... Figure 1: Photoswitchable MEK1 (psMEK1). Is it possible to create other photoswitchable kinases? The... lab present a generic design for creating photoswitchable kinases and use MEK1 as a model kinase. In ... MEK1 activity by attaching two dimerizing photoswitchable Dronpa (dron= a ninja term for vanishing, and...site of a constitutively active MEK1. This photoswitchable MEK1 is called psMEK1. When exposed to 500 ...psMEK1 also worked for creating three other photoswitchable kinases: psMEK2, psRaf1, and psCDK5. MEK2 is... less similarity. For these three kinases, photoswitchable Dronpa domains were inserted at sites homologous...locations they were attached to in MEK. The photoswitchable versions of MEK1, MEK2, Raf1, and CDK5 all ...

...place to provide community protection. Toehold switches for pathogen detection In 2014, as postdoctoral...of programmable riboregulators, called toehold switches, which allow for the rational design of RNA sensors...samples (e.g.: blood, urine). Adapting toehold switches for Zika detection We and other members of the...ordering several dozen versions of the toehold switches and amplification primers. This helped make sure...as one week (Pardee et al, 2016). Using toehold switch-based RNA sensors, our approach uses portions of...we have embedded this next generation of toehold switch-based RNA sensors within an extensive infrastructure...directed isothermal RNA amplification, new toehold switch designs, CRISPR/Cas9-mediated genotyping, as well...

...Cas9, the PITCh-gRNA, and the locus-specifc gRNA. The PITCh-gRNA creates two DSBs in the PITCh plasmid ...the PITCh-gRNA. Step 3: Contransfect the PITCh vector with the vector carrying Cas9 and the PITCh- and...knock-in, termed PITCh (Precise Integration into Target Chromosomes). Using their PITCh plasmids, GFP knock-in...facilitates easier cloning. An overview of PITCh. The PITCh plasmid contains a GFP-Puro cassette flanked...flanked by 5' and 3' microhomology and PITCh-gRNA binding sites. This PITCh plasmic is cotransfected with a ...Abbreviated protocol for PITCh Step 1: Generate microhomologies in the PITCh vector ~20 bp 5’ and 3’ ...These results indicate that PITCh is a robust method for genomic insertion. PITCh can also be adapted for ...

... off-target edits to occur. Although methods to switch on Cas activity using light or drugs have been ...been developed, the field lacked an “off-switch” for Cas proteins. Discovery of anti-CRISPR proteins That...edits. But, just as important is a way to flip the switch and turn off CRISPR activity. The ability to turn...KL, Davidson AR (2016) Naturally Occurring Off-Switches for CRISPR-Cas9. Cell 167:1829-1838.e9 . https...

...night is about you! The FLEx (for flip-excision) switch (aka DIO (Double-floxed Inverse Orientation) or...PubMed PMID: 6319400. Atasoy, Deniz, et al. "A FLEX switch targets Channelrhodopsin-2 to multiple cell types...

...Listen to the photoswitchable protein podcast segment Reversible control of photoswitchable proteins, which... inhibitor CASANOVA for “CRISPR-Cas9 activity switching via a novel optogenetic variant of AcrIIA4.” CASANOVA... Discovery of new protein binder tools for photoswitchable proteins cPYP and AsLOV2 Article contributed... either the light- and dark- states of the photoswitchable cPYP, which changes in conformation in response...bind to only the light- or dark- state of the photoswitchable proteins offers a powerful approach to control...to either the light- or dark- state of the photoswitchable proteins) fused to mVenus linked to a mitochondrial... plasmid tools were also deposited for the photoswitchable AsLOV2. Find the cPYP and AsLOV2 protein binder...

...Neurons in the Periaqueductal Gray Facilitate the Itch-Scratching Cycle via Article Tac1-Expressing Neurons...Neurons in the Periaqueductal Gray Facilitate the Itch-Scratching Cycle via Descending Regulation. Neuron...nucleus accumbens medial shell and behavioral switching. Neurochemistry International 129:104482. PubMed...

...the assembled parts. Tips and Troubleshooting “Stitching” fragments together using oligos When you need...sequence between two PCR products, one method is to “stitch” together several oligos. This technique is especially...Pro-Tip Please note that the way to design the “stitching” primers and the amounts of primers to include...

...Iodixanol Gradient Ultracentrifugation Protocol Mulitchannel Pipetting Technique Learn how to pipette multiple...

...expression of another! What Are FLEx switches? FLEx (or “flip-excision”) switches were designed as a genetic tool...powerful FLEx switch can be created for your specifc experimental needs. How does the FLEx switch work? Let... post will focus on one such strategy--the FLEx switch--which utilizes recombination elements to turn ...vivo using site-specific recombination. The FLEx switch takes advantage of the orientation specificity ...Let’s say you want to design a genetic FLEx switch that turns BFP expression off, while turning on mCherry...mCherry expression. For this FLEx switch to successfully work, the cassette would need to contain the BFP... Schnutgen et al. Nat Biotech 2003): Basic FLEx switch will express BFP in the absence of, or mCherry ...

...reversible PA-FPs (also known as photoswitchers) can be photoswitched from a dark state to a bright fluorescent...color to another, or their fluorescence can be switched on and off reversibly. This ability to modulate...Irreversible photoactivation Irreversible PA-FPs can be switched on from a state of dim or no fluorescence, to ...exposure to a specific wavelength of light. The switch or photoconversion usually takes less than a second... after the goddess of dawn in Greek mythology) switches from green to red fluorescence. mEosFP exposure...dark non-fluorescent state (“quenching”). The photoswitch can occur multiple times by activating with two...reversible PA-FPs also display this “negative” photoswitching. In 2008, scientists used mutagenesis screens...

...Tichy et al., 2019), photoswitchable tethered ligands (PTL), and photoswitchable, orthogonal, remotely-tethered...pathways? Here’s where photoswitchable intracellular proteins come into play. Photoswitches can be used to activate... G-protein coupled receptors (OptoXRs), and photoswitches. Light-gated ion channels directly open and ...potential and a host of other cellular functions. Photoswitches are engineered molecules that induce conformational... special, covalently linked molecules (i.e. photoswitches) attached near the binding pocket of an engineered...activates downstream secondary signaling pathways. Photoswitchable tethered ligands hold the ligand in close proximity....   Controlling secondary pathways with photoswitches OptoXRs are great for activating entire signaling...

...Plasmids: pX330S-2-PITCh - expresses Cas9 & PITCh-gRNA pX330A-FBL/PITCh - expresses Cas9, PITCh-gRNA, & FBL-...97.2 KB Yamamoto PITCh Tagging System The Takashi Yamamoto lab has deposited the PITCh system plasmids ...vectors as well as pX330S-2-PITCh, expressing a generic gRNA targeting the PITCh donor plasmid. Note that...FBL-specific gRNA pCRIS-PITChv2-FBL - PITCh donor vector for EGFP-2A-PuroR knock-in into human FBL locus...cassette to the C-terminus of endogenous proteins. The PITCh system depends on MMEJ (microhomology-mediated end-joining...targeting the genomic site and the donor vector. The PITCh donor plasmid with an EGFP-2A-PuroR cassette, flanked...sequences and gRNA target sites. The first deposited PITCh plasmids were tested by fusing EGFP-2A-PuroR cassette...

...What is an Antibody animation!  Isotype switching Isotype switching occurs when a B cell changes which heavy...there? Why do they matter? And how do antibodies switch isotypes? What is an isotype? An isotype is a class...other isotype heavy chain constant domains. This switching helps B cells adapt during the course of an immune...the effector function of the antibody. Isotype switching is caused by a specialized type of DNA recombination...domain genes in the cluster. However, when B cells switch to expressing a different isotype, the intervening... constant domain gene. For example if a B cell switches to producing IgE antibodies, the gene segments...IgG are deleted.     Figure 2: Isotype switching requires DNA recombination of the antibody gene...

...elevator pitch, talks, the poster session, networking, and social media. The elevator pitch Before the...the conference Practice your “elevator pitch,” the 30 - 60 second blurb you will use to introduce yourself... are familiar with. You can tweak your elevator pitch slightly if you know a bit more about the other ... After the conference After using your elevator pitch several times at the conference, you might notice... some trends. Were there parts of your elevator pitch where the other person seemed confused, bored, or...particularly excited about? If you tried tweaking your pitch throughout the conference, what worked? Use this...

...Their most successful design utilized Magnet photoswitchable proteins derived from the fungal photoreceptor...to Turn Off Genome Editing Single-chain Cas9 photoswitches Photoactivation of Cas9 can also be achieved...cleavage. This technique was applied to create photoswitchable spCas9, dCas9, and S. aureus Cas9 (saCas9) ..., check out our blog post: Anti-CRISPR Proteins Switch Off CRISPR-Cas Systems.  Listen to the anti-CRISPR...resulting system was coined as CRISPR–Cas9 activity switching via a novel optogenetic variant of AcrIIA4 (CASANOVA... be used for in vivo models Single-chain Photoswitchable Cas9 Richter et al., 2016 tsRC9 RsLOV from... (2013) Engineering of temperature- and light-switchable Cas9 variants. Nucleic Acids Research 44: . https...

... wrap the column with blue LED lights. When you switch off the lights, OptoNBs bind YFP and non-specific...designed by the Toettcher Lab, OptoNBs are photoswitchable engineered nanobodies whose binding of their...an OptoNB, the AsLOV2 domain acts like an on/off switch for the nanobody’s ability to bind its target protein...Purifying an untagged protein with a flip of a light switch would improve this process. The team used nickel-coated... or a ligand’s activity, then a flip of a light switch could control a signaling pathway. Ultimately, ...Optogenetic control of protein binding using light-switchable nanobodies." BioRxiv (2019): 739201.  Additional...

...e43072. doi: 10.1371/journal.pone.0043072. Photoswitchable tools for spatial and temporal control of cell...Three years ago, Brian Kuhlman’s lab designed photoswitchable dimers by fusing the SsrA peptide with the ...high throughput binding assays, to engineer photoswitchable dimers which show over a 50 fold change in ... tools which allow you to easily and reversibly switch off and on your favourite signalling pathways (...mitochondrially targeted photoactivatable and photoswitchable FPs that are activated by UV light (300-400nm...mitochondrially-targeted variant of PSmOrange, a photoswitchable fluorescent protein which transitions from ...mitochondrial subpopulations within the same cell; by photoswitching PSmOrange and photoactivating PAGFP in separate...

...has proven useful in the engineering of riboswitches. Riboswitches are naturally occurring regions in the...placing Spinach downstream of a riboswitch, metabolite binding to the riboswitch instead activates Spinach ...ingredients reagents permeate the molecular biologist’s kitchen lab - and scientists using aptamers might know ... the expression of coding RNA. Spinach-based riboswitches can be used as stable metabolite sensors for...

...interest makes FLEx switch a powerful experimental tool. For more information on FLEx switch and what you can...Hu 154018 LiOn-CMV∞Cre leak-proof iOn (LiOn) Cre switch CMV Mammalian Livet loxP (floxed) Constructs These... Cre expression promotes shRNA expression. Gene Switch: These constructs contain two genes of interest...frame to allow in-frame translation of Gene B. FLEx Switch ∗ : This system allows scientists to utilize recombination...while simultaneously turning on another. The FLEx switch system takes advantage of the orientation specificity...Mouse Targeting Shivdasani 18925 pJ241-FLEX FLEX switch empty backbone Sternson 38042 pAAV-CA-FLEX Cre ...Mammalian Cepko 8389 p212 pCMV-EGFP/RFP EGFP-dsRed gene switch plasmid Mammalian Green 22799 Ai9 Rosa26 targeting...

...greatest challenges of neuroscience.”  Hot plasmids: Twitch sensors Griesbeck’s recent contribution to the ...based calcium biosensors. Perhaps most notably, the Twitch calcium sensors, which are available at Addgene...in vivo imaging. Griesbeck explains, while these Twitch sensors are always bright, they change color as...reported in Nature Methods, the FRET changes of these Twitch sensor variants were optimized by testing various... rat hippocampal neuron cultures. The optimized Twitch sensors allowed the researchers to see tonic action...

...optogenetics plasmids such as microbial opsin and optical switch tools to measure and manipulate cells and their... Protein Guide Microbial Opsin Plasmids Optical Switch Plasmids Background Optogenetics plasmids encode...proteins. These fusions can then be used as an optical switch to control protein interactions, dimerization, ...spectra) of different types of opsins and optical switches available in these tables, refer to Addgene's ...Type Promoter PI Publication Return to top Optical Switch Plasmids Browse, sort, or search the table below...

...to the switch and exposes its ribosome binding site, allowing for the gene on the toehold switch to be ...sequence of the toehold switch. For example, Green has also developed toehold switches to detect Zika virus...amplified isothermally, and detected through a toehold switch, an RNA sensor that can be used to detect the presence...

...we discussed inducible promoters, which can be switched from a default OFF to an ON state, and how you...ll look at repressible promoters, which can be switched from a default ON to an OFF state, as well as ...plasmids, see our Tetracycline resource page. Cumate Switch Massie et. al developed the cumate-inducible gene...promoter and turning transcription OFF. The cumate switch system functions well in mammalian cells, and can..., A., Xu, Y., Warren, R. et al. The cumate gene-switch: a system for regulated expression in mammalian...

...overexpressed in cells, microtubule growth can be switched off with blue light which causes LOV2 and Zdk1...cells. These tools consist of cobalamin-binding riboswitches that bind to cobalamin-fused fluorescent dyes...dyes; binding of the cobalamin fusion to the riboswitch results in increased fluorescent signal. Prior ...find plasmids containing the cobalamin binding riboswitches used in the Riboglow platform at Addgene. ...

...your idea is viable.  Go to experts you trust and pitch what you hope to build.  Then ask for their honest...honest opinions -- this part might hurt, but if you pitch to enough people, you will find the answer to your...an impossibility at our early stage.  After each pitch we made, we considered the feedback and adjusted...viable.  This is not to say that we aren’t still pitching for feedback.  Your venture should continuously...

...derivatives of violacein. Using dCas9 as an on/off switch The multiple scRNAs all individually utilize the... can be regulated with dCas9 as a master ON/OFF switch. This will likely prove particularly useful in ...distinct programs controlled by each dCas9 regulatory switch. By no means limited to the field of metabolic ...variants can be used as regulatory ON/OFF master switches to induce multiple transcriptional programs. ...

...Xin Zhou greatly expanded that list by using photoswitchable fluorescent proteins to create enzymes that...turned the enzymes back on. Xin termed these photoswitchable enzymes Fluorescent Light-inducible Proteins...various designs, Xin has been able to develop photoswitchable Intersectin, Cdc42, Raf1, Mek1, Mek2, Cdk5,... You can learn more about Xin’s work with photoswitchable components of the ERK signaling pathway in ...

...infection. Cre or Flp dependent switches control gene expression via switches placed in your cell type of ...specialized promoters, Flp or Cre dependent gene switches, and serotype selection. Promoters and serotype...interest. Careful combination of promoters, gene switches, and/or serotype can also be used to ensure specificity...

... Synapsin1 promoter, Cre mediated flip-excision switch, for AAV production or direct transfection, slow...parapinopsin driven by the CAG promoter. Used for photoswitchable control of inhibitory GPCR signaling cascades...parapinopsin driven by the Ef1a promoter. Used for photoswitchable control of inhibitory GPCR signaling cascades...

... Tian Calcium Sensor: Twitch2B 100040 pAAV.hSyn1.Twitch2B.WPRE.SV40 Syn Twitch2B none Constitutive 1 Griesbeck...ribo-GCaMP8 GCaMP7 GCaMP6 axon-GCaMP6 soma-GCaMP6 Twitch2B Yellow Calcium Sensors jYCaMP1 axon-jYCaMP1 Red...

...function. Adapting Toehold Switches to Detect Zika Virus The RNA-based toehold switches described in this post...binding, produce reporter proteins. These toehold switches can be used to quickly detect the presence of ...

... discussion on the way to the office or in the kitchen, while sipping an afternoon coffee in between experiments...a coffee, or simply moves away from the desk to switch off a few minutes. The host could also set aside...used and could bring back a taste of that casual kitchen chatter, ultimately sparking new ideas. Social ...

...subclass specific secondary antibodies and subclassed switched R-mAbs, multiple targets could now be visualized...Fig. 2. Multiplex immunolabeling with subclass-switched recombinant antibodies in adult rat brain. Figure...Engebrecht J, Trimmer JS. A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced...

...beyond stability, adding color and even on-off switches. Stabilizing gRNA modifications  Sugar phosphate...modifications to improve stability.   On-off switches for gRNAs A major drawback of CRISPR-Cas9 technology..., E., Satyapertiwi, D., Ramel, MC., et al. Photoswitchable gRNAs for Spatiotemporally Controlled CRSIPR-Cas-based...

...to control cells or organisms with the flip of a switch. You may be familiar with popular light-sensitive...activity. Researchers have designed a variety of photoswitchable elements into many other types of proteins....in the Gadella Lab devised a set of optogenetic switches for the GEFs ITSN1, TIAM1, or p63RhoGEF (which...

...were a way to just make one fusion protein that switches to any color or tag you want? It sounds like magic...ticker tape” method for intracellular recordings, switching between different colored ligands at different...increasingly popular. Embrace the flexibility and switch things up for your next experiments! References...

... or activity can be achieved using switches, including riboswitches and optogenetic and chemogenetic tools...tissue or cell type specificity. An on/off genetic switch can be used for expression control. (c) Viral delivery...Dual expression Promoters, miRNAs, enhancers, and switches all take up precious cargo space in AAV vectors...

...Other ways to make the office more eco-friendly The switch to 100% renewable electricity is certainly not ...sustainability effort we’ve implemented at Addgene. The kitchen and office areas provide multiple streams for composting...

...advance your career, promote career growth, and/or switch your field of work, both in academia and in industry...specialized so it might lend itself more readily to a switch. 4. Do you want more autonomy?  Before my master...

...depolarization. They therefore function as a neuron “on switch.” Neuronal inhibition: Hyperpolarization Conversely...the cell. It therefore functions as a neuron “off switch.” Because of the differing peak absorption wavelengths...

...zebrafish embryo over an 80 minute time course.  By stitching together a stable scaffold and HA-binding regions...protein dynamics in vivo. In the future, this stitching technique could be applied to other epitopes and...

...activates transcription with the flick of a light switch and shuts off just as quickly. The original AraC...bacteriograph was created using 160 individual images stitched together. Image from Romano et al., 2021 with ...

...articles. Think we missed something smokin'? You can pitch a Hot Plasmids section (antibodies and viral preps...Andrews NP,  et. al. A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced...

...we’ll discuss inducible promoters, which can be switched from an OFF to an ON state, and how you might ...The Promoter Region Plasmids 101: Cre-lox Light-Switchable Two-Component Systems for E. coli Browse other...

...everything is conceptual, and the team that packages its pitch the best, wins. He explained that entrepreneurs ...the team works well, the idea is solid, and “the pitch is packaged to perfection”, seed money will hopefully...

...At the heart of their method is the RNA toehold switch, a RNA hairpin that can be designed to bind and...interest is a benefit of this method. Find the toehold switch microbiota/biomarker sensors here Studying the ...

...threshold like a switch that turns on or off. CRISPR shows a more continuous response - not switch-like at all...

...Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre–...

...FRET sensors, can reduce these unwanted variables. Twitch is a FRET-based calcium reporter derived from troponin...sensor for neuroscientists is CaMPARI. This sensor switches from green fluorescence to a red fluorescent state...