...biotin, biotinylation, the discovery of the biotin-avidin interaction and the uses of biotinylation in molecular...that are biotinylated. In E.coli for example, the BPL, BirA, is responsible for biotinylating only one...and/or manipulated for a scientist’s intended purpose. Two of the most common uses of biotinylation in the...first must be biotinylated. Scientists have developed several methods to chemically biotinylate macromolecules...specialized chemicals (biotinylation reagents). There are a variety of biotinylation reagents available that... and cleavable biotinylation reagents to help with the specific elution of biotinylated proteins. For ...determining which biotinylation reagent to use check out Thermo Fisher Scientific's Biotinylation Reagent Selection...

...and selected three winners in each category. Congratulations to the winners! Deck the Lab: Classic Edition...Matthijs Dorst (@MattNeuro) December 4, 2020 Congratulations to the winners! We’ll DM you with instructions...

...the process is carefully regulated by a mix of positive and negative regulators. The genome of E. coli...of a cell’s best tools to regulate DNA replication. DnaA negatively regulates its own expression, so high...proteins also contribute to this regulation in different ways: methylating newly synthesized DNA, blocking...These regulatory mechanisms allow E. coli to control chromosomal replication and balance population growth... control, such as pSC101. DNA replication and regulation in bacteria Before looking at an example plasmid...Chromosomal replication in bacteria is carefully regulated to ensure that it occurs at the appropriate time—for...chromosomal replication. Adapted from BioRender template. Since DnaA binding is necessary to initiate...

... the “Three Es of Immunoediting” nomenclature. 1. Eliminate - Latent tumors are recognized by effector...IFNγ, and colony stimulating factor 1 (CSF-1). Additionally, tumor cells recruit regulatory T cells (T-regs...to escape recognition by circulating macrophages. Tumor cells also downregulate the expression of the major... way to grow haywire, access and create more vasculature to feed themselves, use the blood stream to commute... researchers are exploring ways to bolster and modulate our immune defenses to better attack cancer. Combing...TGFβ is also known to change CD4+ Th cells into regulatory cells to suppress function. Creating Tolerance...cell-surface proteome to inhibit expression of co-stimulatory molecules needed for T cell effector function...

... sites. Regulating gene expression with truncated gRNAs An alternative approach is to modulate Cas9’s ...protospacer. The resulting double strand break (DSB) stimulates DNA repair mechanisms that can be exploited to...Podcast Intervew with Alex Chavez Traditional Cas9 regulators In addition to its utility in making targeted...to DNA, Cas9 can be reprogrammed to serve as a regulator of gene expression. Its catalytic domains can ...serve both as a means of modifying DNA and of modulating gene expression makes it an invaluable tool in...simultaneously and selectively induce both cutting and regulation of expression at different genetic loci within...tools for targeted transcriptional and epigenetic regulation have been validated with these proteins. Furthermore...

...Regulating translation is key to cellular function, especially during development or stress. With ribosome...various stimuli on global translation, but a visual technique to study translation remained elusive. Two ...easier to track translation in two different ways: by monitoring the first round of translation or by tracking...destination co- or post-translationally. TRICK: Visualizing the first round of translation While ribosome profiling...in the fluorescent signal: untranslated RNAs appear yellow, and translated RNAs appear red. To enable ...Using translation inhibitors cycloheximide and puromycin, Halstead et al. verified that translation is necessary... was double labeled, indicating translational repression. During later stages, single RFP-labeled osk ...

... in vivo-biotinylated protein (total amount) relative to proteins that have been biotinylated using less...could form tetramers of biotinylated protein. However, oligomerization of biotinylated proteins on avidin ... plasmid for improved biotinylation selectivity A common strategy to biotinylate proteins involves amine-reactive...typically required due to the relatively low efficiency of biotinylation, at least in vitro. Our solution...IPTG results in in vivo biotinylation. The co-expressed ligase will biotinylate the lysine residue in the...This allows washing away of unbound material to calculate the amount of bound ligand (after subtracting ...-in-one plasmid that provided an easy way to biotinylate a fusion protein in bacteria. Our initial attempt...

...non-mutated and methylated template is destroyed leaving behind a pool of mutated plasmids which can later be verified...information. Enzyme Dam methylation Dcm methylation EcoKI methylation ApaI Not affected Blocked ...questions is the same--methylation! Read on to learn about how DNA methylation may affect your restriction...modification systems, DNA methylation also plays an integral role in regulating genome replication, repairing...all prokaryotic DNA is methylated to the same level, the potential for methylation should be considered ...plasmids with no methylation (there are no methyltransferases in the PCR reaction). The template plasmid, on...overlapping methylation Not affected BsaI Not affected Blocked by overlapping methylation Not affected...

...appropriate regulation. To avoid these problems, it is also possible to isolate a phosphorylated intermediate...modified through phosphorylation (1). However, the expansion of studies on protein phosphorylation in an array...databases of post-translational modifications, identifies a staggering ~250,000 phosphorylation events in the...charge associated with phosphorylation. However, these amino acids fail to recapitulate the true steric and...for study. Moreover, the ability to isolate purified phosphorylated products usually depends on tools such...majority of documented phosphorylation sites. If knowledge of a candidate regulatory kinase is known, it’...promotes a robust mobility shift in phosphorylated, but not non-phosphorylated proteins (8). Quantifying the...

...fleeting. “What’s been shown by others is that, in a relatively short amount of time, cancers become resistant... library to include activators for more cancer-related pathways and more nodes within those pathways. ... Research View our Hot Plasmid Pages to See the Latest Plasmid Technologies ...

...dCas9 fused to a biotinylation tag expressed on a plasmid, allowing for in vivo biotinylation through a plasmid-based...the Tapscott Lab identified regulators of DUX4, a transcriptional regulator that when inappropriately expressed...fusion of dCas9 with a protein tag allows for the isolation of a genomic region of interest targeted by a ...targeting the 3xFLAG tag, allowed these researchers to isolate dCas9 with the bound genomic locus, which they ...locus. Prior to the development of this method, isolation of a specific genomic region was an intensive ...genomic region has also recently been adapted to isolate genomic loci in bacteria (Fujita et al., 2018)....2016). 3xFLAG-tag fused to dCas9, along with a biotinylated gRNA, were successfully used in this "in vitro...

...successful streak plate, first the bacteria are inoculated over a quadrant of an agar plate. Then, a stick... using the streak plate technique. Time for the #BioSci3319 #StreakPlateChallenge part 1. Lab B needs...streak plate skills? Find the chromoprotein expressing plasmids here, and check out our streak plate protocol...he knew it would be a perfect fit for the streak plate challenge. Chromoproteins are a subset of the fluorescent...quadrants are used, hopefully revealing single, isolated colonies in the last quadrant after incubation...successful for the students. “They always get a few isolated colonies. I should probably make it a bit more...transformation efficiency.  Aside from the Streak Plate Challenge, Westenberg’s course includes Winogradsky...

... ratios right, you need to do a few calculations. This calculator can do the math for you and it’s simple... download the PEI calculator for AAV packaging Use the PEI transfection calculator for AAV packaging ...spreadsheet will calculate the following μg of each plasmid needed per dish or stack. Calculations are based...corresponding column in the calculator spreadsheet: We hope that this calculator will make it easier to plan...for AAV packaging, considering using this PEI Calculator. AAV packaging typically requires transfecting...whole experiment And here’s the equation the calculator uses to give you the μl needed of each plasmid...experiments. Let us know what you think about the calculator in the comments section below. References 1....

...expression dysregulation event. Diseases are often complex, with multiple layers of dysregulation from either...isoforms, to help researchers understand this regulatory network. The transcription atlas and library...differentiation trajectories and identify both up- and down-regulated pathways. Individual cellular fates were then ...of difficulty with replicating the specific dysregulation in the disease. For example, very few diseases...either genetic mutation and/or TF dysregulation. A targeted TF screening approach can not only lead to...understanding of disease onset, but also better simulate a disease in a model system. TF screens can be...

...interest, and stimulate or manipulate responses through neurological means like electric stimulation, drug administration...response to both of these stimulations. The best part? The chemical stimulation was sucrose…..sounds like...understand my obsession with the combination of chocolate and peanut butter), but it’s an undeniably difficult...them for downstream control – this is important later! Next, additional components are added - the ‘bells... the digestion of food, while brain tissue is relatively static). To operate both types of implanted ...microcontroller, NeuroStack coordinates optical stimulation of its associated fibers across multiple channels...it - the device has a rechargeable battery, is relatively small, and can be detached easily whenever necessary...

...-induced cell ablation with MG-2I-dL5** He et al tested out light-induced cell ablation with MG-2I-dL5...control of protein inactivation/cell ablation while having limited collateral damage to bystander cells. Are...and the MG-2I-dL5** fluorogen that can be used to ablate cells in culture and in vivo.  Read on to learn...wavelength and also produce ROS - targeted cell ablation via ROS is achieved through localized expression...happens to the zebrafish’s heartbeat and blood circulation of following FAP-TAP light activation. Video:...requires mitochondrial targeting to increase cell ablation efficiency, but dL5** phototoxocity is induced...photosensitizers used for cell ablation ...

...humans. Then, one of them heard about our online platform, called Crowdfight, and filed a request for help...Crowdfight-COVID19 Crowdfight was born as a collaborative platform to help researchers working on COVID-19. Its origins...feeling helpless about the situation. But a few days later we had an idea. And this idea rapidly grew and became... became Crowdfight COVID-19, an online platform to connect researchers working on COVID-19 with scientists... designers and other professionals, joined the platform; while they are a minority, their help has been... to push certain projects. The idea behind the platform was simple: any person working on COVID-19 could...simpler and the volunteers helped with things like translations, bibliographic searches, etc.     Expanding...

...developing a moderate throughput functional genomic platform to annotate genetic perturbations that may play...attempting to characterize mutations in a pooled cell population, as mutations with strong phenotypic effects ...therapeutic interventions for cancer. This screening platform is an incredible boost to the cancer research ...

...tagless protein purification as well as reversibly regulation of endogenous signaling activity in cells.  Let...spatiotemporal resolution than pharmacological or genetic manipulation of the signaling pathway.  As a proof-of-principle...Ras and initiates the extracellular signaling-regulated kinase (ERK) signaling cascade. This signaling...results demonstrate that OptoNBs can be harnessed to regulate signaling pathways such as the Ras/Erk pathway...

...Anastasia continued profiling samples, and years later, several whole genome sequencing projects were launched...