... 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 ...

...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...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 ...frequency should be lower than that observed with NHEJ. PITCh: Using MMEJ for gene knock-in Building on the lab...locus, just upstream of a stop codon. Briefly, the PITCh vector should be constructed with 5’ and 3’ microhomology... The first two breaks are induced via a generic PITCh-gRNA; the third break by an insertion locus-specific...

...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...

... 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. PMID...

...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...-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...

...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...

...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...

...plasmids tools (such as microbial opsin and optical switch plasmids) 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...

...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...

... Tian Calcium Sensor: Twitch2B 100040 pAAV.hSyn1.Twitch2B.WPRE.SV40 Syn Twitch2B none Constitutive 1 Griesbeck...soma-GCaMP8 GCaMP7 GCaMP6 axon-GCaMP6 soma-GCaMP6 Twitch2B Red Calcium Sensors CaMPARI jRCaMP1 jRGECO1 sRGECO...

...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...