... for screening protein-protein interactions. In later years, improvements to yeast transformation protocols...can either be applied to the bait in a single population (e.g. transforming both fusion plasmids, or mating... strain for screening), or arrayed on a 96 well plate where each well contains a defined prey fusion.  ...former allows for screening greater numbers, but the latter eliminates the step of identifying prey proteins...protein expression and correct folding, post-translational modification, protein degradation, access to...problem is that your proteins are not post-translationally modified, co-expression of the enzyme responsible...transcription-activating function of a eukaryotic regulatory protein. Science. 1986; 231:699-704. PubMed PMID...

... to copy and modify templates (e.g. http://openwetware.org/wiki/Protocols/Template). Nature Protocol Exchange... up after trying out the new protocols.io beta platform for sharing, annotating, and storing life science...molecular cloning protocols, including making LB agar plates, restriction digest of plasmid DNA, and bacterial...bacterial transformation. And more plasmid-related educational resources can be found in our Plasmids 101 blog...creating and editing Wikipedia pages, OWW will be relatively easy for you to begin using. For the novice, ...the repository. These protocols show up on the related plasmid pages and can help scientists use these...

...in immune cells have identified many putative regulators of immunity. However, in vitro screens cannot... a Thy1.1 selectable marker to facilitate the isolation of modified cells; and (5) is compatible with ...conditions for a screen that assessed genes that regulate antigen-specific CD8+ T cell responses to a murine...responses to cancer. The screen recovered known regulators, such as Ptpn2, Pdcd1, and Zc3h12a, demonstrating...Moreover, the screen identified putative novel regulators of CD8+ T cell responses in tumors, demonstrating...Cancer and the Immune System: Deciphering the Relationship...

...within the cell. Most plasmids encode a negative regulation system involving antisense RNAs or iterons that...indirectly inhibit replication through inhibiting translation of replication machinery proteins, or directly...transcription is repressed and replication is downregulated. In the second model, termed the “handcuff” ...cases of compatible plasmids, different negative regulation systems control plasmid replication of each plasmid... the same origin of replication, the negative regulation system cannot distinguish between the different...different plasmids. The concentration of negative regulation system elements in the cell is artificially high...). However, more recent studies suggest that the late replication that occurs for some types low copy ...

...been battling for centuries. As such, resistant populations can arise very quickly following the tried and...showing bacteria evolve antibiotic resistance on a plate! Antibiotic resistance can arise, and has arisen...antibiotics These scenarios expose bacterial populations to antibiotics at unnecessary times, putting ...pressure on resistant bacteria to over-take the population and making them more difficult to treat in the...susceptible bacteria that will be more difficult to treat later. A similar situation can occur if you treat a bacterial...because scientists in the laboratory will need to isolate and grow whatever bacteria are causing the infection...before they can test for resistance. Growing and isolating the infecting bacteria can take 24 hours or more...

...transcriptional activators. These constructs allow for the upregulation of essentially any gene by fusing mutants of...cause differentiation and to reverse HIV viral latency (Bialek et al., 2016). Additionally, CRISPR-based...factors that may prevent your gene from being up-regulated. For instance, generally, the more highly expressed...Additionaly, cDNAs cannot be used to study in cis regulation and also suffer from an inability to easily deliver...CRISPR-based tools for altering DNA sequence or regulating gene expression.     References Behan FM, Iorio...Stucka R, Hauber J, Lange UC (2016) Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional...for inducible genome editing and transcription modulation. Nat Biotechnol 33:139–142 . https://doi.org/...

...Next-generation sequencing is performed on viral genomes isolated from the final AAV preparation. Sequencing results...of AAV preparations is assayed by comparing the relative stoichiometric ratios of the viral capsid proteins...SYPRO Ruby staining and the molecular weight and relative intensity of the viral capsid proteins are analyzed...Viral capsid proteins VP1, VP2, and VP3 are shown relative to the total protein present in the sample. ...are added to cells in culture. Three to five days later, cell cultures are inspected for sterility. Transducibility...-AAVrg) alone at 1.1E6 vg/mL, or both. Two days later, Cre-dependent GFP expression was detected with ...vectors consist of full particles (white arrowhead) relative to empty particles (black arrowhead). Scale bar...

...dCas13a may be useful for isolating specific RNA sequences from a population (either enriching or depleting... proteins for imaging, tracking, modulating splicing, and regulating expression of specifically targeted...Class 2, candidate 1, 2, or 3). C2c1 and C2c3 are related to Cpf1, but they require both a tracrRNA and crRNA...the spread of infection throughout the entire population. This property of Cas13a opens up the possibility...knockdown to single gRNA expression. Although the collateral RNA cleavage response is well described in bacterial...

...sessions, exhibit booths, happy hour socials, and late, late night socials. As a newbie to the SfN conference... global coffee supply… My back-of-the-napkin calculations put the daily consumption here at ~ 60,000 cups...students and postdocs, which accounts for many, many lattes and espressos. The energy boost is well needed ... Investigator Awards and we couldn’t wait to congratulate him on his successes. We've been lucky enough...SfN14 on Twitter for live tweeting updates and the latest news from attendees. And for those of you presenting...

...have similarly curated their Ebola-related content. -And the latest updates on the ongoing outbreak from... underestimate. Given all the excitement and speculation, we thought we’d do a little digging and assemble...assemble a rough guide to the science of Ebola and related resources. Ebola Basics A few basics: Ebolavirus... data on five strains of Ebola representing the latest outbreak and earlier ones, along with links to ...the current Ebola crisis, Science and Science Translational Medicine have made their complete collection...

...often requiring several rounds of selection to isolate plants with the phenotype of interest. Genome engineering... modification of known or suspected genes that regulate a desired phenotype. In fact, CRISPR has already...variants have been created in an effort to increase translation in a particular plant species or cell type (Belhaj...driven by plant-derived RNA pol II promoters which regulate expression of longer RNAs (such as mRNAs for gene...plant cytorhabdovirus as versatile expression platforms for planthopper and cereal genomic studies. New...Multiplexed Plant Genome Editing and Transcriptional Regulation. Plant Physiol 169:971–985 . https://doi.org/...(2016) Gene-edited CRISPR mushroom escapes US regulation. Nature 532:293–293 . https://doi.org/10.1038...

... target to specific subcellular regions, laser ablation can damage other cellular structures, and genetic...genetic mutations are not easily reversible. Late last year, the Akhmanova Lab published a new optogenetic...for a wide variety of experiments. Opto-RhoGEFs manipulate cell morphology and adhesion But what if you’... Taking control of a RhoGEF is a great way to stimulate and study these physical processes. Joachim Goedhart...of membrane-recruitment optogenetic tools for manipulating Rho GTPases based on BcLOV4. These single-component...membranes) (Figure 4). The authors used SuperPLD to manipulate phosphatidic acid signaling (such as Hippo, AMPK...minutes of blue light illumination, substantial PA accumulates in the plasma membranes, indicated by the PA ...

... be the hot new way to manipulate gene expression, but other gene manipulation systems remain valuable...To achieve even cell labeling, they generated farnesylated derivatives of the fluorescent proteins, which...trafficked to cell membranes. Membrane trafficking of farnesylated derivatives enables labeling of delicate axonal... default fluorescence due to the addition of translation-blocking STOP cassette immediately following ...woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), commonly used to increase transgene...and Flpbow can be used to label distinct cell populations. To decrease the animal breeding necessary to...

... more open to developing a relationship. The time to develop relationships is before you need them (well...doing interesting things. I call this building relationships because networking has gotten a bad reputation...Scientists enjoy learning new things. Building new relationships is all about learning new things from other ...networking for a job connection with ongoing relationship building through Informational Interviews. Making...is always best to meet in person to develop relationships, but if it is not possible consider a phone ...

...formed 20-40 min later. The CASANOVA system is versatile in that it can be adapted to regulate gene editing...modifications by NHEJ and HDR using split systems Later in 2015, the Sato lab unveiled a photoactivatable...opens intriguing possibilities for optogenetic manipulation of CRISPR-based systems in animal models. Use... prevent DNA binding in the dark. Upon light stimulation, the pdDronpa moieties move apart, allowing DNA...LOV2 has a C-terminal helix that unfolds when stimulated by blue light. When fused to the catalytic site...genome and chromatin architecture, it is useful to regulate the time in which dCas9 binding occurs. Bubeck...Niopek D (2020) Optogenetics and CRISPR: A New Relationship Built to Last. In: Methods in Molecular Biology...

...has the capacity to glycosylate proteins and may recapitulate the post-translational modifications added...requires a substantial amount of properly folded, relatively pure protein, but getting to this stage is often...one’s protein of interest can save a lot of time later. Protein purification specialists recommend doing...the MISTIC (membrane-integrating sequence for translation of integral-membrane protein constructs) protein...

...non-coding regulatory elements. Regulatory DNA function is typically inferred based on correlative biochemical...of the method. One observation was an inverse relationship between deletion size and frequency [3]. There...interested in exploring the function of non-coding regulatory elements in their native chromatin context. An...   Like the video? The Orkin and Bauer lab's related paper describes their methods for CRISPR design...

...asked the individuals behind some of these new platforms what their motivations were for developing these...knowledge that could help fight COVID-19. They’ve also collated a list of useful research resources.  "We realized...Canadian volunteers, and features over 200 COVID-19 related research projects, funding opportunities, and community...community response initiatives. Accelerate the translational impact of research Universities have also been...creating research synergies and accelerating the translational impact of research activities. By streamlining...research in their field including datasets and platforms for discussing new developments, and more. As ...

...organisms and cell lines. Although ChIP-seq is a relatively simple and robust protocol it does have limitations...study how differential access to transcriptional regulatory elements is controlled during development using...fused micrococcal nuclease (MNase) to specifically isolate DNA that is bound by a protein of interest. First...First, nuclei from tissue or cell culture are isolated using lectin-coated magnetic beads. Nuclei are then...MNase requires for its nuclease activity and is chelated from the reaction up until this point. The nuclease...simple lab protocols Browse CRISPR plasmids for isolating a genomic locus Browse FRET plasmids for studying...

...Stable Cell Lines with Lentivirus Isolating a Monoclonal Cell Population by Limiting Dilution Lentivirus...high Cas9 MOI considering that A549 cells are relatively easy to transduce. I ended up with too many Cas9...be uniform editing across all the cells in the population, so some of the cells will still appear green...efficiently editing, don’t expect a 100% knockout population. BRAF gRNA delivery  Overview: In practice you...on genomic DNA. The surveyor assay itself was relatively easy. The basis of the Surveyor assay is that...get an idea of the percentage of cells in the population that have an edit. I didn’t do this, but to do...