...protein based research. This year, we put out another call for applications. We had an overwhelming response... variants one at a time is time consuming and unscalable.  Melissa Chiasson, a graduate student in Douglas...of sequence variants on abundance simultaneously called VAMP-seq, for Variant Abundance by Massively Parallel...epoxide reductase (VKOR) variants. VKOR is an enzyme critical for activating clotting factors and is the target...instrumental in interpreting human variation at large scales and making personalized medicine a reality,” Chiasson... Lin Tian’s lab at UC Davis is to generate a genetically encoded fluorescent sensor for serotonin. According...replicate current knowledge, but the sensor can also kinetically outperform current methods. Unger plans to use...

... Biosensor AAV (calcium sensors and GABA sensors) Calcium sensors The latest calcium sensors from Janelia...include: EGFP-expressing AAV for serotype testing Calcium sensors and GABA sensors Optogenetics Recombinases...for monitoring neuronal activity in the form of calcium dynamics. (For more details on this technology,...technology, see our blog post Overview of AAV Encoded Calcium Sensors). We are expanding the inventory of these... publication (Chettih & Harvey, 2019), “this localization should improve the specificity of influence ...Recombinase AAV Targeting and manipulating specific, genetically-defined neuronal populations with intersectional...pAAV EF1a Nuc-flox(mCherry)-EGFP (112677-AAV5) Calcium Sensors pGP-AAV-syn-jGCaMP7b-WPRE (104489-AAVrg...

...To our surprise, we measured identical dissociation rates for catalytically inactive dCas9 molecules. We...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...this publication were performed using a technique called nucleofection, which introduces functional Cas9...

...tools from across the biological spectrum to learn more about the brain and neurological disease. I was surprised...and, like many biological fields the number potential of experiments enabled by physical, software, and...brain functions and vice versa. Even within the biological sciences, there’s plenty of interaction between... microbial DNA they all seem to require some mathematical know-how for analysis. If you’re looking to ...presentation area, I honed in on projects using biological tools. In addition to the researcher using sequencing...but are afraid you don’t have the right kind of biological expertise to get into the field, there’s likely...genes to many different cell types with minimal immunological effects. AAVs are used in experiments targeting...

...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...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...reproduction of the structure in a similar way to the biological protocol, and assembly designs based around the...towards more open research. The EU, for example, is calling for the opening of sources within the next few ...relying on tailored image processing and low-cost optical setups. References Joel T. Collins, Joe Knapper...

... finding new factors in your biological system of interest, the so-called “fishing expedition.” Your first...population (typically cells or small organisms) and then assaying for a functional or phenotypical outcome...that both attempt to identify new players in a biological process. Players can be proteins, genes, enzymes...obligatory growth advantage – that is, you are not specifically looking at cells that can survive. Instead, ..., the entirety of the small molecule realm is typically not feasible to test in any given screening experiment...selection process. Non-functional traits will be automatically eliminated, unlike in screens. Selections begin...the answer that much faster? In reality, many biological questions are not conducive to selections and...

...which let the team detect its co-localization with a membrane-localized mCherry or GFP protein. With this...it co-localizes with its target at the cell membrane and when the OptoNB can’t bind, it localizes to the...The opening and closing of the AsLOV2 domain allosterically alters the shape and binding ability of its...unmodified proteins. Affinity purification methods typically require a protein to be tagged, which limits these...provides greater spatiotemporal resolution than pharmacological or genetic manipulation of the signaling pathway...and differentiation.  Since Ras is a membrane localized GTPase, the team fused a mCherry OptoNB to the...fusion protein in cells that expressed membrane-localized mCherry. With darkness, SOS binds Ras and initiates...

...experimental RNA. To solve this problem, gRNAs can be chemically modified to resist degradation while still effectively...purchasing your gRNA. Pro tip: modifications are typically incorporated at the termini of the crRNA molecule...oligonucleotides. Some of these synthetic modifications, specifically ones which do not contain a 5’ triphosphate ...sequences for your next CRISPR experiment.    Chemical RNA modifications to improve stability.   ...targeted editing events. Both technologies utilize chemical modifications to the RNA to control the entire...visualize transfection efficiency and even track localization in cellular experiments. There are many suitable...References Hendel, A., Bak, R., Clark, J. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome...

...these types of bigger picture problems systematically and logically is as useful for finishing your thesis... but this skill is not just about reacting to technical issues. This skill also comes into play while ...defining problem solving skills as: One’s ability to logically think through a problem/task/situation, come up...Problem-solving overlaps heavily with other skills such as critical thinking, decision making, creativity, or data... really are, while also practicing systematic, logical thinking.  If you are already a lab veteran at ... to troubleshoot; you probably did not just automatically come to a solution. Consider the thoughts that...solutions. Ultimately, problem solving is less about technical know-how and more about how you approach new challenges...

...Stanford University. Sex and gender are critical components of biological research that are often forgotten ...the past decade. For more practical help, there are numerous methodological articles to consider—we have... that of human clinical trials. And, as Janine Clayton points out, late-stage clinical studies that draw...effects of using all female cells and tissues, specifically in stem cell therapies. Let’s go back to why... disease being treated, and other hormonal, immunological, and environmental factors. How to include sex... (NIH) now requires that sex be included as a biological variable in all NIH-funded research. Janine Clayton...

...generation of iPSCs is relatively simple in concept: ectopically express a cocktail of stem cell reprogramming...clones. Retroviruses are not considered safe for clinical applications.     Lentiviral: Lentiviral vectors...lentiviral delivery is not considered safe for clinical applications. Excisable Cre-lox lentiviral vectors...diluted faster when cells are actively dividing. The typical drawbacks of using transfection will still exist...DNA-silencing mechanisms. While minicircles are typically smaller than traditional plasmids, Jia et al generated...determine how stem cell-like they behave. iPSCs are typically tested with molecular-based assays first. These...molecular hallmarks of reprogramming, then they are typically assessed with more rigorous functional tests. ...

...regular part of basic biological research as well as clinical therapy. These biological tools are useful because...  When viruses were first being engineered as biological tools, thought was given on how to keep the replication...encoded in the transfer plasmid, the component that physically integrates into the host genome, the virus is...caution as they are integrating viruses – they physically deposit their cargo into the host’s DNA. These...should be thoroughly decontaminated after use, typically with bleach (plastic) or ethanol (other surfaces...Virus Many virus experiments involve needles or scalpels (sharps). Cuts and scrapes from sharp equipment...specific organization/department/lab’s policies – basically, you’ll need to get trained in each new place ...

...relies on “unmixing.” Unmixing uses a complex mathematical algorithm and single stained reference controls...spectral flow experiment and panel, there are a few critical factors to keep in mind in order to get the best...extraction Cells produce a low level of fluorescence called autofluorescence (you can see this in your unstained...controls). Spectral flow cytometers include a tool called “autofluorescence extraction” which allows the ...autofluorescence across various tissue types varies, it is critical to include an unstained sample for each tissue...detected. Cytek’s website has a convenient tool, called the similarity index, which allows the user to ...and a value of “1” indicating those markers are identical and completely indistinguishable. This tool is...

...methods. Since molecular, ecological, and reproductive confinement typically require very little effort...Development Fellow at the Wyss Institute and Harvard Medical School. Scientists making transgenic organisms ...the entire wild population with unpredictable ecological effects. Because the consequences of such a mistake... to implement and have been tested in yeast5. Ecological confinement involves performing experiments in...experiments attempting to build gene drives in tropical mosquito vectors of diseases such as malaria and...the laboratory. It varies by organism, but your local biosafety officer should be familiar with appropriate...they should not be relied on exclusively because historical studies of pathogen research have conclusively...

...where you assume your technical skills define you and that the other not-so-technical skills you’ve built...industries and work environments. This is why they are called transferable skills. Why are transferable skills... individual with more going for you than your technical expertise in a specific field. While searching...under your belt from your last job. You knock the technical portion of the interview out of the park. It’s...hiring team will also want to examine not just technical skills, but also transferable skills, before offering... make hiring decisions. Though having all the technical skills certainly matters in the job market, it...you possessed. Think about your shining moments. Recall a few times you really felt great about your work...

...library! Now also available as a lentiviral prep. Calabrese: human CRISPRa sgRNA library In addition to inhibiting...lowly expressed genes or genes that are more drastically affected by gene activation rather than depletion... A and B. Sanson et al. demonstrated that the Calabrese library substantially identified more hits than...al., 2015). Root and Doench also compared the Calabrese library to screens performed with open reading...for resistance to MEK inhibition. Although both Calabrese and the ORF overexpression library identified ...in complement to study gene function. Find the Calabrese pooled library and lentiviral prep here! Applications...PMC4744125.  3. Gilbert, Luke A., et al. "Genome-scale CRISPR-mediated control of gene repression and activation...

...club occurs with a small technical team (single lab or team), a deep technical dive on single papers could...number of participants and the standard academic calendar. So you want to start a journal club? Here are...topic, there are a number of administrative and practical decisions to make. How often will you hold it?... the time recurrently carved out. This notice typically should include the paper, any supplemental data...repositories, or other information needed by someone to critically discuss the paper.  Before the first journal ...primary purpose is training graduate students in critical reading. If the aim of the journal club is to ...Addgene, we opted to start with one per month. When to call it a day The most ineffective type of meeting is...

...used to calculate the concentration of the sample. This concentration can then be used to calculate the viral...viable lactic acid bacteria in faecal samples." Journal of microbiological methods 148 (2018): 64-73. PubMed... we have used quantitative PCR to evaluate the physical titer of our preps. We often repeat this assay...factor of 2. This means that if you set up two identical assays with the same sample, you could end up ...would be valid. This is why the AAV reference is critical. It allows you to determine if your titers are...adding your sample to a mastermix. At Addgene, we typically load 3 dilutions onto our ddPCR plate. As the ...Maintaining a high droplet count is important for calculating the concentration of your sample. Even if you...

... but this research is still in the pre-clinical/early clinical stages. Researchers are also working to... applications, and they’ve also had success in clinical gene therapy applications. Read on to learn more...gene therapy. In 2003, VIRxSYS began the first clinical trial with a lentiviral vector. CD4+ T-cells from...load and did not cause severe adverse reactions. Clinical benefits of lentiviral vectors have been observed...genome, they could promote oncogenesis by altering local gene expression. Self-inactivating (SIN) lentiviral...Patient monitoring and follow-up in lentiviral clinical trials. McGarrity GJ, Hoyah G, Winemiller A, Andre...gene therapy of human β-thalassaemia. Cavazzana-Calvo M, Payen E, Negre O, Wang G, Hehir K, Fusil F, Down...

...through mutagenesis in order to improve its physical and biochemical properties. In 1995, Roger Y. Tsien described...scientists to begin to study GFP and deduce its biochemical properties. Now GFP and its fluorescent derivatives...GFP is used in research across a vast array of biological disciplines and scientists employ GFP for a wide...tagging genes for elucidating their expression or localization profiles, acting as a biosensor or cell marker...living cells, enabling the study of dynamic, physiological processes. Re-engineering GFP to increase its...protein that undergoes conformational change. Typically, two fluorescent proteins with overlapping excitation...pH, using a range of strategies such as FRET, calmodulin, and others. Review Addgene's collection of fluorescent...

...analyzing your cells using flow cytometry, you are typically measuring the presence or absence of certain markers...free amines on and inside the cells and are thus typically used for distinguishing live from dead cells. ...atoms to a higher energy state. This process is called excitation. After a short period, the electrons...state, whereby a photon is emitted. This step is called emission. During excitation, some of the photon's... compared to APC. This happens due to a process called Fluorescence Resonance Energy Transfer (FRET). ...PE-CF594PE-Cy5PE-Cy7       Red (640 nm)   APCAlexa Fluor 647   Alexa Fluor 700APC-H7APC-Cy7 ...wavelength of 488 nm (Table 1), which is near a local maximum of PE's excitation spectrum (dotted line...

...Epitopes are between 5-8 amino acids long, with a typical length of 5 or 6 amino acids (Cruse et al., 2004... just as specific for their target antigen as canonical antibodies. They also have the advantage of being...recognize epitopes that full antibodies would not physically be able to reach. While a full antibody is ~150...of light that can pass through each well - the optical density (OD) - of the known and experimental wells...concentration of the known samples is used to calculate the concentration of protein in the experimental...surface of or inside whole cells. Antibodies - typically, primary antibodies that are conjugated to a fluorophore...the presence of specific proteins, but their localization within the cell or tissue as well. Similar to...

... a more-typical scale of tissue culture while still offering the potential for true biological insight...which to test the validity of your biological model and the technical performance of your assay. But, in...components, the model system and the assay are just as critical for success. Sometimes, you might have a few positive...involved. Or you don’t know how well your assay will scale. Or you don’t have enough cells to execute genome-wide...facilitating more rapid follow-up. We hope the biological community will find these reagents to be useful...

... prep library. Addgene’s pooled libraries are typically DNA preps, but we do distribute several of our...larger lentiviral preps can be used for screening-scale transduction of cells, resulting in cell population...The size and composition of your library can drastically impact what conditions it needs to properly amplify...can transduce your cells.  While Addgene uses a physical titer (via ddPCR) to quality control our viral...include a 1.3 mL aliquot with mostlibrary requests specifically to allow scientists to perform functional titering....  Resources and references References Genome-scale CRISPR-Cas9 knockout and transcriptional activation... useful place to ask experimental questions.  Practical Considerations for Using Pooled Lentiviral CRISPR...

...position at Addgene, the skeptical applicant asked if she would partake in “critical thinking” in the position... away from calling non-academic careers and desk jobs “alternative careers.” Realistically, based on the...it. The opportunity to be creative and think critically. One of the very first times I interviewed a ...wish for a stretch of time that doesn’t involve critical thinking. Former Addgenie Jessica Welch says, ...aspect of their chosen career. You can confidently call yourself a Scientist. You work in a lab. You formulate...transferable skills. Transferable skills are the non-technical skills you pick up wherever you work, and you’... communicating. After years of focusing on my technical skills with cloning and lentivirus production,...

... that could bind very specifically to target molecules. Using a technique called systematic evolution ... high specificity and affinity, were eventually called aptamers, from the Latin aptus, meaning “to fit...essentially mimicking GFP.  Though the aptamer, called Spinach, required longer exposure time than its...group selected for a 49-nucleotide aptamer they called Broccoli. As a product of SELEX and FACS, Broccoli..., Akhtar and Marty, Jean L. “Aptamer based electrochemical sensors for emerging environmental pollutants...PMCID: PMC4923196. 8. Sassolas, Audrey, et al. “Optical detection systems using immobilized aptamers.” ...9. Song, Kyung-Mi, et al. “Aptamers and Their Biological Applications.” Sensors (Basel). 2012; 12(1): ...

...institutions set their postdoctoral salary scales to the NRSA scale (Figure 21, National Postdoctoral Association...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...requirements of the FLSA. In the University of California system, the postdoc union has negotiated a raise...likely the number of postdoc positions in the biomedical enterprise in the U.S. will decrease. At Future...has clearly not been a sustainable approach to biomedical research in the U.S. and postdoctoral training...academic system is increasing, and becoming ever more critical. In 2016 not only were postdocs not exempted from...

...preference.   Historically, ICSS was done by implanting an electrode and non-specifically electrically stimulating...the steps and technical considerations are directly applicable to the rat [6]. Technical differences include...[2]. Laser activation is usually controlled automatically using motion capture software such as EthoVision..., regardless of the type of behavior, certain technical considerations still apply. Testing stimulation...is wrong or 2) there is some problem with the technical implementation of your optogenetics setup (viral.... 8.Han, Xue, et al. "A high-light sensitivity optical neural silencer: development and application to...

...signals from these reporters. Lack anatomical context/spatial localization: In vivo imaging with fluorescent... be overlaid onto anatomical ultrasound imaging to show where the bacteria localize in relation to internal...ultrasound fingerprint can be detected. Bourdeau et al calls this method acoustic collapse.  Importantly, acoustic...exciting application for ARG’s: in vivo imaging. Specifically, arg1 expressing E. coli aided in ultrasound...intestinal tract and has been used for 100 years to clinically treat enteric infection and inflammatory bowel..., but lux imaging shows only that the bacteria localize to somewhere in the mouse abdomen. arg1 signal...

...covered in detail here. Classical double-strand break repair (DSBR) In the classical DSBR pathway, the 3’...and Homology Directed Repair (HDR). NHEJ is the canonical homology-independent pathway as it involves the... provided the DNA template used for repair is identical to the original DNA sequence at the DSB, or it...plasmid, etc.), and consists of three pathways: classical double-strand break repair (DSBR), synthesis-dependent...purple arrows) or on the non-crossing strand (vertically at the orange arrows). If resolved dissimilarly...applicable in DSB-induced genome engineering, it has biological importance for the repair of breaks that lack...2281-308; (2013). PubMed. A Mouse Geneticist's practical guide to CRISPR applications. Singh P, et al. ...

...2012) scFv Antibody: Principles and Clinical Application. Clinical and Developmental Immunology 2012:1...being smaller than antibodies. scFvs are often genetically fused to other proteins, which has led to their...with 10-24 copies of the short epitope GCN4, also called a scaffold; and 2) a GCN4 binding scFv that’s fused...is easier and cheaper to generate since it’s genetically encoded.   Translational applications of scFvs...antibodies, there are many scFv-based therapies in clinical trials or already in the clinic (Bates and Power... tumour specific antigen. When these molecules, called Bispecific T-cell engagers (BiTE®s), bind CD3 on...David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody Fragments. Antibodies 8:...

...promoters and Shine-Delgarno sequences used in this so-called bicistronic design (BCD). The 49 vectors in this...: Čelešnik, Helena, et al. "Biosafety of biotechnologically important microalgae: intrinsic suicide switch... Rogers, Jameson K., and George M. Church. "Genetically encoded sensors enable real-time observation ... Ribosome-Controlled Fluorescent Reporter."ACS chemical biology 11.1 (2015): 31-37. pCri system The Gomis-Ruth...latency in matrix metalloproteinases." Journal of Biological Chemistry 290.8 (2015): 4728-4740. You can find...The gene set was carefully chosen to perform statistically relevant benchmarking of cell-free expression...Marx's lab has engineered and validated a new method called FREQ-Seq. This strategy allows scientists to construct...

..., soon directed himself to finding “immunological and virological solutions to crises like those and ended...Therapeutics, which was acquired by Ultragenyx Pharmaceutical in 2017. As one of the original employees at...learned more about Addgene. “The mission to share critical reagents such as plasmids and viral stocks for...depositor. KSQ uses a CRISPR-Cas9 platform to systematically test the function of all 20,000 genes in disease...Target Discovery at the Novartis Institutes for Biomedical Research. Drug development was something Stegmeier...genetics.” As a postdoc in Stephen Elledge at Harvard Medical School, he developed the first lentiviral microRNA-based...Associate Professor at Mass Eye and Ear and Harvard Medical School (HMS) where he leads a lab of 25 people....

...University of California, Irvine University of Massachusetts, Worcester  University of Nebraska Medical Center...University and Medical Center  Weizmann Institute of Science Whitehead Institute for Biomedical Research ...time of request MTAs and deposit agreements to basically instantaneous in many cases.  Suggest that they...your organization below? Don’t worry--we check periodically for possible new awardees, and we’re happy to...Hospital  Brandeis University  Broad Institute  California Institute of Technology Cold Spring Harbor Laboratory...University Rockefeller University  Salk Institute for Biological Studies The Scripps Research Institute St. Jude...

...Python script called SSFinder, but I find its implementation to be too slow for practical use. The R based...causes malaria). I attribute their success to technical expertise, thoughtful single guide RNA (sgRNA)...the piñata builders - the reviewers. After some physical exertion on the part of the party goers, the piñata...the other hand some software tools make use of empirically derived and/or prior knowledge to inform on the... Hsu et al., 2013) used a combination of both empirical and approximate methods. The details of their ...“high mismatch limit”, “approximate design”, “empirical design”, “single-PAM design”, and “multi-PAM design...sites, scores and the statistics that go into calculating them. For this, some online tools such as Cas-OFFinder...

...Talley Lambert, a Research Associate at Harvard Medical School. The need for a community fluorescent protein...one of the most indispensable tools in modern biological research.  Any microscopist will tell you that...maturation, oligomerization), many of which are dramatically affected by environmental conditions (such as... structure was needed.  It is also difficult to scale such a resource when updates depend on a single ...feature filter spectra and collection efficiency calculations.  With ~80 FP spectra (and rapidly growing, ...Talley Lambert is a Research Associate at Harvard Medical School. He manages the Cell Biology Microscopy ...Microscopy Facility, specializing in advanced optical microscopy. Follow him on twitter or github @tlambert03. ...

...form of many, many short DNA sequences called reads. Typically they’ll be in a file in FASTQ format, a...DNA is needed for screening experiments, so the scale of the DNA prep is larger. And since the library...all components of the library are represented. Typically, Addgene uses electrocompetent Stbl4 cells when...ensure a good transformation efficiency. For a typical library, Addgene usually does four to eight electroporation...product because the plasmids in the library are identical except for the insert; there’s no need to sequence...). You can also rank the plasmids by abundance, scaled to 1, to create a “cumulative wealth distribution...distribution” curve, or Lorenz curve. By calculating the area under the curve (AUC), one can gain a sense of the...

...scene. But what about the other yeasts – in the biomedical world, are there even other yeasts? In this blog...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...the expression of recombinant proteins by the pharmaceutical and biotech industries as a protein expression...modification of recombinant protein products, specifically glycosylation. The native glycosylation machinery...human: The important gene co‑expression modules. Biomedical Reports 7.2 (2017): 153-158. DOI: 10.3892/br.2017.941...

...lentivirus and have been difficult to manipulate genetically. To solve this issue, Sarah Teichmann and colleagues...triglyceride or cholesterol contents (what is commonly called bad cholesterol). However, size distribution and...including quantification of abundance, size, and localization in vivo. The LipoGlo system represents a new... (bEBPs) at a sequence upstream of the promoter called the upstream activating sequence (UAS). The Wang...Communications, 2019. PubMed PMID: 31451697. Genetically encoded voltage indicators for use in worms Article... Article contributed by Brook Pyhtila Genetically encoded voltage indicators (GEVIs) change fluorescence...real time when neurons or muscle cells transmit electrical information. Such voltage imaging can be performed...

...Arabidopsis (CIBN), which they call the LADL Anchor. Using specifically designed gRNAs, they can then ...Inducible Degradation (AID) uses plant hormones called auxins to conditionally and reversibly degrade ... for AID. By tagging proteins with a 7kD degron called mAID and expressing the essential plant protein...microorganisms based on metagenomic data. MerMAIDs (Metagenomically discovered Marine, Anion-conducting and Intensely...publicly available metagenomic datasets with biophysical studies and the potential of mining datasets ...transcription factors tethered to dCas9, require long time scales with constitutively expressed proteins that are...nature of genomic looping due to it’s briefer time scale and its inducible and reversible nature. Find the...