...difficult to culture outside of their natural environment. Thus, the Harris Wang lab at Columbia University...gut microbiome members in their native habitat (Ronda et al., 2019). They called their method Metagenomic...19465049. PubMed Central PMCID: PMC2752045. Myhrvold, Cameron, et al. "A distributed cell division counter reveals...PMID: 26615910. PubMed Central PMCID: PMC4674677. Ronda, Carlotta, et al. "Metagenomic engineering of the...

...autophagy. Morphological assays Electron microscopy: Electron microscopy is a traditional method for...at various stages of degradation. Additionally, electron microscopy analysis is time consuming. GFP-tagged...will fluoresce green unless it’s in an acidic environment like the vacuole or lysosome. See Table 2 for...

...capable of producing a visible signal, but it has a strong affinity for avidin and streptavidin, both of which...modifying the antibody sequence or that rely on strong protein-protein interactions with other peptides...of non-canonical amino acids or peptide tags. 5) Strong protein-protein interactions with other peptides... Ashley Waldron...

...effective at editing cytosines preceded by guanine (Thuronyi et al., 2019). Following the development of adenine...created a dual-deaminase editor called SPACE (synchronous programmable adenine and cytosine editor) by ...high product purity and low off-target editing Thuronyi et al., 2019 evoAPOBEC1-BE4max   Improved editing... K. T., Eton, E., Lapinaite, A., Newby, G. A., Thuronyi, B. W., Wilson, C., Koblan, L. W., Zeng, J., Bauer...891. https://doi.org/10.1038/s41587-020-0453-z Thuronyi, B. W., Koblan, L. W., Levy, J. M., Yeh, W.-H....

...and Memory Formation. Neuron 79, 6 P1086-1093. https://doi.org/10.1016/j.neuron.2013.08.032.  Morita S...enzymatic fusions, such as dCas9-TET, can, like the iron ball-and-chain that they resemble, actually impede...unmethylated CpG to behave naturally in the nuclear environment. One good system to balance these needs is the...

...in green monkeys and cultured human neurons, with broader neuronal expression than AAV9. This new capsid...-brain barrier and delivering a transgene, with neuron bias observed in young macaques and vascular bias...Recombinant anti-FLAG tag antibodies By Ashley Waldron Of all the epitope tags we know and love, the FLAG...

.... Pulse generation (TTL pulse) is a standard electronics protocol not specific to lasers or optogenetics... Use of channelrhodopsin for activation of CNS neurons. Current protocols in neuroscience / editorial ... Kim TI, et al. Injectable, cellular-scale optoelectronics with applications for wireless optogenetics... et al. A simple head-mountable LED device for chronic stimulation of optogenetic molecules in freely ...

... relaxed phone call than when first confronted on a stage in front of a crowd. The moderator will then...logistics To make a really lively discussion, I strongly suggest asking the panelists not to use any slides... turnout). This gets people to sit close to the front so the interactions are better. Have a good microphone...

...mitochondrial fission/fusion, nuclear import, and neuronal trafficking. Although FPs have enabled discovery...tagging endogenous proteins with an auxin-inducible degron (AID) tag to generate proteins which can be rapidly...Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors.” Cell Rep.15... Michelle Cronin...

...many researchers remain unaware that EGFP/GFP is prone to forming noncovalent dimers. This property of ... matter of concentration Most FPs in nature are prone to dimerization (i.e. EGFP) [1, 2] or even form ... fused to the POI. These structures are large (microns), bright, non-physiological, and difficult to ignore...CFP and YFP dimers can form in the absence of environmental changes that the biosensor is designed to detect...

...species, such as the human gut bacteria B. thetaiotaomicron, Mycobacterium, Pseudomonas, and E. coli. ...CRISPRi in many types of bacteria: lab-adapted, environmental isolated, and disease-associated. Mobile-CRISPRi...non-model bacteria including pathogenic strains and environmental isolates. For example, Peters and colleagues... remained constant, with fitness effects more pronounced at 12 doublings than at 6. Key takeaways of Mobile-CRISPRi...

... to combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus may acquire mutations...CRISPR/Cas9 constructs. zCas9i is a high efficiency intron-optimized SpCas9-coding gene. SlugCas9 is a compact...selectively in Parvalbumin containing cortical interneurons. We now provide ready-to-use AAV vectors expressing...this promoter. Image calcium transients in PV interneurons with AAV-S5E2-GCaMP6f are now available as a...

...change their excitation/emission behavior upon environmental changes (e.g. pH, Ca2+ flux, etc). The most ...Oligomerization: The first generations of FPs were prone to oligomerize. This may affect the biological function... these FPs cannot be used in oxygen deprived environment. Recently, a new GFP isolated from the Unagi ...you are planning to express the FP in acidic environments (e.g. yeast cytosol, which is slightly acidic...

...DNA dot blot...Transduction assay...SDS-PAGE...Electron microscopy… Any guesses? That's right... ways...genome-containing viral capsids is typically quantified by electron microscopy of a viral vector solution. Since this... this technique is intensive and requires an electron microscope, it is not routinely performed on all...genome-containing (Figure 1).    Figure 1: Electron micrograph of an Addgene AAV viral vector preparation...

...pluripotent stem cells (Chavez et al., 2015) into neuronal cells by activating genes that cause differentiation...CRISPR-based activation can be made inducible upon environmental triggers. This is done by creating a split dCas9...CRISPR activation are also reversible: once the environmental trigger leaves, gene expression is no longer...Dow D, Buser-Doepner C, Bertotti A, Trusolino L, Stronach EA, Saez-Rodriguez J, Yusa K, Garnett MJ (2019...

...experimental systems. They demonstrated LOV-Turbo in neurons, in pulse-chase experiments, and more. Find LOV-Turbo...element (TRE) or the cFos promoter. For example, neurons treated with a phorbol ester activator gave fluorescent...Images of iPAK4 fibers in cultured rat hippocampal neurons. HaloTag dyes are switched from yellow to red at... Teaching an old antibody new tricks By Ashley Waldron While IHC protocols typically call for thin sections...

...Boyden (Addgene viral prep # 84445-AAVrg).   Electron Microscopy The ratio of empty to full (i.e., genome-containing...representative vector preparations was quantified with electron microscopy after negative staining. Empty vector...than full vector particles.     Figure 3: Electron micrograph of AAV vector preparation shows that...psPAX2 and pMD2.G packaging system developed by the Trono lab. Cell culture medium (OptiPro SFM) containing...

...refresher if you ever lose track of which acronym is which. Acronym Full name Notes   Base transition ...close at hand? Once again, this post has a lot of acronyms, so we've included a glossary at the end of this... for saturation mutagenesis, and they provide a strong proof of concept for further development. ...

...contains iterons that serve as recognition sites for replication initiation proteins. These iterons vary ...plasmid with multiple oris that vary in DnaA-box and iteron structure, host-independent   Building broad...Development of constitutive and IPTG-inducible integron promoter-based expression systems for Escherichia...

...either DTT or β-mercaptoethanol (BME). DTT is a stronger reducing agent than BME and has a less… distinctive...life when stored properly. Because BME is not as strong as DTT, you’ll have to use different concentrations...quantifying your blot. CCD cameras have a much higher upfront cost, but require no consumables, produce digital...like Coomassie Blue or Ponceau S. It is far less prone to error than normalizing to so-called housekeeping...happy blotting! Many thanks to Addgenies Ashley Waldron, Meghan Rego, and Amrita Rhodes for their help ...

...reports 5 (2015). BIOFAB plasmid set 3000 Series Confronted with the problem of creating vectors for predictable...Shine-Delgarno sequences used in this so-called bicistronic design (BCD). The 49 vectors in this kit can ...of varying strengths centered within GFP/RFP bicistronic reporters characterized in Cambray et al., 2013...terminators can be used to precisely tune your polycistronic E. coli expression constructs. Some applications...

... methods.   Rapid detection of 2019 novel coronavirus SARS-CoV-2 using a CRISPR based DETECTR assay...in the saliva (Lucia et al., 2020). Detecting coronavirus using CRISPR-based SHERLOCK (Specific ​H​igh ​...ultrasensitive and visual detection of novel coronavirus SARS-CoV-2 Sensitivity: 4.6-11 copies/microliter...2020). An ultrasensitive, rapid, and portable coronavirus SARS-CoV-2 sequence detection method based on...

...aggregation in your prep. If your proteins are the wrong size on your gel or blot, Table 1 (at the end of...and keep an eye on it and the leading line (dye front) visible in the sample buffer. You’ll want to run...interest, but you don’t want to run so long your dye front or ladder runs off the gel. Luckily, this is easily...easily monitored in real time, as the dye front (and many ladders!) are visible to the naked eye.   If ... Troubleshooting Suggestion Proteins are the wrong size Proteins may be aggregating Lyse at lower...

...to reduce false positives within a test.   The Bonferroni Correction and the Benjamini-Hochberg procedure...control for large numbers of comparisons. The Bonferroni does this by establishing a significance threshold...However, if many comparisons need to be performed, Bonferroni and Benjamini will greatly reduce family-wise...Dependent Variables: A Monte Carlo Simulation Study. Front. Appl. Math. Stat. Volume 2 (2016). doi.org/10.3389...

...many scientists operate in a hyper-competitive environment where getting hired hinges on performing near...government scientists to the de-funding of crucial environmental research programs, Canada has been here before...researcher in the University of Michigan Department of Astronomy. She studies how planets form around young stars...community of science supporters is much larger and stronger that I expected. Jennifer Stancill, Ph.D., is ...

... Studying cell mobility within these complex environments has never been easy and, for many years, researchers...same laser) cooperate to cause a higher-energy electronic transition in a fluorescent molecule (see figure...adaptive and innate immunity in diverse tissue environments. These studies provide quantitative measurements...-parasite interactions in the brains of mice chronically infected with Toxoplasma gondii." The Journal...

... (3): An optics system A fluidics system An electronics system  Cells are first resuspended in a pressurized...fluorophore or fluorochrome-stained protein will emit a stronger photon than a cell expressing low levels of the...triton-x, saponin, or tween. Take care when using strong detergents like triton-x and tween as extended ...FACS In addition to the fluidics, optics, and electronics systems, FACS instruments have specialized components...

...in E. coli by co-expressing the molecule chaperone Skp. Front Cell Infect Microbiol 3: . https://doi.org...Antibodies: Historical Perspective and Future Outlook. Front Immunol 8: . https://doi.org/10.3389/fimmu.2017.01589...biosensor biorecognition elements. Biosensors and Bioelectronics 85:32–45 . https://doi.org/10.1016/j.bios.2016.04.091...

...Heinrichs, 2009). When a fluorophore absorbs light, electrons are elevated from a ground state to an excited...After a brief elevation in this excited state, the electron returns to its ground state by emitting a photon...Pyrgiotakis, G., Zhang, Z., Purington, E., Gramlich, W., Grondin, Y., Rogers, R., Bousfield, D., & Demokritou, P...nanocellulose for nanotoxicological studies. Environmental Science: Nano, 6(5), 1516–1526. https://doi....

... present in the sample. Other techniques, like electron microscopy and FRET, can offer more information...well described in the closed conformation, recent electron microscopy studies have also found Cas9:RNA complexes...pairing in the seed sequence.   Figure 6: Electron microscopy structure of S. pyogenes Cas9 bound...structure (left, PDB ID 4ZT0) with Cas9:RNA:DNA electron microscopy structure (right, PDB ID 7S4X), viewed...

...particularly energy-intensive systems in a more environmentally responsible manner.  Other ways to make the...in-depth look at Addgene’s current lab-oriented environmental efforts check out Leila Haery’s blog series,... we're always striving to set an example of environmental stewardship.  Additional resources on the Addgene...

... connections between BLA projection neurons and mPFC interneurons can be labelled with an AAV1 Cre virus...AAV9 tdTomato virus in the mPFC.  The AAV9 is so strong that you can even see dendritic spines!  Curious...

...users. Thanks to our guest blogger Cameron MacPherson! Cameron R. MacPherson is Lead Data Scientist...This post was contributed by guest blogger Cameron MacPherson at the Institut Pasteur CRISPR software...worthwhile buying a dedicated machine. On the offline front, there is a Python script called SSFinder, but I...

...Chinmaya Sadangi, a postdoc at the University of Toronto. The Addictive Brain was founded in early 2018 ...Chinmaya Sadangi, a postdoc at the University of Toronto, created The Addictive Brain to spread awareness...Sadangi is currently a postdoc at the University of Toronto and founder of The Addictive Brain. Prior to this...

...ways that I could change the world around me. My strong interest in the intersections of media, art, and...forward but I have learned to push myself to the forefront, no longer allowing myself to just simply exist... an Addgenie. I thought about all that could go wrong with this internship but in the end it turned out...

...Moriarty, an Associate Professor at the University of Toronto, co-founded the COVID-19 Resources Canada site....groups have focused on iterating on methods for coronavirus research, sharing resources for COVID-19 research...our eye recently (not a comprehensive list). #coronavirus #SARSCoV2 #Covid19researchA thread ⬇️⬇️⬇️ — Addgene...

...image the calcium response in the dendrites of T5 neurons by placing the calcium indicator GCaMP6f in the...optogenetically control the depolarization of R2 neurons using CsChrimson::tdTomato, an optogenetic tool...projection-specific brain-wide reconstruction of single neurons. Nature Methods 15:1033–1036 . https://doi.org/...

...the workforce, but my main concerns were building strong connections with coworkers and doing something ...to engage with the work of others. Having an environment filled with people who are interested in understanding...interested in knowing you as a person, builds a strong sense of trust between coworkers. Since I trusted...

...Your Gene Of Interest (YGOI) in a controlled environment under various conditions. The first thing you'll...you need to know is your expression system or environment. The host organism will determine the type of...overexpressing YGOI in an isolated, controlled environment. In most cases bacterial expression vectors are...

.... In neutral environments, dimeric Keima is excited at 440 nm and in acidic environments at 586 nm. Regardless...mechanism as Rosella: GFP is quenched in acidic environments, while mRFP is relatively stable and fluorescences...