...However, retroviral vectors can still direct the translation of proteins because they integrate this reverse-transcribed...then use cell machinery for transcription and translation. Here, we will discuss several aspects of the...The genome of viral tools are often not virology related at all, and you will instead get expression of ... Cullen, B. R. (1992). Mechanism of action of regulatory proteins encoded by complex retroviruses. Microbiological... sarcoma virus: purification of the virus and isolation of the nucleic acid. Proceedings of the National...Initiation of synthesis with 70 S viral RNA as template. Journal of Molecular Biology, 79(1), 163–183....

...more focused format, where all students work on malate dehydrogenase (MDH), a citric acid cycle enzyme...recently purified MDH to study the impact of phosphorylation on the enzyme’s structure, function, and interaction...offering excellent training in improvisation and the latter making him a more effective motivator (“be like...conducting NIH- and NSF-funded research on planarian flatworms both in and out of the classroom.   References...characterization of a fusion protein: Histidine tag, malate dehydrogenase, and green fluorescent protein. Biochemistry...laboratory – a semester-long research approach using malate dehydrogenase as a research model. Biochemistry...

...vector - overall, I spent two years testing and reformulating our AAV construct to optimize performance in...Working at Addgene has shown me this was far from an isolated case. Our scientific support team answers hundreds... AAV questions every year on a range of issues related to AAV vectors, many from people trying to answer.... (2020). Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally...its occurrence during vector purification and formulation. Molecular Therapy, 12(1), 171–178. https://doi.org...Researcher Perspectives on Data Sharing in Deep Brain Stimulation. Frontiers in Human Neuroscience, 14(December...

... science.                    Two of the platforms we use at Addgene: YouTube, for longer educational...octogenarians is going to be a hard sell!) Digital platforms often have demographic data of their users readily...geographic location. For example, not all social media platforms are available or used in all countries; not all...the limitations and potential of your preferred platform before you start can save a lot of trouble in ...requirements.  No visuals. Variety of hosting platforms can splinter audience. Primarily digital distribution...communicators. Well-developed content creation platforms.  Algorithms can make it difficult to reach people...

...GFP derivatives, but are instead related to the dsRed protein isolated from Discosoma sp. Similar work ...common fluorescent proteins and their mutations relative to wildtype GFP. Although not listed here, many... fluorophores Fluorescent Protein Mutations Relative to Wildtype GFP EGFP F64L; S65T EYFP S65G;...that separates mixtures of cells into distinct populations based on fluorescent signal. Thus, FACS can be...has also been used to identify particular cell populations in drug screens, to visualize micrometastases...

...receiving the translation, Addgene contracted with a professional scientific translator, L. Denise Roche...101 eBook is now available in Spanish!  This translation was gifted to Addgene by Daniel Mejías Sánchez...Roche, to edit the translation. We are pleased to announce that CRISPR 101: Una herramienta de edición ...  Many thanks to:  Daniel Mejías Sánchez, PhD, translator  Denise Roche, PhD, MD, editor, ldeniseroche@...

...the ratio of genome-containing viral particles relative to the total number of viral capsids (which can...vectors consist of full particles (white arrowhead) relative to empty particles (black arrowhead). Scale bar...measured by qPCR.   Physical titer is typically calculated by two popular PCR-based methods: Quantitative...is a factor in AAV titration since the starting template is the purified viral vector solution (which contains...be mitigated by using purified viral DNA as the template instead of the intact AAV particle. Finally, ...absolute titer values may not be as important as the relative titer values. Finally, because physical titer ...

...L-arabinose) without signs of toxicity or burden relative to a fluorescent protein control. We also added.../L yeast extract, and 5 g/L NaCl) or on LB-agar plates containing glucose and L-arabinose at 37oC for ... arabinose and colonies on arabinose-containing plates will be visibly white, and gas vesicles in cells...from left to right: colony whiteness on inducer plates, white inclusions in cells with GVs via phase contrast...heterogeneity required single cell screening to isolate a clonal cell line capable of producing ultrasound...homogeneity, as the cells could be batch-sorted to isolate a polyclonal cell line with strong gas vesicle ...

...space — developing tools for cellular targeting. Lately, they’ve been working on two tools that expand ...safe to say the sample had their attention! They isolated DNA from the sample, amplified the CRISPR locus...most common RNA targeting system, Cas13, the "collateral" activity of the enzyme can lead to off targets...this information to identify a large variety of related bacteria with similar Cas systems. Some of these...knockdown efficiencies. The ability to identify related Cas systems opens up the potential of further development... also compatible with both AAV and adenovirus templates. The system, which is larger than Cas9, can also...

...Transcribing and translating a protein fluorophore also takes time - bypassing translation altogether with...affinity, were eventually called aptamers, from the Latin aptus, meaning “to fit”. SELEX could be used to ... protein fluorophores and quenchers are still relatively bulky. One of the advantages of oligonucleotides... context of a paired fluorophore protein. Years later, Samie Jaffrey’s laboratory took advantage of their...hydroxybenzylidene imidazolinone (DFHBI) with relatively low fluorescence in vivo, the Jaffrey lab used...

...undergo post-translational modification with fatty acyl chains. For instance, myristoylation requires an...Also, in cases where the protein of interest is relatively small, adding GFP may alter diffusion rates. ...Although bioluminescent probes are usually dim relative to fluorescent probes, recent engineering efforts...an N-terminal consensus sequence (MG-) and prenylation requires a C-terminal consensus sequence (-CaaX... that the N- and C-terminus of GFP itself are relatively close, thereby minimizing the disruption of the...

...cells with exogenous biotin. After a few hours biotinylated proteins can be captured on streptavidin affinity... on neighboring proteins resulting in their biotinylation. The advantage of APEX is its speed, labeling...biotin catalytic domain to allow for promiscuous biotinylation. Compared to BioID, BioID2 exhibits enhanced...ways to detect RNA-protein interactions that can regulate multiple biological functions. RapID (RNA-protein...Any proteins that bind the RNA motif will be biotinylated.  Find more details about RapID! EXCELL: Proximity... Plasmid 101 Protein Expression Plasmid 101 Biotinylation Other resources Proximity labeling review:...

...task would take longer than I thought, I’d be too late to meet up, and I’d just end up spending more time... friends, but didn’t realize it until it was too late. I didn’t spend enough time thinking about the possibilities...while the longer tasks will give you larger payoffs later. Don’t be afraid to accomplish some quick but moderately...assess whether that time was sufficient for the task later. If not, then you should definitely allot more time...friends and come back to the lab to split my cells later. Eating with friends has a somewhat immediate deadline...may have prevented me from splitting cells until late in the day anway. There are a few things I’ve found...

...year. Feel the love with MTRIA sensors One of the latest additions to Addgene’s catalog is a new class of...domain of ZAP70 (ZtSH2) which binds only the phosphorylated state of an immunoreceptor tyrosinase-based...only when the RTK is activated and the ITAM phosphorylated. To demonstrate that pYtags can be generalized...smell the flowers - or rather, the lactate! The latest biosensor from Gary Yellen’s lab is LiLac, a fluorescence...concentrations of lactate. The microfluidic screening platform developed here is also a promising approach for...other targets. Figure 6: A) Schematic of BeadScan platform for screening candidate biosensor variants for...

...editing system is regulated not only temporally with doxycycline, but it can also be regulated spatially with...these base editors Spatially and temporally regulated AAV-based genome editing In 2016, Jonathan Ploski... mediated genome editing system that could be regulated by doxycycline. Now, they’ve developed another... that CRISPR efficiency can be controlled by manipulating complementarity between the gRNA and target....

...caused this glowing phenomenon wasn’t until much later when, in the 1960s, Osamu Shimomura studied the ...Washington state. Using these samples, he was able to isolate two proteins from the jellyfish’s photoorgans; ... absorbs that blue light and glows green. In the late 1980s, another researcher, Douglas Prasher, got ...expressed GFP in exogenous organisms (E. coli, and later C. elegans) for the first time. After Chalfie’s ...fluorescent proteins like GFP really are. In this compilation we’ll take a closer look at how they’re used,...

...react and learn. All of this requires genes to be regulated dynamically.” To understand how that works in ...other brain functions, Konermann needed tools to manipulate the expression of specific genes in the brain...and genetically target them to specific cell populations. They demonstrated use of the LITEs to control...transcription in as little as 30 minutes after light stimulation. Under those optogenetic controls, gene expression...chromophores flavin-adenine dinucleotide and methyltetrahydrofolate, which are universally abundant. Second...

...generate a knockout cell line, with the goal of isolating single cell clones to use for multiple downstream...associated with an elevated mutation burden. The CRISPR platform must be optimized to reduce off-targets and patient...Hopefully you can understand that depending on your platform, off-target effects should impact your experimental...major con is that binding by Cas does not always correlate with catalytic activity at those sites, especially...editing events will be on target. Thus, if you isolate/sequence/whatever your editing event outcome is...line to perform downstream characterization of, isolating 2 or 3 distinct clones to perform key experiments...

... colonies to isolate the monomer Sometimes a plasmid can exist in a bacterial population as a mixture ...growing the transformed bacterial culture into the late stationary phase. Growing the culture at a lower...detection methods described above can help ensure the isolation of the monomeric version of the plasmid. Linearize...ligate the plasmid If you are having difficulty isolating a plasmid monomer from a plasmid sample, or if... CP, Carnes AE. Generic plasmid DNA production platform incorporating low metabolic burden seed-stock ...

...long been extremely useful in the lab – they are relatively small and easy to work with, making them the ...much genetic and physiological data have been accumulated over the years using them. Indeed, the future...work is bright as it is now easier than ever to manipulate the mouse genome using CRISPR/Cas9. Similar ...would be great if we could just go in and quickly manipulate individual mouse genes and study their function...successful in my first attempt at using CRISPR? Later on, I found out that my experience was shared by...s41467-017-02706-7 Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-Step...