...been removed/mutated to neutralize the nuclease activity. For strains that still carry this gene, there... purification protocol to remove endonucleases activity. For these reasons choosing the correct solution...any divalent cations like Mg2+, preventing the activity of DNase. While this is very important to preserve... last step will influence the stability of your sample over time and determine which experiments you can...there are usually extra steps like heat inactivation, protein degradation, and/or the use of chelating agents... impact the time you will be able to store your sample without having substantial degradation and which... DNA, it will limit what you can do with these samples. This is because divalent cations are essential...

..., easily purified protein with easily measured activity, MDH is particularly amenable to in-class labs...   It is important to note that research activities in a CURE must be authentic – students should...to take ownership of their work and become more actively involved in the learning process. As trainees ...give up the control embedded in “cookbook” lab activities (follow the recipe, and you get a predetermined...using a 6XHis tag, and compared their specific activities by measuring NADH production.   CUREs are...sciences by making a greater number of students active participants in the research process – so if this...happened in the lab, and he wanted to provide the same kinds of opportunities for his students. But he ...

...using stretches of DNA that were transcriptionally active in retinal cell types in mice. Figure 1:.... The lab deposited AAV plasmids containing the active promoters at Addgene. Performance of synthetic ...borrowed elements from a natural promoter that was active in the outer part of the retina.  Find the AAV ...human retinas Next, the 113 AAV promoters that were active in mouse retinas were injected into macaques and...different synthetic promoter driving expression of the same transgene: an optogenetic reporter, channelrhodopsin...in mice is not a good predictor for targeting the same cell class in NHPs (R=0.38) and humans (R=0.32),...), but the odds of that promoter targeting the same cell class in all three species are still greater ...

...Virus Constitutive  Active in dicots, less active in monocots, with some activity in animal cells. Ubi...eukaryotic cells. Some promoters are constitutively active and on all the time while others are more carefully...Typically contains a minimal promoter with low basal activity and several tetracycline operators. Transcription...Specific Requires the presence of Gal4 gene to activate promoter. Ac5 General expression mRNA Strong...work in prokaryotic cells and typically only in the same or closely related species from which they were ...can be turned on or off depending on what tet transactivator is used. UAS General expression mRNA Drosophila...

...delivery of Cas9 endonuclease, Cas9 nickase, Cas9 activators, Cas9 repressors, select gRNAs, and CRISPR pooled...variety of experiments, while rtTA can be used to activate expression of a gene under the control of the ...available allowing you to chemically control neuronal activity while studying your phenotype of interest. Optogenetic...Optogenetic tools for similarly controlling neuronal activity with light will be available soon. We’re also ...titering, and testing viral preps for them. At the same time, we’re developing ourselves as a knowledge ...

...binding domains and the MSN2 transactivation domain. Transcription factor activation via the appropriate ligand...that TRIM21 overexpression does not aberrantly activate the proteasome. Clift et al. tested multiple methods...variety of protein modules to repress or activate gene expression, sometimes incorporating chemically inducible...binding to the appropriate promoter sequence and activation of gene expression. In addition to plasmids encoding...study epigenetic modulation. While catalytically inactive cas9 (e.g. dCas9) has been fused to a variety ...Factors Podcast Segment Researchers from the El-Samad lab recently deposited plasmids encoding synthetic...encoding these synthetic transcription factors, the El-Samad lab has deposited plasmids encoding fluorescent ...

...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...