...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...

... 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...

...PLAU plasminogen activator, urokinase ATF, UPA, URK, u-PA PLAUR plasminogen activator, urokinase receptor...mitogen-activated protein kinase kinase 1 MAPKK1, MEK1, MKK1, PRKMK1 MAP2K2 mitogen-activated protein ...mitogen-activated protein kinase kinase kinase 14 FTDCR1B, HS, HSNIK, NIK MAP3K8 mitogen-activated protein...Cdc42/Rac)-activated kinase 1 MGC130000, MGC130001, PAKalpha PAK2 p21 protein (Cdc42/Rac)-activated kinase... protein (Cdc42/Rac)-activated kinase 4 - PAK6 p21 protein (Cdc42/Rac)-activated kinase 6 PAK5 PAK7 p21...the immune system triggers several processes to activate cells to recognize and get rid of infectious pathogens...

...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...

... at specific loci. Activate or Repress : dCas9 fused to transcriptional activators or repressors can modulate...processed into individual crRNAs by a special trans-activating crRNA (tracrRNA) with homology to the short palindromic...for gene knockout, as well as transcriptional activation or repression. Combined with the power of next-generation...gRNAs: Truncated gRNAs display less off-target activity. High fidelity Cas9s: Three high fidelity Cas9...eSpCas9, and HypaCas9 display very low off-target activity due to rationally designed mutations. Researchers...CRISPR-Mediated Control of Gene Repression and Activation. Cell . 159(3):647-6. PMID: 25307932 Gilbert ...targeting multiple loci can be easily expressed in the same cell or organism. The ability to precisely target...

...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...

...                 Allewell NM, Sama A (1974) The effect of ammonium sulfate on the activity of ribonuclease A. Biochimica...in cells, and without something to block RNase activity upon cell lysis, RNA is degraded. To effectively...-based solutions that work by inhibiting RNase activity within cells or tissues- they don’t actually chemically... RJ, Rutter WJ (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease...tissues, cells, plants, fungi, or bacteria, keep samples cold and work quickly to mitigate RNA degradation...using DEPC-treated water, autoclave the water to inactivate the DEPC.  Ensure that plasticware or glassware...180°C for at least 4 hours.   If your final RNA sample(s) are resuspended in water or TE buffer, store...

...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 ...

...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 Yeast...