...aggregation tendency, increase its brightness, and decrease the amount of light required for dimerization. She...Fluorescent Proteins Use Light to Control CRISPR Genome Editing Choose the Brightest Fluorescent Protein for...future award opportunities. Xin Zhou: Fluorescent Light-inducible Proteins (FLiPs) Fluorescent proteins ...create enzymes that can be turned on and off with light.  Xin hypothesized that she could make enzymes controllable...known to oligomerize when exposed to ultraviolet light. To do so, she generated fusion proteins in which...enzyme’s active site. In these fusions, ultraviolet light exposure caused oligomerization of the Dronpa domains...shutting off the enzyme. Later exposure to cyan light stopped Dronpa oligomerization and turned the enzymes...

...some light on multicolor imaging with fluorescent proteins. With the right microscope and the right choice...when choosing an individual fluorescent protein (brightness, photostability, and so on; see the previous ...colors and the mCherry spectrum is shown in the lighter colors; excitation spectra are blue and emission...this filter set, we would expect TagRFP to give a brighter signal than mCherry. In general, filter sets designed...supplement your cells with biliverdin for maximal brightness. In mammalian cells, one of the improved folding...fluorescent protein that is supposed to be extremely bright. In S. cerevisiae, we've tested a number of green...proteins with this filter set and have reported brightness measurements. Here, EGFP outperforms the improved...

...are commonly used in the lab as tools and we all might benefit from getting to know them a little better... injury, retroviruses generally have moderate to high immunogenicity, meaning they induce an immune response...transduce (infect) a wide range of cell types with a high efficiency. However, gamma-retrovirus infection ...tools have been derived from. Lentiviruses have slightly larger packaging capacities than gamma-retrovirus...immunogenicity, meaning it does not induce the same high immune response as retroviruses. This virus can ...been identified so far! Adenovirus generally has high immunogenicity, but some strains have been engineered... discussed in this article, adenovirus has the highest maximum titer, a plus for production and infection...

... university. My to-do list might look something like the image on the right (and for those of you who ...Addgene we've set out to fill this gap by both highlighting that researchers do learn MANY transferable ...the lab and by offering advice on areas where you might need some help. Today in our transferable skills...tasks at the expense of time spent on the long and highly useful tasks. The quick accomplishments will likely...you the energy to finish any additional work you might need to do. In most cases, I would recommend the...constantly switching between different projects, you might not be able to make any reasonable progress on any...to-do lists at the end of the day so they can jump straight into tasks the next day. If you work in a job ...

...ideal characteristics (low aggregation, high photostability, and high stability after fixation.) From the ...still being developed today. In this post, I will highlight the evolution of the  Brainbow multicolor labeling...zebrafish and Drosophila. Why Brainbow? Brainbow highlights the trend in biology towards single-cell resolution...development of a new technique. In a structure as tightly packed and interwoven as the brain, robust single-cell..., one would expect ten colors (see table to the right); in various reports, Sanes and Lichtman have observed...observed from 90-160 distinct colors due to higher copy number. The name Brainbow is truly fitting for...fluorescently distinct, their protein sequences are highly homologous, preventing design of antibodies specific...

...bivalent, so may have higher avidity than monovalent Fab’ or Fab fragments. Higher avidity means that F...’ll go over what a Fab fragment is and when you might use it in the lab. What is a Fab Fragment? Consider...chains (two identical heavy chains and two identical light chains) each with a constant region and a variable...heavy chains and between one heavy chain and one light chain. When assembled, the four chains make a Y-...are the “arms” of the Y and comprise the entire light chain and a variable and constant region of the ...important features labeled. V = Variable domain. L = Light chain. C = Constant domain. H = Heavy chain. Created...function but not the Fc region (more on when that might be later). Find scFv plasmids at Addgene Depending...

...the contents. Tightly coupling barcodes and containers might not be such a bad idea, tightly coupling them...we’ll highlight some of the lessons we’ve learned over the years with the hope that our insights can help... help steer you in the right direction when writing your own software. None of this is gospel, but we ...help tell this story. In this first example to the right (# this will make you sad),  someone has put in ...numbers. Loosely couple containers and contents Right from the get-go be as clear as you can be (there...them to their contents might leave you sad and confused when a tricky procedure failed and one of your...In the first example we’ve used inheritance to tightly couple the contents of a tube with the tube itself...

...easily detect and measure the light they emit. Proteins that give off light include chemiluminescent proteins...victoria there is a ring that glows with a faint green light. To investigate this green glimmer, Shimomura collected...which he called aequorin, gives off a faint blue light when it binds calcium ions, and the second, which...which we now call GFP, absorbs that blue light and glows green. In the late 1980s, another researcher, Douglas...protein localization. And as it turns out, he was right! He began to study the A. victoria gene responsible... fell into place, as they, taken alongside the insights provided by the studies in Tsien lab, covered ...their work on GFP. This great honor serves to highlight how useful fluorescent proteins like GFP really...

... and Mark Brigham have taken the technology to another level with the addition of two light-sensitive ...occurs within minutes of light exposure. Third, the response can be graded with light dose and is fully reversible...reversible after light retraction. Finally, because light can be applied non-invasively, its use is not...borrowed from Arabidopsis thaliana. The results are light-inducible transcriptional effectors (LITEs) designed...them on or off, literally at the flip of a (blue) light switch. Zhang’s team devised the plasmids now available... transcription in as little as 30 minutes after light stimulation. Under those optogenetic controls, gene...several advantages of the new system: "First, the light-responsive molecules of cryptochrome 2 are the chromophores...

...error — both human and biological. Even the most high-fidelity polymerases can make mistakes and lead ...these errors. These processes allow us to have a high standard of sequence quality across all plasmids... same QC process to ensure all plasmids pass our high-quality standards and contain the expected plasmid...accurate The gene/insert or any major feature highlighted by the deposited scientist  The backbone elements...deposited to Addgene. This review process also highlights other QC topics that may require additional attention...Illumina MiSeq platform, such as GC-rich regions and highly repetitive sequences and/or features. Most of these...assembly in Geneious can still provide additional insights to resolve the assembly and determine if there...

...only scientists with an MSc or higher apply. It was important to weigh the cost to benefit of studying... I didn’t need to explain why I changed jobs. I highly recommend taking on a similar position in your ...your field of interest before you dive straight into a full-time position. For my part, I really enjoyed ...for a disease that traditional medicine couldn’t fight effectively. I had to learn how to cope with a different... companies which are rapidly expanding, as this might not be sustainable. After working at this company... of different types of pancreatic cancer and highlighting which treatment would produce the best outcome... does not work for all patients, leading to very high mortality. I really loved the fact that my work ...

...understand how chromatin might influence editing results at your target. Figure 3: High-throughput screening...comes in! Evaluate prime editing efficiencies in high-throughput libraries My labmates and I wanted to...of pegRNA designs based on patterns observed from high-throughput experiments, where we can evaluate tens...of designs in parallel. To do this, we performed high-throughput ‘self-targeting’ screens with oligo libraries... edit types and cell contexts. Figure 1: High-throughput screening of pegRNA efficiency and building...screens such as PRIDICT/PRIDICT2.0 provide valuable insights into pegRNA editing efficiencies for specific ...research by the Shendure lab (Li et al., 2024) have highlighted how chromatin substantially affects prime editing...

...needed, without a high number of samples (~5). Tukey’s test – Most conservative test. Higher false negatives...select the right test for the job (and, uh, gentle reminder: keep your friendly neighborhood biostatistician... can tell you, and how to identify which one is right for you. Statistics – The basics Statistical analysis...ANOVA. Isn’t that what a two-way ANOVA test is you might ask? Not exactly! A MANOVA test assesses the effect...could test the impact of temperature, humidity, sunlight exposure time, and watering volume on total crop...total), your likelihood of a false positive is much higher than if you only look at 2 or 3 pairwise comparisons...range test – Most powerful. Low false negatives, high false positives. Control vs. all samples Oftentimes...

... colony formation on agar plates, less stable at high temperatures and in acidic environments.   Carbenicillin...better’ antibiotic than amp: it is more stable in high pH environments and is less susceptible to degradation..."fontawesome-5.14.0" name="Check" style="SOLID" height="16" purpose="decorative" title="Check icon" %}...

...common reagents, straightforward protocols, and standard microscopes sounds great, right? But there is always...         Why Expansion Microscopy?  Traditional light microscopes are limited in the level of resolution...everything needed for synaptic transmission, are right around 250 nm, making it difficult to study the ...the inner workings of the synapse by conventional light or fluorescence microscopy (Dani, et al., 2010)....What is Expansion Microscopy?  ExM provides a straightforward answer to the space problem. Instead of imaging..., biological samples are enmeshed in a gel of a highly absorbent material that is able to expand evenly...points with a conventional, diffraction-limited light microscope.  Along with using more accessible imaging...

... the assayed PPI. This high-throughput application is much more straightforward to carry out because it... This is straightforward in easy-to-transfect cell lines where plasmid dosage can be tightly controlled...the non-radiative transfer for energy between neighboring fluorophores, FRET can be used to detect the ...subcellular localization of the studied PIPs. The straightforward nature of BiFC also makes it easier to generate...generate a large number of constructs for high-throughput screening of PPIs. For example, Bischof et al....Hu, 2012). Having a rough idea of how your PIPs might interact will allow you to determine where to fuse... Proteins 101: GFP Fusion Proteins - Making the Right Connection. Cellular expression Whether BiFC constructs...

...protein at higher levels, you risk observing artifacts only present when the protein is at a very high concentration...membrane-targeted CAAX-SunTag was 18-fold brighter than sfGFP! The high SunTag signal allowed them to cut their...transcription factor binding sites in a promoter lead to higher transcriptional activation. Multiple nuclear localization...explosion SUperNova," SunTag can help you turn up the brightness in your fluorescent imaging experiments. Fluorescent...their laser power by over 80% and still obtain a higher signal-to-noise ratio with a lower photobleaching...mitoNEET-SunTag-GFP. As expected, they obtained bright images of mitochondria without organelle toxicity...to traditional fluorescent proteins Improved brightness and signal-to-noise ratio Less chance of phototoxicity...

...elevated plus maze, the light-dark box is a simple device comprised of a brightly lit (risky) and a dark... a camera or tracking software. Locomotion is a highly complex process on the level of individual neurons...findings by controlling neurons in the mPFC with light. Activation of these neurons via the channel Channelrhodopsin...inhibitory mechanism of locomotor sensitization and might open new ways for a novel therapeutic approach against...to stay in dark areas (protected) as opposed to brightly illuminated ones (risky). They also prefer to ...diseases, animals have reduced anxiety levels and a higher tendency for risk taking. These animals also show... arm (risky), or a closed arm (safe). Mice with higher IL-1 beta production spent more time in the open...

...make sure people pay attention. Highlight your lowest points and highest points and make clear what was...I’m working with a talented journalist to shine light on living as a scientist in Beijing, talking to ...judgement. Eventually, we published the paper in a high-impact journal and it worked out for all of us.”...cases, the protagonist will be you. Make sure to highlight what lessons you’ve learned and what knowledge... into your research at an outreach event as you might in a scientific paper. Build lots of versions of...difficult and what was easy when appropriate. It might not be scientific to bring emotional discovery into...people pay attention. Practice Nobody gets good overnight. The only way to improve is to keep working on...

...dye-nitrilotriacetic acid (NTA) conjugates (Kapanidis, Ebright, & Ebright, 2001). These Cy/NTA conjugates work the ...6. Kapanidis, Achillefs N., Yon W. Ebright, and Richard H. Ebright. "Site-specific incorporation of fluorescent... James D. Fessenden, an Assistant Professor at Brigham and Women’s Hospital. Biochemists often struggle...mailbox: How to get your fluorophore delivered to the right place? FRET experiments require targeting the donor...the proper mailbox (i.e., a binding site) in the right city (protein). In a cell, this task is even harder...acceptor molecules are targeted nearby (such as a neighboring house on the same street as the donor), then ...specificity Donor fluorophores need to go to the right place. Mis-targeted donors never “see” FRET acceptors...

... is the cr:tracrRNA approach highly efficient, it is also straightforward as the RNA molecules can be ...events. In the light of these results, we re-analyzed our data and found that 75% of our highest knock-in efficiency...of interest are pulled-down and later coupled to high-throughput sequencing to identify genome-wide binding...Telmer 1998). Indeed, this method can be used at high throughput because numerous cell lines can be generated...challenges, we set out to develop a hassle-free, highly efficient Cas9 protein-based method for epitope...which, in our hands, is both cost-effective and highly active. After csRNP complex formation, a single-stranded...transfection/delivery step needs to be optimized to achieve high knock-in efficiency. To calculate the tagging efficiency...

...aberrant light chain sequence, but was predicted to occur at a low frequency within functional light chain...a degenerate primer set to amplify IgG variable light (VL) and variable heavy (VH) chain sequences. Fusion...plasmid backbone for expression of the mAb heavy and light chain, each from a constitutively active CMV promoter... expression plasmid involved in coexpression of light (green) and heavy (blue) chains as driven by two...express the target antigen could be screened in a high-throughput manner. Cells were labeled with both ... R-mAbs. It was recognized that an aberrant IgG light chain transcript was expressed by some of the hybridomas...cloning, which in most cases left the functional light chain intact, the authors were able to greatly increase...

...Addgenies support a high collaboration, high communication, high efficiency, high quality, low meeting...getting ready to embark on new exciting projects. Right now we don’t have to worry about succeeding at our...materials to better serve the community. We are delighted at the early outcomes and the ecstatic reception...by the scientists who request from us but also by high school students and industry scientists as a universal...21,806th most popular website in the world. I know, right? Upgrades to the internal LIMS pages and excellent...excitement of what we are accomplishing and what more we might do. I am grateful every day for their dedication...viral_sharing=False, embed_button=False, width='1280', height='720', player_id='7039679868', style='' %} Resources...

...two recombinases. How might one read the state of an RSM? The most straightforward way to read the state...state machines in living cells” (1), into a more straight-forward, concise explanation of how to adapt our...exposition, and detail, please refer to the paper. Why might one be interested in state machine technology? Biological...sent the PCR product for sequencing.  A less straightforward, but more population-wide (as opposed to single-cell...method since it lends itself to cheap, quick, and high throughput state detection with flow cytometry. ...cells; the sequencing and qPCR methods do not. How might one implement RSMs? In our study (1), we implement...Figure 3 below): Input Plasmid: The input plasmid (high copy, ColE1 origin of replication), contains large...

...Atsushi Miyawaki and colleagues identified a highly bright and stable fluorescent protein, StayGold. Although...Every few months we highlight some of the new plasmids, antibodies, and viral preps in the repository...uses a much shorter crRNA. Cas12-RNP-PAGE achieved high editing frequency (nearly 100% in some cases) and...break up the dimerization without losing that brightness and stability. Here’s a quick overview of these.... This variant has similar photostability and brightness compared to the original, though its maturation...sufficient to disrupt dimerization while maintaining the bright and stable fluorescence of the original (Ivorra-Molla....rs-2684100/v1.  Piatkevich, K., et al. (2023). Bright and stable monomeric fluorescent proteins derived...

... recommended.  Environmental influences like bright light or noise should be avoided as this will also...consideration for behavioral testing in a laboratory, a high level of experimental control is required, because...frequencies (> 20 kHz, produced from fluorescent light bulbs, for example) that are outside the humans’...optogentics to control neuronal activity, commonly via light-activated ion channels. Similarly, chemogenetic ... trigger neuronal excitation or inhibition upon light (or drug) application.  Genetic manipulation can... generally mediates broad cell tropism and has a high percentage of neuronal targeting after stereotaxic....  Viral vector injection Viral vectors allow a tight spatial and temporal control of gene expression ...

...concentrations of IL-3. Cells grown in high concentrations of IL-3 show a high proportion of blue fluorescence...Every few months we highlight a subset of the new plasmids and viral preps in the repository through ...Split-wrmScarlet is a new split red FP that is three times brighter in worms than split-sfCherry3. It was created ...in live cells. The fluorescent timer emits blue light around the time of synthesis and, after a few hours...cells. Cells undergoing rapid cell cycles have a high blue to red ratio, whereas cells with long or slow...cells as measured by flow cytometry. Cells grown in high IL-3 showed mostly blue fluorescence, indicating...CRISPR corner By Jennifer Tsang Here are a few highlights from recent CRISPR plasmids. To find all of the...

... an MRI, you know that magnetic fields can pass right through our tissues unimpeded. Andrew and Maria ...for a magnetic response among other proteins that might serve as a starting point for developing useful ...Andrew and Maria explained, “to our surprise and delight,” the very first protein they tested — E. coli ...coli expressing good ol’ EGFP — showed a slight but detectable change in fluorescence when a small magnet...team was careful to test the possibility that this might just be an artifact and eliminate sources of interference...expressed and purified. Although a ~1% change in signal might not make a very powerful tool, it was promising ...enhanced for future MagLOV-based tools, just as light-induced conformational changes are the basis of ...

...antibody 9E10 displays highly variable epitope recognition dependent on neighboring sequence context. Sci...However, it was (and still is) infeasible to generate highly specific antibodies for every potential new target...interest with unrelated peptides that already had high quality antibodies available - aka peptide tagging...tagging.  Key to this strategy was the existence of high quality antibodies for well defined peptides. Munro...the neuropeptide substance P. This protein had a highly specific antibody to the C-terminal five amino ...sounds less than ideal.  Unexpected connections Right around the same time that Munro and Pelham were ... (1) Locate a promising monoclonal antibody with high specificity. (2) Narrow down the specific epitope...

...Every student about to graduate shares the same nightmare: the question “So, what comes next?” For those...education or ending a Master’s program, the answer might be to continue in academia and pursue a PhD. However... such as Zoom, to determine if a program is the right fit for you without the financial and logistical...to around twelve professors and heard back from eight. These emails don’t have to be long; the idea is...you a chance to get to know the faculty that you might be working with for the duration of your program...that will help you determine if the program is the right fit. Reaching out to PIs also means doing plenty...close to your computer with things you want to highlight in the interview is another way to make the virtual...

... and image analysis software are highly sensitive to detecting light signals not visible to the human ... that the resolution of light microscopy is limited by the wavelength of light and, practically speaking...and diseased states can also provide interesting insights into disease mechanisms and protein function. ...well-characterized marker proteins tagged with FPs to highlight various subcellular structures. Please see Figure...visualize subcellular structures. For example, you might use your fluoresent protein fused to a signal sequence...complexes with. These “colocalization” studies give insights about the proximity of two proteins within the...PMC3074624. 5. Bindels, Daphne S., et al. "mScarlet: a bright monomeric red fluorescent protein for cellular ...

...keep in mind that UV light only kills microorganisms that it strikes directly. UV light exposure is also ...ensure nothing is covering it. Turning on the UV light 3-4 times a week for up to half an hour can help...also harmful for eyes and skin so keep the light off when the hood is in use. Wipe the hood with a disinfectant...source of contamination for your experiments, the bright side is that you also have the power to take the...morphology: Shape and size matter You should have a light microscope close to the hood so you can view your... morphologies and doing a quick check under the light microscope will help you identify bacterial and ...are sensitive to pH. Anything between orange and light orange usually indicates that the cells have grown...

...verify loss of minicircle RNA Delivery DNA-Free High High Transgene-free and vector-free Multiple transfections... Pros Cons MMLV-derived Retrovirus Integrating High Low Efficient and Stable Genome integration Insertional...reactivation, residual expression Lentivirus Integrating High Low Efficient and Stable Genome integration Insertional...transfections required Protein Delivery DNA-Free Low High Transgene-free and vector-free. Slow and inefficient...reprogramming efficiencies ranking as low, medium or high is based on the reprogramming efficiencies reported...pMXs or pMSCV, infect dividing cells, and have a higher efficiency of generating iPSCs compared to other...integrate or replicate. Their small size leads to higher transfection efficiencies and they tend to express...

... cell or neuron type may not work well for a neighboring cell type, even within the same brain region ...Low-magnification images showing viral spread. (D-F)  High-magnification images immuno-stained against dopamine...frustrating and often months-long quests to find the right AAV for your system or to successfully replicate...sharing of negative data.  Negative AAV data is highly desirable  In a survey we conducted to improve ...and that it often provides incredibly valuable insight into one’s work. As a community, we must therefore... descriptions, a minimum n = 2, and well labeled high- and low-magnification images demonstrating vector... do you have sitting in computers and notebooks right now?, I’m willing to bet the answer would be along...

...from Addgene right on our plasmid pages and provide a citation that you can put straight into your reference...our website using the search bar found at the top right of every page. You can search for plasmids by name... number, or keyword. Although an initial search might give you many results, you can narrow your search...search results using the filters found on the right side of the search page. A great breakdown of our search...special collections pages. These pages serve to highlight plasmids that can be used for specific purposes...Every quarter we release a Newsletter where we spotlight some of the plasmids that have come through our...page - each day we showcase a new plasmid that you might otherwise miss. We hope this feature will draw attention...

...Every few months we highlight a subset of the new plasmids, antibodies, and viral preps in the repository...Improved voltage indicator: JEDI-2P Novel class of light-gated potassium channelrhodopsins Myc-tag antibody...indicator (GEVI) called JEDI-2P. Using a custom, high-throughput screening platform to apply saturating...10.1016/j.cell.2022.07.013.   Novel class of light-gated potassium channelrhodopsins by: Mike Lacy ...Xue and John Spudich identifies a novel class of light-gated potassium channelrhodopsins, named Kalium ...2022). The authors show that HcKCR1 and HcKCR2 have high selectivity for K+ over Na+ and rapid photocurrent... optogenetic use.   Fig. 2: A) HcKCR1 is highly selective for K+ over other Na+ and other cations...

...serum. Harrington et al suspected GeoCas9 might have higher stability since prior research had shown that...zebrafish, frogs, some bacteria; but not thermophilic (high heat loving) bacteria. Until recently the only available...s SpCas9. These Cas9 proteins don’t work well at high temperatures, so to use them in thermophiles, bacteria...approach only works for facultative thermophiles (high OR medium heat loving), but not obligate thermophiles... RNP activity or longevity. GeoCas9 is active at high temperatures Figure 1: Thermostability of...tested the ability of GeoCas9 RNPs to be incubated at high temps but still maintain activity following heat...temperatures. In both cases, GeoCas9 outperformed SpCas9 at high temperatures. See Figure 1 for a summary of the ...

...Every few months we highlight some of the new plasmids, antibodies, and viral preps in our repository...different genomic loci — in some cases, over 20-fold higher editing than PEmax. The authors hypothesized that...immunoblotted against vinculin as a loading control (right panel). Image from Addgene. These antibodies... subthreshold voltage fluctuations, while having high fidelity during action potentials of fast-spiking...j.neuron.2023.03.009. PMID: 37015225.  Simple and highly specific targeting of microglia with AAV By Alyssa...human IBA1 promoter (hIBA1a, 466 bp) that drives high microglia-specific gene expression when incorporated...hIBA1a promoters in mice. B) Quantifications showing high microglia specificity of GFP expression for the ...

...upper right) and a mixed population of B cells and dendritic cells (DCs) in the lower middle right. DCs...generated often looks like the graph on the left, it might make more sense once you realize events are plotted...plotted on an invisible Cartesian plane (right.) Each event is plotted by the amount of signal read from...cells. If you look at the dot plot in Figure 2, you might assume that all cell populations are positive for...another two are B+. Our A-B+ cells are in the lower right corner.      Figure 2: This flow plot has... (SSC) and forward scatter (FSC), which measure light that “scatters” past the cell, instead of fluorescent...DCs), mDCs and pDCs. Let's follow it from left to right.  First, SSC and FSC are measured to differentiate...

...neuron-optimized viral vectors, and high-throughput screening approaches. Here, we’ll highlight these recent advancements...least, more straightforward than generating cDNA for each gene isoform individually. To highlight this advantage... The Day Lab used their optimized vectors to highlight a key advantage of CRISPRa over traditional cDNA...targeted anywhere in the genome, it is relatively straightforward to use CRISPRa to selectively target these ...-optimized lentiviral vectors can be found here. High-throughput screening strategies Although the tools...