... RNA targeting system, Cas13, the "collateral" activity of the enzyme can lead to off targets and result...protein, and a guide RNA. The enzyme has dual activities: processing pre-CRISPR RNA into mature CRISPR...:spacer duplex without detectable non-specific activity. Its major advantage is the significantly reduced... the cell’s intrinsic machinery being already activated to work. (It was also found to be independent ...thousands new integrases with efficient integration activity in human cells. Moreover, they optimized the specific...engineering, enabling even more efficient integration activity. They now had a system that could integrate genes...lab often tests samples of new bacteria from anywhere they can find, whether the samples are from a rain...

... T cells activated ex vivo are also contestants for cell-based immunotherapy. Using activated DCs ex vivo...from attack. It is a region of incessant chemical activity, and consists of other cell types, especially ... immune cells that restrict the production and activity of other effector immune cells. TGFβ is also known...death ligand (PD-L1) that inhibits the cytotoxic activity of CTLs and NK cells. These cells would normally...signaling cascade, where lack of oxygen in the tumor, activates hypoxia inducing factor (HIF-1), which promotes...direct proximity to the tumor; chemokine-aided activation of APCs produces modified dendritic cells (DCs...when strengthened through efforts to boost the activity of the immune system, the therapeutic ability ...

...enriching your active RNAi subpopulation can be easily achieved through fluorescence-activated cell sorting...your gene or genes of interest to trigger RNAi activation. A typical RNAi experiment can be divided into...phenotype. Although all these steps are areas of active development, the first step necessarily dictates... RNA) and the initial state of the dsRNA upon activating the RNAi machinery (long dsRNA, typically hairpin... mammals are available at Addgene. Identifying actively silenced plants Once you’ve introduced your RNAi...RNAi into your organism, how do you identify the actively silenced ones? Here, I’ll cover some screening...benefits from an easy method to identify and quantify actively silencing plants. Non-destructive methods, most...

...phosphomimetic mutations on the activation loop do not fully activate MEK1. In fact, replacing just one...MEK1 (5). MEK1 is inactive without requisite dual phosphorylation events on its activation loop, but can ... of any of these phosphorylation events on the activity of a given protein? Classical methods for studying...lack knowledge of the regulatory kinase, access to active kinase for the reaction, or have difficulties with...can be made constitutively active through phosphomimetic mutation of these residues. Due to the limitations...pSer/phosphomimetic dual mutant) elevated kinase activity over 70-fold over dual phosphomimetic mutation... purified from the system, researchers can run samples on SDS-PAGE gels containing Phos-Tag, a small molecule...

...Dronpa domains flanked either side of an enzyme’s active site. In these fusions, ultraviolet light exposure...the Dronpa domains thereby occluding the enzyme active site and shutting off the enzyme. Later exposure... amino acid loops on either side of an enzyme’s active site and still block enzyme function upon exposure...variety of enzymes and proteins with inducible activity and recently presented her work at the 2017 Winter...down the HMO of interest, the lactose released activates expression of GFP from the lac operon. GFP expression...results in an increase in fluorescence of cells that actively produce the HMOs. Fatima has demonstrated the ...to determine the amount of HMO originally in the sample. The second sensor is a fusion protein consisting...

...Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA Targeting RNA Targeting...protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both the...

...Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA Targeting RNA Targeting...protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both the...

... make this STEM activity fun,” but rather “let’s see how we can make this fun activity relate to STEM....their expertise with the kids through some fun activities. If your school doesn’t have a Math & Science...parents from any background, it really helps if activities are run by parents who can answer some of the... a lot of kids, you’re going to want a lot of activities (try for a dozen!). The best approach may not...STEM.” Figure 1 shows the activities we had for my school’s Math & Science Night. There are plenty of places... places to get ideas for science activities. I did some crowdsourcing on Twitter and got lots of suggestions...Steam Powered Family  Biology and Chemistry activities from Quatr.us Foldscopes Steve Spangler Science...

...dip in plasmid request activity around March. A rough estimate shows that activity level in March was around...the effect of COVID-19 on worldwide scientific activity. However, this type of open-ended question is ...plasmid requests from Addgene, as a proxy for global activity in our particular scientific subfields.  Plasmid.... Values are the inferred percentage of normal activity (the average rate of unique PIs/labs placing orders.../15/20 (the supposed end of lockdown), request activity appears to return to normal. The model fit these... compatible with an infinity-fold reduction in activity due to COVID-19 to zero. Thus, depending on when...our analysis of COVID-19’s effect on scientific activity. The effects of COVID-19 on many other scientific...

...binding of an aptamer to its target small molecule activates the fluorescence of the small molecule. Fluorescent... metabolite binding to the riboswitch instead activates Spinach fluorescence. In the search for something...directed evolution. First, a pool of RNAs that activate the fluorescence of DFHBI were identified using... transformed, and then sorted by fluorescence-activated cell sorting (FACS) in the presence of DFHBI to...2017). Corn naturally dimerizes, and this dimer activates the fluorescence of 3,5-difluoro-4-hydroxybenzylidene-imidazolinone... quantify the effect of mTOR inhibition on the activity of RNA Polymerase III. Red and orange fluorescent...Katherine Deigan, et al. "Structural basis for activity of highly efficient RNA mimics of green fluorescent...

...Rinehart lab synthesized the activated form of human mitogen-activated ERK activating kinase 1 (MEK1) with either...Opto-RTKs have a similar level of background activity and activation as their wild-type counterparts. The Janovjak...optogenetics guide. More optogenetics tools – light-activated receptor tyrosine kinases Receptor tyrosine kinases...engineering approach, they designed Opto-RTKs, which activate signaling cascades on exposure to low-intensity...clonogenic neural stem cells concomitant with activated DsRed2 expression. Brown et al., Genesis 2014...first-generation APEX suffered from inconsistent activity levels and limited sensitivity under the stringently...distributing his Designer Receptors Exclusively Activated by Designer Drugs (DREADD) plasmids on his own...

...detect PKA activity can be generalized to measure phosphatase activity, second messenger activity, and protein-protein...biosensors for live-cell imaging of biochemical activity Article contributed by Angela Holmes Jin Zhang... imaging of the dynamic changes in biochemical activity within the cell. Their Fluorescence fLuctuation...tracked protein kinase A (PKA) activity at the plasma membrane in living cells using a construct in which...different time points, the authors tracked protein activity while also capturing information about spatial...living biosensor for measuring caffeine content in samples. The Barrick lab and the UT Austin undergraduate...fluorescence intensity due to interactions with photoactivatable fluorescent protein Dronpa. The authors tracked...

...Mobile CRISPRi blog post. CRISPR activation (CRISPRa) While the activation of transcription is a more challenging...required for the activation of sigma-54 type promoters. The system was functional in activating promoters in...Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA (2013) Programmable repression and activation of bacterial...polymerase, the lab   successfully repressed or activated transcription of select genes in E. coli. Furthermore...Streptococcus thermophilus, Cas9sth1 as a robust and highly active enzyme for transcriptional silencing Mycobacterium...controlled, Tet responsive promoters to avoid leaky activation of the system. Mobile CRISPRi for Gram-negative...SoxS based CRISPRa In the search for a CRISPR activation method in E. coli, Jesse Zalatan’s lab used an...

...outside of the mitochondria, the same inflammatory response can activate. These types of cellular dysregulation... nucleic acid can build up. The foreign dsRNA activates the innate immune response machinery of the host...molecules designed to degrade mRNA transcripts. RNAi activates the RNA-induced silencing complexes (RISCs), which...consequences arise. dsRNA An immune response is activated if and when dsRNA levels reach a given threshold...reach this threshold and thus do not typically activate an inflammatory response. However, if dsRNA sensors...systems can lead to local dsRNA production. In the same vein, genotoxic stress, such as chemotherapy treatment...

...Di Ventura. It relies on the sugar arabinose to activate transcription by causing conformation changes ... one that represses transcription to one that activates transcription.However, this system lacks spatio-temporal...light, the VVD domains of the BLADE dimerize and activates transcription. The team created two constructs... cannot do with AraC,” says Di Ventura. BLADE activates transcription with the flick of a light switch...images, you can see just how spatially precise the activation is.   Figure 2: A bacteriograph of Michelangelo's...References Romano E, Baumschlager A, Akmeriç EB, Palanisamy N, Houmani M, Schmidt G, Öztürk MA, Ernst L, ...

... understand their activity and visualize the effects of alterations to this activity. Learn more about...protein-protein interactions, visualizing promoter activity, and much more. Read on to learn more about GFP...additional co-factors, substrates, or enzymatic activity – it only requires the presence of oxygen during...almost completely required to maintain fluorescent activity; very little truncation is tolerated, however,...conjuction with FACS (see below). Fluorescence-activated cell sorting (FACS): This is a type of flow cytometry...developmental stage at which these promoters are active. Further, GFP can label transgenically modified...that of the gene of interest, or expressed from the same transcript as the gene of interest, but after an...

...characterization of two Cpf1 orthologs that display cleavage activity in mammalian cells. Like Cas9 nucleases, Cpf1 ...see if any Cpf1 nucleases would exhibit in vivo activity in mammalian cells. From 16 diverse Cpf1 candidates... et al. found two that display robust cleavage activity similar to that of Cas9. These two nucleases, ...editing efficiency of two Cpf1 orthologs known to be active in mammalian cells, LbCpf1 and AsCpf1. In both ...-base sequence. Double mismatches ablated Cpf1 activity, except when they were present in the 3’ end of... as Kleinstiver et al. saw no decrease in Cpf1 activity with 4-6 base deletions at the 3’ end of the target...found that both orthologs show lower off-target activity than that previously observed with SpCas9. Kleinstiver...

...and mCherry fluorescence is activated when APOBEC-Cas9n-UGI-mediated activity edits out a frame-shift mutation...this episode of Hot Plasmids! Light controls the activity of a microtubule-associated protein to regulate...quantification of base-editing and Cas9 cleavage activity Article contributed by Angela Kaczmarczyk  Listen... utility in quantifying multiple gene editing activities. Click here to find the ACE reporter plasmid...and as a consequence, EB1 protein function is inactivated by the separation of its synergistic domains....imaging with broccoli aptamers or MS2 tags using the same number of fluorophores. You can find plasmids containing...

...genome editing tool. However, Cas9’s endonuclease activity can be limited by the need for a specific PAM ...SpCas9, they also fused the SpCas9-NG with the activation-induced cytidine deaminase  Target-AID-NG, which...are ion channels that can be used to silence the activity of neurons. Earlier this year, the Hegemann lab...valuable optogenetics feature when studying neuronal activity. The researchers identified MerMAIDS from their... the Phillips-Cremins lab developed the light-activated-dynamic-looping (LADL) system. The group fused...SpCas9) requires an NGG PAM to recognize the target. Osamu Nureki’s lab engineered SpCas9 (SpCas9-NG) with ...

...roseus, but processes specific to photosynthetic active tissues cannot be studied in hairy roots, as functional...been transformed. But caution: check for GUS-like activity of your tissue. Run a control with Agrobacterium...gene since plants can have endogenous GUS-like activity (Hu et al., 1990). We commonly use pSB161 - pL2...therefore good temporal reporters to measure promoter activity, for example. Useful positive controls could include...115. Hu, Ching-yeh, et al. "Intrinsic GUS-like activities in seed plants." Plant cell reports 9.1 (1990...thanks to our guest blogger Samuel Mortensen from Northeastern University. Samuel Mortensen is a PhD candidate...This post was contributed by Samuel Mortensen, a PhD candidate at Northeastern University. Working with...