... 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...

...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...

...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...

...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...

...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...