...AAV plasmids contain several important elements, but among the most important are the inverted terminal ... ITR integrity and prevent unwanted mutations?  During propagation, mutants rapidly replicate in a growth...Next-generation sequencing is also helpful in quantifying ITR mutants since analysis of individual reads allows...Adeno-associated viruses (AAV) are an important biological tool that allow scientists to express a gene...and the ITRs are retained with the gene in a recombinant AAV (rAAV) vector (Figure 1). Everything else...outside the ITRs gets left behind.    Figure 1: Recombinant AAV vector with ITRs that flank the gene cassette...modifications and types  Since ITRs are one of the most important elements of AAV and rAAV vectors, modifications...

...might want to pick a yeast strain best suited to it. Putting K. phaffii to work   The yeast giants - S....alternative model yeast strain and discuss why you may want to consider a yeast off the beaten path.   Yeast...have a human counterpart, enabling study of human-relevant pathways (Liu et al., 2017). Beyond human relevance...well as glucose or glycerol). K. phaffii is only distantly related to S. pombe and S. cerevisiae; evolutionarily...attention for its powerful promoter of a gene important for methanol usage. This gene promoter, AOX1, ..., was co-opted for the expression of recombinant proteins by the pharmaceutical and biotech industries...efficient at protein secretion, making harvesting recombinant proteins a cinch. This is because it doesn’t ...

...incompatibility with antibiotic-resistance plasmids, scientists have expelled antibiotic resistant plasmids from...encode a negative regulation system involving antisense RNAs or iterons that inhibits replication when...number in the cell falls too low (Novcik, 1987).  Antisense RNAs encoded on the plasmid either indirectly ...plasmid increases, increased amounts of these antisense RNAs inhibit plasmid replication.   Figure...incompatibility going to affect my cloning project?  If you want to clone two plasmids within the same bacterial ... uncommon, or perhaps a synthetic biologist who wants to develop a plasmid-based tool that is easy to ...Using plasmid incompatibility for therapeutic advantage Although it may seem like plasmid incompatibility...

... AAV9 mutants that had a small randomized peptide loop inserted in the AAV9 capsid. This mutant library...reach all regions of the CNS. PHP.B, a neurotropic variant of AAV9, is better than AAV9 at gaining access ...sequence of DNA and both identified lymphocyte antigen 6 complex (LY6A) as a receptor for PHP.B. Read ...PHP.B viruses. Creating PHP.B, a neurotropic AAV9 variant PHP.B’s specificity for C57BL/6J mice is likely...screened in vivo in C57BL/6J mice strains to identify variants that reached the CNS following IV injection. Compared...efficiency, but at a log lower dose than PHP.B.  A Ly6a variant is linked to PHP.B CNS permissiveness Both the ... labs determined LY6A, also known as stem cell antigen-1 (SCA-1), is the receptor used by PHP.B to reach...

...nature can inform us on how to create better recombinant vectors. In this blog post, we’ll define plasmid...harness those features to create broad host range recombinant vectors.  A plasmid’s host range is determined...phylogenetic groups (Jain and Srivastava 2013).  Recombinant vectors that are made from broad host range plasmids...host-independent   Building broad host range recombinant vectors Replicons, which consist of a plasmid...broad host range plasmids can be used to create recombinant vectors that can replicate in different bacteria...methanolicus, Methylosinus trichosporium, Myxococcus xanthus, Neisseria spp., Paracoccous denitrificans, Pseudomonas...Rhodospirillum spp., Shewanella spp., Thiobacillus spp., Xanthomonas campestris IncQ RSF1010, R300B, R1162 ...

...by guest bloggers Erik L. Snapp and Lindsey M. Costantini. "You underestimate the power of the Dark Side...non-fluorescent, and misfolded FP-fusions on quantitative imaging experiments and cell physiology in general... the cytoplasm? Or more troubling, what if a significant fraction of the fusion protein molecules fail...population is not readily apparent and can confound quantitative imaging experiments or even negatively impact...fusion based on your experimental needs. Equally important, before cloning you must consider whether and ...resident proteins within these compartments undergo significant post-translational modifications including glycosylation...misfolded dark FP fusions including erroneous quantitation of fusion protein levels. It remains unclear...

...The FLAG antibody has been well characterized and the system allows you to add this same antibody to any...known to have good antibodies. The results look almost identical. Addgene: What advantages does your system...established method called ChIP-seq. ChIP-seq relies on antibodies to recognize and bind transcription factors directly...the transcription factor. It also gives cells antibiotic resistance to neomycin so that you can select...of the proteins inside a cell have a commercial antibody against them. There’s also a question about how... solved in this CRISPR system with a universal antibody. It’s an epitope not found in human or other cells...transcription factors, but there are many reasons you might want a system like this. It’s actually surprising to ...

...Salary –not relevant to most postdocs. Commercial successes in the performing arts- not relevant at all. ...options discussed in this post benefit from the applicant thinking about the process early, keeping good... minimum (8 U.S. Code § 1184 - Admission of nonimmigrants). The foreign worker must possess at least a... cap subject H-1B (last year less than 30% of applicants received sponsorship) then he or she can start... facility management, collaboration management, grant administration, clinical operations, and so on. ...1B petitioners are exempt from the cap if the applicant works at institutions of higher education, a related...US Immigration officers use to determine if an applicant qualifies for the National Interest Waiver. The...

... our ready-to-use recombinant antibodies service, part of the Neuroscience AntiBody Open Resource (NABOR...library of antibody educational resources, including our new Antibodies 101 eBook, the Antibody Guide, and...could use the same principles to provide recombinant antibodies, another plasmid-based product that, with...front of us. So in our last celebratory moment, we wanted to walk through some of our favorite milestones...designing a website users could easily navigate. This meant we could grow accessibility alongside our repository...so easy!” — Nina Jain, Harvard Medical School. We want to give a special thanks to the early-adopter scientists...beginners” on r/labrats, Reddit.  Part of that work meant facilitating the Material Transfer Agreement process...

...are numerous GFP variants. Here I use the name ‘GFP’, which refers to mEGFP. This variant was made by introducing...contributed by guest blogger Joachim Goedart, an assistant professor at the Section of Molecular Cytology...jellyfish GFP (AvGFP) to create enhanced GFP (EGFP), a variant with improved brightness (Tsien, 1998). Another...necessary to generate the strictly monomeric EGFP variant, mEGFP (Zacharias et al., 2002). GFP needs oxygen...that generates the chromophore (Tsien, 1998). Importantly, this reaction requires molecular oxygen and ...Cranfill et al., 2016 and FPbase.org). A number of variants remain fluorescent under acidic conditions, with...N-terminus, but this hardly reduces its size. The substantial size of GFP may interfere with the activity or...

... community which is really important to her. Addgenie Shreya Vedantam with our resident dinosaur...school. QC Scientist Alyssa Cecchetelli knew she wanted to get her PhD. “Going back to school was a pretty...in Addgene since he started, said, “the thing I wanted more of [after undergrad] was to learn and speak...may open doors for you, but depending on where you want to go in your career you may not need those doors...her first job at Addgene as an Order Processing Assistant. After a few months, she left school when she ...Technician. “I was so tired of being in school so I just wanted to work,” she said. “School will always be there...mean you’ve failed. “I got a job as a research assistant in a lab at the university that I went to. Making...

...Notably, the SpRY variant didn’t display higher off-target effects than SpCas9, a pleasant surprise since... PAM requirement, and how these proteins are advantageous in today’s genome editing landscape. The need...anywhere within the RNA will do. Similarly, if you want to knock out a gene, a frameshift mutation introduced...that don’t utilize NGG   There are plenty of Cas9 variants which utilize alternative PAMs. These alternative...looking for more pan-genome PAM flexibility, you’ll want to consider one of several Cas9s that recognize ...compatibility. This alternative PAM requirement adds a significant amount of flexibility compared to a strict single-compatibility...rounds of engineering, they developed the SpRY Cas9 variant (Walton, et. al, 2020). SpRY uses NRN PAMs and ...

...recombination software looks for and quantifies the presence of recombinants in recombinase-dependent sequences...experiments to confirm their findings. But no scientist wants to repeat an experiment because of poor reagent ...production process? These questions are especially important when producing multiple vectors in parallel because...serotype of the packaged DNA and look for potential contaminants. The analysis is a 2-step process using Geneious...total reads are most likely to correspond to the contaminant. Each contig is then BLASTed against the NCBI...contaminated. For AAV-68544, the best match for the contaminant is Addgene plasmid #44362. Method validation,... were easily able to identify AAV-44632, the contaminant we mixed into the original AAV sample at 5%. ...

... to reassess what I wanted to do and so I applied for a role in Industry. I wanted to get "a real job"...then, and still feel now, that it is important to take advantage of the fact that science is a global ...staff hired in anticipation of the contract suddenly have less work to do than was anticipated and there can... Sutton, located on the edge of London to gain relevant experience. I applied for this summer position...abroad in rural Africa Once this position ended, I wanted to explore another area which I was really passionate...Procedures (SOP’s) and every item I tested was constantly tracked with barcodes and spreadsheets. It was...to be in the lab doing experiments  without the constant fear of failure because I knew the tests worked...

... tissue temperature proximal to the implanted fibers. As important as its capabilities are the practical...Sands, J., Zhu, W., Tabet, A., Manthey, M., Allen, H., Loke, G., Antonini, M.-J., Rosenfeld, D., Park, ...gut-brain axis (admittedly for self-serving reasons – I want to understand my obsession with the combination ...connection to study on a molecular level. The brain is constantly influenced by signals it receives from the entire...included in them for downstream control – this is important later! Next, additional components are added -... be, as well as other features necessary for implantation in brain tissue. The gut, on the other hand...hand, was the wild west for fiber implants. Due to the soft nature of the intestines and the requirement...

...to aid in the expression and purification of recombinant proteins. Tags for stability and solubility ...hurdles to overcome in order to overexpress a recombinant protein? It is not generally in a cell’s best...glutathione S-transferase (GST), which can be fused to recombinant proteins for one-step purification with glutathione...uncharacterized protein, or a protein for which a good antibody has not been developed (and just because your ...your protein has a commercially available antibody, that doesn’t mean it’s a good one), then your first step... an affinity tag to it. The FLAG, hemaglutinin antigen (HA), and c-myc tags have been the workhorses of...depend on your application (see table below). The antibodies available for these tags really are good and ...

...editing by APOBEC-Cas9 Though the ACE reporter quantifies APOBEC-Cas9 efficiency in real-time, it relies...Harris Lab created a panel of eGFP reporters to quantify the on-target DNA editing efficiency of APOBEC-Cas9...would restore eGFP fluorescence, making it easy to quantify single base editing efficiency by dividing the... et al., 2016). For TREE they engineered a BFP variant that undergoes conversion to GFP after being targeted...deaminase base DNA editor. Specifically the BFP mutant (BFPH66) contains a histidine at amino acid 66....changes the histidine to a tyrosine generating a GFP variant (GFPY66) that has a shifted emission spectra. Thus...the test by infecting HEK-293 cells with the BFP variant, a BE4am-Cas9 base editor, and sgRNA vectors for...

...for every application. Disadvantages include their duplicated (i.e., redundant) genome, their long three-month..., including investigations into environmental pollutants and health, embryo growth, brain function, and...straightforward husbandry and ability to produce abundant, externally fertilized, transparent progeny (Figure...visualize expression data in useful ways. Zebrafish mutants In the 1990’s, two revolutionary large-scale random... mutagenesis screens produced ~1,500 zebrafish mutants that are still used in labs across the globe (Nüsslein-Volhard...which I will come back to) is the one-eyed pinhead mutant, which regulates a developmental pathway implicated... in congenital heart defects and cancer. These mutants are used extensively to study development and disease...

...Westwood) Dodders are parasitic plants that steal all their food from other plants and are unusual enough to...efficiency to reduce the number of plants you need to grow to get a recombinant because this process is expensive...between gene function and phenotypes in diverse plants, animals, microbes, viruses, and fungi for which...next project. Gymnotiformes and Mormyridae (Jason Gallant) Weakly electric fish are excellent systems to ...are now studied in 40 labs as a great model for plant adaptation and speciation. Their phenotypes are ...great for studying parenting strategies and make important ecological indicators since species live in water...Aiptasia - algae Breviolum minutum symbionts are important models for solutions to the coral reef crisis....

...repression using Cas9 Don’t want to permanently modify the genome but still want to affect gene expression...post was originally written by Joel McDade and significantly updated in 2022 by Susanna Stroik.  The advent...your experimental target, SpCas9 and its enhanced variants, eSpCas9 and HypaCas9, are great choices. Check...regions of the genome and consider using a Cas9 variant with higher specificity (eSpCas9 and HypaCas9) ...added bonus of low off-target effects. The SpG variant, discovered by the Kleinstiver group has a PAM ...Further, this group also characterized a second Cas9 variant, SpRY, which cuts at NRN PAMs with high efficiency...that the desired edit will be performed on all relevant bases within the editing window, for example all...