...improved sensors for these and other targets. Figure 1: MTRIA biosensors are generated by inserting a circularly-permuted...fluorescent biosensors. Nat Commun. 2022 May 25;13(1):2919. doi: 10.1038/s41467-022-30685-x. Feel the ...new class of sensors for neuropeptides and neurotransmitters. Motivated to develop a biosensor for oxytocin...devised a “Modular fluorescence unit fused with TRansmembrane region-to-IntrAcellular loop linkers” (MTRIA...sensors based on GPCRs recognizing various neurotransmitters, neuropeptides and other ligands - almost ...kinetics and localization for imaging synaptic transmission. bioRxiv 2022.02.13.480251. doi: 10.1101/2022.02.13.480251...2022.02.13.480251 JEDI-2P voltage sensor The François St-Pierre lab and colleagues have developed a new...

...infections, cancer, and other diseases. References 1. Harrington, Lucas B., et al. "Programmed DNA destruction...that Cas14 could indiscriminately cleave ssDNA in trans like Cas13 and Cas12 do for RNA and dsDNA, respectively...can cleave fluorescent probes of dsDNA and RNA in trans. Fluorescent signal indicates the presence of this...

...fluorescent in acidic organelles (Figure 1), showing that its acid tolerance is maintained in cells.   GFP is ...References 1. Zacharias, David A., et al. "Partitioning of lipid-modified monomeric GFPs into membrane microdomains...of different fluorescent protein are available (Cranfill et al., 2016 and FPbase.org). A number of variants...specific RNA stem loop-structure (Querido and Chartrand, 2008). Finally, a nuclease-deficient Cas9 can...clearly the case when proteins undergo post-translational modification with fatty acyl chains. For instance...28360214. PubMed Central PMCID: PMC5385933. 6. Cranfill, Paula J., et al. "Quantitative assessment of ...PMC2644460. 12. Querido, Emmanuelle, and Pascal Chartrand. "Using fluorescent proteins to study mRNA trafficking...

...education, government, and civilization in general (1). Science and politics? Science is an apolitical process...consequences of a march. Three of these risks include: 1) Lost trust Perhaps the greatest risk is that the ...working full-time, and have received a research grant in the last five years as polled in the American...climate change page on whitehouse.gov, freezing grants, and proposing to defund aspects of research at...following subjects: Climate change Vaccines GMOs Transgender issues Reproductive health Immigration Pollution...while many also seek to honor and credit women, immigrants, and minorities who have had great impacts on...encouraged by “Broader Impacts” requirements for certain grants) (28). As one young scientist, Matthew Niederhuber...

...by insertion and deletion frequency. Fig. 1: Overview of TIDE. The input is sequencing trace files...keep your product to knock-in size ratio below 10:1. Small knock-ins Is your knock-in edit small, with...optimizing this step, or consider enriching for transfected cells by flow sorting or drug selection, assuming...

...at a low angle (FSC) or large angle (SSC) (Figure 1). FSC values depend on the cell's size, while SSC ... or ask experienced colleagues.   Figure 1: The laser pulse illuminates the cell and is scattered...software in order to generate signals in a reasonable range. If colleagues in your group already have experience...experimentally-specific gates. Two-dimensional staining (quadrant gating) For our last example plot, I will show...cells (CD8+) from dendritic cells (CD11b+) using a quadrant gate. Within my “lymphocyte + single cell + live... two stains in relation to each other. Using a quadrant gate, I can divide my cell population into DN,...for each stain respectively). (B) Instead of a quadrant gate, I am free to use multiple individual gates...

...with the Gibson assembly master mix for 1 hour, and then transform into competent cells. The success rate...you should consider giving it a try! References: 1. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison...generating long overhangs which allows the single stranded regions with homology to anneal a polymerase, ...hour or so, the sample is immediately ready to transform into competent cells. The master mix of enzymes...if the ends of the fragments have stable single stranded DNA secondary structure, such as a hairpin or ...would directly compete with the required single-stranded annealing and priming of neighboring assembly ...

...compare the experimental group.     Figure 1: A, Most biological experiments will benefit from ... Histochemistry & Cytochemistry, vol. 59, no. 1, SAGE Publications, Jan. 2011, pp. 6–12. Crossref,...across samples and equal protein transfer from the gel to the membrane. A loading control is critical for...identically, used to assess naturally occurring random biological variability. If one were studying the...purple) and nonspecifically through the Fc region (orange). The Preimmune serum control will not bind to .... Always probe the loading control on the same membrane that the target protein was measured on.    Flow...

...downstream experiments. You only successfully generate 1 null clone and proceed with it. In this case, the ...there are 10 putative off-target locations you can 1) determine if editing at those sites would confound...While this may effectively serve as a transfection or transduction control, a different gRNA would have..., you’ll want to select a Cas enzyme with low tolerance for mismatches within the gRNA sequence. ...near each other, this will result in a DNA double strand break (DSB), the event required for frameshift ...

...in the crystal jellyfish Aequorea victoria (Figure 1) in molecular detail. On the underside of the “umbrella...glowing protein could be used to measure gene transcription, as well as to track protein localization. And...bioluminescent, some types of corals and anemones are vibrantly colored. He hypothesized that GFP-like proteins...

... science 1)      In February, the Office of Science and Technology Policy issued a memorandum calling ...(66 percent) gave preference to journal access granted via a public repository or authors’ own web sites...

... same output regardless of who or which computer ran the process. Another design choice we appreciated...drives our bioinformatics needs       Fig. 1: QC'ing data at Addgene is like standing under a Niagara...automating the bioinformatics steps needed to transform the raw sequencing data into a complete plasmid...appropriate place in our workflow without having to translate or convert programs to a uniform language.  The...concepts and vocabulary of Nextflow so that we could translate our pipeline steps into processes and then connect...

... and a reverse transcriptase (RT) template including the desired edit. Figure 1: Prime Editing ...doi.org/10.1038/s41587-021-01039-7 Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I....nicking of the unedited strand and encourage the cell to use the edited strand as a template during DNA...on a fusion enzyme of Cas9 nickase + reverse transcriptase and a prime editing guide RNA (pegRNA). The ...although sequences with G/C content outside this range can still be optimized (Anzalone et al., 2019). .... Test different lengths of reverse transcriptase template, starting with about 10–16 nucleotides. For...pegRNA primer binding site (PBS) and reverse transcriptase template (RTT). Created with BioRender.com. ...

...changes.   1. Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Transfection by Alyssa...editing. Using a fusion between Cas9 and reverse transcriptase and a prime editing guide RNA, it can introduce...

...other. Figure 1: Base transition editors catalyze purine-to-purine or pyrimidine-to-pyrimidine base...SpCas9 base editors with editing windows as small as 1-2 nucleotides. To reduce off-target effects associated...engineering and protein delivery. Nature Communications, 8(1), 15790. https://doi.org/10.1038/ncomms15790 Richter...the opposite strand). ABEs make an A to G change (or a T to C change on the opposite strand). Both are ...place on the free strand of DNA and converts a C to U without producing a double strand break. UGI = uracil...repair (HDR), the pathway for resolving DNA double-strand breaks (DSBs) preferred by researchers. Compared...are base transition editors: converting one purine base to the other or one pyrimidine base to the other...

...Consistency, and Transparency. This label scores products by their environmental impact on a scale of 1-10: the... but with the right incentives (for example, by grant agencies), we can shift toward a more sustainable...your RMV to the landfill, it’s collected and transferred to the Red2Green processing facility, where it...

...organized and boost your productivity in 2014. Tip 1: Use color coding, labels, and post-its to make finding...pile of lab notebooks and binder on your desk. Arrange them in chronological order (or by project, if ...Organize you experiments, keep track of upcoming grant deadlines, and sync everything to your calendar....lists of your upcoming tasks, set deadlines, and arrange automatic email reminders. These programs can be...

... western, proteins are: (1) separated by size, (2) transferred to a membrane, and  (3) detected using ...using antibodies.   Figure 1: Overview of the Western blot process. The sample, a mixture of protein...systems efficiently transfer a range of protein sizes in minutes without any transfer buffer. These systems...protein is separated in a gel, transferred to a membrane, stained with antibodies, and finally visualized...a broad size range to be separated on a single gel.  Immobilizing proteins on a membrane Once separated...migrate out of the gel and attach to the membrane.  In a wet transfer system the sandwich is completely submerged...filled with a transfer buffer formulated to conduct the electric current. The wet transfer process is very...

...also available for lentiviral transduction. Lysine-specific Demethylase 1 (LSD1) Tatjana Sauka-Spengler's...P2A-PuroR for lentiviral expression. Figure 1: dCas9-p300 adds H3K27ac marks to histones in promoter...light to regulate transcription, also includes light-regulated histone methyltransferases and deacetylases...CRISPR Epigenetics resource. Transcriptional activation p300 acetyltransferase dCas9 fused to the catalytic...other Tet1 delivery systems. Transcriptional repression DNA Methyltransferase 3 Alpha (DNMT3A) Vlatka Zoldoš... PuroR enable sorting and selection of transduced cells. Grant Challen’s lab also created constitutive...associated with transcriptional repression. Created with BioRender.com. DNA Methyltransferase MQ1 Margaret...

...synapses and deep-brain fiber photometry.  Figure 1: Characterization of iGluSnFR4 in mice. A) Scheme ...CycT1 or PolII-CTD antibodies and unlabeled MS in DLD-1 cells (colorectal cancer). Image courtesy of Brianna...al., 2025). In retrotransposon insertion, a combined endonuclease-reverse transcriptase uses homology ...fused to a shortened R2Tocc transposon protein, and can "retarget" the transposon to new loci in the genome...genome. The modified transposon RNA has untranslated regions (U, blue) on the outside of the homology arms...-associated membrane protein 5 (LAMP5) — as well as cholinergic (choline acetyltransferase, ChAT) neurons...is one of the most important and abundant neurotransmitters, but it is challenging to monitor because ...

...Presented in figure 1). More information on Agrobacterium-mediated transformation can be found in this...interest. Figure 1: Simplified schematic of agrobacterium-mediated transformation in plant cells. Agrobacterium...variants (hereafter referred to as Cas9) and a single stranded guide RNA (gRNA) is sufficient to modify the genome...variants have been created in an effort to increase translation in a particular plant species or cell type (Belhaj...CRISPR components can be expressed stably or transiently depending on the delivery method and cell type...CRISPR components can be delivered and expressed transiently using a standard detergent, Polyethylene Glycol...plant genome. B) The Ti plasmid and helper are transformed into agrobacterium tumefaciens and exposed to...

...furimazine by Promega’s Chemistry group is a good story (1). From a deep sea shrimp to a compact powerhouse of...cellular analysis products at Promega.     References 1. Hall, M.P., et al. (2012) Engineered luciferase reporter...process known as bioluminescent resonance energy transfer (BRET). The fluor then emits light at a tissue-penetrating... consists of NLuc fused to the more penetrative orange fluorescent protein fusion (OgNLuc). Both fluorescent...for excitation. The result was the cyan-excitable orange fluorescent protein, CyOFP1. CyOFP1 was molecularly...and improved maturation time compared to similar orange fluorescent proteins. In their work, the authors...great imaging tools generate cyan, yellow, green, orange and red fluorescence. The paper reports a wide ...

...relations, advocacy, and writing legislation (see Figure 1 for career map). The beautiful part of science policy...about science policy as a profession. Figure 1: Science policy can lead you to many paths! For a ...AAAS Science and Technology Policy Fellow at the Veterans Health Administration. “But, seriously, what even...Science and Technology Policy Fellow (STPF) at the Veterans Health Administration, I could never get one straight...variety of backgrounds with technical expertise that ranges from astronomy to bioethics to chemical engineering...happen at the national level within the executive branch or in Congress, or can also happen at the state... early career researchers who have decided to transition out of academia from graduate school or a postdoc...

...communication check out these SciComm blog posts. References 1. Bourdeau, Raymond W., et al. "Acoustic reporter genes...topics both interesting and approachable!   The grand finale of visual impressions was delivered by the...

...five methods for extracting DNA without a kit too. 1. Early Career Researcher Toolbox: Free Online Molecular...sharing even broader and began working with tech transfer offices to make reagents available to scientists...

...individually or as a complete kit from Addgene. References 1. Goulas, Theodoros, et al. "The pCri system: a vector...contains the MISTIC (membrane-integrating sequence for translation of integral-membrane protein constructs...glycosylate proteins and may recapitulate the post-translational modifications added to proteins in other eukaryotes...includes a special feature for difficult-to-purify membrane proteins. pCri vector pCri-13a, designed for use...protein tag. MISTIC drags tagged proteins into the membrane and may improve expression of proteins that natively...

...Molecular and cellular endocrinology 215.1-2 (2004): 1-10. PubMed PMID: 15026169. Soo, Rochelle M., et al...ve also carved pumpkins (yes, Blugene made an appearance there too) and shared fascinatingly creepy science...algae cell is just the cell wall and cytoplasmic membrane. Vampirovibrio chlorellavorus will not grown in... the snail, the larvae reach the eyestalk and transform them into a pulsating display imitating a moving...grasshoppers and crickets. When it’s ready to transform into an aquatic adult, it forces its insect host...uninfected grasshoppers, including those linked to neurotransmitter activity and geotactic behavior (Biron et ...

...proteins, DNA, and RNA used in CRISPR.   Figure 1: Overview of the parts of CRISPR. The bacterial chromosome...target and nontarget DNA strands upstream of the PAM site found in the nontarget strand. Protein Cas: ...sequences and keeping the array organized. Target strand: The strand of foreign DNA that Cas proteins recognize...recognize using an RNA guide. Non-target strand: The paired strand of foreign DNA cut by Cas9, whose sequence... and variable spacer sequences. The array is transcribed and processed into crRNAs, each including one...protospacer-adjacent motif” present in the non-target strand of DNA to be cut, which varies between CRISPR systems...from Addgene!   RNA crRNA: “CRISPR RNAs” are transcribed from the CRISPR array and include a spacer and...

...of experiments they are commonly used for.  Table 1. Points to Keep in Mind When Performing Biosensor ...enzyme or transcriptional activation, conformational changes, translation, and protein translocation. Targeting...triggers CaM/M13 interaction that increases FRET transfer between the fluorescent proteins. FRET is highly...characteristics like pH, redox state, and voltage across membranes. These sensors generally follow the same pattern...an organelle-specific targeting signal or a transmembrane domain, biosensors can be targeted to a particular...endoplasmic reticulum (ER), vacuole or cellular membranes. Examples include blue flame mitochondria-targeted...

...strategies in the comments section!   References 1. Yearly Planning Meetings: Individualized Development...then formulate a plan to further develop your transferable skills and reach your career goals.      Although... What Makes a Good Manager Anyway Developing Transferable Skills During Science Training ...

...fingers of a completely normal size.      References: 1. Doench, John G., et al. "Optimized sgRNA design to...end with little more than an Excel spreadsheet of random numbers that’ll leave you, well, #sad. Learn...

...et al., 2019; McLaughlin, 2019).    Figure 1: Schematic of multiplex immunofluorescence approaches...). Springer New York. https://doi.org/10.1007/978-1-4939-8935-5_26 Jambor, H., Antonietti, A., Alicea,...palette for display (like green-magenta-blue or orange-blue rather than red-green). Even better, show ....2846 Radtke, A. J., Kandov, E., Lowekamp, B., Speranza, E., Chu, C. J., Gola, A., Thakur, N., Shih, R...

...Kan, SpHIS5, or CaURA3 selection marker. Lee et al.(1) assessed many of these fluorescent tags in S. cerevisiae...life repair genomic damage, specifically double strand breaks. Researchers have long taken advantage of...product is introduced into the cell by direct transformation. A given insert will typically contain both...selectable gene product for isolation of successful transformants. Addgene distributes several ready-to-use, modular...

...plasmids CRISPRi in Caulobacter crescentus Figure 1: Serial dilutions of CRISPRi in Caulobacter cells ...The system consists of dCas9, the mammalian transcriptional repressor Mxi1, and an optogenetic tool that...CRISPRi plasmids A multiplexed gRNA piggyBac transposon system for human pluripotent stem cells Lindy...and gRNAs whose expression varies depending on transgene design and delivery. They generated hPSC lines...

...that work for our experiments!)     Figure 1: The IBEX Imaging Community front page. That’s...data, more discussion, and more functionality. Transparency, both in what works and in what doesn’t, and...Ichise, H., Gola, A., Kabat, J., Lowekamp, B., Speranza, E., Croteau, J., Thakur, N., Jonigk, D., Davis...00644-9 Radtke, A. J., Kandov, E., Lowekamp, B., Speranza, E., Chu, C. J., Gola, A., Thakur, N., Shih, R...

...social media? No problem - see our recap below! Day 1: gRNA design tools: How to Design your gRNA for CRISPR...or A:T->G:C edits in DNA without causing double-strand breaks. Our new page shows you base editing plasmids...certainly taken notice! Our newest page in the Transcriptional Regulation category already has five blue flame...with eSpCas9, SpCas9-HF1, & HypaCas9 Day 10: Transcriptional regulation: Truncated gRNAs for Regulating ...

...save that application for another day! References 1. Tanenbaum, Marvin E., et al. “A protein-tagging system...better. More transcription factor binding sites in a promoter lead to higher transcriptional activation....for single molecule imaging, finding that plasma membrane-targeted CAAX-SunTag was 18-fold brighter than...

...depends on what you are looking to study. Figure 1: A schematic showing the differences in chemogenetics... have pushed the boundaries in neuroscience by granting targeted control over neuronal activity. While... Mouse studies, however, require a permanent intracranial implant to be surgically installed to allow ...receptors Invasiveness Mice require permanent intracranial implant Noninvasive Controlling Stimulation...

... towards understanding this disease. Figure 1: Scientists are continuing to do essential research...community response initiatives. Accelerate the translational impact of research Universities have also been...creating research synergies and accelerating the translational impact of research activities. By streamlining...

... everyone was very approachable.     Fig. 1: Examples of DNA sequencing QC work    In addition...with a detailed training schedule that helped me transition easily. Training and responsibilities Through... the “9-5” was not as scary as it seemed. The transition into a traditional work environment was aided...