...years since its discovery, researchers have done significant work to improve the Adv genome’s utility in research... quickly due to immune response against AdV. Importantly, recombination-competent adenovirus (RCA) can...formation assay on A549 cells . Alternatively, antibody-based assays are commercially available for faster...more complex and the presence of helper virus contaminants have slowed the use of this system. New and ...detailed instructions on how to generate the final recombinant adenoviral plasmid construct, please see Addgene... references 1, 2.*Pro-Tip*: once the correct recombinant pAdV plasmid is identified, re-transform into...2-3 weeks) - Here you’ll produce the primary recombinant adenoviral stock (rAdV-S). HEK293 cells are transfected...

...degradation of caffeine (1,3,7-trimethylxanthine) and its conversion into xanthine. The majority of this operon...enzyme required for conversion of xanthine into guanine, and without xanthine supplied in the media, these ...desired due to its very low quantum efficiency. ShadowY has a lower quantum yield and higher absorption...material for the bioproduction of therapeutic methylxanthine drugs like theophylline. To facilitate future... glutathione S-transferase homolog from a Janthinobacterium sp. Moreover, a caffeine “addicted” E. coli... of this knockout strain can be rescued by the xanthine produced via complete demethylation of caffeine... assays. ShadowG is darker than other dark YFP mutants, it reduces the risk of spectral contamination ...

...seriously hinder gene therapy outcomes. Anti-AAV antibodies developed by the immune system – patients...To date, gene therapy has been used to treat 10 infants with this disease. To do so, their T-cells were...and the T-cells were transferred back into the infants. Almost all patients have achieved persistent immunological...immunogenic profile. AAV was first discovered as a contaminant of adenovirus preparations. It is an icosahedral...disease, and HIV. A notable landmark study showed significant clinical efficacy against a form of blindness... of LCA and mutations in RPE65 (n=3), showed significant improvements in vision over a period of 1 to ...infection. For example, prevalence of neutralizing antibodies against some AAV types, AAV1, 2, 3 and 5 are ...

...to solve, we want to focus our research on worm locomotion, but our worms are constantly moving so we ...complexity using existing C. elegans mutants. For instance, we use a mutant known as CX51 that exhibits motor...real mutant worm. Like many academic labs, we have the capability to study many different mutants because...educational institution meaning that we can obtain known mutants at $7.00 a strain. Makerspaces like ours can also...model  so that we can use CRISPR/cas9 to build the mutants, analyse their motor behaviour, and ultimately ...month - with only occasional supplementation with grant funds, money is always tight. Of course, unlike ...monitoring homemade microfluidic chips using the fantastic optics and fluidics platform it contains. Biotech...

...transform your resultant plasmid libraries into chemically competent E. coli and analyze transformants for appropriately...characterization, these proteins could function as surrogate antibodies for identifying and characterizing sugars and...Essentially, any method that results in the bulk of resultant fragments falling between 1 and 3 kb results in...culture, dilute and plate the complete library of transformants for single colonies (this usually requires 10...DNA directly from the colonies. Transform the resultant plasmid DNA into E. coli BL21 (DE3) cells (these... protein production), then dilute and plate transformants for isolated colonies (150-200 per plate) across...then be exposed to a lysis buffer to release recombinant proteins directly onto the nitrocellulose for...

... used to identify antibiotic resistance genes. Genes that are targets of antibiotics are often essential...for CRISPRi transfer Mobile-CRISPRi offers two advantages over existing bacterial CRISPRi methods. First...customization with strain-specific promoters, antibiotic resistance selection markers, gRNAs, and dCas9...downstream of the glmS gene in the recipient's genome. Antibiotic plates lacking DAP select against donor bacteria...The ICE integrates into the trnS-leu2 locus. Antibiotics select for recipient bacteria with an integrated...experiments where maintaining library diversity is important, while lower transfer rates are only suited for...researchers to study if that gene is a target of an antibiotic. For example, let’s take a look at the essential...

...to try developing a variant with an enhanced response. Evolution of a new variant Maria and the team set...project, Maria’s screens of AsLOV2 variants showed some significant gains! After a few more rounds of ...mutagenesis and screening, the EGFP and mScarlet variants were showing no obvious signs of improved response...the magnetic field (Figure 2); they named this variant MagLOV. Andrew was thrilled to see that MagLOV’...: Fluorescence intensity and ΔF/F of MagLOV, a variant of AsLOV2 with mutations C450P, L496V, Q513K, G528K... Andrew and Maria expect that further-improved variants of MagLOV will be possible with other engineering...other researchers from a range of fields, like quantum chemistry and protein biophysics, as well as inventors...

...noncovalent dimers. This property of EGFP can lead to significant artifacts. If you're using green fluorescent ..., and behavior of a POI. As an investigator, you want a fusion tag to be inert, to not produce artifacts...making FPs monomeric, yet many investigators remain ignorant of FP dimerization. Equally problematic, several...its molecular affinity, termed the dissociation constant or Kd, and its concentration. The smaller the ...volume. In many biological research situations, the relevant volume is that of the cell. For the physical behaviors... Fusion of an FP to a transmembrane protein significantly increases the effective concentration of the...biosensors were made with CFP and YFP, cyan and yellow variants of EGFP equally capable of dimerizing as EGFP....

...example we’re going to be looking for recombinant monoclonal antibody plasmids from the James Trimmer Lab...better. Here, I wanted to find a specific plasmid from the NeuroMab collection for an antibody against PARIS... terms used, so you will only be shown filters relevant to your current search. We’ll take a closer look...look at filters in a moment. First, I want to highlight the convenient ways our new search tool lets you...distributing 94 plasmids expressing validated antibodies for neuroscience research (see the NeuroMab Collection...plasmid that would be useful in an experiment, and want to see if someone has already created and deposited...and filters change as we now only show options relevant to your current search terms and potential results...

...either transcript variant led to robust and specific upregulation of the targeted variants without inducing...activation? To summarize, CRISPRa uses a nuclease-dead variant of Cas9 (dCas9) and engineered combinations of ...Figure 1a for the transgenic construct). More importantly, the expression of this construct is Cre-dependent...used their optimized vectors to highlight a key advantage of CRISPRa over traditional cDNA overexpression... gene isoform individually. To highlight this advantage, they selected the immediate-early gene Bdnf, ...inducing expression of the non-targeted variant. Moreover, isoform-specific expression of Bdnf resulted in... between the Bdnf I and Bdnf IV overexpression variants were other immediate-early genes associated with...

...genes important for mesoderm differentiation Now, let's turn tinman and bagpipe, two genes important for...protein Groucho, a transcriptional co-repressor important in development. Groucho family proteins are found... in neural development, sex determination, and anterior-posterior segmentation (Jennings and Ish-Horowicz...Dana Farber Cancer Institute! Sonic Hedgehog, an important regulator of embryonic development The largest...video game character but rather to an extremely important gene in animal development. To understand where...name but some of our Addgenies had an idea: sqh mutant fly ovaries had “squiggly” actin fibers that resemble...spermatocytes to enter meiosis. Testis of cookie monster mutant flies are filled with early mitotic cells as well...

...aptamers hold a few important advantages over their amino acid-based counterparts, antibodies and enzymes, such...applications, including detection of environmental pollutants, drug discovery, and disease diagnosis. As biosensors...commercial synthesis (those of us who have used antibodies for diagnostics know how frustrating it can be...hybridization (FISH) where fluorophore-linked antibodies are used to visualize RNA. Low signal-to-noise...only in this case ligand binding would bring the relevant ends together rather than separate them (Fig. ...quenchers are still relatively bulky. One of the advantages of oligonucleotides over proteins is their modularity... Years later, Samie Jaffrey’s laboratory took advantage of their deep knowledge of the nature of GFP fluorescence...

...expression mRNA Plant promoter from maize ubiquitin gene Constitutive  Gives high expression in plants. H1 small...look at some of its parts. Elements such as the antibiotic resistance gene and the origin of replication...with the type of RNA that needs to be made: if you want mRNA (for gene expression) you need to use an RNAP...various eukaryotic cell types (mammalian, yeast, plants, etc) require unique promoters and there is very...expression. CaMV35S General expression mRNA Strong plant promoter from the Cauliflower Mosaic Virus Constitutive...transcription, the promoter drives either the sense OR antisense transcript depending on its orientation to your...

... and not in academia is run by grant funding. Learning to write grants quickly, efficiently and effectively...apply to or offer to help with lab grant writing. You may not get the grant or your PI may edit everything...mentors don’t need to be more senior or more “important”, they just need to know you well enough to ask...they can share on their resumes have a definite advantage in our process. We see candidates who have done...thinking about future career options. 5. Write a grant - even if you don’t need one A lot of science in...valuable skill. There are many different kinds of grants that you can apply for. If you are in a lab, your...Here are some sites that have compiled lists of grants: Science: Where to Search for Funding The Ohio...

... released a new variant of jGCaMP7 (keep reading) and will be releasing more variants in the next few ...few months (see “coming soon”). The 7b variant (pGP-AAV-syn-FLEX-jGCaMP7b-WPRE) exhibits the brightest resting...processes like dendrites and axons. Find the 7b variant of jGCaMP7 in AAV1 at Addgene! See all calcium ...membrane localization and brightness. The F102G variant (pAAV.hSyn-FLEX.iGABASnFR.F102G) is noticeably ...responses to GABA. Find the iGABASnFR and its F102G variant in AAV1 at Addgene! Archon1, a voltage sensor AAV...electrical activity in the brain was measured with implanted electrodes. The Archon1 voltage reporter allows... vectors can be used to visualize cells and to quantify targeting of particular cell types. For example...

...working full-time, and have received a research grant in the last five years as polled in the American...climate change page on whitehouse.gov, freezing grants, and proposing to defund aspects of research at...scientists if the march highlights data supporting unwanted results (14). Some loss of trust, I fear, has ...m not in the job of having evidence”, scientists want to make sure having evidence is the job of everyone...while many also seek to honor and credit women, immigrants, and minorities who have had great impacts on...on your state in this map. Finally, the march is meant to inspire scientists to be more involved in their...encouraged by “Broader Impacts” requirements for certain grants) (28). As one young scientist, Matthew Niederhuber...

.... "Detection and Quantification of Ribosome Inhibition by Aminoglycoside Antibiotics in Living Bacteria...used to purify a variety of peptides including antibiotic efflux pumps and ankyrin seven-repeat proteins...expressing two genes from a single transcript, or if you want to prevent read-thru transcription from resulting...Helena, et al. "Biosafety of biotechnologically important microalgae: intrinsic suicide switch implementation... was carefully chosen to perform statistically relevant benchmarking of cell-free expression systems, ...Protein Expression Kit has been used to produced recombinant proteins for in-vitro assays: Koch, Daniel, ...By counting DNA sequence reads, FREQ-Seq can quantitatively determine allele frequencies across timepoints...

... in advance Before validating your edit, it’s important to know a few things about your target cells and...for, you are ready to begin the screening process. Want to set yourself up for a screening success? Before...clones, it’s wise to quickly assess whether a significant number of cells have been edited. For many experiments...goal is to generate a clonal cell line, so it’s important to verify that you won’t need to screen hundreds...in Sanger sequencing and a simple algorithm to quantify editing efficiency by insertion and deletion frequency...that you can easily visualize the size shift you anticipate. A good rule of thumb is to keep your product... effects  While pricier, NGS can be used to quantitatively assess genome edits in your target sequence...

...lentivirus HIV-1. Today, HIV-1 can be controlled with antiviral therapies such that the virus is undetectable ...infections not usually seen in healthy individuals. Antiretroviral therapy (ART) can virtually eliminate plasma...populations were also immune to HIV-1 reinfection. Importantly, no off-target effects were detected. Kaminski...-1 strains, Cas9/gRNA lentiviral expression significantly reduced the HIV-1 copy number, although the ...patients. Although this is an exciting finding, it’s important to note that these HIV-1 patients were ART-naive... be more successful. “Shock and kill” has the advantage of killing cells infected with HIV-1 to deplete...functional Cas9 nuclease, so there’s no potential for aberrant DNA cleavage. Direct HIV-1 cleavage allows T-cells...

..., discovered in the 1980s, is one of the most important ways to spatially and temporally control gene ...expression; these deletions are defined using 2 loxP variants (only identical loxP sites can mediate recombination...either deletion removes one of the other loxP site variants, preventing further recombination. Brainbow-1.1...with recombination events specified by three loxP variants, but the principles remain the same. The first...transient Cre recombination is required to prevent constant inversion of the construct. Sanes and Lichtman...sequences are highly homologous, preventing design of antibodies specific to each fluorophore. Third, Brainbow...mKate2.) They then successfully generated custom antibodies to each of these proteins and confirmed that ...