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...transfected (see Technical Tips). Minigenome replication and transcription In order to replicate the minigenome...transcription and replication strategies of Marburg virus and Ebola virus by using artificial replication systems...allowing researchers to study aspects of the EBOV replication cycle under BSL-2 conditions (4). What is a ...that are crucial cis-acting signals for genome replication and viral gene transcription (6). The EBOV genome...5’ ends of the genome which are required for replication and transcription (Figure 1). All viral genes...cis-acting signals necessary to direct minigenome replication and reporter gene transcription by the EBOV polymerase... in 2, 5). The minigenome is capable of being replicated and transcribed in the cell when the appropriate...

...resistance-mediating polymorphisms in African samples of Plasmodium falciparum." Journal of clinical microbiology 51.8 (...furiosus." Acta Crystallographica Section F: Structural Biology and Crystallization Communications 61.12 (2005... In vitro, ligases (notably T4 DNA ligase) are critical reagents for many molecular biology protocols,...or DNA base pair mismatches (8-11). For some applications, there cannot be any end-joining activity, and...regions. Why use a high fidelity ligase? Numerous applications have been developed to take advantage of the...Taq and other thermostable DNA ligases. These applications detect specific nucleotide sequences with high...rounds of melting and annealing allows linear amplification of the probe ligation product. With the right...

...D) teams at pharma companies carry out preclinical and clinical research to discover and test the best...years. Complicating these challenges is the fact that they occur in the context of significant clinical...training in reviewing literature, and critical analyses skills were applicable to my cases. I can directly use...molecular behaviors of chemicals and proteins to learn how a particular drug or medical device works. This... consulting? Healthcare consultants advise pharmaceutical companies, biotech start-ups, health insurance...efficient ways of delivering healthcare. Clients typically engage consultants as short-term experts to work...acquisitions of or collaborations with other companies. Medical Affairs groups work on building awareness among...

... receptors which play a critical role in development and are often implicated in disease progression. ...inability to replicate the spatio-temporal precision with which signaling events occur in a physiological setting...brighter mutant S65T-GFP, and the subsequent biochemical characterization, has enabled scientists to engineer...observations: Rapamycin is a membrane-permeable pharmacological agent that can rapidly induction heterodimerization...flexible cloning process provides improvements to classical cloning methods, particularly for complex transgenesis...enabled the group to perform independent two-color optical excitation of neurons. The paper describes five...setting. Genetic techniques typically rely on overexpression or knock-down, where signaling dynamics are...

...rather than being geographically constrained to the brain. In cases where it’s critical to minimize or eliminate...recommended for all systemic capsid applications. For example, AAV-PHP.eB is typically administered at a total dose...your specific application and animal model.  Other model organisms Tropism and efficacy can vary depending... 2023 * indicates viral preps available. Mechanisms of crossing the BBB are indicated in parentheses...crossing into the brain. While helpful from a biological standpoint, it makes delivering AAVs to the brain...throughout the organism to target large or diffuse biological structures. Of primary interest here are systemic...suitability of a systemic capsid for an individual application is dependent on a number of factors, including...

...FAIRE-seq, and CHIP-seq), genome-wide identification and quantification of enhancer activity have remained...Identifying new ligands for orphan GPCRs is of significant medical interest, however efforts to do so have been...GMAP-compatible vectors for a wide variety of biological applications. First, GMAP-compatible backbones for lentivirus...mutant library generation in several biotechnologically and clinically relevant bacterial species. pORTMAGE...backgrounds can lead to disparate physiological effects and complicate any metabolic study. The Ralser ...brief articles highlight the main features and applications of a partiular plasmid or set of plasmids. We...Wu Lab library can be found in the associated publication. Ma, et al. Cell Rep. 2015. PubMed PMID: 26190106...

...protein site-specifically to alter its function, mimic a biologically relevant modification, or add in ...function relationships or, in more practical applications, allow a protein to be more effectively used in ...presents a significant barrier for those wishing to use it as a tool. In short, while the technical skills...proteins, you’re probably quite interested in the canonical 20 amino acids. But in your quest to learn more...code expansion (GCE): techniques in which non-canonical amino acids (ncAAs, also sometimes called unnatural...in fluorescent or chemically reactive side chains, all of which can help determine structure and function...was developed into an E. coli system, with the noncanonical amino acid in the media and the tRNAs and synthetase...

.... "A genetically encoded fluorescent probe in mammalian cells." Journal of the American Chemical Society...). GFP is the most popular, most widely used genetically encoded fluorescent probe. Several factors contribute...probe, it is the first choice when selecting a genetically encoded fluorescent tag. There are, however, ... proteins.Therefore, GFP is not suitable for applications that require anaerobic conditions. In experiments...of these strategies works best for a specific biological system under anaerobic conditions needs to be...adding GFP may alter diffusion rates. Smaller genetically encoded probes are based on cofactor binding ...tags are peptides that are engineered to bind specifically and with high affinity to fluorophores. Examples...

...or feedback. One of the most significant shifts in scientific communication is the rising use of preprints...arXiv.org), but have been only adopted by biological and biomedical scientists (see: bioRxiv.org or medRxiv.org...the most important part of your research is communicating the results of the work. Yes, those control ...any impact! There are lots of ways scientists communicate with each other: from formal mechanisms like ...poster or talk, or submitting patent or grant applications; to informal settings like lab meetings, social...publishing in a journal.  Accelerating scientific communication Preprints have been common in fields like math...increasingly growing time from submission to publication. It takes many months, often more than a year...

...people will simply load an extra ladder or technical replicate (ie, leftover sample) to fill the relevant...background, your issue is likely technical. White circles, for example, could indicate that you had air trapped...Sensitivity in Western Blotting. Journal of Pharmacological and Toxicological Methods, 64, 233–237. https://doi....to get right. If you’ve thoughtfully done the technical design of your blot, but are still finding yourself... be done at 4 °C or on ice, whichever is more practical. If your lysing method is not resulting in your...nuclear- or DNA-binding proteins, you may need to sonicate your lysates in order to release the proteins ...you may want to consult Tables 1 and 2 in our Technical Design of a Western Blot post to make sure you...

...you.  Starting from plasmids requires amplification, verification, and lentiviral production steps before...the same proportion after amplification as they were prior to amplification. And as anyone who has worked...library can drastically impact what conditions it needs to properly amplify.  Amplification protocols therefore... prep library. Addgene’s pooled libraries are typically DNA preps, but we do distribute several of our...process for using pooled lentiviral libraries. A) Amplification, validation, and lentiviral generation of a ...pooled library is transformed into E. coli for amplification. Transformed E. coli are allowed to grow on ...into HEK cells allows production of lentivirus. Purification of this lentivirus results in the pooled library...

...Diecke, S., et al., Recent technological updates and clinical applications of induced pluripotent stem...opened up vast possibilities for the clinical and therapeutic applications of iPSCs. The generation of human... patients, specifically children and patients with skin disorders for research and clinical trials, as...derivation of clinical grade hiPSCs, reprogramming methods that do not involve modification of the original... OriP and induces amplification of the episomes concurrently with DNA amplification in the host cell. ...system thus provides a practical way to generate transgene-free, virus-free clinical grade hiPSCs from easily...therapy trials and remains a safety concern in the clinical field [4]. Three major reprogramming methods are...

...experiments with too few replicates leading to suspect statistical significance (you can read more about...someone has replicated it precisely. You can find a great discussion of different types of replication in Nosek...formulating new biological concepts. However, rates of reproducibility for preclinical studies, as reported...perfect story for a “high impact” publication can lead to the publication of rare flukes showing positive...more practical) is pre-registration and review of experimental design as part of the publication process...on solutions to reproducibility issues in the biological sciences and featured speakers from academia,...Overview - Jeffrey S. Flier, Researcher at Harvard Medical School, former dean of the faculty of Medicine ...

...Series 4. Become a science communicator More and more fellowship applications are asking for a 200-300 ...good quality application. Even following this advice, only about 50% of my grant applications have been ...your grant application to your peers and collaborators can also help you get some critical insight into...do need to be able to research and write your application, then give it to someone else to read and / or...it). It’s easy for reviewers to spot when an application is rushed (whether the idea isn’t properly researched...researched / developed or the application is full of typos) and these are unlikely to get past the initial...someone else in the department) to read over your application and give you feedback. They’ve most likely been...

...production and purification that yield high titers (see also AAV protocols for production, purification and titration...higher specificity and reduced technical variability compared to chemical tracers. rAAV2-retro also has...ability to spread trans-synaptically on to subsequent neurons. AAV vectors are replication incompetent and cannot...intracerebral application, which is more invasive and has a much higher risk of complications. References...between different brain regions. For example, communication between the hippocampus and the prefrontal cortex...memory, a special type of memory which includes autobiographical events (see Jin & Maren, 2015). Directional...intracellularly spread away from the site of application and aid in visualization of the morphology of...

...research? I grew up knowing little to no biological or biomedical scientists and had no idea what the daily... global problems. Biomedical research, for instance, contributes heavily to medical practice, and understanding...comes your way. Think critically about whether it’s something you want to dedicate your time and energy...These conferences have a lot of programming specifically dedicated for career advancement, and for students...entire page dedicated to different career options from science policy to science communication and much ...the Daily Psychological Experiences of First-Generation College Students. Social Psychological and Personality...hazardous liquids. Working in a lab is nothing like a typical part-time or 9-to-5 job. I had to ask to find out...

...get there and more practically, when do you need to be there? A typical biomedical postdoc fellowship ... findings that fail to replicate. Plan experiments logically and strategically to increase your rate of...theJanelia Research Campus of the Howard Hughes Medical Institute. Today's postdoctoral fellows (PFs) face...need? Multiple first or corresponding author publications (published, not in preparation) A history of...submitted manuscript in a high-profile journal typically takes 6-12 months depending on the revisions and... to be ready to write by Year 3.5. Thus, it is critical to get an experimental system up and running very...The other items will follow. If you get more publications, you will be more competitive for fellowships...

...Waugh, 2002). The tag can also be proteolytically cleaved after purification, so as not to perturb protein...for downstream applications. Tag cleavage must be performed after primary purification and requires a ...thoughtful planning, affinity tags can make protein purification a cinch. Types of affinity tags, the proteins... Affinity tags are used primarily as protein purification tools (and can have secondary functions as detection...single protein is expressed, with the intent of purification, it will be expressed along with all other cellular...available antibodies, resins, beads, and other purification aids readily available. Choosing the best tag...tag to remain on your protein for downstream applications Below, we describe a few of the most common...

...the sandwich and typically takes less than an hour. However large proteins typically do not transfer as...validated applications, pay close attention to the controls listed on the vendor’s website. Typically, the ...downregulated in a system, detect post-translational modifications, quantify protein levels relative to standards... boiling in the presence of a reducing agent, typically containing thiols, to cleave disulfide bonds. ...gel on top of the resolving gel. A stacking gel typically has a different ionic strength and lower pH and...are “sticky” and have the potential to bind nonspecifically to the antibodies used for staining. To address...search, first pay attention to the validated applications listed on the vendor’s website and choose an...

...would be a good time to talk about the amplification and verification processes we use to ensure high-quality...your own experiments. Library amplification 101 Library amplification seems straightforward: E. coli ...represented. Typically, Addgene uses electrocompetent Stbl4 cells when performing amplifications, and we use...transformed with the library DNA, they grow and replicate, and then their DNA is harvested, much like one...library has its own depositor transformation and amplification protocol, but the overall workflow is similar...ll need to remember when undertaking library amplification and sequencing. Maintain representation of the...ensure a good transformation efficiency. For a typical library, Addgene usually does four to eight electroporation...

... expression for experimental, biotechnological, and medical applications. Researches can visualize proteins...of complex post-translational modifications. Extraction and purification of recombinant proteins from ...of proteins including antibodies and pharmaceuticals, specifically interleukins. Yeast expression systems...downstream applications. Cell-free expression systems however, are expensive and very technically challenging...modified via an array of post-translational modifications including protein folding, formation of disulfide...mRNA and the addition of post-translational modifications Learn more molecular biology in our Plasmids...expression systems used for protein production and purification. These include mammalian, insect, bacterial,...

...colleagues have developed a new genetically-encoded voltage indicator (GEVI) called JEDI-2P (Liu et al...Verkhusha’s lab has generated a new genetically encoded calcium indicator using small monomeric NIR fluorescent...Tuning the Sensitivity of Genetically Encoded Fluorescent Potassium Indicators through Structure-Guided...colleagues present an improved genetically encoded temperature indicator B-gTEMP designed to meet those... B-gTEMP itself is a significant improvement over previous temperature indicators, it also demonstrates...doi: 10.1038/s41592-022-01597-x. New glutamate indicator iGluSnFR3 Improving on the glutamate sensor iGluSnFR...They generated variants optimized for different applications (v82 has higher affinity for glutamate but slower...

...or macromolecule. Biotin can be added enzymatically or chemically. Enzymatic biotinylation occurs naturally...molecular biology applications for biotin and avidin. A short list of some other common applications and links...downstream application. Biotin labelled oligonucleotides can be used in a multitude of applications including...post-translational modification of exceptional specificity.” Trends in Biochemical Sciences, 24.9 (1999b... M. “Biotin: The forgotten vitamin.” American Journal of Clinical Nutrition 75.2 (2002): 179–180. PubMed...dye-labeled molecules on surfaces.” Journal of the American Chemical Society 125.27 (2003): 8074–8075. PubMed PMID...this highly specific post-translational protein modification. Most organisms have less than five enzymes ...

...present.  Purification reagent contaminants The reagents used during nucleic acid purification can leave...thiocyanate (used in many plasmid purification kits), which can dramatically reduce the A260/230 ratio at ...Selecting Your Plasmid Purification Kit Resources on Addgene.org DNA Quantification Protocol Recovering... and are ready to move on to your downstream application — but wait! Do you know how much plasmid DNA ...(Figure 1B). The A260/230 ratio can be a good indicator of protein contamination, but a number of other...researchers traditionally use the A260/280 ratio as an indicator of protein contamination. However, this ratio ...suggest that the A260/280 ratio does not drop significantly until protein levels exceed 70% of the sample...

...process allows you to physically magnify a sample so you don’t have to optically magnify quite so much...compatible with conventional antibodies, chemical dyes, and genetically encoded fluorescent proteins (Gambarotto... and the physical properties of the sample itself. For example, cultured cells are typically easier to...promises an easier, more accessible way to image biological features previously only visible via techniques...with an abundance of protocols and examples of applications from across disciplines now available. In this...experiment or evaluating ExM images in a new publication.         Why Expansion Microscopy?  Traditional...techniques also require specialized instruments and technical expertise, limiting their accessibility (Prakash...

... and LV have been used successfully in preclinical and clinical settings for ocular conditions. However...used in clinical trials, a variety of engineered ones have shown great promise in preclinical studies ...that I got into the field of gene therapy, more specifically AAV-based retinal gene therapy. The year was... Gelsinger in an adenovirus-based gene therapy clinical trial gone horribly wrong. It brought the whole...scientists, doctors and patients hope are many more clinical successes for gene (and cell) therapies. One may... over 25 years ago when completing her PhD and medical training in Ophthalmology. Gene therapy was in ... time advances in human genetics led to the identification of several disease-causing genes, including...

...dCas9-fusion proteins has dramatically increased the potential applications of CRISPR/Cas9 in genome-engineering... tools for all-optical electrophysiology A major challenge in the field of all-optical electrophysiology... the perfect voltage indicator and optogenetic actuator pair. Ideally, indicators should possess fast ...sensitivity. This hierarchical screen led to the generation of two enhanced voltage indicators: QuasAr1 and...temporal control over CRISPR/Cas9 mediated genome modification and gene expression. The plasmids associated... system widely accessible for many potential applications. Polstein LR & Gersbach CA, Nat Chem Biol ...expression Brilliant technologies adopt basic biological concepts and employ them in creative and innovative...

...SR, Bird A (2017) Radically truncated MeCP2 rescues Rett syndrome-like neurological defects. Nature 550...awareness and published his observations in a German medical journal. Decades later and after years of painstaking...silenced by a Lox-Stop cassette, resulting in the neurological deficits observed in Rett Syndrome. Restoring... tamoxifen-inducible Cre not only halted the neurological damage but completely reversed it, regardless...this interaction to varying degrees determine the clinical severity of different MECP2 mutations. More recently...the MECP2 protein is necessary to rescue the neurological defects of Rett syndrome (Tillotson et al., ...detrimental as too little, resulting in MECP2 duplication syndrome or Rett Syndrome, respectively. One ...

... editing in Escherichia coli. Acta Biochimica et Biophysica Sinica. https://doi.org/10.1093/abbs/gmab036...far, we’ve used the MUCICAT system in two ways: In our initial paper, we used MUCICAT to optimize the gene...industrial strain. Not only is MUCICAT better, it is faster. Using MUCICAT only needs 2 newly constructed...engineered cells for green chemistry and biomedical applications.   Yiwen Zhang entered the Sheng Yang Lab...integration using CRISPR-associated transposases” or MUCICAT. Here, we’ll describe each of these CRISPR systems...providing users with a complete editing process. The MUCICAT system While the above systems are great for experiments...chromosomal integration (ex: genome editing), our MUCICAT system which is based on the CRISPR-associated ...

...regulator. Other methods for physical shearing exist as well, such as sonication and needle shearing. Essentially... whole genome amplification (by a mechanism known as multiple displacement amplification) even in the ...proteins! Many thanks to our guest blogger Jessica Sacher! Jessica Sacher is a microbiology PhD student at...Their Viruses." Chemical Reviews (2016). PubMed PMID: 27319741. 3. Simpson, David J., Jessica C. Sacher, and...This post was contributed by guest blogger, Jessica Sacher, a microbiology PhD student at the University...bacteria) may be the most numerous and most diverse biological entities on our planet, but we still know collectively...adding methyl groups, glycosyl groups, and other modifications to their bases in order to protect their DNA...