...in their ability to be specifically and potently activated by pharmacologically selective effector molecules...chemogenetics for research and potential clinical applications. Science 6436: eaav5282. https://science.sciencemag.org...wiring of the brain and the underpinnings of neurological disease, neuroscientists need to be able to ...What is chemogenetics? Chemogenetics relies on genetically introducing engineered receptors that are only...et al., 2019). Using PSAMs PSAMs PSAMs, or pharmacologically selective actuator modules, were developed... such as varenicline, are already approved for clinical use. Varenicline has been further modified to ... create ultrapotent PSEMS (uPSEMs) that have drastically increased potency. Download our chemogenetics...

...is complex Plasmid classification has shifted over the years, and new classifications are still being created... Characteristics and Potential Applications in Biotechnology. Critical Reviews in Biotechnology, 24(4)...us! A plasmid is a small piece of DNA that can replicate independently from its host’s chromosome. Although...genes between bacteria can also have serious implications for public health. Bacteriocin plasmids: microbial...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...

...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...performed on one register at a time. If a particular application makes it difficult to isolate single registers...state read-out method may not be suitable for applications that require minimal cellular perturbation. ...The input plasmid (high copy, ColE1 origin of replication), contains large serine recombinase genes downstream...contains the register. Depending on the desired application, the inducible promoters we used to drive recombinase...

...relationships as part of the job application process can dramatically increase the chances of being seriously...demonstrate in your application). Beware the myths of scientist “overqualification” but it may not be ... of a resume can be crucial for a successful application. It takes time to prepare an excellent resume... Many job postings will result in hundreds of applicants the day they are posted.  If your resume is not... here for more on timely submission of a job application. 3) Read job descriptions carefully Be open minded...bold. You don’t have to have all of the listed qualifications to apply for a position.  A job description...wish list and the hiring manager knows that no applicant is likely to meet all the criteria. Note the criteria...

... many biological research situations, the relevant volume is that of the cell. For the physical behaviors...experimental study of GFP-based FRET, with application to intrinsically unstructured proteins. Protein Sci, ...noncovalent dimers. This property of EGFP can lead to significant artifacts. If you're using green fluorescent ...problem for fusion proteins. One of the major applications of FPs is to visualize the localization, dynamics...monomeric FPs are not actually monomeric, at least in practical terms. The propensity of a particular type of ... Fusion of an FP to a transmembrane protein significantly increases the effective concentration of the...structures are large (microns), bright, non-physiological, and difficult to ignore [1, 2]. 2. Fusion to...

...Kusumika (Kushi) Mukherjee, editor of Trends in Pharmacological Sciences, a Cell Press reviews journal. Stem...stem cells are: Their ability to self-renew or replicate while maintaining the undifferentiated “stem” ...differentiate only into myogenic precursor cells [5]. Classification of stem cells Stem cells are classified into...differentiate into cells of peripheral blood. Typically, there is a very small number of ASCs in each ...Limited. Only specific cell types can be produced Ethical concern High. Embryos are destroyed during ESC ...of post-transplantation rejection when used therapeutically High Low (if ASCs are isolated from the patient...bypass the transplantation compatibility issues and ethical concerns of ESCs. As iPSCs can be sourced directly...

...must make is which system you will use, or more specifically, the type of cells that will be used to make...spanning both prokaryotic and eukaryotic organisms. Typically, what system is used is dictated by the specific...so most labs opt to purchase adenoviruses from dedicated adenoviral production labs or companies. Production...something you may have to optimize for your specific application. With mammalian systems, it is standard to do... one passage (transfection). This differs from typical insect cell preparations, which we talk about below... below. Processing The viral supernatant is typically collected over the course of a couple days to increase...supernatant will depend on the virus type. AAV is typically purified using methods like ultracentrifugation...

...strengthened my critical thinking and problem solving abilities, increased my technical know-how, and helped...the bench, look for “technical or product support specialist,” “field application scientist,” or “scientific...scientific or technical writer,” among others. Don’t discount job postings looking for PhD applicants since many... helped develop my science communication skills. Ultimately, my degree prepared me to be exactly the type... many will also accept applications from master’s candidates with experience.  Use LinkedIn to see what... you’re interested in (i.e. biotechnology, pharmaceuticals, etc.) Do their jobs sound interesting? Is ...deeper foundation of scientific knowledge and technical know-how that increased my autonomy and satisfaction...

...outcomes of the screen for improved genetically-encoded voltage indicators (GEVIs) based on the ASAP sensors...what you'll find in this post: Improved voltage indicator: JEDI-2P Novel class of light-gated potassium...-Myc [9E10] now available! Improved voltage indicator: JEDI-2P  by: Brian O'Neill  The François St-Pierre...have successfully developed an improved voltage indicator (GEVI) called JEDI-2P. Using a custom, high-throughput...predecessor GEVIs (called ASAP 1 and 2), they found an indicator that is optimized for 2-photon imaging of voltages...Sustained deep-tissue voltage recording using a fast indicator evolved for two-photon microscopy. Cell, 18, 3408...optogenetic silencer in experiments with mouse cortical neurons, far out-performing previous attempts ...

...The fix/perm solutions can make a significant difference in the efficacy of your antibody. When compared...procedure enables quantification of antibody performance in major research applications. eLife, 12, RP91645...that the antibody being published doesn’t always indicate the presence of supportive data. To be supportive...that the antigen being detected in the intended application — here, flow cytometry — is the desired target... saponin. 400,000 cells were stained with the indicated Synaptotagmin-1 antibodies and corresponding Multi-rAb...multiple timepoints. When the target of interest is critical to cell survival or proliferation, an additional...this approach should also allow for the clear identification of the cell types of interest. By correlating...

...therapeutics. We’ll have more to say about clinical applications of CRISPR in the next section. In order ...and aceylation, respectively (these are chemical modifications, or functional groups, found on lysine)..., start, and stop. For instance (chr3, 1, 10) indicates a stretch of DNA at the very beginning of the ... Table 1. Because of DNA’s 3D geometry and the chemical properties of modified histones, a genomic region...(ESCs). Your ChIP-seq finds just over 13,000 significant binding peaks for NANOG in the human genome. ...maintaining ESCs. So which of these 13,000 regions are critical? One hypothesis: the enhancers! This leads us ...approach for designing an experiment to identify the critical NANOG binding sites: 1. Find Enhancers that have...

...containing a phage origin of replication that can be packaged into replication-incompetent phage particles...bacterial host range. To further potential clinical applications, Ando et al. recently engineered synthetic...the bacterial population. But what if we could specifically target only virulent or antibiotic resistant...essential processes: cell wall synthesis, DNA replication, or protein synthesis. Because they target such...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...

...drives and their ramifications. Readers unfamiliar with academia’s challenges with publication and funding ...and CRISPR of course address some of the ethical implications of using these technologies, but also the...Connecticut College who has been teaching and communicating science for over two decades, attempts to answer... primarily on fluorescent proteins and their applications, tackles much broader issues in this brief volume...sexism in science, problems with peer-reviewed publications and funding, bioethics in the age of CRISPR,... and her essential-but-unlauded role in the identification of DNA’s double helix. Perhaps to the dismay...dismay of middle-aged scientists, the identification of Green Fluorescent Protein (GFP) and its development...

...networks in biological systems. NGS will also play significantly into the realm of clinical therapeutic...detection of KRAS mutations: diagnostic and clinical implications." The Journal of Molecular Diagnostics 12.4...validation process, also known as CRISPR genotyping, is critical to demonstrating causal relationships between ... Figure 1: Schematic representations of the indicated unbiased genotyping options. Figure reproduced...those relating to DNA structure (e.g. epigenetic modification, bulges) that may also impact off-target edits...cell-based methods are genome-wide, unbiased identification of double-strand breaks (DSBs) evaluated by... amplicon sequencing (Hendel et al. 2015), a significant difference as CRISPR experiments move closer ...

... We’ve also created constructs with containing chemical tags such as the SNAP-tag, Halo-tag, and CLIP-...animals and fix the samples. When you use AAVs, it typically takes a few weeks to achieve optimal expression... Any longer and the cells may start to show morphological abnormalities due to the excessive amount of... levels of XFPs - Reduce the amount of tTA. Paradoxically, too much tTA leads to a reduced expression ...most likely by suppressing transcription. It is critical to express a minimal amount of tTA to achieve ...sensory systems focusing on the functional and anatomical maturation of the olfactory system.      Takeshi...University. His lab has been studying functional and anatomical aspects of sensory circuit development using ...

...basics but are here because you want to know the intricacies of transductions, we've got you covered there...The good news is that viruses are very valuable biological tools, and with the right safety considerations...infiltration into a cell. The twist is that the typical genetic cargo — the viral genome — is swapped, ...vector containing the cargo of interest is then typically transfected along with viral packaging plasmids...risk management when working with viral vectors. Typically, one day after transfection into the production...expression or integration resulting from transduction typically plateaus within a few days to a few weeks, depending...viral preps available for a variety of broadly applicable and popular viral vectors. Check out our viral...

...Effective verbal and written communication skills are key to publication and acquisition of funding. If...  Writing Communication Publicity Consulting internships Communication Teamwork Management...Models Communication Regulatory knowledge Networking Blogging Writing Communication Networking... skills such as leadership, management, and communication. A great approach to fully understand and acquire...find open positions and view their required qualifications and skill sets Taking the time to identify... science class: Science Club for Girls) Communication to diverse audiences Organization Mentorship...  Leadership Management Marketing Communication Business models Conference or club organizer...

...Marker-independent identification of glioma-initiating cells. The International Cell Line Authentication Committee... the Biotechnology and Biological Sciences Research Council (BBSRC) and Medical Research Council (MRC)...have tended to avoid cell culture in favour of biochemical assays followed by in vivo testing. The mantra...propagation of the line, and can include assays for authentication and contamination) and cell lines can become...Estimates of the amount of money wasted in basic oncological research due to contamination are in the tens...let alone the human cost. Here are just a few publications which have since been retracted due to cell ... and most diverse of these repositories is the American Type Culture Collection aka ATCC where I work....

...position at Addgene, the skeptical applicant asked if she would partake in “critical thinking” in the position... of time that doesn’t involve critical thinking. Former Addgenie Jessica Welch says, “[It] seems counter-intuitive...collaborating, managing, and communicating. After years of focusing on my technical skills with cloning and...involves a few key features that can be difficult to replicate in other work settings: The sense of being the...it. The opportunity to be creative and think critically. One of the very first times I interviewed a ...transferable skills. Transferable skills are the non-technical skills you pick up wherever you work, and you’...careers and desk jobs “alternative careers.” Realistically, based on the numbers of PhDs and available ...

...has and will continue to dramatically increase the number of CRISPR applications. The first non-Cas9 CRISPR... D1135E variant is far more selective for the canonical S. pyogenes PAM sequence NGG compared to wild-...variant may increase the specificity of genome modifications at DNA targets adjacent to NGG PAM sequences...VRER variants is similar to wild-type SpCas9, indicating that the variants are likely just as selective...Wolfe SA, Sontheimer EJ (2018) NmeCas9 is an intrinsically high-fidelity genome-editing platform. Genome...doi.org/10.1038/nbt.3609 Find plasmids from this publication at Addgene. Kim E, Koo T, Park SW, Kim D, Kim...10.1038/ncomms14500 Find plasmids from this publication at Addgene. Kleinstiver BP, Prew MS, Tsai SQ...

...addition to certain staff scientists and those in technical roles. Therefore, institutions are responsible...suggested that the salary level should now be automatically adjusted every 3 years. In May 2016, Secretary...centrally administered, which leads to many complications, including the inability of some institutions...paid. Institutional salary policies are one indicatator of how salaries may changes due to the FLSA. ...from $43,692 to $48,216, an increase of 10.4%. The Icahn School of Medicine at Mount Sinai in New York is...of the postdoctoral workforce. Based on our communications with and research on these institutions, we...to the new level:  We are still waiting for an indication of what (most of) Harvard, Johns Hopkins, (most...

...on DNA, or are enzymatically active. For this reason, recombinant proteins are typically expressed in E...expression vector cannot have p15A origin of replication F- ompT lon hsdSB (rB- mB-) gal dcm (DE3) pLysS...expression vector cannot have p15A origin of replication F- ompT lon hsdSB (rB- mB-) gal dcm (DE3) pLysE...Expression vector cannot have p15A origin of replication F- ompT hsdSB(rB- mB-) gal dcm (DE3) pRARE2 (...Expression vector cannot have p15A origin of replication fhuA2 [lon] ompT gal (λ DE3) [dcm] ∆hsdS/ pLemo...Expression vector cannot have p15A origin of replication F-, Φ80ΔlacM15, thi, lac-, mtl-, recA+, KmR Expression...medium. Induction timing is important, as you typically want to make sure your cells have first reached...

...to actually hold all those seemingly abstract biological reactions that I learned about from textbooks...physics class to learn about electronics or mix chemicals together in chemistry to see what happens, but... expensive tiny amounts of reagents are, how complicated (and again, expensive) the different machines...proteins outside of a cell - cell free extracts Typically, if you want to make a protein – say green fluorescent...proteins without the need to handle a living, replicating organism. Furthermore, cell free systems can ...they can see, enzymes that can convert certain chemicals into fragrances that they can smell, or enzymes...central dogma while also giving them practice with practical lab skills like developing a hypothesis, precisions...

...To our surprise, we measured identical dissociation rates for catalytically inactive dCas9 molecules. We...altering its function). HDR is crucial for certain applications, for example, correcting the allele that causes...these editing reactions is low. Understanding the biological cause of this repair bias is a fascinating (and...(Richardson et al 2016) revealed some of the biophysical parameters that can influence the HDR/NHEJ decision...key scientific principle: that understanding a biological process in detail often suggests new strategies... dissociation rates and discovered that the catalytically active form of the nuclease dissociates very...Our in vitro results also demonstrated that catalytically inactive dCas9 produced a “bubble” structure...

...than the classical purification protocol involving iodixanol gradients. The current purification protocols...particles accumulate inside the producer cells and purification protocols often describe a process that begins...lysis to release AAV particles with subsequent purification steps. It’s also possible for AAV vectors to...from Massachusetts General Hospital and Harvard Medical School took a closer look at the AAV vectors that... This is particularly useful for therapeutic applications in vivo where endogenous anti-AAV antibodies...antibodies often neutralize the therapeutic efficacy of AAV vectors. How are exosome AAVs produced? Purifying ...exo-AAVs seem to target cell types with a similar specifically as conventionally produced AAV particles. But...

...taraxacifolia (Willd), an annual herb of Western Tropical Africa typically known as wild lettuce. Aside from their...diabetics nephropathy, one of the major pathophysiological complications of diabetes mellitus. Recombinant ...researchers in resource-limited settings. More specifically, the Addgene-Seeding Labs plasmid grant provide...formation. The quest for plant compounds to treat complications of diabetes Gloria Otito Izu, PhD Candidate...flow. Novel treatment options, as well as non-pharmacological solutions to modify the course of this condition...scanning microscope will allow Tsakanova to image cortical neurons and study changes in neural activity after... employing synthetic biology resources and microfabrication technologies to generate a self renewable ...

..., Nature Communications, 2019. PubMed PMID: 31451697. Genetically encoded voltage indicators for use ...Article contributed by Brook Pyhtila Genetically encoded voltage indicators (GEVIs) change fluorescence in ...lentivirus and have been difficult to manipulate genetically. To solve this issue, Sarah Teichmann and colleagues... many aspects of ApoB-LP biology, including quantification of abundance, size, and localization in vivo...Pubmed PMID: 23068583 Thierer et al, Nature Communications 2019. Pubmed PMID: 31366908 Adapting sigma54...real time when neurons or muscle cells transmit electrical information. Such voltage imaging can be performed...measurements such as patch clamp recordings. However, technical difficulties such as small fluorescence changes...

...for post-translational modification of recombinant protein products, specifically glycosylation. The native...scene. But what about the other yeasts – in the biomedical world, are there even other yeasts? In this blog...2017). Beyond human relevance, yeast have many applications in food science and biotech due to its fermentation...The most commonly used yeast by biotech and pharmaceutical companies is K. phaffii (formerly known as ...the system. Since then, multiple biotech and pharmaceutical companies (along with some research laboratories... bread. The following table lists some of the biological commonalities and differences between K. phaffii... ancient yeast strains and metazoan cells.   Applications for K. phaffii There’s a number of different...

...together can inform decisions about in vivo application and clinical testing. This would pave the way for improved...helps researchers understand a drug’s safety and efficacy in vivo.  ADAs and Adverse Immune Reactions Anti-drug...drug and increase drug clearance, lowering its efficacy and increasing the risk of an adverse immune reaction...silico approach The in silico approach cannot replicate the complexity of antigen processing events that... evaluation of ‌therapeutic immunogenicity is typically performed using traditional 2D assays with immune...drug product while ensuring the safety and the efficacy.  Better together Combining in silico and in vitro...This system would somewhat act like a 2-step verification. All approaches have their own limitations. ...

... has been critical to the Broad’s success over the last 11 years and was certainly critical to drawing...background in history and specifically the history of RNAi left him a bit skeptical of the technology at the...cancer biology in Ed Harlow’s group at Harvard Medical School and then applied to work at the Broad Institute...towards the Broad. In order to ask interesting biological questions, you need a tremendous amount of technology... in true collaboration with scientists asking biological questions. Doench loves that he still gets to...terms of determining what genes are involved in biological process X, you can do with CRISPR technology....with Addgene - he says he’s “known of Addgene basically since the beginning and [has] been using Addgene...

...your application a whole week before the deadline. The day of submission, review your application with... their dream.  To help students focus on the application process, and simplify access to academic resources...Scrutinize every opportunity prior to starting an application to assess your eligibility, interest, and career... detailed. Beyond the resume, a cover letter communicates your interest in a specific program, and institution...professors, and mentors. Ask for specific guidance on grammatical errors, sentence structures, and overall format...February represent peak season for internship applications. Thus these noble individuals are inundated ... with a stellar letter of support.        5. Application submission  Set a calendar reminder for every...

...ITRs serve as a type of origin of replication to initiate replication of the dsDNA virus. Viral packaging... endogenous E1 gene, making it replication deficient. This is typically supplied by the production cell...plasmid while the other plasmids with packaging or replication components are called helper plasmids. Below,...viral experiments is to deliver a genetic cargo, typically an expression construct, to cells/organisms. This... production, they can be helpful for certain applications and aid in ease of use. Our lentiviral guide...yield dsDNA. Unlike LTRs, ITRs are perfectly symmetrical to allow for this pairing to occur. Like LTRs...AAV are rep and cap. Rep is required for viral replication and also aids in infrequent genome integration...

...addition of a helper virus or plasmid allows it to replicate episomally, essentially remaining an extrachromosomal...extrachromosomal part of the host genome that can replicate and divide along with the endogenous DNA. AAV also...human gene therapy and is currently being used in clinical settings. AAV is also a cool tool in the laboratory...cell type doesn’t always lend itself to direct application of virus. However, the cargo capacity of AAV ...gamma-retro viruses. This is large enough for many applications, but in some cases multiple AAVs (co-infection...that we’ve reviewed the common viruses used in biological research, you might have an idea which ones could...and AAV. If you’re wondering more about what applications these viruses are good for, we have plenty of...

...promising therapeutic applications; it can enhance therapeutic delivery across broader physical areas of the brain...driven by neurons. By studying how neurons are physically connected, we can begin to understand how the...Therefore, by simply looking at how neurons are physically connected we can begin to understand how the ...region (anterograde tracer, Fig. 1B). Tracers are typically delivered to a particular location and they either...neuronal signaling, or manipulating any other genetically controlled function. By using AAVrg in Cre transgenic...Cre-dependent AAV into Broca’s area of a mouse specifically expressing Cre in the hippocampus (Fig. 3). ... pigs (work you can find in AAV Data Hub!) The cortical pathways to the IC were well transduced by AAVrg...

...with biological protocols and revolutionary physical approaches. However, with each new technical achievement...aim to make microscopy as a tool accessible to practically everyone.     Fig. 1: Open source hardware...convinced that most microscopy techniques can be replicated with commercially available consumer-grade components...modify and optimize the code, find and fix security-critical bugs, and contribute to constant updates of the... OpenFlexure microscope or open-source modular optical toolbox openUC2 are trying to overcome this hurdle... tools has allowed the community to regularly replicate designs worldwide. Distributing hardware via globally...reproduction of the structure in a similar way to the biological protocol, and assembly designs based around the...

...limited understanding of plant biology is a significant technical obstacle to plant synthetic biology. We ...biotechnologists face is public perception of genetic modification (GM) and synthetic biology. The mistrust and...emerging engineering technology. Monoculture and chemical spraying, mainstays of modern agriculture, are... by developing a plant biotechnology product specifically for consumer sales. Flowers that change color...a new conversation about GMOs.  Challenge #2: Technical obstacles to plant synthetic biology In addition...cannot build what we do not understand, and the complicated and unique metabolic processes of plants are ...creative problem solving like the Howard Hughes Medical Institute have begun to pivot towards plant biotechnology...

... apply it in a clinical setting. CRISPR is often used for animal germline modification, to repair or add... important proof of concept for future preclinical and clinical work. Find Plasmids for Disease Research...exon skipping is a good option for DMD treatment. Clinical trials have used oligonucleotide exon skipping...estimated that exon skipping therapies would be applicable to 80% of DMD patients. Figure 1: Exon...that AAV-CRISPR does not cause obvious germline modification, an important finding given the controversy ...six months. Future directions and obstacles to clinical CRISPR editing Given the success of the CRISPR-mediated...researchers are very excited. This approach may also be applicable to a number of rare genetic diseases caused by...

...mice at a time. Because mice are more genetically and physiologically similar to humans than many other model... of mice as model organisms is that they are genetically tractable. Mice can be easily manipulated with...also relatively straightforward to manipulate genetically. Scientists can make a new line of flies in only...reasons above but because they actually share many biological properties and processes with our own cells. ...important experimental model in the study of neurological diseases, such as Parkinson’s disease (PD). ... throughout their lifetime. Thus any genetic modification, such as expression of fluorescent protein or...embryonic development to adulthood. This ability to genetically manipulate and visualize cells and tissues allows...

...immunofluorescence? Generally speaking, IF is an application in which antibodies that have been joined to ...molecules, knowing that where you see fluorescence indicates your target’s location. Some other terms you might...ICC or a tissue in the case of IHC. IF is used specifically when we visualize the antibodies using fluorescence...bind to a single primary, resulting in signal amplification. Another reason is cost effectiveness. Generating...are not exclusive to IF, but they can have a significant impact on the outcome of your IF experiment. ...controls to show that your antibodies are binding specifically. For positive controls, aim to use samples that...antibodies you choose for your experiment will have a significant impact on the protocol you use and the ultimate...

...through our lab using barcoded tubes and plates. The critical pieces of information for a given plasmid are ...plasmid sequence. Other information may also be critical to the maintenance and use of certain plasmids...access and use the materials they need, and avoid duplicating your effort. In addition, if you should choose...for the process. Always sequence verify Once the physical sample and its associated information are at Addgene...recover around 750 bp of clean sequence from a typical reaction, or about 250 amino acids worth, so make... for more tips on sequence analysis, plasmid verification, and using NCBI BLAST. Tips for high-throughput...comparable success rates and sequence quality, which is critical for the efficient handling of large numbers of...