the Myc1-9E10 variable regions ...

...domain fragment derived from a camelid antibody called a heavy chain antibody (HCab). Because of their...their small size, nanobodies can be synthetically expressed off of plasmids in bacteria, and they can target...enables rapid selection of synthetic nanobodies, called sybodies, against membrane protein targets in as...with a large and diverse pool of sybodies that hypothetically contain ones that bind strongly to your target... identifying sybodies that bind strongly and specifically to the protein of interest. The Seeger lab used...van Schie L, Hoffmann M, Pöhlmann S, Graham BS, Callewaert N, Schepens B, Saelens X, McLellan JS (2020) ...

...protein must be expressed (inducibly, ectopically, stably… it’s your call). The TIR1 F-box protein will associate...their fused protein under certain conditions (typically the addition of an exogenous small molecule). ... size from several kDas up to ~35 kDa and are typically knocked-in via gene targeting or cloned in-frame...proteasomal degradation. Adapted from Nabet et al., Chemical Biology, 2018.    HaloTag The HaloTag was ...protein structures, interacting partners, and localization. Thankfully, this is a hot topic with many resources...

... editing. Specifically, these types of CRISPR systems work using a multi-Cas complex called Cascade (CRISPR-associated... Cas3. Cas3 can induce genomic alterations on a scale that other Cas effectors can’t, while maintaining...recruit Cas3, which carries out the brunt of the mechanical work for editing. Class 1 CRISPR systems make...unraveling the double-stranded DNA (dsDNA) in what is called a reeling mechanism (see next section for details...debilitating disease! This is, of course, only hypothetical at this point. More time and energy must be ...

... the pharmaceutical industry for years. They have proven very difficult to target therapeutically due ...however, have yet to slay the beast. On a global scale, tens of millions of new cancer cases arise and ...fit. Kevan Shokat’s group at the University of California, San Francisco, is approaching the Ras problem... cancers, and synthesizing compounds to bind specifically to their mutated residues. For Ras researchers...represented, making the results of any such study as clinically relevant as possible. Connecting the research...

...delivery methods below: Chemical transfection   Lipid mediated Cationic polymers Calcium phosphate Cells... some CRISPR mammalian expression systems, the typical applications for each, and potential delivery methods... or introducing mutations and tags, while a catalytically dead dCas9 can be used in conjunction with activator... be constitutive or inducible. U6 promoter is typically used for gRNA. May contain reporter gene (e.g....Medium Medium Medium Difficulty Easy Easy Easy Physical transfection   Electroporation / Nucleofection...

...is certainly not the first means to carry out so-called “forward genetic screening experiments”, it is ...in a particular phenotype. CRISPR can be readily scaled up for genome-wide screening due to the broad range...experiments you use them for! A well-developed biological question and experimental system are absolutely... to skewed results. Selecting a cell type - Theoretically, any cell type can be used in a CRISPR screen...screens check out Addgene’s 2016 publication “Practical Considerations for Using Pooled Lentiviral CRISPR...McDade JR, Waxmonsky NC, Swanson LE, Fan M (2016) Practical Considerations for Using Pooled Lentiviral CRISPR...

...neuroscience research is the lack of a method for genetically manipulating neurons throughout the whole brain...CNS and PNS.That is until the Gradinaru Lab at Caltech stepped up to the challenge. Find the PHP Plasmids...astrocytes. Why screen for IV deliverable AAVs? Typically AAVs are directly injected into the brain, but...throughout the body and is often difficult to surgically access. Therefore an IV injection is an ideal...pictures (see figure 4). It also enables single-cell anatomical study and neuronal tracing, aka determining where...mechanistic studies and gene therapy treatments for neurological diseases). The Gradinaru Lab has a lot going...

...immunoprecipitation (enChIP) using CRISPR." Biochemical and biophysical research communications 439.1 (2013):...genomic alterations. These studies utilize a catalytically inactive form of Cas9 known as dCas9 (Jinek ...interactions at this site (Fujita and Fujii, 2013). They call this method engineered DNA-binding molecule-mediated...begun to make important discoveries in various biological fields. In 2015, the Fujii Lab identified non-coding... Fujita, Toshitsugu, et al. "Identification of physical interactions between genomic regions by enCh IP‐Seq...

... non-scientist friends and family is Part Five, called simply “Bad Science.” Zimmer kicks off the section...science, or misuse science, and that is what I would call bad science.”  The three chapters in this section...Zimmer provides several. In Part Three of the book, called simply “Old Science,” he describes the now infamous...are “old,” they lead Zimmer to one of his most critical observations: “Today the most successful scientists...optogenetics and CRISPR of course address some of the ethical implications of using these technologies, but also... certainly still is and will continue to be a “critical consequence of ...the marginalization of science...

...using a mammalian production system, cell lines typically used include Human Embryonic Kidney (HEK) cells...also need to know the extinction coefficient to calculate the concentration. At 280 nm, the extinction coefficient...protein standard and the concentration may be calculated using imaging software. Any bands on the gel ...validated to ensure they bind their antigens specifically. Possible applications for validation include...Sellick C, Varley P, Rockberg J, Chotteau V. Small-scale bioreactor supports high density HEK293 cell perfusion... MS, Carpenter JF, Lengsfeld CS. Do not drop: mechanical shock in vials causes cavitation, protein aggregation...

...  Ampicillin Ampicillin, often just called amp, is the historical king of the antibiotic resistance realm... antibiotic resistance genes so that you can specifically isolate bacteria containing your constructs ...immediately after it has been heat-shocked or electrically zapped. As a result, a shorter recovery period... of antibiotics, which induce cell death by intercalating into DNA and generating DNA breaks. The Sh ble... antimicrobial agents in MIC trays. Journal of Clinical Microbiology, 23(5), 959–961. https://doi.org/...

...the bacterial population. But what if we could specifically target only virulent or antibiotic resistant...antibiotics by their suppliers, and public health and medical organizations are working to help educate the public...encoded by a high-copy plasmid. In the case of the genomically encoded quinolone resistance, mediated by a single...represented here by cells of different colors, specifically targets bacteria with the given genomic or plasmid...Yosef et al. suggest that it could be valuable in medical settings where antibiotic resistant bacteria have...narrow bacterial host range. To further potential clinical applications, Ando et al. recently engineered ...

...scientists often refer to as “retroviruses” are technically gamma-retroviruses - another, separate member...differences may seem subtle they result in a key physiological difference between lenti- and retroviruses. ...transfer vectors for transient transfections? A2: Technically, yes. Will it actually work? That depends. The...when using a plasmid backbone that was designed specifically for transient expression (find some useful backbones...RRE, and psi packaging signal. Viral components typically total <1.5kb, which is rarely more than 30% of...terms of biosafety? A8: According to the American Biological Safety Association, "the two major risks of lentiviral...

...asbestiform minerals such as erionite, are the only scientifically proven causes of the disease. There is no question... question on this point within the medical and scientific communities, and the link between asbestos and.... New techniques are currently being tested in clinical trials to detect mesothelioma earlier than ever...the high mobility group box 1 protein. Normally localized to the nucleus, if the cell dies from injury, ...brought a lot of promise to a field that has historically been full of bad news and dreadful prognoses...Study Cancer Drug Resistance Plasmid Grant to Aid Cervical Cancer Screening in Ghana Additional Resources...

...improved, by adding a nuclear localization sequence (SV40 Large T nuclear localization sequence) and expressing...asymmetric target site core but its 8 bp length that is critical for Cre and FLP function. Because recombination...Recombination between the two target sites with identical sequence leads to inversion of the flanked DNA...this plasmid mix results in sparse labeling of cortical neurons and their synapses.   How can you...control   Figure 2: Expression of a morphological marker (eYFP) and synaptic marker (PSD95-mcherry...

...detection is crucial for clinical diagnostics, genotyping, and biotechnological advancements. Many methods...be used for an array of applications including clinical diagnostics (e.g. pathogen or virus detection)...points out that the protocol is not approved for clinical use at this point, they hope that the protocol...this ability to create a DNA detection platform called DETECTR. How does DETECTR work? DETECTR works similarly...mixed population for an array of molecular and clinical diagnostic applications. The company Mammoth Biosciences...

...defined chunks, called stories, and work on stories during short periods of time called sprints. The team...code, we might be calling something a sample and in other places, we might be calling it an aliquot. This...follow an agile methodology of software development called Scrum. The Scrum framework is based on an iterative...ourselves. We are also able to consider different technical approaches that may come up during discussion....

...a reporter or tag that can then be detected.  Typically, the reporter is an enzyme such as horseradish...level of antigen present in the sample and is calculated by extrapolating from the standard curve. Because...include undesirable molecules that may bind non-specifically to assay components and increase background ...amount of antigen present in the sample and is calculated by extrapolating from the standard curve. Scientists...antibodies are used.  Competitive ELISAs Scientists typically use a competitive or inhibition ELISA when measuring...While the precise setup of the method can vary, typically users first coat the multiwell plate with a reference...

...DnaA binds multiple specific sites within the ori called DnaA boxes. These boxes come in both high- and ...Created with BioRender.com. Once DnaA breaks the local hydrogen bonds holding the DNA together, it recruits...itself. RepA binds to directly repeated sequences called iterons located near the ori and directly interacts... the protein to be translated. An antisense RNA called Inc, also encoded by the ColIb-P9 plasmid, is complementary...between iterons on different plasmids in a process called "handcuffing," which blocks replication. ColIb-...Replication Browse other Plasmids 101 Posts Get practical molecular biology help with our Molecular Biology...

...range of logistical barriers. Addgene worked to identify, reduce, and remove these logistical barriers...’ve turned the available into the accessible. Physical access Addgene is located in the USA, but our ...Beijing Zhongyuan 2012 China Summit Pharmaceuticals International 2013 Japan Leehyo Bioscience...Digest Analysis We also introduced guides — practical primers to help users get started with tools like...increasing the accessibility of our repository, from physical materials to useful information, but we’re not...

...cryogenic electron microscopy (cryo-EM) protocols typically require high concentration and purity of the target...limitations, the Funabiki Lab developed a technique called Magnetic Isolation and Concentration (MagIC)-cryo-EM...circles), magnetic bead (*), and spacers are labeled; scale bar: 100 nm. D) 3D model of the obtained structure...right). B) Protein samples protected from AWI by helical LEAs. C-E) cryo-EM single-particle analysis of ...orientations, this could be overcome with methods such as chemical crosslinking or tilted-stage imaging. A truncated...evolved recombinases and prime editing. Nature Biomedical Engineering, 10.1038/s41551-024-01227-1. doi:...

...combining these two strategies allows you to specifically incorporate homo- or heterozygous mutations....in the field at the time but had never been systematically analyzed: introducing silent CRISPR/Cas9 blocking...DNA region? To this end we developed a technology called CORRECT, which, by undergoing two rounds of gene-editing...efficiently incorporated during HDR. We decided to systematically characterize this phenomenon and found a general...two successful mutations in a single cell. We calculated the likelihood of obtaining homo- and heterozygous...University. He currently works to understand pathological mechanisms of Alzheimer’s disease by developing...

...transformative biological discoveries and life-saving medical breakthroughs and undermine critical research ...Addgene as a trusted partner for archiving their biological materials during funding transitions. Enabling...private partners to create additional pathways for critical scientific work to continue.  Moving Forward ...

... first, which he called aequorin, gives off a faint blue light when it binds calcium ions, and the second...second, which we now call GFP, absorbs that blue light and glows green. In the late 1980s, another researcher...gene transcription, as well as to track protein localization. And as it turns out, he was right! He began.... He even obtained samples of certain ones from local pet shop aquariums in Moscow where he lived! His...

...Translocation Reporters (KTRs) whose cellular localization serves as a proxy measurement of kinase activity...KTRs) Kinase Translocation Reporters (KTRs) are genetically encoded fluorescent kinase reporters. A KTR is...which is further fused to a bipartite nuclear localization signal (bNLS) and a nuclear export signal (NES...phosphorylatable. Prior to phosphorylation, the KTR localizes to the nucleus, but following phosphorylation ...breakdown (without the nucleus, there’s no nuclear localization to monitor). Whether you’re looking for a ready-to-use...

...one be interested in state machine technology? Biological research has produced a massive amount of information...processes that are influenced by temporally ordered biochemical events. How do RSMs work? The state of an RSM...prescribed region of DNA termed the “register”. Each chemical input to an RSM drives expression of a distinct...to record all permuted substrings of a set of chemicals. Specific examples of arrangements that should... record all permuted substrings of up to seven chemical inputs are given in Table S2 of our paper. The... He is broadly interested in biocomputing and biological circuits.     References 1. N. Roquet et al.,...

...re tired of calculating dilutions or you can’t quite remember the hazards of that chemical in your obscure...surpassed 60,000 views for the month of September! Historically we do better in September than in the summer..., Lianna Swanson, and researcher at Sanofi Pharmaceuticals. This post highlights a rare disease, Fibrodysplasia...can find the rest of our September posts in chronological order here: Four Things I Learned at the 10...

...by the use of AAVs? 1) Combined functional and anatomical mapping of circuits In our lab, we use AAV-DREADD...these complexes are so robustly expressed that anatomical assessments can be easily performed too. While...identified this feature of AAVs as a new tool for neuroanatomical tracing (11). With the ability to produce neuron...Retrograde AAV Preps at Addgene 2) Comparison of genetically engineered receptors (i.e. chemogenetics vs. ...should strengthen its appeal for the scientific and medical communities. Many thanks to our guest blogger ... Floris G., et al. "A fourth generation of neuroanatomical tracing techniques: exploiting the offspring...

...depositors; Gizela, the lab member who knows how the physical kits are made and shipped; Chiara, the tech transfer...repository, performing quality control, and providing technical customer service. Team members are highly trained...problem? Test a hypothesis? Whether you officially call it a “project team” or not, most scientific collaborations...But this is a blog post, so here are just a few critical features based on my own experience: Leadership...should be on the team. As a team leader, I’ve (diplomatically) booted people off teams. I’ve also kicked ...like meetings, but run well, meetings can be a critical part of any successful team. For reasons I don...

...human speech near the laminar airflow, the human scalp, sneezing, coughing, etc. Also, a constant in-flow...contamination Detection of mycoplasma by naked eyes or optical microscope can be very difficult, so how do you...cells detect mycoplasmas, they trigger a series of chemical reactions and this induces a noticeable color ...contamination, before using the cell lines for assay or analytical purposes. Use 0.1-micron filters as are they ...Fumigate the laminar airflow and the lab facility periodically. The CO2 incubators, used for maintaining cell...cleaned regularly and checked for contamination periodically. For more tips, check out Addgene's video Getting...

...The biomedical field is often concerned with understanding the cause of diseases and how to treat those...optimization is performed to take the drug to the pre-clinical and human trial phases.   So how does CRISPR factor...enables researchers to select and generate physiologically relevant models. In complex diseases, it can...standard is to test the drug on models which are genetically proficient and deficient for the target. CRISPR...discovery – it’s an exciting time to be in the biomedical research field! References and Resources References...

... developed an improved voltage indicator (GEVI) called JEDI-2P. Using a custom, high-throughput screening... the protein sequence of the predecessor GEVIs (called ASAP 1 and 2), they found an indicator that is ...approach and outcomes of the screen for improved genetically-encoded voltage indicators (GEVIs) based on the...optogenetic silencer in experiments with mouse cortical neurons, far out-performing previous attempts ...selective for K+ over other Na+ and other cations. B) Cortical slice of HcKCR1-EYFP and tdTomato expressed layer...