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...ultraviolet light. In this post, we will describe the mechanism of homology directed repair, one of the repair...template for repair and is thus the more accurate mechanism for DSB repair. The process is generally error-free...the template DNA. There are several unique HDR mechanisms, but all templated-repair pathways have these...process Homology directed repair pathways HDR mechanisms can be divided into two main camps: non-conservative... conservative. The conservative methods are characterized by use of a homologous donor (e.g., sister chromatid...single strand annealing (SSA), an error prone mechanism that notably does not require a donor template... (SDSA) pathway SDSA is a conservative repair mechanism which exclusively yields non-crossover events....

...founders Podcast breakdown Intro Chapter 1 - Who are Addgene's Founders? Chapter 2 - The process of founding...ensuring that success Chapter 6 - Developing Addgene’s Brand and culture Chapter 7 - Building Addgene ...distribution!) Chapter 8 - Opportunities to create repositories with other materials Chapter 9 - The Founders... came from and how the idea got off the ground Chapter 3 - Addgene as a leader in standardized tech transfer...transfer and the adoption of the UBMTA Chapter 4 - Developing Addgene as a modern e-commerce company and... and establishing credibility Chapter 5 - Realizing that Addgene would be successful and the importance...and beyond - Ginkgo, Tufts Launchpad, and more Chapter 10 - Addgene's Future, the research team, and the...

...identify how MagLOV’s structure changes under magnetic field. Such changes could be harnessed or enhanced... Entropy as a Proposed Gating Mechanism for Magnetogenetic Ion Channels. Biophysical Journal, 116(3), ... good ol’ EGFP — showed a slight but detectable change in fluorescence when a small magnet was passed ...Fluorescence intensity of EGFP-FlavinTag in E. coli changes in response to a ~25 mT magnet. Gray regions indicate...expressed and purified (Figure 1). Although a ~1% change in signal might not make a very powerful tool, ... Q513K, G528K, D540M) that shows a fluorescence change of ~75% in response to the magnetic field (Figure...MagLOV-based tools, just as light-induced conformational changes are the basis of many optogenetic tools. And, since...

...jellyfish Cassiopea xamachana. Marine Biology 94:423–430 . https://doi.org/10.1007/bf00428249 Chan MCY, Karasawa...-colored protein from the jellyfish Cassiopea xamachana (Blanquet and Phelan, 1987). Eight years later...of chromoproteins were identified in sea anemone (Chan et al., 2006). Why do these organisms have chromoproteins...specific purposes.     Chromoproteins for allelic exchange Figure 3: pTOX plasmids expressing amilCP... create negative selection markers for allelic exchange. These vectors contain a toxin gene (regulated...rhamnose-inducible promoter) and a chromoprotein gene. Allelic exchange occurs through two sequential homologous recombination... the tsPurple chromoprotein.  Find the allelic exchange vectors here! ShadowR, a novel FLIM-FRET acceptor...

...flank the desired change with 4-5 silent changes in the wobble codons, i.e. make changes to the third base... translated protein.  These changes can be 5’ or 3’ of the desired change.   Table 1: Overview of ds- ...sequences, point mutations or other small base pair changes, and the addition of protein tags. It also has ...to restriction enzyme cloning where the genetic changes occur in a test tube. The donor DNA substrate only...ssDNA substrate is best for point mutations or changes of only a few base pairs.  dsDNA Substrate dsDNA...prone to mutations and will have more unintended changes to their genomes. 2) Designing ssDNA oligos that... to generate insert ssDNA Point mutations or changing a few nucleotides 0.1-1% or 25-50% when activation...

...bacteriorhodopsins, halorhodopsins, and channelrhodopsins, the first two found in the Archaea, the third in green algae...). These proteins control the flow of electric charges across cell membranes and maintain membrane potential... 2012). For example, blue light activates channelrhodopsins that, in turn, trigger neural excitation, ... promoter upstream of an opsin gene (e.g., channelrhodopsin-2, ChR2) fused to a reporter gene (e.g., mCherry...vectors to study the neural circuitry behind the mechanisms of sleep and anesthesia. In Cre transgenic mice...vectors to answer this question, mapping and characterizing the activity of the DA neurons of the VTA. ...optogenetics to understand the cellular and molecular mechanisms of the brain in vivo. Many thanks to our guest...

...constant domains, the plasmids for the heavy chain and light chain are also available from Addgene!   ...disruption in M. abscessus  Bioluminescent reporter of mechanical forces   Epitope tag antibody collection grows...K., & Russell, W. C. (1987). Isolation and characterization of monoclonal antibodies to simian virus 5...of its biology, pathogenicity, and resistance mechanisms.     Figure 4. Overview of the dual-plasmid...10.1128/jb.00335-23.  Bioluminescent reporting of mechanical forces with PILATeS By Emily P. Bentley Time ... lab and collaborators have deposited a new mechanosensor called PILATeS, Pulling-Induced Luciferase Association...different contexts.   Figure 5: Left: Mechanism of the PILATeS tension sensor. The LgBiT (cyan...

...this is not a direct reference to the video game character but rather to an extremely important gene in animal...1980, when Christine Nusslein-Volhard and Eric Wiechaus first identified the hedgehog gene in Drosophila...that resembled a hedgehog (Nusslein-Volhard and Wiechaus, 1980). The mammalian hedgehog protein, sonic ...into vertebrate chromosomes via a cut and paste mechanism. Scientists reconstructed a transposase system...squash (Sqh) is the Drosophila Regulatory Light Chain (RMLC) of nonmuscle myosin II (Karess et al., 1991...plasmids in our inventory from the lab of Eric Wieschaus that express sqh.   cookie monster, a meiotic...required for spermatogenesis after a Sesame Street character? The scientists who discovered comr thought the...

...body monomers respond to pH changes by undergoing local conformational changes. These tiny movements are... much force can be generated by conformational changes in proteins? How can information propagate through... repeatedly extend and contract in response to changing pH, and they can rupture E. coli spheroplasts ...and retract over many microns in response to pH changes without the need for ATP or other chemical fuel...vasculature or extracellular matrices) in response to pH changes. Beyond these applications, purified R bodies ...bodies could also perform as actuators in microelectromechanical systems (MEMS) devices. They could be functionalized...

...introduction of specific sequence changes. To generate a specific sequence change the cell must undergo homology-directed...extensively for the purpose of making sequence-specific changes in human induced pluripotent stem cells (iPSCs)... (ssODN), which contains the intended sequence changes to be incorporated into the edited genome. In mammalian...CRISPR/Cas9 blocking mutations in either the PAM (changing one of the G’s to an A,T or C – but avoid NAG)... to be silent. In our view, it’s always best to change the PAM site, however we have also demonstrated...mismatch in the binding sequence, we recommend changing several bases as close to the PAM as possible ...blocking mutation together with the intended sequence change and screen a few hundred clones to find the right...

...warming The New York Times: With Trump in Charge, Climate Change References Purged from Website The Washington...reality – a government cannot declare that gravity change direction and make things fly up when we drop them...outcomes are actually possible for a given policy. In exchange, the government protects (and often funds [3])...scientists has been rising as evidence of climate change, the safety of genetically modified organisms (...90% of active research scientists believe climate change is mostly due to human activity while much less...administration’s actions, including taking down the climate change page on whitehouse.gov, freezing grants, and proposing...advocating for science education and literacy that this changes.  2) Lumping all scientists (and consequently their...

...Engineered and evolved optimizations for prime editing CHARM: A compact epigenetic silencer Recombinant antibodies...in Xenopus nucleosomes and in complex with its chaperone NPM2, but the technique should be widely applicable...more complex protective methods or detergents like CHAPSO, while requiring a lower concentration of sample...  https://doi.org/10.1038/s41551-024-01227-1   CHARM: A compact epigenetic silencer By Emily P. Bentley...for Autoinhibition Release of Methyltransferase (CHARM), with several advantages over the earlier system...methyltransferase for epigenetic silencing, which can be toxic, CHARM uses a cofactor fused to a histone tail to recruit...potentially with multiplexed targets.   Figure 4: CHARM uses a fusion of the DNMT3L cofactor (D3L) with ...

...precision medicine a reality. Challenges for cancer variant characterization For many years, we’ve hoped...report includes rare variants that haven’t been characterized? Some have developed exciting computational...algorithms, such as SIFT or PolyPhen to approach this challenge, using attributes of cancer variants such as evolutionary... or predicted deleteriousness of the amino acid change based on maps of protein structure. Sometimes this...Collection, that we hope researchers can use to characterize these variants in a high-throughput manner and...validation experiments. But, there have been two huge challenges preventing us from testing this hypothesis: ...Accelerator Plasmid Collections To overcome this first challenge, we’ve developed the Broad Institute Target Accelerator...

...antibody.     Figure 2: Changes to the heavy chain or light chain genes within a hybridoma impacts...off-target effects. This is because there are more chances for these antibodies to recognize epitopes that...monoclonal antibodies. Genetic drift refers to changes in the nucleic acid sequence of the antibody-encoding...over time as the hybridoma cells divide. These changes can alter the antibody that is actually produced...produced by the hybridoma, meaning that there will be changes between lots over time. Specifically, the antigen-binding...antigen-binding sites of the antibody - the paratopes - may change and impact the specificity and avidity of the antibodies...be identified by sequencing the heavy and light chains. To circumvent any possible genetic drift, scientists...

...cholinergic (choline acetyltransferase, ChAT) neurons. The authors characterized these tools and validated their...important and abundant neurotransmitters, but it is challenging to monitor because only a small number of glutamate...deep-brain fiber photometry.  Figure 1: Characterization of iGluSnFR4 in mice. A) Scheme for two-photon...GPC4). All IPI antibodies undergo extensive characterization at IPI as well as community-led evaluation...retrotransposon elements they identified and characterized (Fell et al., 2025). In retrotransposon insertion... a wide variety of genomic edits — single-base changes, small deletions, and large insertions up to 12...to specific cell types in the brain can be a challenging — but critical — factor for successful experiments...

...the ion pore domain (IPD) of a channel, the PSAM acts as a gating mechanism to control ion flux. The system... endogenous gene expression, which can lead to changes in normal cell physiology and function (Saloman...to one receptor, so it is important to fully characterize the compound, or compounds, you choose to ensure...system allows you to directly manipulate ion channels. When working with PSAM receptors, your impact on...with. Combining a PSAM with the IPD of a cation channel, such as the serotonin 3 receptor, will promote...Alternatively, combining with the IPD of an anion channel, such as the glycine receptor, will promote hyperpolarization...doi.org/10.1523/ENEURO.0171-19.2019Jendryka M, Palchaudhuri M, Ursu D, van der Veen B, Liss B, Kätzel D,...

...donor-acceptor pair and a change in FRET efficiency correlates with a change in the distance and/or orientation...to a change in the environment which alters the FRET efficiency. Detecting certain cellular changes with...studying protein-protein interactions by measuring the change in FRET efficiency, from which conclusions regarding...error in suboptimal or undetectable FRET, well-characterized FRET reference standards can be used to validate...rearranging the start and end positions without changing the order of the amino acids in the protein) may...cellular processes and signaling cascades, quantify mechanical tension (a molecular “spring”) (Meng et al., ... et al. "A fluorescence energy transfer‐based mechanical stress sensor for specific proteins in situ."...

...., Bussey, H. et al. Functional characterization of the Saccharomyces cerevisiae genome by gene deletion...students (1, 2), but educators face significant challenges in designing suitable projects. In the O'Connor...methionine synthesis (5). The budding yeast, Saccharomyces. cerevisiae, makes a great reference organism...cerevisiae (Figure 1) occurs through a well-characterized pathway with multiple enzymatic steps (5). In... or not proteins from the fission yeast, Schizosaccharomyces pombe, have the same function as their counterparts... the Advancement of Science. (2011). Vision and Change in Undergraduate Biology Education: A Call to Action...yeast for budding geneticists: A primer on the Saccharomyces cerevisiae model system. Genetics 197, 33-48...

...contributed by guest bloggers Alissa Lance-Byrne and Alex Chavez, researchers at the Wyss Institute for Biologically...double strand break (DSB) stimulates DNA repair mechanisms that can be exploited to either inactivate a ...alteration. Listen to Our Podcast Interview with Alex Chavez Figure 1: Schematic of native (nuclease-...different perturbations. Consider, for instance, the challenge of attempting to simultaneously and selectively...importantly, most Cas9 orthologs are less well characterized than the conventionally used SpCas9 (derived...transcriptional regulator without making any functional changes to Cas9. For instance, when native (nuclease-competent... our guest bloggers Alissa Lance-Byrne and Alex Chavez! Alissa Lance-Byrne is a graduate student at the...

... the production of Fab fragments where variable chains are secreted into the periplasmic space to be oxidized...transfection, depending on whether the heavy and light chain genes are contained to one plasmid or if they’ve...preferred buffer. If that’s the case, a buffer exchange can be performed using a desalting column or an...concentration to give your downstream applications the best chance at being successful.  Using a spectrophotometer...SDS-PAGE gel. Once stained, the heavy and light chains become visible alongside your ladder and a protein...Any bands on the gel beside the heavy and light chains can also be measured. Contaminating bands lower...western blot. This can be due to conformational changes in the antigen or lower detection sensitivity of...

...help. Here, we will outline some fundamental characteristics to consider when selecting a dye or ligand ...Fluorescence microscopy takes advantage of the characteristics of these fluorophores to enable highly specific...Hayashi-Takanaka et al., 2014). Other dyes may prove challenging to stain intracellular compartments in in vivo...impacted by both the excitation coefficient, characterizing the strength a species absorbs or reflects ... mitochondrial homeostasis maintained through mechanisms like fission, fusion, biogenesis, and mitophagy... as Stimulated Emission Depletion (STED), and Stochastic Optical Reconstruction Microscopy (STORM). These...key to observing interactions and conformational changes in proteins. Both short distances of molecules ...

... Research is Advocating for Change Within the Sciences Learn How Changing Labor Laws Can Effect Postdoc...editors) can move slowly, news about possible broad changes to policies seems to break everyday. Many researchers...active and involved if we wish to make positive change. Below I present perspectives on the March for ...Addgene. Boston, MA, USA 42.3601°N 71.0589°W – Alina Chan The job of scientists to observe how things work...generation is in the veracity of its work. Alina Chan is a postdoctoral researcher at Harvard Medical ...cause autism to politicians saying that climate change is a hoax. This has culminated in the current President...interested in global health policy.  He is also chair of the postdoctoral engagement committee and active...

...separates the cells mechanically in a vibrating nozzle, imparting a positive or negative charge to cells that...GFP and its derivatives by the late Roger Tsien changed everything. For the first time, fluorescent proteins... lymph nodes at different times after antigen challenge, scientists were able to track where GFP labelled...two-photon microscopy enabled scientists to see how chaotic and random the scanning was and how fast T cells...et al. 2016, Cavanagh et al. 2008)? Technical challenges of two photon microscopy Intravital imaging is... at steady state or under physio-pathological challenge. However technical specificities have to be taken...imaging studies with blue or cyan proteins without changing excitation wavelength (Salomonnson et al. 2012...

...solutions to complex challenges. As a cross-disciplinary researcher, the challenge of keeping up on studies... Sharing your research with the world can be challenging. After months, or years, of grueling effort to... personnel, and sociology journals when I get a chance, but I know that there is much more research that...biology or international relations.  Many of the challenges in science today are too complex for any single...disciplines. Included in the article is an interactive chart where you can see the yearly interdisciplinarity...Out Addgene Videos Over at the Addgene Youtube Channel   After watching these trends for a few years, ...making publishing their research all the more challenging even when it is of quality). On the site you ...

...will always be that way. Things change and processes often have to change with them. If you can, set aside... needing to brainstorm with a team. One major challenge with these brainstorming sessions is avoiding ...person who submitted the idea did not have to be in charge of implementing it. The purpose of this form was...creative when you don’t believe you have the power to change anything. You may not have the highest position...received, understand that people can be resistant to change. Ask your peers or your superiors to explain their...unworkable idea into a viable solution.  Anticipate Change Just because a process is working, doesn’t mean...aside time to prepare for future challenges. This doesn’t have to be time-consuming or even very frequent...

...tags, FLAG is the most charged, with 5 negatively charged and 2 positively charged amino acids. FLAG tag...tag has a net negative charge with 4 negatively charged and 1 positively charged amino acids. Myc tags ...The tag has two negatively charged amino acids for an overall negative charge. This tag has been used successfully...doesn’t come with a high background can be a huge challenge. Epitope tags eliminate the need for target-specific...

...This post was contributed by guest blogger Chris Richardson, a Postdoctoral Researcher in Jacob Corn’s lab...and yet unanswered) question. Our recent paper (Richardson et al 2016) revealed some of the biophysical ...binding to the cleaved DNA duplex. Image from Richardson et al., 2016. We next wondered if we could...Example single stranded donor DNA and a presumptive mechanism of action are presented in Figure 2. Figure...interaction with the Cas9-DNA duplex. Image from Richardson et al., 2016.   dCas9 enables sequence replacement...accompanies Cas9 cutting. We still don’t know the mechanism underlying dCas9 editing, but it could be very...Results from our lab and others (Lin et al 2014, Richardson et al 2016, and Kim et al 2014) have shown that...

...The late Michael Davidson and Roger Tsien played enormous roles in fluorescent protein development by...community. To honor their legacy, we launched the Michael Davidson and Roger Tsien Commemorative Travel Awards...happy to announce the three winners of this year’s Michael Davidson and Roger Tsien Commemorative Travel Awards... Awards: Melissa Chiasson, Elizabeth Unger, and Michal Bernach. Melissa Chiasson: Quantifying the abundance... in vitro against different drugs and dosages.  Michal Bernach: Developing an artificial leaf replica ...plant-bacteria and bacteria-bacteria interactions  Michal Bernach is a graduate student in Mitja Remus-Emsermann... But the shape and metabolism of leaves can be changed by light and temperature and leaf autofluorescence...

...new light on mechanisms of miRNA-mediated gene regulation Article contributed by Michael Lemieux  Listen...longstanding challenge. Using shRNA, TALEN, or ZFN to edit immune cells poses challenges such as the inhibition...synapses was developed by Matthew J. Kennedy’s and Chandra L. Tucker’s labs from UC Denver. At the core of...researchers split the proteolytically active light chain of BoNT/B into two fragments (BotNT-N and BotNT-... of blue light, iLID undergoes a conformational change allowing high affinity binding to SspB, brings ...host organism, the expression of some genes can change depending on the location. Current methods for ...pathways. Despite several years of research, the mechanisms of miRNA regulation have yet to be fully elucidated...

...discussion and for being “coachable”. That means the mentee needs to be open to change and constructive suggestions... “Learning is not attained by chance, it must be sought for with ardor and attended to with diligence...current position Understand your interpersonal challenges if they know some of the people you work with...Help identify the skills you will need to make a change Provide perspective on core values at another company...excellent group mentoring program in many of its chapters. Find out what other groups have going on locally.... Alumni who enter their information in these searchable databases are usually offering their contact ...their time. There are many professional coaches who charge for their advice and time. Consider this option...