...and is active over a range of elevated temperatures (37–75 °C). T4 DNA ligase is highly active in nick... applications, there cannot be any end-joining activity, and for others, there is a need for the exclusive...ligate mismatches to a significant degree, and very active ligases, such as T4 DNA Ligase, can ligate nicks...and A:C mismatches. Thermostable DNA ligases are active at elevated temperatures, allowing further discrimination...combination of the intrinsic selectivity of the ligase active site and careful balance of reaction conditions...Bruce Wallace. "Specificity of the nick-closing activity of bacteriophage T4 DNA ligase." Gene 76.2 (1989...Watson-Crick base pair, are joined with nearly the same efficiency as a correct base pair. DNA ligases have...

...practices in the field, and even getting into some active science communication research.   This summer,... really, the possibilities are endless.    Activity Example audience  Talking about your career...defining your audience for science communication activities  Many people are concerned that by defining a...good communication is making sure everyone’s on the same page. So for our first post, I thought it best to...communication by and to scientists in (roughly) the same field, often through manuscripts, seminars, or posters...PCR test” to mean “a test that uses a biological sample to look for presence of a virus.” A scientist would...consistent and clear.   And now that we’re all on the same page, we’re ready to get into the more exciting ...

...contributed by Aliyah Weinstein Sparse Predictive Activity through Recombinase Competition (SPARC) is a fully...788679 A destabilized GFP reporter for calcineurin activity in yeast Article contributed by Leah Schwiesow...propidium iodine (PI), allowing for calcineurin activity measurements in stressed or aging cell populations...al., 2020. Find the GFP reporter calcineurin activity here Diessl et al., Microbial Cell, 2020. https...performance of each gRNA multiple times within the same experiment by counting both the gRNAs and their ...required for virulence in pathogenic fungi, it is an attractive drug target. Calcineurin reporters allow scientists...

...chemogenetics are powerful tools to modulate the activity of neurons and other brain cells. Since the opsins...receptor, one of the Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) (Armbruster et al....need to be located on the plasma membrane to be activated by the DREADD actuator after it has been administered...family of G protein-coupled receptors potently activated by an inert ligand. Proceedings of the National...protein will be expressed in the same cell, but not necessarily at the same subcellular compartment as the...

... it can rotate about 140 degrees between inactive and active conformations (Bravo et al., 2022; Sternberg...cutting efficiency and are important for robust activity in cells (Jiang & Doudna, 2017). Within an interior...non-target DNA strand is important for stabilizing the active conformation of the HNH domain, ensuring all binding...the HNH domain also allosterically controls RuvC activity through a hinge region between the two (Jiang ...RuvC can cleave DNA, which helps limit off-target activity. What next? It’s not actually clear! In vitro,...finish:   Figure 9: Cartoon summary of Cas9 activity. The apo enzyme is shown in the open conformation...a valuable laboratory tool are still an area of active research. New details are still being uncovered...

... on bacterial expression systems here) and the activity of the plant compounds on these proteins will ... be tested. Investigating changes in neuronal activity after ischemic brain injury Dr. Gohar Tsakanova...image cortical neurons and study changes in neural activity after ischemic brain injury. Additionally, optogenetic...construct plasmids containing promoter regions that activate fluorescent protein transcription in response ...robot that will “swim” in the river, take water samples at specific locations, measure fluorescent signals...

...other proteins, it may still block the function or activity of the tagged protein because of its size. This...Determining other aspects of functionality (catalytic activity or interactions) can be challenging. If a fusion... visualization of heterotrimeric G protein Gq activation in living cells." BMC biology 9.1 (2011): 32.... Jin, and Peter Devreotes. "Receptor-mediated activation of heterotrimeric G-proteins in living cells....al. "A FRET-based biosensor for measuring Gα13 activation in single cells." PloS one 13.3 (2018): e0193705...for robust measurement of Gαi1, Gαi2 and Gαi3 activation kinetics in single cells." PloS one 11.1 (2016...conclusive evidence that the fusion operates in the same way as the native protein. Although a fluorescent...

... is a cofactor that in its active form is covalently attached to the active site of key metabolic enzymes...is covalently bonded to a specific lysine at the active site of a newly synthesized enzyme. Biotin protein...polypeptides to a substrate Flow cytometry/fluorescence-activated cell sorting (FACS) In situ hybridization Methods...2388593 7. Holden, M. A., & Cremer, P. S. “Light activated patterning of dye-labeled molecules on surfaces...Wang, Y.-H., Lee, C.-Y., & Li, P.-C. “Buoyancy-Activated Cell Sorting Using Targeted Biotinylated Albumin...side chain that can be easily manipulated to make reactive species without affecting the avidin binding site...pre-incubated with free avidin can be added to your sample. Any free biotin-binding sites on this complex ...

... A, et al. Glutamate indicators with improved activation kinetics and localization for imaging synaptic...signaling receptors, but the options for measuring activity of specific RTKs are limited. In a new preprint... a modular system for monitoring specific RTK activation in vivo (Farahani et al. 2022). The authors take...phosphorylated state of an immunoreceptor tyrosinase-based activation motif (ITAM). By fusing ITAM to an RTK of interest...localizes to the plasma membrane only when the RTK is activated and the ITAM phosphorylated. To demonstrate that...pYtag fluorescent biosensors for monitoring RTK activation by shift in cellular localization. Image adapted...to the range of concentrations expected in your samples. Higher affinity isn’t always better, especially...

...knockdown, it still displays substantial off-target activity. By mutating residues in ADAR2 that interact with...the RNA substrate resulted in 15% C-to-U editing activity. They then performed sixteen rounds of directed...ADAR2DD to further optimize cytidine deaminase activity. While RESCUE can perform C-to-U edits, the authors...editing efficiency. They note that RESCUE is most active in a 30 nt guide when C or U base-flips are present...East-Seletsky, Alexandra, et al. "Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and...editing. To increase A->G editing, they employed hyperactive ADAR constructs, like ADAR2DD(E488Q). They also...deaminate cytidine and used dRanCas13b (catalytically inactive Cas13) to target the cytosine deaminase. Three...

...processes and an antitoxin that suppresses the toxin’s activity. The key to these systems is that the toxin is...death. Conversely, CcdA binds to and blocks the activity of CcdB. Applications of ccdB in plasmid cloning...continual production of MazE, the toxin becomes active. Shen Yang’s lab made use of mazF as a counterselectable...systems consist of two enzymes that have opposing activities, it is not surprising that they also play toxin-antitoxin...toxin-antitoxin system, they were able to monitor lacZ activity over 48 hours in vitro without antibiotic selection... introduce multiple genetic modifications in the same strain. MazF expression is under control of the ...

... of robust CRISPR activators We previously reported on a novel Cas9-based activator developed by the Church...facilitates robust activation of target genes (20-40x greater than standard dCas9-VP64 activators) when introduced...Although the VPR activation domain is an incredible tool for researchers trying to activate target genes,..., which then recruit an MS2-fused hyperactive variant of activation-induced cytidine deaminase (AID). ...(Q3) | December 2016 (Q3) MARCH 2016Enhancer activity mapping with STARR-seq   Although many technologies... identification and quantification of enhancer activity have remained challenging due to the variable ...alternative method, called STARR-seq (self-transcribing active regulatory region sequencing) that tests enhancer...

...These can be used to investigate the effects of activating particular signaling pathways in specific cell... et al., 2003), which can then be exclusively activated by the small molecule clozapine N-oxide (CNO) ...regions of mice, rats, or non-human primates. To activate the DREADD, CNO is delivered either through drinking...Maratos-Flier E, Roth BL, Lowell BB. Rapid, reversible activation of AgRP neurons drives feeding behavior in mice...systems typically express Cas9 and a gRNA from the same plasmid. LentiCas9-blast virus can be used to generate...

...gRNAs that were used for activation in a human system, search for ‘sapiens activate’ to filter the table ...applications we currently have data for are: cut, activate, interfere, visualize, nick, purify, tag, scaffold... consider testing multiple gRNAs targeted to the same locus to ensure their effects are specific to that...

...phage display assays, ubiquitination activity assays, toxin activity, small molecule binding, growth rate...discover unexpected epistatic changes in protein activity. See Natarajan et al for an example of this. .... Stephany, and Stanley Fields. “Measuring the Activity of Protein Variants on a Large Scale Using Deep...These enzymes, Nt.BbvCI and Nb.BbvCI, recognize the same restriction site, but nick opposite strands of DNA...: The second mutant strand is synthesized in the same manner as the first mutant strand, but a universal...followed by the top strand. Both produced about the same number of positive clones. See Supplementary Note...primers? The key is to design primers that are the same sense as the strand of DNA degraded in the first...

... Cas9 activity in my stable A549 cell pools. Using this virus, it’s expected that Cas9 activity, guided...green despite having active Cas9 (see tip 2). Pro-Tips 1. To quantify Cas9 activity using this assay, you...Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Doench JG, Hartenian ...described in Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9...make my first CRISPR/Cas9 gene edit. I wanted to inactivate the gene BRAF (a kinase implicated in several... bottom of the gel in the BRAF gRNA-transduced samples (data not shown). Pro-Tips: 1.Designing primers...

...where it is cleaved to generate an active enzyme. Pho8 phosphatase activity or the molecular weight shift ...degraded) and it is slow. Alkaline phosphatase activity: In yeast, the PHO8 gene encodes a vacuolar alkaline...secretory pathway, and then cleaved to generate an active form of the protein. To monitor autophagy, the ...induced and quantified by measuring the amount of radioactivity in the culture supernatant (this is indicative... practice to compare degradation rates between samples treated with or without an autophagy inhibitor....expressed. Pho8Δ60 localizes to the cytosol in an inactive form unless autophagy is induced. Then non-selective...

...Addgene. 4. Kiani, Samira, et al. “Cas9 gRNA engineering for genome editing, activation and repression.”...induce genome editing. If used with catalytically active Cas9, the short, CRISPRainbow gRNA will permit ...been previously described for transcriptional activation/repression and genome editing. Ready to make...When Using Fluorescent Proteins Control Protein Activity with GFP Resources on Asddgene.org Find Fluorescent...

...in the helical structure of spider silk, or the activity of a protein in a synapse. What kinds of tools...to measure specific small biomolecules or gene activity are often intramolecular probes, as the linker...protein-protein interactions, measure the concentration or activity of small molecules, detect cellular processes ...spring”) (Meng et al., 2008), and monitor neuronal activity (voltage sensors), to name a few.   Find Many ...contain the donor and acceptor fluorophore on the same protein and are useful when a process affects the...

...optical stimulation will be erratic, opsin activation/inactivation inconsistent, and behavioral results impossible...each opsin variant has slightly different activation/inactivation kinetics. For example, the light-gated ...receive sufficient quantities of photons to become activated. Light is able to penetrate brain tissue a short... Britt JP, et al. Use of channelrhodopsin for activation of CNS neurons. Current protocols in neuroscience...be at least 85% of the initial laser output. The same considerations apply to the polishing and preparation...