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...various teal, green, yellow, orange, and red fluorophores. The spectra for mOrange2 (scaffold of OCaMP) are...are shaded in orange. Data from FPbase.org. Scientists have developed calcium indicators spanning ...complement these tools, we recently developed a new orange calcium indicator, OCaMP (Aggarwal et al., 2025...2025), based on the fluorescent protein mOrange2 (Figure 2). Figure 2: OCaMP structure and mechanism...CC-BY-NC-ND 4.0 International license. Why orange? OCaMP’s emission peak at 565 nm fits neatly between...for two-photon imaging with newer lasers in the range of 1000–1080 nm. These industrial fiber lasers are...References Aggarwal, A, et al. (2025). A sensitive orange fluorescent calcium ion indicator for imaging neural...

...lactose analogue, IPTG (isopropyl β-D-1-thiogalactopyranoside). (To be completely accurate, IPTG binds...x-gal (5-bromo-4-chloro-3-indolyl-β-D-galacto-pyranoside) into galactose and an insoluble blue pigment...α-peptide) to a lacZΔM15-mutant bacterial cell in trans complements the mutation allowing for a functional...site (MCS) into the α-peptide (represented as an orange wedge in the figure on the left) and inserted it...produces pigment from x-gal (included in the transformation plate medium), turning the bacterial colony...IPTG is also included in the medium to ensure transcription of the lac operon.   Tips for blue-white screening...screening Use a good control: Transform the backbone plasmid without insert. All colonies on this plate...

...split-vector systems’ by separating viral genes and transgene expression across several plasmids. The most commonly...separate envelope and packaging plasmids as well as transfer plasmids. This concept ensures safe handling and...Gamma-retroviral vectors have a relatively high transduction efficiency, but in contrast to lentiviral vectors... are very versatile tools as they are able to transduce dividing and non-dividing cells. More detailed...Gamma-retroviral vectors Popular gamma-Retroviral Transfer Vectors   Expressing your gene of interest pMKO...events and is widely used in the field of mouse transgenics. Experimental uses include: Cre-dependent gene... lab. Learn more about CRISPR plasmids for transcriptional inhibition. Retroviral Plasmid Kit Retroviral...

...students first transform the S. cerevisiae met deletion strains and then isolate transformed strains on media...organism into overexpression plasmids. Students next transform deletion strains from the reference organisms ...to a whole genome duplication and extensive rearrangements in the S. cerevisiae lineage (7). In our class...class research project, students transform S. cerevisiae met deletion strains with overexpression plasmids...expression of the plasmid-encoded MET gene allows the transformed strain to grow in the absence of methionine (... to select yeast that have been successfully transformed with the plasmids, because the S. cerevisiae ...of MET/Met genes from the plasmids allows the transformed strains to grow in the absence of methionine....

...and spread over the second quadrant. The process continues until all quadrants are used, hopefully revealing...students transform them into different strains of E. coli,” says Westenberg. During the transformation experiment...plate, first the bacteria are inoculated over a quadrant of an agar plate. Then, a stick is dragged through...revealing single, isolated colonies in the last quadrant after incubation. The challenge is usually successful...genetic backgrounds of the host strains affect transformation efficiency.  Aside from the Streak Plate Challenge...

...growth rate/transfection efficiency is not known, this is easily achieved by splitting transfected cells into...specific cell line, and its transfection efficiency. We often trypsinize the transfected cells on the day after...after transfection into plates containing selection agent at different densities, then dispose of the plates...under-expressing clones. If GFP-encoding cDNA is co-transfected at a favorable ratio to the gene of interest ...such as the Viaflo from Integra Biosciences) to transition from a 48-well cell plate to a 96-well assay ...expression characteristics from a population of transfected clones. The technique is applicable to a wide...

...non-selective anterograde transport to a lack of genetic control. ATLAS (anterograde transsynaptic label based on...of the color spectrum more powerful, the lab of Francois St-Pierre recently developed a pair of new yellow...in other plant species, thanks to the broad host range of TRV. Find TRV-TnpB plasmids here! Weiss, T.,...Viral delivery of an RNA-guided genome editor for transgene-free germline editing in Arabidopsis. Nature Plants...lung cells via airway administration. AAV11 can transduce neurons for retrograde labeling, making it a powerful...allows VAMP2-At to remain on the presynaptic cell membrane while AF, fused to a payload, is released into...). ATLAS: a rationally designed anterograde transsynaptic tracer. Nature Methods, 22(5), 1101–1111. https...

...for a growing organism to make the complicated transition from a clump of cells to a fully differentiated...cooperate to cause a higher-energy electronic transition in a fluorescent molecule (see figure to the ...past 15 years efforts have been made to create transgenic mice expressing fluorescent protein reporter ...microscopy enabled scientists to see how chaotic and random the scanning was and how fast T cells could jump...Blue-green fluorescent proteins like EGFP Yellow-orange fluorescent proteins like TagRFP, tdTomato, DsRed...tdKatushka2 (Drobizhev et al. 2011) The commonly used orange and red fluorescent proteins are excited by 750...essential role for neutrophils in leishmaniasis transmitted by sand flies." Science 321.5891 (2008): 970-...

...epithelial origin. Suppressing function, creating tolerance, and evading recognition are a few methods by ...regulatory cells to suppress function. Creating Tolerance T-regs in the tumor microenvironment produce CTL... This in turn regulates the expression of the tolerance-producing antigens enunciated above. Finally, ...released from the microenvironment help create tolerance beyond direct proximity to the tumor; chemokine-aided...relapse. Chemical and radiation therapies cause a transient shutdown of the immune system, hampering its ability...microenvironment. Antibodies that block cell-surface tolerance-producing proteins are one of the most commonly...cells to knockout proteins involved in immune tolerance. Recently, China spearheaded the first ever CRISPR-based...

... of fluorescence: For applications that involve ranges of pH, and significant lengths of time, the stability...receptor conformation and signaling activity​​ (Sridharan et al., 2014). It's also crucial to evaluate how...the background. Dyes that can pass through cell membranes can prove useful for live-cell imaging. Experimental...considerations regarding the choice of fluorescent dyes (Sridharan et al., 2014). Manufacturers will typically recommend...accurate tagging. In Fluorescence Resonance Energy Transfer (FRET) applications, the dyes need to have compatible...and emission profiles to enable effective energy transfer, which is key to observing interactions and conformational... specifically to the AT-rich regions of double-stranded DNA and exhibits a blue-fluorescent light when...

...applicant to the Addgene, Seeding Labs Plasmid Grant. For this grant, we’re working with fellow nonprofit, Seeding...about the Addgene, Seeding Labs Plasmid Grant Read about Plasmid Grant Recipient, Gladys Kaba Resources on...their research. Kwabena came across the plasmid grant in Seeding Lab’s Newsletter and recently visited...Kwabena applied for the Addgene, Seeding Labs plasmid grant. 7:12 - 7:11 : Kwabena's work in Christopher E. ... recipient of the Addgene, Seeding Labs Plasmid grant and is working on a variety of projects in Ghana...

...fluor. Transcribing and translating a protein fluorophore also takes time - bypassing translation altogether... could be screened for their affinity to a wide range of non-nucleotide targets. These RNA molecules, ...The key to aptamers’ flexibility is their broad range of targets, including proteins, peptides, amino ...ions, and cells (even pathogenic bacteria). That range is possible because aptamers can either fold to ...biosensors. Aptabeacons Fluorescence resonance energy transfer (FRET) technology has been studied and used in...fluorescence-activated cell sorting (FACS) of E. coli transformed with aptamer libraries, the Jaffrey group selected...

... the P and V proteins of Simian virus 5 (SV5) (Randall et al., 1987). The epitope (GKPIPNPLLGLDST) has...10.57733/addgene.l0ulu4. Hanke, T., Szawlowski, P., & Randall, R. E. (1992). Construction of solid matrix-antibody-antigen....org/10.1099/0022-1317-73-3-653. PMID 1372038. Randall, R. E., Young, D. F., Goswami, K. K., & Russell...functional properties, and is amenable to a wide range of biophysical assays (Kroupova et al., 2024). They...using mCherry for selection of sgRNA plasmid transformants, they avoid M. abscessus’ high rate of spontaneous...workflow in M. abscessus. A) Sth1Cas9 mediates double strand break at the target site, which results in either...indels after DNA repair. B) M. abscessus is first transformed with Cas9 on an integrative plasmid containing...

...the proteins cooperatively can separate the DNA strands. Created with BioRender.com. Once DnaA breaks...it recruits helicases to continue unwinding the strands. This creates two replication forks that move in...elongation phase, a DNA polymerase synthesizes a new DNA strand at each replication fork. Termination sequences...act as a feedback mechanism to reduce further transcription. Replication is also regulated by the balance... concentration is controlled at the level of translation. RepZ mRNA must fold into a “pseudoknot” held...intramolecular base pairs for the protein to be translated. An antisense RNA called Inc, also encoded by...prevents the mRNA from folding and blocks RepZ translation (Asano & Mizobuchi, 1998). This system keeps ...

... and inhibit its own transcription. As copy number increases, RepA transcription is repressed and replication...indirectly inhibit replication through inhibiting translation of replication machinery proteins, or directly... division. They don’t simply rely on chance (or random diffusion) to make this happen.  There are several...CBPs and NTPase to go around, the plasmids are randomly positioned, leading to plasmid loss (Diaz et al... if these do not provide clarity, a wet lab transformation experiment will ultimately inform whether your... Partition-associated incompatibility caused by random assortment of pure plasmid clusters. Molecular ...classification and identification based on replication and transfer systems and host taxonomy. Frontiers in Microbiology...

...of customizable DNA-binding domains based on transcription-activator-like effectors (TALEs) as anchors ... thaliana. The results are light-inducible transcriptional effectors (LITEs) designed to bind specific.... Genes under LITE control showed a rise in transcription in as little as 30 minutes after light stimulation... universally abundant. Second, induction of transcription occurs within minutes of light exposure. Third...really flexible and modular,” Konermann said. “Transcriptional activators are easy to expand or combine with...For those who do want to apply LITEs for the transformation of neurons, she offers a tip: save yourself...al. "Optical control of mammalian endogenous transcription and epigenetic states." Nature. 500, 472–476...

...Hola! Que tal? My name is Brandon Solis, and I am a rising senior studying Sociology and Dance at Skidmore...interests are very clear, I had no idea how to translate them into real world careers. I figured this internship...a way of challenging what I am used to. Brandon Solis, marketing intern. Something that I struggle...world, I was worried about how my ideas would be translated through Zoom calls and Google docs. I pondered... do was create content that was unique but also brand friendly. I introduced the team to a new design ...program, Canva, and I was able to create cute and on-brand templates for these posts. Some of recent ... Brandon Solis...

...scientists, tech transfer offices, research foundations, and funding initiatives such as Fast Grants that support...worked closely with the legal partners and Tech Transfer Offices at universities to make COVID-19 research-related...United States, and many universities and Technology Transfer Offices were, at least, temporarily closing, these...Texas at Austin University of California, San Francisco  University of Pennsylvania Stanford University...them available to industry, please reach out at techtransfer@addgene.org and we will initiate a conversation...conversation with you and your technology transfer office to discuss the possibilities for expanded sharing. ...

... tissues are nearly transparent. As Verkhusha explained it to me, the transparency window of mammalian...tumor. They wanted to find something better. Transparency window That’s exactly what they reported doing...proteins that would fall within that near-infrared range, and that’s exactly what iRFPs achieved. Those proteins...corals, but all of them fluoresce outside the transparency window of mammalian tissues,” Verkhusha said...near-infrared fluorescent proteins within this transparency window, so now, we could see deeper.” Adding... proteins that operate within the 750 to 800 nm range, and his team is also looking in the direction of...

...is composed of single stranded DNA, which is particularly well suited for strand invasion, a key step ... are then packaged into AAV virions via transient transfection of 293T cells with helpers, and used to...non-sequence specific frameshift mutations through aberrant double strand break repair, it has proven more challenging...identical targeting vectors delivered through transfection of naked DNA. Other investigators who had been...AAV-based gene editing via the introduction of a double strand break by using a targeted nuclease. Asuri et al...

...emphatically deny that I have a magic formula to guarantee a reader the faculty job of his/her dreams. There...currency of scientific success is publications and grants. 2. Write succinct and easy to understand documents...years. You will discuss the focus of your first grant, how you will overcome certain obstacles, alternative...sentences) What will be the focus of your first grant? (1-2 sentences) Simply stated, what are your...It's not about you and how you will finally be granted the faculty position you have craved since grad...how YOU will fit into a department and bring in grants. What kind of colleague will you be? Do you understand...takes to be successful in running a lab, getting grants, and writing papers? This means that once you get...

...dopamine (DA), a well-known reward chemical and neurotransmitter. But aside from DA’s famous role as the “happy...standard method of measuring DA and other neurotransmitters in the brain has been in vivo microdialysis...performed with a probe surrounded by a semipermeable membrane, which is inserted into the region of the brain...passive diffusion of DA across the semipermeable membrane from the extracellular fluid into the perfused... to a threatening looming stimulus in vivo in transgenic zebrafish Report DA release in live mice over...developments in the detection and measurement of neurotransmitters in various species and while exhibiting complex...Dopamine-sensitive adenyl cyclase: possible role in synaptic transmission." Science 174.4016 (1971): 1346-1349. PubMed...

...reverse transcribe their gRNA into DNA, which is then transcribed into mRNA before being translated into ...genome types that can be either double stranded (ds) or single stranded (ss) (ie, dsDNA, ssDNA, dsRNA, positive... of virus. RNA genomes can be transcribed into mRNA, directly translated into proteins, or, in the case...

...bound to anything? Is it a transmembrane protein, or is it know to translocate or fold in certain conditions...as part of performing a specific function. A transmembrane protein that is being used as a marker for flow...cell, instead of when it is attached to a cell membrane.  Picking the Right Antibody  It can get even ...looking for a protein of interest while it’s in a transition state or specific configuration. So how do you...

...factors that could be transferred between cells. At the time, these extranuclear agents of heredity were...was proposed a few years later by Élie Jacob and François Wollman and became the widely adopted name for...-factors. Like F-factors, R-factors could be transferred between bacteria via cell-to-cell contact; however...pSC102, with EcoRI and selected for E. coli transformants that were resistant to both. When this proved...Chizuko Ogata, and Sachiko Sato. “EPISOME-MEDIATED TRANSFER OF DRUG RESISTANCE IN ENTEROBACTERIACEAE VIII:...PubMed Central PMCID: PMC314833. Datta, Naomi. “Transmissible Drug Resistance in an Epidemic Strain of Salmonella...

...field is a series of fluorescence resonance energy transfer (FRET)-based calcium biosensors. Perhaps most ...developed and published in 2010 for visualizing the transcription factor cAMP-responsive element-binding protein...really important and when? That is not known.” Transition Metals Maarten Merkx of Technische Universiteit...particularly for the intracellular imaging of transition metal ions, such as zinc, copper, and iron. “...the sensors involves plenty of trial and error. Transition metals also present challenges in that they are... way to make a FRET sensor with a large dynamic range,” he says. Merkx recently applied this “trick” to...red-shifted FRET sensor for zinc using variants of mOrange2 and mCherry as donor and acceptor domains. He ...

...contamination from uncut plasmid. 4. Transformation Transform your ligation reaction into your bacterial...perform, at minimum, two transformations after a ligation: 1. Control Transformation containing the ligation... how many colonies you should expect in the transformation due to background re-circularization and contamination...competent cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent...ligation mixture with backbone alone; 2. Transformation containing ligation mixture with insert and backbone...conduct a positive control to ensure that your transformation worked. You should also verify that you are...

...more detailed level, the Gal4/UAS system is a transcription activation system co-opted from yeast (1). It...workhorse to turn genes on or off. Gal4 is a transcriptional activator that binds to UAS enhancer sequences...sequences found in DNA. It then recruits transcription machinery to the site to induce gene expression. Thus...reads “#” as a “pound symbol”. References 1. A. H. Brand, N. Perrimon, Targeted gene expression as a means...15262411. 4. T. D. Southall, D. A. Elliott, A. H. Brand, The GAL4 System : A Versatile Toolkit for Gene ...Lai, T. Lee, Genetic mosaic with dual binary transcriptional systems in Drosophila., Nat. Neurosci. 9, 703...The Q system: a repressible binary system for transgene expression, lineage tracing, and mosaic analysis...

... seems straightforward: E. coli bacteria are transformed with the library DNA, they grow and replicate... Each Addgene library has its own depositor transformation and amplification protocol, but the overall...Maintain representation of the library during transformation First, use enough DNA and competent bacteria...and we use electroporation to ensure a good transformation efficiency. For a typical library, Addgene ...growing the cells on solid plates, where each transformant forms its own colony, uninfluenced by the growth...concern, but Addgene still recommends plating the transformation mixture on solid media. The colonies are then...that were amplified preferentially). You can also rank the plasmids by abundance, scaled to 1, to create...

...responses are varied; one outcome is directed transcription from a given promoter. To harness the power ...the RR (OmpR) via phosphorylation, promoting transcription (see figure below). Subsequent exposure to red...response regulator via phosphorylation, promoting transcription of a GFP reporter. Subsequent exposure to red...activity persisted in the inactive state. The dynamic range, defined as the difference between the lowest and... reduced system leakiness and increased dynamic range to around 100-fold. These improvements will allow...Applications of these systems The large dynamic range of these systems ensures that they can be used with...types of proteins, including Cas9 and dCas9-transcription factors, for which low expression levels are...

...Wondering how you can develop more transferable skills? See Developing Transferable Skills During Science Training...of the University of British Columbia. When transitioning from an academic science career path to a non-academic...advantage over other candidates. Write about your transferable skills Some of the skills you developed might...research. For example, did you help put together a grant application? Are you familiar with the publishing...have presented in some public lectures? These transferable skills could be meaningful for your potential...Originally on track for a research career, she transitioned into a communications role instead after she...

...brightness When a dish of mammalian cells is transiently transfected with a plasmid containing a fluorescent...fluorescent proteins, e.g. see Chudakov et al (2010), Cranfill et al (2016) or Thorn (2017). However, the theoretical...developed in our lab uses a reference protein that is translated from the same open-reading frame and separated...reference protein) under identical conditions, a ranking of the practical brightness can be made (Goedhart...repeating this with another fluorescent protein, a ranking of practical brightness can be made. This strategy...the comparison of the practical brightness of two orange fluorescent proteins, differing by only a single...

...synaptic transmission. Cleavage of VAMP2 renders it inactive, thus inhibiting neurotransmitter release....Plasmids podcast Light-mediated block of synaptic transmission Article contributed by Klaus Wanisch  Listen...enable VAMP2 cleavage, and suppresses neuronal transmission within minutes. Constructs using the photodimerizer...utility of light-mediated suppression of synaptic transmission in brain slices from mouse, and showed that ...recognized as important regulators of post-transcriptional gene expression across the majority of cellular...an unregulated fashion and thus a marker for transfection. The other (mKate2) is regulated in the 3’ UTR...

.... elegans. These dsRNAs target specific mRNA transcripts for degradation. Despite how easy these experiments...often completely ineffective or results in low penetrant phenotypes that are hard to study. To overcome...target gene between two inverted T7 promoters. Transcription from each promoter progresses past the specific...podcast segment A fundamental approach to studying membranes, organelles, and other subcellular compartments...enzymes function across a much broader temperature range than BioID, which only functions optimally at 37...and C. elegans.   Find the TurboID plasmids here! Branon et al. Nat Biotechnol. 2018 PubMed PMID: 30125270...This tetramer cleaves chlorophenol red-b-D-galactopyranoside on the assay paper turning it purple, allowing...

...luminescent) worlds Forster Resonance Energy Transfer (FRET) is the transfer of energy from an excited donor fluorophore...Resonance Energy Transfer (BRET) works the same way, except that energy is transferred from a luminescence... elements — such as promoters, enhancers, or untranslated regions — on gene expression. The regulatory... fluorescent protein close enough, energy is transferred by BRET and the fluorescent protein emits light...as the most efficient means to disrupt the cell membrane and deliver the substrate; however, secreted luciferase...

... has recently published techniques to follow translation in living single cells. However, before CRISPR-Cas9...the Max Planck Institute for Brain Research in Frankfurt am Main, Germany. She presented a novel technology...to be invited to learn from them at the MPI in Frankfurt am Main and perform similar experiments. Visualizing...Both antibodies have a short antibody specific DNA strand added to them. Only if the proteins of interest...are in close proximity to each other can the DNA strands take part in rolling circle DNA amplification. ...microscope she likes to travel, discover new restaurants, go to concerts and bake. you can contact her...neurons reveals a mechanism for regulating its translatability." Science 343.6169 (2014): 419-422. PubMed ...

...Cas9 fused to the reverse transcriptase (RT), which induces two double-strand breaks and deletes the intervening...They further develop a version of SEND for gene transfer with pseudotyped viral particles, expanding options...enzyme from the Liu lab to target opposite DNA strands and specify a large intervening deletion. Not only...deletions and introduce epitope tags and RNA transcription start sites. The lab further demonstrated that...uses paired pegRNAs targeting complementary DNA strands to program large genomic deletions with or without...PEDAR is capable of correcting larger genomic rearrangements as demonstrated in a mouse model of tyrosinemia...

...the filament from (a-b), showing the appearance and disappearance of 649-mDia1 or 549-CP (upper, middle...takes is a bit of chemistry magic to produce a wide range of useful molecules linked to that substrate. If... with SiR-CA (magenta) and outer mitochondrial membrane labeled with SNAP-OMP25 with Atto590-BG (green...separately with different dyes. b) mDia1 and CP appearance at the filament tip from (a). c) Intensity traces... probes are not very good at crossing the cell membrane. So, you should investigate your options and weigh...are typically 230–250 aa. Smaller size isn’t a guarantee that a tag won’t disrupt the function of the target...https://doi.org/10.1038/nmeth749  Strebinger, D., Frangieh, C. J., Friedrich, M. J., Faure, G., Macrae, R...

... efficient double stranded templates, rather than the more efficient single-stranded oligodeoxynucleotide...generally very small, and can contain different neurotransmitters. Using genetically encoded fluorescent proteins... them to localize to the synapse and minimize extraneous fluorescence from other regions of the cell. ...synaptic maturation pattern of hippocampal adult-born granule cells over time that were not previously possible... al., 2020). Figure 3: BaF3 cells were transduced with H2B-FT virus and grown in varying concentrations...lentiviral CRISPR library that targets human glycosyltransferase genes and other proteins related to cellular...

...their mutations corrected, and the T-cells were transferred back into the infants. Almost all patients have...repertoire (1). In this example, it was possible to transduce the T-cells outside the body and thereby correct...techniques may hold the key. In the simplest form, translating gene therapies from conceptual design to clinical...that carries a small, approximately 4.7 kb single stranded genome. AAV belongs to the family Parvoviridae...AAV vectors are also capable of infecting a wide range of cell types including but not limited to muscle...disease-associated mutations. AAV vectors maintain persistent transgene expression over many years in postmitotic, long-lived...targeting and enhances endonuclease delivery for double-strand break repair." Gene therapy 17.9 (2010): 1175-1180...