...pAAV-hSyn-DIO-EGFP   Biosensor AAV Calcium sensors Tracking calcium dynamics that govern such activities... from Christopher Harvey’s lab. These plasmids localize opsins to the soma to avoid off target photostimulation...need to determine these transduction patterns empirically for their own experiments. To this end, we offer...studying neural networks. We have expanded our calcium sensor collection to include our first Flp-dependent...Several new options are also available for the calcium sensor jGCaMP7 from Janelia, including three jGCaMP7...technology, see our blog post Overview of AAV Encoded Calcium Sensors). Plasmid Serotype Name 104489 AAVrg...

... alumni as you can or schedule video/calls if they are not local. Alumni meetings can be the most informative...alumni, don’t ever disregard warning signs of unethical behavior, harassment or bullying. Even with the...innuendo and sarcasm, persistent teasing, name calling, insults, intimidation, sexual harassment Isolation... including preventing access to opportunities, physical or social isolation, withholding necessary information...administrative responsibilities? Is the work (topic and/or technical aspects) applicable to industry if this path interests...Questions to ask: Will someone(s) train you technically? Will the PI/supervisor look at your data and... if you take the time to evaluate the options critically. How will it feel when you find it? If you find...

...bacteria) may be the most numerous and most diverse biological entities on our planet, but we still know collectively... refractory to enzymatic digestion, however, a physical fragmentation strategy is preferable. Nebulization...tank with a pressure regulator. Other methods for physical shearing exist as well, such as sonication and...transform your resultant plasmid libraries into chemically competent E. coli and analyze transformants for...reduce the number of cells that will bind non-specifically to either the membrane itself or to any “sticky... antibody or other protein on hand that will specifically bind your target organism, it can be helpful...2015): 185-191. PubMed PMID: 25708933. 2. Cooper, Callum J., Mohammadali Khan Mirzaei, and Anders S. Nilsson...

...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...proteins outside of a cell - cell free extracts Typically, if you want to make a protein – say green fluorescent...incorporated into a host cell (through a process called transformation), essentially giving the cell all...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...Ally Huang is a 4th year PhD student at MIT in Biological Engineering and is interested in developing synthetic...

...taraxacifolia (Willd), an annual herb of Western Tropical Africa typically known as wild lettuce. Aside from their...researchers in resource-limited settings. More specifically, the Addgene-Seeding Labs plasmid grant provide...ameliorate diabetics nephropathy, one of the major pathophysiological complications of diabetes mellitus. Recombinant...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...working in a wide range of research fields and geographical locations. In addition to providing reagents...

...physiology. Ribosome profiling Ribosome profiling, a biochemical method developed in 2009, takes a “snapshot” ...Recent work has adapted this technique to monitor local translation and determine if proteins are targeted...translation While ribosome profiling co-opts the physical location of the ribosome, TRICK (Translating RNA... the reporter. Because it contains a nuclear localization signal, NLS-PCP-GFP then returns to the nucleus...recruit scFV-GFP, generating green puncta that colocalize with the red reporter mRNA. Yan et al. used harringtonine...inhibitor that stalls ribosomes after initiation, to calculate a ribosome translocation rate of ~3.5 codons/second...studying translation in various cell types and physiological states. References 1. Ingolia, Nicholas T.,...

...agreement on the transfer of biological materials in 1995: the UBMTA. Because biological materials are widely ...many different flavors of MTAs, but the Uniform Biological Material Transfer Agreement (UBMTA) is one of...process was time-consuming and lengthy. So the NIH called a meeting in 1990 to discuss the process, which...MTA for that plasmid facilitated by Addgene — basically, instead of having to sign individual MTAs for...agreements allow for centralized tracking when biological reagents are shared — both at what time and with... options for the terms you want to share your biological materials under! While most plasmids at Addgene...

... like writing. It’s basically the equivalent of crossing off days on the calendar after completing your... Writing schedule tools How to write a lot: A practical guide by Paula J. Silvia  This book has tips for...take a 5-minute break. Each 30-minute interval is called a “Pomodoro.” After four Pomodoros, you can take...suggestions Hemmngway makes (it wasn’t designed for technical writing), it’s great at flagging passive voice...jargon, this tool can help you avoid overusing technical terms when writing for a general audience. The...goal of the phrase, such as being cautious, being critical, or defining terms. The phrases in Phrasebank ...

...neuronal membrane trafficking and localization. Improved photophysical properties The improved variants...iGluSnFR3 variants that are more sensitive and better localized for synaptic glutamate response than its predecessors...researchers did the following: Improve sensor’s photophysical properties such as kinetics, ∆F/F0, SNR, dynamic...Supplementary Figure 2.   Improved trafficking and localization through different C-terminal anchoring domains...Blog Learn about other Fluorescent Biosensors A Practical Approach to Choosing the B(right)est Fluorescent...indicators with improved activation kinetics and localiation for imaging synaptic transmission” bioRxiv (2022...

...presented chronologically. With a resume however, most often people choose a reversed chronological style ... job asks for experience in “statistical analysis”, mention “statistical analysis” (word by word) in your... applying for. Also remember that a resume is typically no more than 2 pages long. Write your resume for...handled large data sets and are familiar with statistical analysis. If you have done fluorescent imaging...Some universities even have officers working specifically to assist graduate students in job searching...

...Luciferases are a class of enzymes capable of catalyzing chemical reactions in living organisms resulting in the... is measured resulting from the catalysis of a chemical reaction requiring luciferin, ATP and oxygen as...Fluorescent Protein (GFP) because of the nature of the chemical reaction compared to using a high-intensity laser...Luminescence Fluorescence Source of Emitted Photons Chemical reaction High-energy photons Kinetics of Photon...High-throughput assays Improving Well-established Biological sources of luciferase Although luciferase isolated...with a peak emission at 480nm upon catalyzing a chemical reaction with the substrate coelenterazine and...expressing luciferase Luciferase reporter assay The typical luciferase-containing plasmid is most often used...

...all. Base editing to the rescue! Base editors chemically modify DNA bases without breaking the DNA backbone...elements. They are, broadly: A Cas protein (catalytically dead or nickase) fused to a deaminase that makes...al., 2016). Target-AID acts similarly but not identically to BE3, modifying a 3-5 base window 18 bases ...expressed well. The Liu lab modified the nuclear localization signals and codon usage BE4 to create BE4max...al., 2023; Neugebauer et al., 2023). These CBEs (called Td-CBEs, CBE-Ts, or TadCBEs) are smaller and less...an adenine base editor with improved nuclear localization and expression. This base editor was named ABEmax...Keith Joung’s lab created a dual-deaminase editor called SPACE (synchronous programmable adenine and cytosine...

...systems are available at Addgene! Chemically inducible promoters Chemically regulated promoters are among ...sensitive expression systems are typically less leaky than chemically induced promoters; they show near-zero...transcription is turned ON. Types of inducible promoters Chemical agents, temperature, and light are all examples...decreasing promoter leakiness.   Other examples of chemically induced promoters include positive inducible ... promoters only work in prokaryotic cells and typically only in the same or closely related species from...

...Immunity, as well as various combinations thereof (so called “Immuno-Oncology”) is exploding. Researchers are... in the presentation, starvation or the use of caloric restriction mimetics trigger autophagy and, in ...Binghamton University! Cancer therapeutics The so called field of Immuno-Oncology, a sizzling hot topic,... and correlating them with tumor mutations and clinical responses. This platform has the potential to ...the use of optogenetic tools is expanding to immunological research. Here the Zhou group presented Opto-CRAC...cells that are not excitable but responsive to calcium. We’d love to expand our cancer and immunology ... References: 1. Pietrocola, Federico, et al. "Caloric restriction mimetics enhance anticancer immunosurveillance...

...and are often impractical for small projects. Most researchers instead turn to so-called “mismatch nuclease...involves splitting up a sample into thousands of physically isolated partitions (such as droplets or wells...Since there are many thousands of partitions, statistical modelling can be used to determine the actual..., does not require standard curves, and can theoretically detect a single target molecule in a sample....wild type target in each sample can be quickly calculated (details in [2]) and can be used to quantify ...mutations versus those with the bi-allelic mutations typically desired when screening clones for complete functional...

...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...understanding of plant biology is a significant technical obstacle to plant synthetic biology. We cannot...creative problem solving like the Howard Hughes Medical Institute have begun to pivot towards plant biotechnology...organism for plant research for decades, and the biochemical pathways involved in central anthocyanin metabolism... – our project wouldn’t be possible. A second technical hurdle is the lack of resources associated with...

...mammalian cells often relied on chemical (CaCl2, cationic liposome) or electrical (electric current forming ...research since their first use in the early 90’s to genetically modify primary cells. Amongst the different vectors...its safety was a disadvantage particularly for clinical development. In addition, MoMLV is inefficient...lentiviral vectors was developed for safe use in a clinical setting. In this generation, the HIV Tat gene ...lentiviral vectors can be used for research and for clinical purposes, however improving the safety of these...than with the 2nd generation. Knowing this you can scale up your production set-up and purify and concentrate...virus in order to aid viral binding. This method, called spinoculation, has been found very effective for...

...HE, Lee C-YS, Calidas D, Lu T, Smith J, Schmidt H, Krause MW, Seydoux G (2014) Scalable and Versatile ...is an assistant professor at the University of California, Santa Cruz. Emerging CRISPR/Cas9 editing technologies...melting and rapidly cooling dsDNA repair templates dramatically boosted editing efficiency (Ghanta et al., 2020... more efficient. Pure PCR repair templates are critical for efficient knock-ins (Ghanta et al., 2020)....Farboud et al., 2019). Work from the Mello lab has dramatically improved RNP editing with linear repair templates...is an assistant professor at the University of California, Santa Cruz where his group focuses on gene regulation...Tzur YB, Friedland AE, Nadarajan S, Church GM, Calarco JA, Colaiácovo MP (2013) Heritable Custom Genomic...

...simulate physiological gene expression regimes that cannot be easily achieved with classical gene overexpression... super-resolution microscopy.” Endosomes are historically difficult to study in living cells using conventional...system to study epigenetic modulation. While catalytically inactive cas9 (e.g. dCas9) has been fused to...activate gene expression, sometimes incorporating chemically inducible approaches to provide temporal control...kinetics of chromatin regulation and subsequent mathematical modeling. Its reversibility allows precise and...hER or progesterone for hPR - leads to nuclear localization, binding to the appropriate promoter sequence...

...Therefore, it is not possible to visualize the localization of protein synthesis. Alternatively, fluorescent...graduate studies I attended a scientific symposium called Horizons in Molecular Biology. This annual symposium...PhD students at the Max Planck Institute for Biophysical Chemistry, Göttingen in Germany. I attended this... symposium seeking inspiration to resolve the technical hurtle of monitoring protein turnover of specific...interesting talk by Prof. Erin M Schuman, a former Caltech researcher, now Director at the Max Planck Institute...technique applies metabolic labeling using a noncanonical amino acid, azidohomoalanine (AHA). In the technique...application of AHA labeling is known as fluorescence noncanonical amino acid tagging (FUNCAT, Dieterich et al....

...pathways and how they lead to neurological phenotypes observed.” Using genetically-encoded biosensors and iPSC-derived...Scientific Advisor. It’s possible that solute channels typically expressed on other cell types can compensate for...understand so that we could possibly develop pharmaceutical interventions that would inhibit IL-36 release...oligomerization, signalling complex assembly, and localization to the nucleus,” says Dr. Terry Hébert, a professor...professor and assistant dean of biomedical science education at McGill University.  These protein-receptor... iPSC-derived cortical neurons from patients with GNB1 mutations, the lab hopes to capture the signalosome...

... The previous post in this series described a practical approach to selecting a bright fluorescent protein...amounts of excitation power (Shaner et al, 2008). In confocal laser scanning microscopy, fluorescent proteins...selective plane illumination, TIRF or spinning disk confocal use entirely different excitation power regimes...performed with soluble fluorescent proteins or localized fusion proteins and do not require dedicated plasmids...Amsterdam). He develops, characterizes and uses genetically encoded fluorescent probes. You can follow him...(University of Amsterdam) where she develops genetically encoded FRET based biosensors.     Additional...Fluorescent Tags Find Plasmids for Subcellular Localization Browse the entire Fluorescent Protein Collection...

... the flick of a light switch. Neurons are not typically persuaded to fire when light is shined on them...channelrhodopsins. Their new channelrhodopsins, called ChRgers, make the brain a little easier to access...determine membrane expression and plasma membrane localization of transmembrane proteins, like rhodopsins, ...directed evolution, because channelrhodopsins are typically evaluated with low-throughput methods such as ...properties of a channelrhodopsin, such as cell localization, kinetics, and spectral properties, must be ... midbrain, fiber-optic cannulas were still stereotaxically implanted into the mice’s brains so that light...optogenetics.  TL;DR The new channelrhodopsins, called ChRgers, make it easier and less invasive to use...

...are commercially available. Titer calculation (1 or 10 days) Physical titer (particle count, rAdV genomes...repeated multiple times (2-4 rounds) at increasing scale over the course of 1-2 weeks. Each round of amplification...genomes/mL) can be measured by: Optical density: particle concentration is measured by looking at absorbance...260nm corresponds to 1.1 x 1012 particles/mL. To calculate the particle number: OD260 reading x dilution ... days later. Plaques are counted and titer is calculated in terms of plaque forming units (pfu), taking...1236. PubMed PMID: 17546019. 2. He TC. et al. A practical guide for using the AdEasy System. 3. Jager, Lorenz...

...mass spectrometry (AP-MS), lack the ability to specifically identify proteins that interact with a particular...lysed before the bait protein is pulled down, typically with an antibody. This can artificially select...controllable version of the BioID protocol. The critical component for both BioID and Split-BioID is BirA...functional BirA* enzyme. This type of experiment is called a protein fragmentation complementation assay (... via Western blot, and that result in proper localization of the fusion protein. Advantages of Split-BioID...Further characterization of these candidates is critical to understanding how they interact with the bait...Varnaitė, R. (2016). Meet the neighbors: Mapping local protein interactomes by proximity‐dependent labeling...

...science, dating back several hundred years, with historical ties to psychology and philosophy (the mind-body...chemogenetic tools are designed channels that are chemically activated. Both are introduced in neurons and...modifications at the DNA level in a temporally and locally defined manner. This allows scientists to draw ...and not targeted to a specific area. Some other technical considerations for stereotaxic injections are:...be high enough to maximize possible outcomes. Typically, AAVs have to be minimum around 1012-1013 vg/ml...has to be large enough to target larger areas, typically a few hundred nl. Alternatively, for larger brain...expression levels of the viral vector trangenes typically occur two weeks after injection. Therefore, it...

...embryos. But once you generate a growing colony of genetically engineered mice, what can you do? In this post...Cre-lox lines, and how to properly control for genetically engineered mice in your experiment. As you’ve...ve learned in Part 1, there are many types of genetically engineered mice: transgenic mice, knockin and...getting a small piece of tissue from each mouse (typically via either a blood draw, tail clip, or toe clip...Hemizygous mice can be included in breeding strategies identical to those used for breeding heterozygous mice. ... cases Whether your mouse colony consists of genetically engineered mice with a single mutation or strains...prepared to plan an experimental strategy that uses genetically engineered mice. Best wishes on your experiment...

...visualizing ncRNA localization and function, but directly incorporating very long ncRNAs is impractical. Instead...the full ncRNA locus (Ho et al. 2012). For large-scale screening, sgRNA libraries are useful for targeting...short when it comes to targeting precision and practical application. If the CRISPR/Cas9 system is not ...interact with specific chromatin structures or other local genomic elements (Hazan and Bester 2021). Other ...can cleave single-stranded RNA using gRNAs to specifically edit or degrade targeted lncRNAs. The Cas14 ...15476286.2021.1899500 Engreitz JM, Haines JE, Perez EM, et al. Local regulation of gene expression by lncRNA promoters...

...designed by synthetic biologists to perform large scale, one-tube cloning reactions. In this blog, we’ll... Gate cloning is MoClo. That’s because MoClo specifically refers to the organizational schema described...organization schema MoClo can be broken down into three hierarchical levels: individual DNA parts, transcriptional...together. They can express one or more gene(s), and typically begin with a promoter and end with a terminator...sounds really neat — but when is this type of large-scale assembly useful, you may ask? You might be surprised...microbially or in cell-free systems. These pathways typically involve multiple steps in which a precursor molecule...and subsequent necessary modification of these chemical products. The flexible nature of MoClo allows ...

...generated in vitro using synthetic genes that are typically expressed from a plasmid or from an integrated...share, making recombinant antibodies the most practical choice. Hybridomas are much tougher to ship, often... often lack sequence data, and may genetically drift over time. Antibodies made from animals are limited...the ease with which scientists can now improve genetically-encoded research tools, it is shocking that the...Begley CG, Ellis LM (2012) Raise standards for preclinical cancer research. Nature 483:531–533 . https:/...Crosnier C, Staudt N, Wright GJ (2010) A rapid and scalable method for selecting recombinant mouse monoclonal...for the Selection of Monoclonal Antibodies for Clinical Use. In: Antibody Engineering. InTech Additional...

...likely that the shifts caused to the practical and philosophical landscape of the scientific enterprise...precipitated increases to the NIH recommended pay scale – used by many institutions to set postdoc wages...focus toward advocacy and action with sessions specifically chosen to better define productive routes for...Riglar. David T Riglar is a postdoc at Harvard Medical School. In the lab, he uses synthetic biology to...References: 1. Alberts, Bruce, et al. "Rescuing US biomedical research from its systemic flaws." Proceedings...Pubmed Central PMCID: PMC4304227. 4. Revitalizing biomedical research: recommendations from the Future of ...

...What’s behind these sights are an abundance of biological molecules like chromoproteins, fluorescent proteins...proteins and luminescence have been widely used in biological research for a long time, chromoproteins are ...just now being developed for a broad range of biological inquiries.  What is a chromoprotein and how ...photosynthetic activity. This still allows photosynthetically active wavelengths to reach zooxanthellae...the Sea AnemoneCnidopus Japonicus. Journal of Biological Chemistry 281:37813–37819 . https://doi.org/10.1074...Pigments of Pocilloporid and Acroporid Corals. The Biological Bulletin 189:288–297 . https://doi.org/10.2307...chromoproteins for bacterial synthetic biology. Journal of Biological Engineering, 12(1). https://doi.org/10.1186/s13036...

...reverse-transcribed piece of DNA called a “provirus.” This DNA is synthesized inside of what is called the “target cell...The Retroviridae (commonly called retrovirus) family — of which HIV is a member — may seem like an unlikely...retroviral vectors and 2.) a subclass of retroviruses called the lentiviruses (like HIV-1) — that have been ...(Prussin et al., 2015), only a small fraction typically cause disease. The gamma-retroviruses have their...transduction by vectors (which we sometimes still call “infection” as a shorthand, although it is not quite... proteins encoded by complex retroviruses. Microbiological Reviews, 56(3), 375–394. https://doi.org/10.1128...

...kind of biological framework, suggested to them that their findings may be the result of a technical artifact...Biomedical research has a long and well-documented history of racial bias, which still impacts and influences..., by extension, race. Many researchers do large-scale CRISPR screens in cancer cell lines, looking to ... Aaron knew that their study had significant statistical limitations, and expected to find only a few,...Seeking an explanation  First, they looked for a biological explanation, a relationship between the genetic...far larger and more complex problem endemic to biomedical research: pervasive bias that is easy for researchers...

...candidate in the Sternberg Lab at Columbia University Medical Center. DNA transposons are ubiquitous genetic ...a multi-spacer CRISPR array. Workflow and technical considerations Making insertions in bacterial ...into cells and plate overnight Screen colonies phenotypically or by PCR/sequencing While CRISPR-transposons...few important details. For transposons to be specifically recognized during transposition, genetic cargos...Additionally, these transposon also exhibit something called target immunity, whereby multiple proximal insertions...Leveraging a unique marriage between distinct biological elements, CRISPR-transposons excel at programmable...candidate in the Sternberg Lab at Columbia University Medical Center. He currently studies CRISPR biology and...

...purification (TAP) once referred specifically to a combo tag comprised of a calmodulin binding peptide (CBP), a...to) purification, detection, solubilization, localization, or protease protection. Thus far Plasmids 101...overexpress and purify proteins can face any number of technical challenges depending on their protein of interest...consider junk protein. Though there are a number of chemical and peptide-based proteosome inhibitors, glutathione... relatively small sequence of amino acids, is basically a molecular leash for your protein. If you’re ...want to combine a fluorophore with a tag that localizes your protein to the nucleus? Or you want multiple...

...Harvard Medical School, and is currently a fellow in clinical genetics at the University of California, Los...Vincent and Niroshi Senaratne, from the Biological and Biomedical Sciences program at Harvard University...the Program in Genetics and Genomics at Harvard Medical School. He studies developmental gene regulation...

...results (i.e. mistakenly calling a gene a hit when really it’s not). In practical terms, this means that... approach for identifying genes involved in a biological process. These screens provide an alternative...cutting than full-length guides (19-20bp)?  Small-scale studies (Tsai et al., Fu et al.) suggest 17-19 bp...non-targeting guides on screen analysis and hit calling. 5,644 non-targeting guides and 6,750 safe-targeting...ricin screen. When safe-targeting guides are used to call hits from the growth screen, p-values were less ...References 1. Morgens, David W., et al. "Genome-scale measurement of off-target activity using Cas9 toxicity...

...Emily Bentley Building on their CROPseq method for optical pooled CRISPR screening, Paul Blainey’s lab has...multiplexing approach to Cas9-based pooled CRISPR screens called CROPseq-multi (Walton et al. 2024). This platform...compatible with enrichment, single-cell sequencing, and optical pooled screens in human cells. Addgene offers vectors...one of the virus’s most abundant proteins and a critical component for viral genome packaging. Addgene ...Genetic modification of microglial immune cells is critical for central nervous system research; however, ...cells.      Figure 5: A) Representative confocal images showing GFP expression from ssAAV5s with...gene of interest, reducing the immunogenicity typically associated with viral vector-based therapies. ...

...Harvard Medical School, and is currently a fellow in clinical genetics at the University of California, Los...Niroshi Senaratne and Ben Vincent from the Biological and Biomedical Sciences program at Harvard University...the Program in Genetics and Genomics at Harvard Medical School. He studies developmental gene regulation...